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F A C U L T Y O F S C I E N C E U N I V E R S I T Y O F C O P E N H A G E N
PhD thesis Sigyn Jorde
Functional analysis of proteins involved in actin cytoskeleton organization in Ashbya gossypii and Candida albicans
Academic advisor: Carlsberg Laboratory and Department of Biology, University of Copenhagen
Submitted: 04/11/10
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Table of Contents
1 Preface ........................................................................................................................... 3
2 List of Abbreviations .................................................................................................... 4
3 Abstract ......................................................................................................................... 5
4 Resumé .......................................................................................................................... 6
5 Introduction .................................................................................................................. 7
5.1 Fungi as model organisms .......................................................................................... 7
5.1.1 Saccharomyces cerevisiae .......................................................................................... 7
5.1.2 Comparative genomics ............................................................................................... 8
5.1.3 Ashbya gossypii .......................................................................................................... 9
5.1.4 Candida albicans ...................................................................................................... 10
5.2 Penelope Research Training Network and foci of this thesis ................................... 12
5.3 Endocytosis .............................................................................................................. 13
5.4 Protein-protein interactions ...................................................................................... 14
5.4.1 SH3 domains ............................................................................................................ 15
5.5 The actin cytoskeleton .............................................................................................. 15
5.5.1 Actin polymerization ................................................................................................ 16
5.5.2 The Arp2/3 complex ................................................................................................. 17
5.6 Polarized growth ...................................................................................................... 18
5.6.1 Polarization via Rho-GTPases ................................................................................. 18
5.6.2 Hyphal growth .......................................................................................................... 19
5.6.3 The Spitzenkörper .................................................................................................... 20
5.7 The endocytic machinery ......................................................................................... 21
5.7.1 Clathrin-dependent endocytosis ............................................................................... 22
5.7.2 Sla2 and Sac6 are key components in endocytosis of S. cerevisiae ......................... 25
5.8 Eisosomes ................................................................................................................. 25
5.8.1 Lipid rafts ................................................................................................................. 26
6 Methods ....................................................................................................................... 28
6.1 Strains and media ..................................................................................................... 28
6.2 Transformation ......................................................................................................... 28
6.3 Generation of GFP tagged C. albicans strains ......................................................... 29
6.4 Yeast Two Hybrid assay .......................................................................................... 31
6.5 β-galactosidase assay ................................................................................................ 33
6.6 Generation of mutant and GFP strains in A. gossypii .............................................. 33
6.7 Plate assays and germination of spores .................................................................... 36
6.8 Microscopy and staining procedures ........................................................................ 36
7 Results ......................................................................................................................... 37
7.1 Generation of heterozygous C. albicans strains ....................................................... 37
7.2 Localization of C. albicans SH3 domain proteins ................................................... 38
7.3 Yeast two hybrid assay ............................................................................................. 39
7.4 SLA2 is ascent from the A. gossypii genome ............................................................ 41
7.5 Generation of Agsac6 ............................................................................................... 42
7.6 Characterization of Agsac6 ...................................................................................... 43
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7.7 Deletion of LSP1, SUR7, PKH1 and YPK1 .............................................................. 46
7.8 Characterization of lsp1 and sur7 ............................................................................. 46
7.9 Deletion of PIL1 ....................................................................................................... 49
7.10 Characterization of the pil1 phenotype .................................................................... 50
7.11 Localization of AgPIL1-GFP ................................................................................... 54
7.12 PIL1-GFP does not colocalize with actin patches .................................................... 55
7.13 Heterologous expression of AgPIL1-GFP in S. cerevisiae ...................................... 56
8 Discussion .................................................................................................................... 58
8.1 Localization of C. albicans SH3 domain proteins ................................................... 58
8.2 Functional relation between genes regulating actin filamentation in
S. cerevisiae, C. albicans and A. gossypii ................................................................ 59
8.3 Vrp1-Wal1-Myo5 complex in C. albicans ............................................................... 60
8.4 Ligand binding of SH3 domains is weak ................................................................. 61
8.5 SLA2 is absent from the A. gossypii genome............................................................ 61
8.6 Agsac6 has a similar phenotype to Agwal1 .............................................................. 63
8.7 A. gossypii pil1 germlings cease to grow before reaching maturation ..................... 63
8.8 PIL1-GFP does not localize with cortical actin patches .......................................... 64
8.9 Sur7 is not necessary for eisosome formation but affects vacuolar fusion .............. 65
8.10 A link between lipid rafts and endocytosis .............................................................. 65
9 Summary ..................................................................................................................... 66
10 Prospects ..................................................................................................................... 67
11 Acknowledgements ..................................................................................................... 69
12 References ................................................................................................................... 70
Appendix I – Verification PCR on C. albicans strains ........................................................ 78
Appendix II – Verification PCR on A. gossypii strains ....................................................... 79
Appendix III – Plasmids ........................................................................................................ 80
Appendix IV – Strains ............................................................................................................ 82
Appendix V – Primers ............................................................................................................ 83
Scientific publications ............................................................................................................ 86
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1 Preface
This thesis presents the results of my PhD internship at Carlsberg Denmark. The project was
supervised by Jürgen Wendland, Professor of Yeast Biology at Carlsberg Denmark, and Steen
Holmberg at the University of Copenhagen and was funded by Marie Curie Programme. My
work resulted in three publications which are listed at the end of this thesis. I begin with an
introduction of fungal biology and the topics of this thesis and continue with a detailed
Material & Methods part. My research has focused on several topics and they are presented in
the same order in all parts. Results are followed by a discussion and a short summary and
finally I mention the prospects of what I would have done if I had only more time in the lab.
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2 List of Abbreviations
AFM Ashbya Full Medium
ANTH AP180 N-Terminal homology
bp base pair
Cd Candida dubliniensis
CFW Calcofluor White
clonNAT nourseothricin
Cm Candida maltosa
Co-IP Co-Immuno Precipitation
CSM Complete Synthetic Media
DiOC6 3,3′-dihexyloxacarbocyanine iodide
ENTH Epsin N-Terminal homology
G-protein Guanine-nucleotide binding protein
GAP G-protein Activating Protein
GEF Guanine-nucleotide Exchange Factor
GPI-anchored Glycosyl Phosphatidyl Inositol-anchored
LY Luciferase Yellow
NPF Nucleation Promoting Factor
MAPK Mitogen Activated Protein Kinase
MCC Membrane Compartment occupied by Can1
ONPG ortho-Nitrophenyl-β-galactoside
ONP ortho-Nitrophenol
PCR Polymerase Chain Reaction
PEG400 Polyethylene Glycol 400
SDS Sodium Dodecyl Sulphate
SH3 Src homology 3
TE Tris EDTA
YNB Yeast Nitrogen Base
YPD Yeast Peptone Dextrose
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3 Abstract
This thesis deals with some of the aspects of endocytosis in fungi. The human pathogen
Candida albicans and the filamentous Ashbya gossypii were used as models when
investigating some of the core mechanisms in this process. The virulence of C. albicans is
dependent on its ability to switch between yeast and hyphal growth, which is why these
dynamic processes are of special interest. A. gossypii has been used in comparison to study
the extended polarized growth in hyphae. First, a set of SH3-domain containing proteins in
C. albicans were tagged with GFP to allow for visualization and localization. Three proteins
were successfully visualized with a C-terminal GFP, namely Bbc1, Sla1 and Cyk3. Cyk3
localized at septal sites and Bbc1 and Sla1, proteins involved in endocytosis, were seen in
cortical patches. Second, this PhD thesis addressed interactions formed in C. albicans among
the core proteins in actin filament nucleation. For this we used the yeast two-hybrid assay,
which indicates physical interactions between Vrp1 (WIP homolog) and the Arp2/3 nucleators
Wal1 and Myo5 (WASP and myosin I homologs, respectively); a similar complex is formed
in S. cerevisiae. Also, functional analysis of the A. gossypii fimbrin mutant sac6 was
performed, a gene that is synthetic lethal with SLA2 in S. cerevisiae. SLA2 is essential for
endocytosis in other studied fungi but the gene is missing in A. gossypii. Deletion of SAC6
leads to impaired endocytosis, greatly affected organization of the actin cytoskeleton and
swelling hyphae when grown at elevated temperature. These phenotypic characteristics are
similar to the wal1 (WASP) mutant implying that the two proteins take part in the same
pathway. Finally, proteins of the eisosomes, Pil1, Lsp1 and Sur7 in A. gossypii were
investigated. As in S. cerevisiae deletion of PIL1 produces the more severe phenotype. Agpil1
is barely viable under the growth conditions tested in this assay but investigation of a few
germinating mutants showed no obvious defects in endocytosis or actin organization. A GFP
tagged AgPil1 localized in a punctate pattern beneath the plasma membrane but in separate
structures from cortical actin patches. The same localization was observed in all eisosome
mutants as well as in sac6 and wal1. This implies that eisosomes are not directly involved in
the endocytosis process but might have a regulatory function.
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4 Resumé
Denne afhandling omhandler nogle aspekter af endocytose i svampe. Ved undersøgelse af de
centrale mekanismer i denne proces, blev det humane patogen Candida albicans og den
filamentdannende svamp Ashbya gossypii anvendt som modeller. Virulens af C. albicans er
afhængig af dens evne til at skifte mellem gær- og hyfevækst, hvilket er grunden til at disse
dynamiske processer er af stor interesse. A. gossypii har været anvendt til at undersøge den
udvidede polariserede vækst i hyfer. Først blev et sæt af proteiner, som indeholder et SH3-
domæn i C. albicans, markeret med GFP for at muliggøre visualisering og dermed
lokalisering. Tre proteiner, nemlig Bbc1, Sla1 og Cyk3, blev visualiseret succesfuldt med et
C-terminalt GFP. Cyk3 findes på septale tværvægge, og Bbc1 og Sla1 som er involveret i
endocytose findes i aktin-klumper i hyfespidsen. For det andet behandler denne Ph.D.-
afhandling interaktioner blandt de centrale proteiner i dannelsen af aktinfilamenter i
C. albicans. Her blev der anvendt gær 2-hybrid assay, der viser den fysiske interaktion
mellem Vrp1 (den WIP homolog) og de Arp2/3-initiativtager Wal1 og Myo5 (henholdsvis
WASP og myosin I homologe). Et lignende kompleks dannes i S. cerevisiae. Derudover blev
den A. gossypii fimbrinmutant sac6 undersøgt. SAC6 er et gen, der er syntetisk dødelig i
forbindelse med SLA2 i S. cerevisiae. SLA2 er afgørende for endocytose i de øvrige
undersøgte svampe, men genet mangler i A. gossypii. Fjernelse af SAC6 fører til forringet
endocytose, forandringer i actinorganisation især i hyfespidsen og hævelse af svampetråde
ved forhøjet temperatur. Disse fænotypiske kendetegn, som er identiske i wal1- (WASP)
mutant, antyder at de to proteiner deltager i den samme signalproces. Endelig blev
eisosomproteiner, nemlig Pil1, Lsp1 og Sur7, undersøgt i A. gossypii. Fjernelse af PIL1
medfører en meget alvorlig fænotype, som også ses i S. cerevisiae. Agpil1 er næsten ikke
levedygtig under de vækstbetingelser, som blev testet i denne analyse, men en undersøgelse af
nogle spirer viste ingen åbenlyse mangler i endocytose eller aktinorganisation. Et GFP
fusioneret AgPil1-protein findes i et punktformet mønster under plasmamembranen, men det
blev aldrig lokaliseret samme sted som aktinklumperne. Det forholder sig på denne måde i
alle eisosom-mutanter samt i sac6 og wal1. Dette indebærer, at eisosomer ikke er direkte
involveret i endocytose-processen, men de kunne have en regulerende funktion.
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5 Introduction
5.1 Fungi as model organisms
Fungi can be found in most environments; from the surface of sweet fruits and symbiotic
lifestyles as mycchorizas and lichens to pathogens of plants, mammals and even other fungi.
They can grow as individual cells, yeasts, or as filamentous fungi producing mycelia,
sometimes building up a fruit body, mushroom, that you can pick in the forest. Some fungi are
dimorphic and upon environmental stimuli are able to switch between the yeast-like and
filamentous growth mode (reviewed by Wendland 2001). Many species produce industrially
important metabolites such as antibiotics, ethanol and enzymes that degrade complex organic
compounds (Duff and Murray 1988; von Nussbaum et al. 2006). Fungi has recently emerged
as a major cause of human disease, especially among immunocompromised patients (Pfaller
and Diekema 2007). The ability of some pathogenic fungi to produce very resilient biofilms
on medical implants causes problems to get rid of the infection (Chandra et al. 2001). An
important feature of fungi is that they are eukaryotic; they have a nucleus containing
chromosomes just like in mammalian cells, this makes them especially fitted as model
organisms.
5.1.1 Saccharomyces cerevisiae
Saccharomyces cerevisiae, also known as Bakers’ or Brewers’ yeast, has been studied for a
long time in the field of molecular biology. Many useful molecular tools have been developed
within this organism in order to explore the basic molecular biology of eukaryotes. By
exploiting the amazing power of yeast genetics, pathways that are conserved from yeast to
man can be unveiled and lay the foundation for understanding the rules of life. The yeast cells
propagate by budding, i.e. by the emergence of a daughter cell that is finally pinched off by a
process called cytokinesis, completely separating the progeny from the mother. This budding
yeast is easy to manipulate genetically and is generally not harmful to man. S. cerevisiae
occurs as a diploid in nature; its nucleus contains a double set of chromosomes, as in humans.
These cells harbour both mating types, a and α, and in response to starvation the yeast cell
produces four haploid spores through meiosis, two a and two α spores. These spores can
easily be separated and crossed, or mated, with a spore of the opposite mating type. The yeast
genome is spread on sixteen chromosomes with just over twelve million base pairs and
contains about 6000 genes. Sequencing of the yeast genome was completed in 1997 and
several projects exist to determine the function of all of its genes (Zagulski et al. 1998). About
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100 million years ago, the S. cerevisiae ancestor underwent a whole genome duplication,
WGD, followed by a massive gene loss (Fig. 1). Most of the extra copies of genes became
redundant and were lost rapidly but several duplications remained and were allowed to evolve
divergently. This event may have enabled new ways of surviving and adapting to the
environment. For example, many of the gained genes in S. cerevisiae have a function in
ethanol production, these genes are lacking in species that diverged before the duplication
event (Thomson et al. 2005; Gordon et al. 2009). Some genes are still found in duplicates in S.
cerevisiae even though their sequences have deviated, these twin genes often have
overlapping functions.
5.1.2 Comparative genomics
Yeasts like S. cerevisiae and mammalian cells share many basic biological properties and
many pathways involve homologous genes. As a result, yeast can be exploited to investigate
the function of genes involved in very basic cellular processes, such as DNA repair, cell
division or gene regulation and to test new drugs. In more closely related species like among
vertebrates, the sequences are very similar and the gene order, synteny, is highly conserved.
Figure 1. Phylogenetic tree of the Candida and Saccharomyces clades, arrows, showing the whole genome duplication, WGD, and the point from when CTG was used to encode serin insead of leucine. Modified picture from Butler et al. 2009.
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The gene map of one species can then be used to help find genes in a related species with a
poorly developed map.
The two fungi that have been used in this study are the filamentous fungus Ashbya gossypii
and the human pathogen Candida albicans. The genomes of these two species are sequenced
and readily available in genome databases. S. cerevisiae, A. gossypii and C. albicans belong to
the family Saccharomycetaceae of the phylum Ascomycota but they represent different
genera. They also present different ways of growth and comparative functional studies of
processes involved in for example endocytosis and polarized growth may aid in revealing
important and evolutionary conserved mechanisms.
5.1.3 Ashbya gossypii
Ashbya gossypii (also known as Eremothecium gossypii) is a plant pathogen that grows
exclusively as a filamentous fungus. It was isolated from cotton plants (Gossypium hirsutum)
and described by Ashby and Nowell in 1926. The fungus was recognized as an overproducer
of riboflavin (vitamin B2) which is responsible for its yellow colour (Wickerham et al. 1946).
A. gossypii is an attractive model to study filamentous growth since it has a small haploid
genome that is easy to manipulate. Homologous recombination occurs with a high frequency
and it can propagate plasmids (Wright and Philippsen 1991; Steiner et al. 1995). The nine
million base pair genome of A. gossypii with 4700 protein encoding genes is divided on seven
chromosomes. The A. gossypii lineage diverged from S. cerevisiae before the WGD but
despite their different life styles they are very closely related. The sequencing and annotation
of the genome revealed that 95% of the A. gossypii genes have orthologs in S. cerevisiae and
the two species share a high degree of synteny (Dietrich et al. 2004). This knowledge was
useful in the annotation of the S. cerevisiae genome and to reconstruct the evolution history
from their common ancestor.
A. gossypii grows as a multinucleated mycelia divided by septa and sustains a polarized
growth for almost all of its life cycle (Fig. 2). The life cycle starts with a germinating spore; a
short period of non-polarized growth produces a spherical germ cell. Switch to polarized
growth leads to formation of a germ tube. A second germ tube emerges on the opposite side
of the germ cell and the young mycelium continues to branch laterally. About twenty hours
after germination the hyphae will start to branch dichotomously, producing V-shaped tips, and
the speed of growth will increase about twenty-fold. The growth rate of the mature mycelia
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reaches a maximum of 200 µm/h (Knechtle et al. 2003). Upon sporulation the compartments
defined by septa will swell and produce sporangia containing the needle shaped spores. Each
spore can germinate and start a new life cycle. To produce a null mutant of A. gossypii a
heterokaryotic mycelium is first generated containing both wild type and mutant nuclei. This
strain will grow under a selective pressure and yet behave as the wild type. When sporulated,
each spore will contain one nucleus and homokaryotic null mutants can be selected for during
germination.
5.1.4 Candida albicans
The yeast Candida albicans is one of the most important human fungal pathogens. It occurs
as a relatively harmless commensal organism in the nasal, digestive and vaginal tract of
mammals but a small set-back in the protective immune system can allow C. albicans to
cause vaginitis and urinary tract infections. As an opportunistic fungus it can invade tissue,
spread and cause life-threatening systemic infections when the immune system is severely
compromised (Odds 1988). Its ability to form highly resistant biofilms on implants like
catheters and heart valves propose difficulties to rid the pathogen permanently from the host
A
B
CE
F
D
A
B
CE
F
D
Figure 2. Lifecycle of A. gossypii. A The germcell swells during a short phase of polarized growth, B switch to polarized growth produces first one germtube and then, C, another on the opposite side of the germ cell. D The hyphae continues to grow and begins to branch laterally. E When the mycelium matures the tips starts to split dichotomously and growth speed increases. F The septa defines the compartments that will enclose the spores and produces asci. Spores are set free and can start a new cycle.
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(Chandra et al. 2001). C. albicans is termed a dimorphic fungus because it proliferates either
in a yeast form or a hyphal form (Fig. 3) (Berman and Sudbery 2002). The invasiveness and
virulence of C. albicans is dependent on its ability to switch between the two growth modes
(Lo et al. 1997). Addition of serum to the growth media and elevated temperature (37 C) will
induce production of hyphae which consist of continuous elongated uni-nucleated cells
separated by septa. Formation of hyphae is followed by coexpression of other virulence
factors such as degrading enzymes and adherence factors in order to enhance the overall
virulence of C. albicans (Naglik et al. 2003). Further, hyphal growth is a response to nutrient
deprivation, especially low nitrogen, and filamentous growth enables the fungus to forage for
nutrients more effectively. However, C. albicans can also form opaque cells required for
mating, pseudohyphal cells like those formed in S. cerevisiae (Fig. 3), and chlamydospores,
all are distinct cell types that form in response to genetic or environmental conditions
(Whiteway & Oberholzer 2004). Biofilms of C. albicans consist of layers of yeast and hyphal
cells embedded within a polysaccharide matrix providing resistance to antifungal drugs
(Baillie and Douglas 2000; Chandra et al. 2001).
C. albicans was initially classified as asexual because no direct observation of mating or
meiosis had been reported. Sequencing of the genome revealed mating related genes and the
existens of a and α mating types (Hull and Johnson 1999). The yeast cells are always diploid
containing both a and α mating type loci and only if one set of mating type locus is removed,
by deletion or induced chromosome loss, the cells are able to mate with the opposite sex (Hull
et al. 2000). Mating diploids produce tetraploid cells, following mitosis and chromosome loss
the cells return to a diploid state. This parasexual cycle of C. albicans involves white-opaque
A B
C
A B
C
Figure 3. A. C. albicans budding yeast cells, B pseudo-hypha and C hyphal filaments.
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switching, where the white cells is the default yeast state and opaque cells is the mating-
competent form of C. albicans (Miller and Johnson 2002).
C. albicans uses a non-canonical genetic code that translates CUG into a serine instead of a
leucine. However, the translation of this codon is ambiguous and C. albicans allows
incorporation of leucine at this position, dramatically increasing the number of different
proteins encoded by the C. albicans genome (Ohama et al. 1993; Santos and Tuite 1995;
Gomes et al. 2007).
C. albicans is distantly related to S. cerevisiae, with approx 90 % of their genes in common,
and gene products and pathways are sometimes used in different ways. Many of the genes in
C. albicans that are lacking in S. cerevisiae are coding for secreted hydrolytic enzymes and
adhesins implicated to be virulence factors (Naglik et al. 2003). C. albicans has a rather large
genome, fourteen million base pairs with 6200 protein-coding genes on eight chromosomes.
The genome of C. albicans is highly dynamic and the occurrence of numeral chromosomal
rearrangements and high heterozygosity increases the diversity and dynamics of the
C. albicans populations. Due to C. albicans diploid nature and lack of a complete sexual
cycle, gene disruption mutants must be constructed through knockout of both alleles. This,
together with an unstable genome, can make it difficult to construct a homozygous mutant and
to determine whether a gene is essential (Enloe et al. 2000).
5.2 Penelope Research Training Network and foci of this thesis
Penelope is a Research Training Network within Marie Curie Programs with an overall goal
to gain understanding of the evolution of protein interaction networks in eukaryotes. The main
topic of Penelope is focused on the interplay between SH3 domains and their binding partners
in four different ascomycetous yeasts, Saccharomyces cerevisiae, Candida albicans, Ashbya
gossypii and Schizosaccharomyces pombe. Penelope has aided in the funding of this study
with the aim on SH3 domain proteins in C. albicans and proteins involved in the endocytosis
pathway in A. gossypii. Localization of a set of SH3 domain-containing proteins from
C. albicans was investigated for comparison with the known homologs of S. cerevisiae
(Tab. 1). Analysis of these genes had previously not been done in C. albicans which is why
they were selected for localization studies. The yeast two-hybrid method was used to explore
the physical interactions of the total set of SH3 domains in C. albicans against Vrp1, homolog
to human WIP. Also, association between Vrp1 and the different domains of the WASP-like
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protein Wal1 was assayed with the two-hybrid system. Wal1 and Vrp1 play a fundamental
role in the endocytosis process and actin cytoskeleton organization and interactions between
these and SH3 domain proteins in S. cerevisiae are known to exist. Further, the functional role
of SAC6 in A. gossypii was explored in this study. The SAC6 gene encodes the yeast fimbrin
homolog that enforces and stabilizes the actin cytoskeleton. SAC6 in S. cerevisiae is synthetic
lethal with SLA2, a gene that is absent from the A. gossypii genome. Sla2 is a cortical actin
patch component with essential functions for endocytosis in other fungi. The last part of this
thesis investigates the function of eisosomes in A. gossypii. These fungal specific structures
were first described in S. cerevisiae and are implied to be involved in endocytosis (Walther et
al. 2006).
5.3 Endocytosis
All heterotrophic cells need a constant supply of precursor material and energy rich molecules
to keep up an active metabolism. Water and dissolved salts and smaller particles can readily
pass the cell membrane through pores or via transporters. Molecules that are too large to
diffuse through the cell membrane need to be internalized by an active process called
endocytosis (from greek endo: within, and cyto; cell). There are several types of endocytosis
but they all function to recycle plasma membrane components and regulate cell surface
Information about S. cerevisiae genes was derived from the Saccharomyces genome database, www.yeastgenome.org (September 2010).
nucleus and cortical
actin patches
required for assembly of the cortical
actin cytoskeleton3orf19.1474SLA1
plasma membrane
transmembrane osmosensor,
participates in the HOG pathway1orf19.4772SHO1
NOHBY1orf19.4742RVS167-2
nucleus and
cytoplasminvolved in the HOG pathway1orf19.6588NBP2
nucleus and
cytoplasm
involved in actin patch assembly and
actin polymerisation1orf19.5956PIN3
bud neckinvolved in cytokinesis1orf19.6240CYK3
Incipident bud sites,
bud neck and sites
of polarised growth
involved in polar growth and bud-site
selection1orf19.3555BUD14
cortical actin patches
involved in assembly of actin
patches1orf19.2791BBC1
localization in
S. cerevisiaefunction in S. cerevisiae
# of SH3
domains
systematic
name
gene
name
Table 1. SH3 containing genes of C. albicans to be analysed in this study
nucleus and cortical
actin patches
required for assembly of the cortical
actin cytoskeleton3orf19.1474SLA1
plasma membrane
transmembrane osmosensor,
participates in the HOG pathway1orf19.4772SHO1
NOHBY1orf19.4742RVS167-2
nucleus and
cytoplasminvolved in the HOG pathway1orf19.6588NBP2
nucleus and
cytoplasm
involved in actin patch assembly and
actin polymerisation1orf19.5956PIN3
bud neckinvolved in cytokinesis1orf19.6240CYK3
Incipident bud sites,
bud neck and sites
of polarised growth
involved in polar growth and bud-site
selection1orf19.3555BUD14
cortical actin patches
involved in assembly of actin
patches1orf19.2791BBC1
localization in
S. cerevisiaefunction in S. cerevisiae
# of SH3
domains
systematic
name
gene
name
Table 1. SH3 containing genes of C. albicans to be analysed in this study
14
expression of signalling receptors. It is a complex process that involves a large number of
proteins and requires the timing and regulation of many successive events (Kaksonen et al.
2003). The cell wall invaginates and is pushed into the cell to finally be pinched off as a small
vesicle. These vesicles fuse to form endosomes that are transported to other cell
compartments like lysosomes, vacuoles and the Golgi network. The reverse process when a
vesicle from inside the cell fuses with the cell membrane and releases its content on the
outside is called exocytosis. In this way the cell deposits new cell wall material and
incorporates receptors and other cell wall components. The cell also excretes waste products,
extra cellular material and signalling molecules. Exocytosis is critical for fast growth of
fungal hyphae and the rate of delivery of exocytic vesicles greatly influences the rate of
growth. Cells may ingest dissolved molecules from its environment and membrane-bound
receptors via constitutive or ligand-induced endocytosis. Fluid-phase endocytosis is a non-
specific and constitutive process where the cell engulfs small portions of its environment and
cell membrane and anything that is attached to it. Often, however, the cell needs to selectively
ingest a molecule and therefore produces specific cell surface receptors for each target
molecule, or ligand. When the ligand has bound its receptor, a signal is conveyed to the inside
of the cell and the membrane with receptor and target molecule is invaginated and
internalized. Light microscopy and electron microscopy have been the tools to study these
dynamic events, providing temporal and spatial resolution (Kaksonen et al. 2003; Idrissi et al.
2008). The actin cytoskeleton plays a fundamental role in both endo- and exocytotic events,
providing rigidity, mechanical forces and the tracks for transport of vesicles. This thesis will
explore the dynamics and organization of the actin cytoskeleton during endocytosis with
emphasis on the similarities and differences between the two fungi A. gossypii and
C. albicans and the well known model yeast S. cerevisiae.
5.4 Protein-protein interactions
In order for a cell to survive, propagate and to execute its function it needs to respond to
changes in its environment, to sense developmental cues and environmental stresses. The
signal needs to be relayed from the source of input to a cell function, often the regulation of a
gene. Signal transduction is propagated through close interactions of proteins, inducing
modifications of a protein or enzyme to change its conformation and binding capacity or to
activate or inactivate it. Most processes in the cell involve scaffolds and protein-protein
interactions; complexes of proteins that work in concert or functions as inhibitors or
activators. Several binding motifs are known to promote these interactions, for example
15
Src Homology 3 domains, SH3, that connect to other SH3 domains, associating different
proteins. A scaffold is a protein whose major function is to serve as a structural platform
which recruits and connects multiple members of a signalling pathway, bringing them close
together and orienting them in a preferred way.
5.4.1 SH3 domains
The SH3 domain is a well-characterized family of protein interaction modules involved in a
variety of biological processes. SH3 domains are conserved from yeast to man. The human
genome contains nearly 300 different SH3 domains dispersed in about 200 proteins, some
proteins containing up to six individual SH3 domains (Kärkkäinen et al. 2006). The genomes
of C. albicans and S. cerevisiae encode a total of 29 and 28 SH3 domains respectively in 24
genes. The domains are 50–70 amino acids long and often present in eukaryotic signal
transduction and cytoskeletal proteins. SH3 domains lack enzymatic activity but bind with
moderate affinity to proline rich motifs, the core consensus motif is PxxP. The basic fold of
the SH3 domain is formed from five or six β-strands arranged in two anti-parallel sheets
producing a characteristic β-barrel (Fig. 4). The strands are connected by short loops or
helices that together with a conserved hydrophobic binding site define the ligand-binding
properties of the motif (Yu et al. 1992). Weak SH3 interactions are functionally important and
contacts between the SH3 loops and residues outside the PxxP motif of the target ligand can
greatly enhance specificity and affinity of binding (Mayer and Saksela 2005).
5.5 The actin cytoskeleton
Many fundamental processes of eukaryotic cells, such as cell motility, organelle movement,
cytokinesis and endocytosis, require reorganization and polarization of the cytoskeleton.
Figure 4. Ribbon diagram of the SH3 domain of chicken alpha spectrin. A β-barrel is formed by five anti-parallel strands connected by loops. Made with MOLMOL.
16
Assembly and disassembly of microtubule and actin filaments is required to orchestrate these
changes. In S. cerevisiae a large number of genes have been identified encoding proteins that
are involved in actin polymerization and an overview of the budding yeast functions will be
described here.
5.5.1 Actin polymerization
The actin cytoskeleton is one of the most dynamic and complex systems in eukaryotic cells
and rapid actin assembly and turnover are required for diverse cellular processes (Tang et al.
2000). Monomeric actin units polymerize into chains of filamentous actin which in turn builds
up actin patches, cables and rings (Fig. 5). Actin patches are spots that localize in the cell
cortex and whose distribution is polarized towards sites of growth and endocytosis. The actin
cables consist of thick bundled actin filaments attaching at their ends to the cortical patches
and rings localize to bud necks and septal sites (Adams and Pringle 1984; Amberg 1998).
Initiation of polymerization requires nucleation factors such as the Arp2/3 complex and the
formins Bni1 and Bnr1. Actin monomers can be added to a growing filament in the slow
growing, pointed end and in the fast growing, barbed end. Polymerization is accompanied by
capping of the newly formed end in order to regulate the length of the filament. Actin patches
are nucleated by Arp2/3 complex and the barbed end of the growing filaments is capped by
the diheteromer Cap1/Cap2 in yeast. The tight capping of this complex stimulates nucleation
of new actin branches and the formation of short filaments, producing an intensely branched
actin network in a small patch (Kim et al. 2004). The formins are leaky cappers of the barbed
end that allows for elongation at the same time as it protects the end from tight cappers. This
will increase the duration of elongation and promote formation of long unbranched actin
cables (Zigmond et al. 2003).
B CA B CA
Figure 5. Actin structures visualised with rhodamine staining. A Actin patches and cables in S. cerevisiae. B Actin patches clustered in the hyphal tips of A. gossypii and actin cables extending from the tip. C Acting rings at septal sites and nonpolarized patches in A. gossypii hyphae.
17
5.5.2 The Arp2/3 complex
Arp2/3 complex is an actin filament nucleation machine consisting of seven proteins, highly
conserved from yeast to mammals (Fig. 6). Two of the proteins, Arp2 and Arp3, are actin-
related and build up the two first blocks of the new filament (Machesky et al. 1994). The
Arp2/3 complex itself has a low intrinsic actin nucleation activity and need nucleation-
promoting factors, NPF’s (Winter et al. 1999). One of the strongest and best characterized
nucleation promoting factors of Arp2/3 complex in yeast is Las17, the Wiscott-Aldrich
Syndrome (WASP) homolog. The C-terminus of Las17 contains an acidic domain that binds
Arp2/3 complex and a verprolin homology (VH) domain that brings the first actin monomer
together with Arp2/3 complex. Actin filaments themselves act as promoters of actin
nucleation as the Arp2/3 complex often associates to the sides of pre-existing actin filaments
(Machesky and Insall 1998).
Other NPFs include the type I myosins, also strong activators, and the weaker Abp1 and Pan1,
all of which bind Arp2/3 complex via an acidic domain similar to that of Las17 (Lee et al.
2000) Abp1 contains two acidic domains and does not nucleate Arp2/3 complex by providing
actin monomers like the other NPFs do. Instead Abp1 associates tightly with Arp2/3 complex
and attach it to the side of a preexisting actin filament (Goode et al. 2001). Having only weak
nucleation-promoting activity, Abp1 has a suggested role as an antagonist to other NPFs,
Figure 6. Arp2/3 complex nucleating a branch on an actin filament. Arp2 and Arp3 (light blue and green) initiates the new filament, the actin monomers (dark blue) are brought by the NPF’s and are added to the barbed end (Boczkowska et al. 2008).
Arp2 Arp3
actin
18
serving as a competition mechanism that helps balance out NPF activities in space and time
(D'Agostino and Goode 2005).
5.6 Polarized growth
The yeast cells of S. cerevisiae and C. albicans grow isotropically to increase in size. At this
stage the actin patches are randomly distributed beneath the plasma membrane. In the initial
phase of budding in yeast and in germ tube and branch formation in filamentous fungi actin
patches polarizes and aggregates at a confined site. This defines a growth zone and allows for
unidirectional growth for as long as the polarization is sustained. In yeast cells this phase is
very brief and switching back to isotropic growth produces an ellipsoid shape. In hyphal cells
growth is locked in a polarized mode for longer periods. External signals promote polarization
of the actin cytoskeleton via G-protein coupled receptors and enables cells to move and
respond to their environment.
5.6.1 Polarization via Rho-GTPases
Proteins of the Rho family belong to the small G-protein (GTP-binding protein) superfamily
and regulate various cell functions through the reorganization of the actin cytoskeleton (Hall
1994). These Rho-GTPases, and in particular Cdc42, are essential for guiding the polarization
of actin to sites of growth. The recruitment of Cdc42 to growth sites on the plasma membrane
activates effectors that signal to the actin cytoskeleton (Pruyne and Bretscher 2000). Fig. 7
shows the G-protein induced pathways leading to polarization of the actin cytoskeleton in
yeast. Like most small GTP-binding proteins, Cdc42 is active in the GTP-bound state and
inactive in the GDP-bound state. The cycling between these two states is tightly regulated by
GAPs (GTPase-activating proteins) and GEFs (guanine-nucleotide exchange factors) (Zheng
et al. 1994). At the start of a new cell cycle a site of bud emergence is established by
recruitment of Cdc42 and actin patches cluster at the incipient budding site. During a brief
period of polarized growth at this site, actin cables extend from the mother cell into the bud as
it grows. Redistribution of Cdc42 over the bud surface disperses the actin patches into the bud
as it continues to grow isotropically into an ellipsoid shape (Pruyne and Bretscher 2000). A
cytokinetic ring of filamentous actin will form at the bud neck, which contracts during
cytokinesis (Field et al. 1999). Then the actin patches appear at the bud neck on both sides,
directing cell wall synthesis between the new cells to separate them completely. The mother
cell resumes budding immediately but the daughter will grow isotropically and increase in
size before entering a new cycle (Pruyne and Bretscher 2000). The establishment,
maintenance and termination of cell polarity require a feedback regulation at each stage, to
19
coordinate and reinforce the ordering of events. Cdc42 will be activated by its GEF and a
series of events will lead to its deactivation by its GAP, ending the polarized growth phase
and initiating the switch to isotropic growth. Locking Cdc42 in an active state results in
hyperpolarization of cortical actin patches and an elongated bud morphology (Richman and
Johnson 2000). Both A. gossypii and C. albicans are dependent of the Rho-like GTPase
modules for establishment and maintenance of polarized hyphal growth (Wendland and
Philippsen 2000).
5.6.2 Hyphal growth
The yeast cells of diploid S. cerevisiae can undergo a morphological transition in response to
nitrogen starvation. Cells become elongated and incomplete cytokinesis results in chains of
cells. This pseudohyphal growth allows yeast cells to forage for nutrients as they grow away
from the colony and invade the substrate (Gimeno et al. 1992). The morphology is made
possible by sustaining polarized growth for a longer time than in normally budding cells.
A. gossypii represents a very different life style in that it is an obligatory filamentous fungus,
Ste
5
Arp2/3
Ras1
polarisome
actin patches
actin cables
Cdc42
Bni1
Cdc35
cAMP
Protein kinase A
Sok2Ste12
Wal1 Ste20
Ste11
Ste7
Fus3Kss1
endocytosis secretion
Polarised growth/hyphal growth/mating
Ste
5
Arp2/3
Ras1
polarisome
actin patches
actin cables
Cdc42
Bni1
Cdc35
cAMP
Protein kinase A
Sok2Ste12
Wal1 Ste20
Ste11
Ste7
Fus3Kss1
endocytosis secretion
Polarised growth/hyphal growth/mating
Figure 7. Model of signal transduction pathways in yeast leading to polarized growth. Homologs to the S. cerevisiae proteins are present in A. gossypii and C. albicans. Activation by Ras1 of the Rho-GTPase Cdc42 affects via a MAPK cascade and via regulation of the actin cytoskeleton. Ste5 acts as a scaffolding protein, tethering kinases and substrates of the MAPK pathway in close proximity. Ras-GTPase also directly regulates the cAMP pathway via the adenylate cyclase, Cdc35. Modified picture from Martin et al. 2005.
20
sustaining polarized growth for most of its life cycle. Maintained polarized growth leads to
the production of long tubular cells that can extend considerable distances enabling the fungus
to explore and penetrate its environment. Hyphal cells are not separated but septa define the
compartments of the hyphae. The cytoplasm is connected along the filaments through pores in
the septa allowing for transport and distribution of nutrients and organelles within the
mycelium (Alberti-Segui et al. 2001).
C. albicans cells normally bud as a yeast but is able to form both pseudohyphae and true
hyphae in response to its environment. Hyphal growth is even a requirement for the
pathogenicity of the organism. The morphogenetic switch in C. albicans is of special interest
when trying to unlock the key events that confer polarized and non-polarized growth.
Budding and pseudohyphal formation in both C. albicans and S. cerevisiae is distinct from the
hyphal phase of C. albicans why the filamentous A. gossypii may serve as a better model to
understand the underlying mechanisms of constant polarized tip growth in fungi. Hyphal
growth requires maintenance of tip directed growth and a constant delivery of vesicles to the
extending surface, a process dependent on the actin cytoskeleton and microtubules (Horio and
Oakley 2005). Filamentous fungi manage to sustain multiple axis of polarized growth and a
much higher growth rate than yeast cells. The rate-limiting steps of hyphal elongation in
mature mycelia are the transport of vesicles to the tip and the subsequent incorporation of new
material into the expanding membrane and cell wall. At constant growth conditions the rate of
supply of exocytic vesicles to the apex will exceed their consumption. The build-up of excess
vesicles will eventually trigger a branch formation generating two new hyphal tips with
different axis of growth (Trinci 1974; Watters and Griffiths 2001).
Endocytosis is also required for fast hyphal growth suggesting that a constant flow of new cell
wall and plasma membrane material from recycled membrane components is important.
When secretory vesicles fuse with the plasma membrane, excess membrane and components
need to be recycled by endocytosis. In this way endocytosis and exocytosis are tightly coupled
via early endosomes (Wedlich-Söldner et al. 2000).
5.6.3 The Spitzenkörper
The Spitzenkörper was first described as a phase-dark structure located in tips of growing
hyphae of fungi (Fig. 8). Spitzenkörper (“apical body”) is a zone at the apex which is
dominated by vesicles and nearly devoid of other cell components (Grove and Bracker 1970).
21
This complex structure is associated with the direction of tip growth. Vesicles produced in the
subapical region become concentrated in the apex where they are incorporated at the
expanding surface. The Spitzenkörper disappears when hyphal extension ceases and reappears
as tip growth resumes (Grove and Bracker 1970).
A complex called the polarisome, is required for apical actin organization and is only present
at actively growing sites. The complex comprises Spa2p, Pea2p, Bud6 and the formin Bni1p
and is localized to the very tip of buds and hyphae as long as polarized growth is sustained.
Bni1p plays a central role in linking polarisome components to RhoGTPases such as Cdc42
and Rho1, to promote actin filament assembly in response to G-protein signaling (Kohno et
al. 1996). Components of the polarisome were found to localize to the Spitzenkörper of
filamentous fungi (Sharpless and Harris 2002) suggesting that the polarisome is a component
of the Spitzenkörper. Microtubular cytoskeleton plays a major role in the formation and
positioning of the Spitzenkörper by providing the tracks for supplying vesicles. The finding
that actin localizes to the Spitzenkörper suggests that vesicles might switch from microtubule-
based to actin filament-based transport within this structure (Harris et al. 2005). The
microtubules would primarily be responsible for the long-distance transport of secretory
vesicles to the Spitzenkörper, while actin filaments control vesicle transport to the plasma
membrane.
5.7 The endocytic machinery
Many genes are known to be involved in the processes that orchestrate the endocytic events.
An outline of some of the factors in S. cerevisiae will be shown here, focusing on the clathrin-
mediated endocytosis pathway with some of the key components further elaborated.
Homologous genes to most of these factors exist in A. gossypii and C. albicans and pathways
are expected to function similarly. Naturally, these organisms have evolved divergently and
Figure 8. Electron micrograph of a hyphal tip of Aspergillus niger showing a dense zone of microvesicles forming the Spitzenkörper. Larger vesicles (V) surrounds the area. Golgi (G) and mitochondria (M) are visible. Scale bar is 1 µm (Grove and Bracker 1970).
22
found new ways of coping with their environment and both similarities and differences are
targets for investigations in this study.
5.7.1 Clathrin-dependent endocytosis
Ligand-induced and fluid-phase endocytosis are also called clathrin-dependent endocytosis
because the invaginating vesicle is coated and stabilized with a lattice of clathin required for
proper recruitment of later endocytic proteins (Fig. 9) (Kaksonen et al. 2005). In both cases,
ligand-activated or unbound receptors set off the initial steps of the internalization process at
the plasma membrane. Several early endocytosis factors assemble at the site in a fashion
independent of the actin polymerization machinery and initiate the invagination by bending
the cell membrane inwards. Proteins are recruited and disassembled in a sequential manner
during the whole process of internalising the forming vesicle, first in a slow-movement phase
representing the invagination, but later in a faster moving phase coinciding with the release
and inward transportation of the endocytic vesicle. Actin plays a fundamental part in this
movement as its polymerization generates the mechanical force for pushing the membrane.
The vesicles are transported into the cell where they fuse with endosomes and are then
targeted to other compartments. Fig. 10 shows a sketch of some of the key components in the
endocytic process.
As a first step of forming the endocytic vesicle, clathrin assembles at the endocytic site
beneath the plasma membrane. The clathrin lattice is made up of triskelions of heavy and light
chains with ability to self assemble into a cage like structure (Ungewickell and Branton 1981).
Early (34-240s)
Clathrin (60-120s)
Coat (20-40s)
Wasp/Myo (10-40s)
Amphiphysin (10s)
Actin (10-15s)
Variable Phase Regular phase Early (34-240s)
Clathrin (60-120s)
Coat (20-40s)
Wasp/Myo (10-40s)
Amphiphysin (10s)
Actin (10-15s)
Variable Phase Regular phase
Figure 9. Temporal organization at the site of clathrin mediated endocytosis in S. cerevisiae. Clathrin and early endocytosis factors assemble at the incipient site of invagination. These proteins initiate bending of the membrane and aid the recruitment of other coat proteins. At the arrival of actin and actin binding proteins the vesicle elongates slowly inwards the cell and is finally pinched off by amphiphysin-like proteins. The coat module disassemble and the vesicle is transported into the cell. Modified picture from Stimpson et al. 2009.
23
The lattice confers an initial curvature to the membrane and also provides anchorage for the
endocytic machinery. Clathrin is dependent on Ent1/2 and AP1801/2 for recruitment, adaptor
proteins harboring plasma membrane binding ENTH/ANTH modules. These modules are
located at the N-terminal of clathrin binding proteins involved in signaling and actin
regulation. ENTH/ANTH domains are built up by several alpha helices that bind lipids in the
membrane and could aid in the bending of the membrane at this initial stage of endocytosis
(Stahelin et al. 2003). Las17, a strong promoter of Arp2/3 nucleated actin polymerization, is
recruited early in the endocytosis process, soon followed by other early coat proteins like Sla2
and the Pan1-Sla1-End3 complex (Tang et al. 2000). Sla2, homologous to the human Hip1/R,
contains an ANTH domain that may help the localization to the plasma membrane, a coiled-
coiled central domain that binds directly to clathrin and Pan1, and an actin-binding domain
(McCann and Craig 1997). Sla1 contains three SH3 domains and is crucial for actin patch
regulation and Sla2 localization (Ayscough et al. 1999). Pan1 is another activator of the
Arp2/3 complex, while Sla1 functions as an inhibitor of Las17 activity (Rodal et al. 2003).
Vrp1 and Bzz1 are next recruited to the site. Bzz1 relieves Sla1p inhibition of Las17 so that
actin may start to assemble in patches marking sites of endocytosis (Sun et al. 2006). Vrp1, a
key regulator of cortical actin-patch distribution, contains several actin-binding modules and
is required for Arp2/3 activation by Las17 and the type I myosins (Anderson et al. 1998). In
the following stages, actin, Arp2/3 complex and the actin binding protein Abp1 are recruited,
coinciding with a slow membrane invagination. The coated pit will extend into a tubular
shape, about 50 nm wide and up to 180 nm long. Sla2 is an important factor during this
movement; it couples the vesicle via clathrin to the polymerising actin cables, providing the
force to push the invaginating vesicle inwards the cell (Kaksonen et al. 2003). Myo5, one of
the type I myosin motor proteins and an NPF of the Arp2/3 complex, arrives and further
promotes actin polymerization to drive the vesicle rapidly inward (Sun et al. 2006). Vrp1 is
very rich in prolines and is therefore a likely target for SH3 domain binding. Myo3 and Myo5
in S. cerevisiae interact with both Las17 and Vrp1 via their SH3 domain (Evangelista et al.
2000). Bbc1, another SH3-protein, functions as a negative regulator of both Myo5 and Las17
(Rodal et al. 2003). As the clathrin coated pit internalizes, the amphiphysins Rvs161 and
Rvs167 are recruited to the neck of the invagination contributing to the release of the forming
vesicle (Kaksonen et al. 2005). Sla1, Pan1 and Sla2 disassemble and the vesicle will
internalize in a fast-moving step. While Las17 executes its function mainly during the slow
movement and stays at the plasma membrane Myo5 is the prime nucleator of actin
polymerization during the rapid inward movement and follows the tip of the invagination.
24
Myo5 appear at cortical patches immediately proceeding the fast movement of the actin
structures away from the plasma membrane, correlating with the vesicle scission event
(Jonsdottir and Li 2004). A pool of Myo5 remains at the membrane base of the invagination
and could aid in the constriction of the neck (Idrissi et al. 2008).
Abp1 and the actin bundling protein Sac6 contribute to the dynamics of the actin network in
the later stages and follow the patch as it moves inward. Sac6, or fimbrin, contains two sets of
an actin binding domain, each comprised of two calponin homology, CH, domains. Sac6
bundles actin filaments into tight bundles as well as stabilizes them against depolymerization
(Goode et al. 1999). Several factors aid in the uncoating of the vesicle after it is pinched off,
among them the kinases Ark1 and Prk1, and Abp1. The SH3 domain of Abp1 associates the
kinases with the actin patch, enabling phosphorylation, inactivation and disassembly of the
Pan1-Sla1-End3 complex. This step is pivotal for the recycling and reorganization of actin
(Zeng et al. 2001). During internalization the small vesicles become associated with actin
cables, tight bundles of actin filaments. As the bundles elongates the associated vesicles are
moved further into the cell. It has been shown that early endosomes are attached to actin
cables and is transported along them towards sites of internalization at the membrane
(Toshima et al. 2006). Newly formed endocytic vesicles and early endosomes are in this way
directed towards each other, facilitating their fusion.
clathrin
Pan1, Sla1, End3
Sla2
Las17
Abp1
actin
Arp2/3 complex
Vrp1, Bzz1
Myo5, Bbc1
Sac6
Rvs167, Rvs161
Figure 10. Components of the endocytic machinery in yeast, SH3 domain proteins are written in bold. Clathrin is recruited in the initial phase along with the Pan1 complex, Sla2 and Las17. Arrival of actin and other factors regulating actin polymerization results in a slow invaginating movement of the clathrin coated pit. Rvs161/Rvs167 aid in the scission of the vesicle, allowing for a fast internalization and fusion with early endosomes.
25
5.7.2 Sla2 and Sac6 are key components in endocytosis of S. cerevisiae
Sla2 belongs to a conserved family of actin-binding proteins; the human homologs Hip1 and
Hip1R bind to huntingtin, the protein whose mutation results in Huntington’s disease. In the
model of S. cerevisiae Sla2 arrives next after clathrin at the endocytic site and provides a
direct link between the clathrin coated pit and polymerizing actin filaments (Fig. 11). The
N-terminal ANTH domain binds to the plasma membrane and could together with the clathrin
lattice aid in the initial bending of the invaginating plasma membrane. The central domain
binds, in addition to clathrin also Pan1, Rvs167, Sla1 and it self; Sla2 is found as a dimer in
vivo (Wesp et al. 1997). The C-terminus harbors an F-actin binding talin-like motif that is
important for endocytosis only at elevated temperatures. This domain could regulate Sla2
function by folding itself, occluding actin binding.
The fimbrin Sac6, member of a family of actin-bundling proteins, is important in the actin
dependent movement of the endocytic patch and might also have a function in scission of the
vesicle. Actin binding is mediated by two pairs of calponin homology domains (Fig. 11),
conserved motifs that are found in a set of cytoskeleton organizing proteins. The two actin
bind domains associates actin filaments closely together and Sac6 can be found in both
patches and along cables (Drubin et al. 1988). The S. cerevisiae and A. gossypii Sac6
homologs have a high degree of similarity, 80 % identity at the amino acid level. They share
conserved residues in the actin binding sites with the mammalian dystrophin and actinin,
members of the same superfamily containing calponin homology domains.
5.8 Eisosomes
Eisosomes are newly described large immobile protein assemblies that localize in a punctate
pattern beneath the plasma membrane of fungi (Fig. 12). These structures are mainly
Figure 11..Functional domains of yeast Sla2 and Sac6. Sla2 is composed of the lipid binding ANTH domain, a central coiled-coil region that contributes to dimerization and binding to several other proteins, including clathrin, and a C-terminal actin binding domain. Self regulation of Sla2 could be achieved by the ABD folding upon itself, preventing actin binding. The two actin binding domains of Sac6 constitute double calponin homology domains.
CH1 CH2 CH3 CH4
ABD1 ABD2
ScSac6
642 aa
ANTH coiled-coil ABD ScSla2
968 aa
CH1 CH2 CH3 CH4
ABD1 ABD2
CH1 CH2 CH3 CH4
ABD1 ABD2
ScSac6
642 aa
ANTH coiled-coil ABD ANTH coiled-coil ABD ScSla2
968 aa
26
composed of two highly similar proteins, Pil1 and Lsp1, in equimolar proportions. Pil1 and
Lsp1 have no recognizable domains but colocalize with a plasma membrane protein, Sur7.
Eisosomes seem to be static with no exchange with free cytoplasmic pools of Lsp1 or Pil1
(Walther et al. 2006). Pil1 and Lsp1 are phosphorylated by Pkh1 and Pkh2 in S. cerevisiae
and this phosphorylation is critical for eisosome organization. In addition, Ypk1 and Ypk2
kinases, involved in sphingolipid-mediated signalling, are necessary for maintaining
eisosomes. Eisosomes are subsequently dynamic structures whose formation and turnover are
regulated by the sphingolipid-Pkh1/2-Ypk1/2 signalling pathway (Luo et al 2008). Pil1
expression in S. cerevisiae is regulated by the cell cycle and determines size and number of
eisosomes (Moreira et al. 2009). Deletion of PIL1 leads to clustering of eisosome remnants,
and cause a reduction of the endocytic rate (Walther et al. 2006). The discovery of eisosomes
linked them to endocytosis for several reasons; deletion of PIL1 results in impaired
endocytosis, eisosome components show synthetic lethality and interaction with several
proteins known to function in endocytosis, and the colocalization of eisosomes with early
endocytic endosomes and a subpopulation of actin patches (Walther et al. 2006). Lsp1 is not
required for localization of Pil1 and despite their similarity in sequence they have distinct and
non-redundant functions.
Sur7 was originally identified as a multicopy suppressor of RVS161 and RVS167 mutants,
actin patch components involved in endocytosis (Sivadon et al. 1997). Sur7 itself does not
localize to actin patches but is involved in sporulation and sphingolipid content of the plasma
membrane (Young et al. 2002).
5.8.1 Lipid rafts
Lipid rafts are detergent-resistant assemblies laterally distributed in the plasma membrane.
They are involved in many cellular processes such as protein sorting and trafficking. In yeast
they are enriched in ergosterol and sphingolipids and they recruit specific membrane proteins
into distinct domains (Bagnat et al. 2000). Typically, GPI-anchored proteins and transporters
PIL1-GFP LSP1-GFP SUR7-GFPPIL1-GFP LSP1-GFP SUR7-GFP
Figure 12. Proteins in the eisosome complex localize in a punctate pattern beneath the plasma membrane. Z-stack image from Walter et al, 2006.
27
associate with these rafts. On of these domains called MCC, membrane domain occupied by
Can1, colocalizes with Sur7, linking them to the static eisosome structures. The MCCs are
very stable and immobile; they are not dependent on the cytoskeleton for formation and do
not colocalize with sites of active endocytosis (Malinska et al. 2004); Walther et al. 2006;
(Grossmann et al. 2008). These structures are not turned over by endocytosis but could
instead act as protective areas. As long as a protein is associated with the MCC the turnover
by endocytosis is avoided (Grossmann et al. 2007). Deletion of the eisosome component Pil1
leads to clustering of both Sur7 and Can1 in a manner similar to Lsp1. Disruption of the core
components of MCCs, including Pil1, causes disassociation and faster degradation of marker
proteins in the complex (Grossmann et al. 2008).
28
6 Methods
6.1 Strains and media
Plasmids, strains and primers used in this study are listed in Appendices III-V.
Saccharomyces cerevisiae BY4741 (his3Δ1; leu2Δ0; met15Δ0; ura3Δ0) was used for in vivo
recombination and generation of GFP fusions. For the yeast two-hybrid assay the following
strains were used: PJ69-4a: trp1-901; leu2-3,112; ura3-52; his3-200; gal4Δ; gal80Δ;
lys2::GAL1-HIS3; GAL2p-ADE2; met2::GAL7-lacZ and PJ69-4α: trp1-901; leu2-3,112;
ura3-52; his3-200; gal4Δ; gal80Δ; lys2::GAL1-HIS3; GAL2p-ADE2; met2::GAL7-lacZ.
Candida albicans and Saccharomyces cerevisiae were grown in rich media (YPD; 1 % yeast
extract, 2 % peptone, 2 % dextrose) or minimal media (6.7 g/L YNB w/w ammonium
sulphate w/o amino acids, 0.69 g/L CSM, 20 g/L glucose) in 30 C. Hyphal formation of
Candida albicans was induced in YPD with 10 % newborn calf serum in 37 C for 2-4 hours.
Ashbya gossypii was grown in rich media (AFM; 1 % yeast extract, 1 % peptone, 2 %
dextrose and sporulated in minimal media (1.7 g/L YNB w/o ammonium sulphate w/o amino
acids, 0.69 g/L CSM, 20 g/L glucose, 2 g/L Asparagin, 1 g/L Myo-Inositol). Efficient
sporulation of Ashbya gossypii was achieved by inoculating 5 ml fresh mycelia from an AFM
culture into 200 ml sporulation media. After 3-7 days of vigorous shaking in 30 C, spores
were harvested by gentle centrifugation. The pellet was resuspended in 30 ml TE buffer and
incubated with 5 mg zymolase in 37 C until spores were set free and remaining mycelium
was degraded, 1-2 hrs. Spores were washed repeatedly in water with 0.03 % Triton X-100 and
frozen down in 25 % glycerol at -80 C. Antibiotics where used for selection with additions of
200 µg/ml G418/geneticin or 100 µg/ml clonNAT/nourseothricin to rich or minimal media.
Escherichia coli strain DH5α was used for propagation of all plasmids generated and
electroporation was used for transformation.
6.2 Transformation
S. cerevisiae was transformed with the lithium acetate procedure (Gietz and Schiestl 2007).
Transformation of C. albicans was preformed with a modified lithium acetate protocol
(Walther and Wendland 2003) with o/n incubation at 30 C in PEG4000 followed by a 15 min
heat shock at 44 C. Transformation of A. gossypii was achieved by using electroporation
according to (Wendland et al. 2000). After transformation mycelia were plated on AFM plates
and incubated for 6 hrs in 30 C to allow for expression of the resistance marker, then the
29
mycelia were overlaid with 8 ml 0.5% agarose with 6.6 mg G418 or 3.3 mg clonNAT (final
conc. of 200 µg/ml resp. 100 µg/ml).
6.3 Generation of GFP tagged C. albicans strains
The Candida albicans SN148 strain (arg4; leu2; his1; ura3, Noble and Johnson 2005) was
used as the progenitor strain for all GFP fusions of specific genes in this study. First,
heterozygous strains were generated were the complete ORF of one gene allele was deleted.
Then, GFP was fused to the 3’ end of the second allel. To this end, specifically designed
cassettes were constructed with the 3’ end of each gene cloned separately and later fused with
GFP via in vivo recombination in yeast. The cassettes were constructed with long homologous
flanks to C. albicans sequences for proper targeting. The procedure is shown in Fig. 13 and
eliminates expression of any wild type copies of the gene. Eight genes of C. albicans were
GFP tagged following this procedure, BBC1, BUD14, CYK3, NBP2, PIN3, RVS167-2, SHO1
and SLA1 (see Tab. 1).
S1 S2
~1kb
A4A1
S2S1-GFP
A
B
C
D
ORF
CdHIS1
ORF
3’
GFP
GFP3’
ORF
CmLEU2
CmLEU2
G4
G3G2
G1
S1 S2
~1kb
A4A1
S2S1-GFP
A
B
C
D
ORF
CdHIS1
ORF
3’
GFP
GFP3’
ORF
CmLEU2
CmLEU2
G4
G3G2
G1
Figure 13. Generation of GFP-tagged C. albicans strains. A A deletion cassette with 100 bp flanking regions will exchange the complete ORF of one allele of the target gene with the CdHIS1 marker, generating a heterozygous strain for that locus. B A 1 kb fragment covering the 3’ end of the gene was cloned in pRS417. C A GFP cassette was amplified by PCR using primers with homologous flanks to sequences in the target gene in C. albicans. Co-transformation of S. cerevisiae BY4741 with the pRS417-3’-end plasmid and the GFP cassette generates an in frame fusion of the 3’-end of the gene to GFP by in vivo recombination. D Transformation of C. albicans heterozygous strains with the fusion cassette tags the remaining allele with GFP.
30
Deletion cassettes for the first allele were created using PCR based methods. The marker
cassette was amplified from pFA-CdHIS1 (Candida dubliniensis HIS1), with S1/S2 primer
pair for each gene. The primers contain 100 bases of homologous regions to the target gene
UTR flanks, enough to ensure for integration into the target locus. C. albicans SN148 was
transformed with the cassettes and mutant colonies were selected on CSM-plates lacking
histidine, generating heterozygous knockout strains. Integration of the marker into the wright
locus was verified by PCR of 5’ and 3’ ends with primer pairs G1/G2 and G3/G4,
respectively. G1 and G4 binds to sequences outside of the replaced gene in the genome, G2
and G3 binds to the HIS1 marker. Two heterozygous strains where produced for every gene.
The GFP-fusion cassettes for C-terminal tagging of the corresponding proteins had to be
constructed in several steps. The 3’ end of all genes where cloned and fused to GFP, deleting
the stop codon of the gene, before integration into the genomic locus. A fragment of about
500 base pairs up- and downstream of the stop codon was cloned in either of the standard
vectors pDrive Cloning Vector, Promega, (BBC1, CYK3, NBP2, RVS167-2) or pGEM-T Easy
Vector, QIAGEN, (BUD14 ,PIN3, SHO1, SLA1) using gene specific primers A1 and A4. The
fragments were cut out with restriction enzymes, XhoI/BamHI for pDrive fragments and
SacII/SalI for pGEM fragments, and re-cloned into pRS417, a plasmid which was made by
exchanging the LEU2 marker of pRS415 to GEN3. The pRS plasmid with the cloned 3’ end
was co-transformed into S. cerevisiae BY4741 with a GFP-cassette amplified from pFA-GFP-
CmLEU2 (Candida maltosa LEU2) with the primer pairs S1-GFP/S2. The S1-GFP primer
will exclude the stop codon and add 45 bases at the 5’ end of the cassette which is enough for
homologous recombination in yeast. The S2 primers are the same as for making the deletion
cassette, adding 100 bases at the 3’ end of the cassette. The GFP is optimized for C. albicans
with S65A, V68L and S72A (Morschhäuser et al. 1998). The efficient homologous
recombination machinery of S. cerevisiae will integrate the GFP cassette immediately after
the cloned 3’ end of the gene on the plasmid, excluding the stop codon. Yeast clones were
selected on both the plasmid (GEN3) and the inserted GFP-cassette (CmLEU2) on CSM
media lacking leucine with G418. Plasmids were harvested and further propagated in E. coli.
Sequencing of the GFP fusion plasmid with primer 392 ensures correct in-frame fusion of the
gene to GFP after homologous recombination. The whole GFP fusion cassette was cut out
from the plasmid, using the same enzymes as for the initial cloning of the 3’ end into pRS417,
providing 500 base pairs flanks for homologous recombination in C. albicans. Transformation
of the cassette into the corresponding heterozygous strain tagged the remaining allele with
31
GFP, clones where selected on CSM lacking leucine and histidine. Integration of GFP was
confirmed by PCR using a specific primer set G1-GFP/392 for each gene. G1-GFP binds
upstream the 5’ homologous region, in the ORF, and 392 binds to GFP itself. SLA1-GFP and
CYK3-GFP cassettes where also transformed into the progenitor strain SN148, producing
strains with one wild type copy of the gene left in the genome. Before microscopy, o/n
cultures where diluted in YPD and grown to exponential phase in 30 C. Hyphal formation
was induced as previously described.
6.4 Yeast Two Hybrid assay
A yeast two hybrid assay with various fragments of the ORF of WAL1 or VRP1 and a set of
SH3 domains of C. albicans genes was performed as described in Borth et al. 2010 (Fig. 14).
Wal1 was truncated at its N- and C-termini and the central proline-rich region was removed.
The different truncated versions of WAL1 and the full length gene were fused to the DNA
binding domain, DBD, of the galactose induced transcription factor GAL4 on plasmid pGBT9.
VRP1 was divided in two parts, the C-terminal was fused to the activation domain, AD, of
GAL4 on pGAD424 and the N-terminal was cloned in both vectors (Fig. 15A). 24 sets of SH3
domains from 23 C. albicans genes (originally isolated by Reijnst, unpublished) where fused
to the AD on pGAD424 (Fig. 14). The SH3 domains were in most cases isolated individually
with ~100 nucleotides up- and downstream of the domain. The two SH3 domains of ABP1
and BEM1 and the two first SH3 domains of SLA1, named SLA1-1, were cloned as one
fragment. The third SH3 domain of SLA1 is referred to as SLA1-2.
Figure 14. Truncated versions of Wal1 and Vrp1 and SH3 domains from C. albicans used in the two-hybrid assay. Fragments are fused to either the DBD or the AD of the transcription factor GAL4. WH1: WASP-homology 1; WH2: WASP-homology 2, P1-P2; proline-rich regions; VCA: verprolin-central-acidic domain; LBD: Las17-binding domain; HOT: Hof1-trap.
DBDbait prey AD
BWH1 P1 VCAP2 P3 P4
VCAP3 P4
WH1
LBDWH2 HOTWH2
Wal1
Wal1ΔN-term
Wal1Δpro
Wal1ΔC-term
Vrp1 N-term
Vrp1 N-term
Vrp1 C-term
WH2 LBDWH2
HOTSH3
SHO1
CDC25
RVS167-2
Q59U90
NBP2
Q5AAN3
MYO5
CDC25L
BEM1L
FUS1
HOF1
PEX13
LSB1/2
BUD14
RVS167-1
LSB3
ABP1
SLA1-1
SLA1-2
BOI1
CYK3
HSE1
BBC1
BEM1WH2 LBDWH2
LBDWH2 HOTWH2
DBDbait DBDbait prey ADprey AD
BWH1 P1 VCAP2 P3 P4
VCAP3 P4
WH1
LBDWH2 HOTWH2
Wal1
Wal1ΔN-term
Wal1Δpro
Wal1ΔC-term
Vrp1 N-term
Vrp1 N-term
Vrp1 C-term
WH2 LBDWH2
HOTSH3
SHO1
CDC25
RVS167-2
Q59U90
NBP2
Q5AAN3
MYO5
CDC25L
BEM1L
FUS1
HOF1
PEX13
LSB1/2
BUD14
RVS167-1
LSB3
ABP1
SLA1-1
SLA1-2
BOI1
CYK3
HSE1
BBC1
BEM1WH2 LBDWH2
LBDWH2 HOTWH2
BWH1 P1 VCAP2 P3 P4
VCAP3 P4
WH1
LBDWH2 HOTWH2
Wal1
Wal1ΔN-term
Wal1Δpro
Wal1ΔC-term
Vrp1 N-term
Vrp1 N-term
Vrp1 C-term
WH2 LBDWH2
HOTSH3SH3
SHO1
CDC25
RVS167-2
Q59U90
NBP2
Q5AAN3
MYO5
CDC25L
BEM1L
FUS1
HOF1
PEX13
LSB1/2
BUD14
RVS167-1
LSB3
ABP1
SLA1-1
SLA1-2
BOI1
CYK3
HSE1
BBC1
BEM1
SHO1
CDC25
RVS167-2
Q59U90
NBP2
Q5AAN3
MYO5
CDC25L
BEM1L
FUS1
HOF1
PEX13
LSB1/2
BUD14
RVS167-1
LSB3
ABP1
SLA1-1
SLA1-2
BOI1
CYK3
HSE1
BBC1
BEM1WH2 LBDWH2
LBDWH2 HOTWH2
WH2 LBDWH2
LBDWH2 HOTWH2
32
The yeast strains used for the two hybrid assay (PJ69-4a/α) are deleted for the GAL4
transcription factor involved in galactose metabolism. If the fusion proteins interact with one
another they form an active transcription factor that induces the expression of several reporter
genes (Fig. 15B). S. cerevisiae PJ69-4a or PJ69-4α were transformed with two plasmids, one
expressing the DBD and one with an AD construct. pGBT9 and pGAD424 harbors the
selective markers TRP1 and LEU2, respectively, and yeast transformants were selected on
CSM lacking tryptophan and leucine for maintenance of both plasmids. Interactions between
protein fragments expressed from the plasmids were analyzed qualitatively and quantitatively
exploiting the reporter genes lacZ and ADE2. lacZ codes for β-galactosidase that can break
down lactose substitutes like X-gal and ONPG to colourful products that are readily seen or
measurable by their light absorption. Growth on plates lacking adenine and production of blue
color on X-gal plates was indicative of an interaction. The conversion of ONPG in a
β-galactosidase assay was measured photometrically.
GAL4AD
PADH1
TADH1
LEU2
amp
pGAD424
MSCGAL4DBD
PADH1
TADH1
TRP1amp
pGBT9
MSC
prey AD
DBDbait
promoter reporter gene (ADE2/lacZ)
transcriptional
activator (Gal4)
A
B
GAL4AD
PADH1
TADH1
LEU2
amp
pGAD424
MSCGAL4AD
PADH1
TADH1
LEU2
amp
pGAD424
MSCGAL4DBD
PADH1
TADH1
TRP1amp
pGBT9
MSCGAL4DBD
PADH1
TADH1
TRP1amp
pGBT9
MSC
prey AD
DBDbait
promoter reporter gene (ADE2/lacZ)
transcriptional
activator (Gal4)
prey AD
DBDbait
promoter reporter gene (ADE2/lacZ)
transcriptional
activator (Gal4)
A
B
Figure 15. A Maps of the two plasmids that were used to clone fragments for the two hybrid screen. pGBT9 contains the DNA binding domain, DBD, which was fused to the bait protein. The prey protein was cloned in pGAD424 which harbours the activation domain, AD. The plasmids can be selected for by the TRP1 and LEU2 markers. B The two fusion proteins were expressed in a yeast strain deleted for a transcription factor involved in galactose metabolism. If bait and prey proteins interact with one another they form an active transcription factor that can activate the expression of several reporter genes. The reporter gene lacZ produces the enzyme ß-galactosidase. The lactose substitutes X-gal and ONPG can serve as substrates for ß-galactosidase. The enzyme cleaves X-gal into galactose and an indigo blue dye which is readily visible. Colour less ONPG will be degraded to galactose and yellow ONP and the absorption can be measured at 420nm.
33
6.5 β-galactosidase assay
The β-galactosidase assay exploits the enzymatic activity of β-galactosidase, the product of
the lacZ gene. The enzyme degrades the colourless substrate ONPG into galactose and the
yellow ONP (o-nitrophenol) which absorption can be measured at 420 nm. Yeast strains
containing plasmids with a DBD fusion and an AD fusion were grown o/n in selective media
at 30 C. 1 ml of the culture was diluted 1:4 in YPD and grown for another 4 hrs. OD600 was
measured and cells from 2 ml culture were harvested. To obtain comparable data in the assay
all strains should have reached the same cell density. Cells were washed and resuspended in
300 µl Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4), 100 µl
was used in duplicate tests. Cells were broken by freeze/thawing and then incubated with
150 µl 6 mg/ml ONPG solution at 37 C for 30 minutes. The enzymatic reaction was stopped
with 400 µl 1 M Na2CO3 and cell residues were spun down. The exact timing of the reaction
of all samples is of great importance for the calculated values of the assay. 500 µl of
supernatant was mixed with 500 µl water and absorption at OD420 (yellow dye) and OD550
(cell debris) was measured. Enzyme activity, A, in Miller units was calculated with t= 30 min,
V= 0.1 ml, according to the formula
A = 1000 * OD420-(1.75*OD550)
t * V * OD600
6.6 Generation of mutant and GFP strains in A. gossypii
Disruption of genes in A. gossypii was achieved either by long-flanking deletion cassettes
(Noble and Johnson 2005), PCR generated S1/S2 cassettes (Walther and Wendland 2008) or
by insertion of a cloned marker cassette into the ORF. In all cases the kanMX marker derived
from the plasmid pFA-kanMX, was used. It contains the kanamycin resistance ORF regulated
by the promoter and terminator of the AgTEF1 gene. Two strains were produced for each
genotype and all were made in the Agleu2 background, the strain that also acts as wild type
control in all assays in this study. In a first step a heterokaryon was generated by
electroporation (1.5 kV, 100, 25 μF in the Equibio EasyjecT electroporator, Wolf lab) producing
a mycelium containing both wild type and transformed nuclei. Upon sporulation, germination
under selective pressure and micromanipulation homokaryotic strains were isolated in cases
where the deletion was not lethal. An MSM Micromanipulator (Singer Instruments) was used
to pick spores that had produced only a germ cell, larger germlings with germ tubes and a few
lateral branches could be isolated with a Patchman NP2 micromanipulator (Eppendorf). Only
34
germlings where the spore remnants and germ cell could be identified were lifted and moved to an
isolated spot on the plate, thereby making sure that the mycelia produced will be originated from
one spore and thus homokaryotic.
Long flanking deletion cassettes were generated by a two step PCR (Fig. 16) and were used to
delete SAC6, LSP1, SUR7, PKH1 and YPK1. 400 base pairs long fragments of the upstream
and downstream flanks of the target gene and the kanMX marker were amplified by PCR. The
primers for generation of the gene flanks added regions overlapping with the marker cassette
so that the flanking fragments could be fused to the marker in a second PCR, providing very
long regions for homologous recombination and reliable integration of the marker into the
target locus. Flanks were generated with primer pairs 5’a/5’b-S1 and 3’a-S2/3’b, and the
marker cassette with S1/S2 primers in the first PCR step. S1 and S2 regions are the
overlapping parts, 22 bases long. The whole deletion cassette was amplified in the second step
using primers 5’a and 3’b together with both flanks and the marker. The deletion cassettes
were cloned into a pSK background (XhoI/EcoRI cut C351 for pkh1, XhoI/XbaI cut C424 for
sac6 and XhoI/SacI cut C424 for the others). Cassettes were then cut out with the same
enzymes as were used for cloning and transformed into A. gossypii. Integration of the deletion
cassette into target locus was confirmed by PCR with G1/G6 and G4/G5 primer pairs for the
pkh1 mutant and G1/G2 and G3/G4 primer pairs for the other mutant strains, where G1 and
G4 bind outside of the targeted locus and G2/G3/G5/G6 binds in the kanMX marker.
Heterokaryotic strains were generated for pkh1 and ypk1 and they would also give a PCR
product with primers I1/I2 annealing inside the ORF. The homokaryotic null strains of sac6,
lsp1 and sur7 did not give an internal PCR product with I1/I2 primers.
target gene
5’b-S1 3’a-S2
S1 S2
5’a 3’b
3’b5’a
kanMX
B
A
S1 S2
target gene
5’b-S1 3’a-S2
S1 S2
5’a 3’b
3’b5’a
kanMX
B
A
S1 S2
Figure 16. Generation of a long flanking deletion cassette. A 5’ and 3’ flanks of the target gene are and a marker are amplified by PCR, producing fragments with regions that overlap (S1/S2). B In a second PCR step, the flanks are fused to the marker, generating a deletion cassette with long homologous flanks, ~400 base pairs.
35
Disruption of PIL1 was achieved in two different ways and the gene was tagged with GFP, as
shown in Fig. 17. PIL1-S1/ PIL1-S2 primers were used to amplify the kanMX marker to
delete the PIL1 ORF. Since the promoter region of PIL1 could not be amplified, the S1 primer
was placed in the very beginning of the ORF, thereby leaving the first 55 nucleotides intact in
the genome of the pil1 strain ASJ24. Integration of the deletion cassette in pil1 heterokaryons
was confirmed by PCR using primers G5, annealing in the 3’ end of the marker, and G4,
annealing downstream of the integration point. Disruption mutants of PIL1 were also
generated by inserting the marker in the middle of the ORF as follows, generating ASJ21. A
part of the PIL1 gene was first cloned in pGEM-T Easy Vector, QIAGEN, using primer pair
I1/I2, producing pGEM-AgPIL1-A. The marker as a blunt ended PvuII/EcoRV fragment from
pFA-kanMX was ligated into an EcoRV site in the PIL1 ORF of pGEM-AgPIL1-A. The
disruption cassette was cut out with NotI and transformed into leu2. I1/G2 and G3/I2 primers
confirmed integration of the cassette. A GFP was fused to the 3’ end of PIL1 via in vivo
recombination in S. cerevisiae. First the whole ORF of PIL1 was cloned in pGEM, producing
pGEM-AgPIL1-B, with primers I3/G4. The fragment was cut out with PmeI, provided with
the I3 primer and NdeI, cleaving at an endogenous site just upstream of G4, and ligated to a
pRS417 backbone (from 651). The GEN3 marker was exchanged to NAT5 from pFA-NAT5
with restriction enzymes BstZ171/PacI, generating pRS418-AgTEF1p-AgPIL1. Both GEN3
and NAT5 contain the ScTEF2 promoter and terminator, BstZ171 and PacI cleaves in these
regions.
PIL1 GFP
3’ PIL15’ PIL1
kanMX
kanMX
I1 I2S1 S2
S1-GFP S2-GFPEcoRV
B
A
C
D
I3
G4
AgTEF1p PIL1 GFP
3’ PIL15’ PIL1
kanMX
kanMX
I1 I2S1 S2
S1-GFP S2-GFPEcoRV
B
A
C
D
I3
G4
AgTEF1p
Figure 17. A Annealing sites of primers in PIL1 for generation of ORF disruption and 3’ end GFP fusion. B Almost all of the ORF was replaced with a marker cassette amplified with S1/S2, leaving the first 55 nt in the genome. C A part of PIL1 was cloned in pGEM with primers I1/I2 and the kanMX marker was inserted into the EcoRV site. The whole disruption cassette was cleaved out and transformed into A. gossypii. D PIL1 was cloned behind an AgTEF1 promoter on a pRS418 plasmid with I3/G4. A GFP-marker cassette with homologous flanks to the end of PIL1 was amplified with primer pair S1-GFP/S2-GFP. The PIL1 ORF and GFP were fused in an in vivo recombination event in yeast.
36
pRS418-AgTEF1p-AgPIL1was co-transformed into yeast with a GFP cassette amplified from
pFA-GFP-kanMX with primers PIL1-S1-GFP/PIL1-S2-GFP, producing the final GFP-tagged
gene construct, pRS418-AgTEF1p-AgPIL1-GFP. This plasmid was used for complementation
of the pil1 mutant but also transformed into leu2, lsp1, sur7, sac6 and wal1. GFP was
integrated into the the PIL1 locus of leu2 with an AatII/NotI fragment of pRS418-AgTEF1p-
AgPIL1-GFP, cutting inside the ORF of PIL1 and after the GFP cassette.
6.7 Plate assays and germination of spores
To see how the growth of deletion strains was affected, 5 µl drops of mycelia were inoculated
on AFM plates and colony diameters were compared to leu2 after a week at different
temperatures. Germination efficiency of sac6 was investigated by spreading diluted spore
solutions on AFM plates and counting visible colonies that had formed after tree days. 1 µl of
a sac6 spore solution with OD600 of 0.1 was spread on plates with 200 µl water. leu2 spores
were diluted another 100x before plating.
6.8 Microscopy and staining procedures
Microscopy was performed with an Axio-Imager microscope (Zeiss, Jena and Göttingen,
Germany) and images processed with Metamorph 7 software (Molecular Devices Corp.,
Downington, PA). Fluorescent microscopy was performed with the appropriate filter
combinations for GFP, FM4-64, Alexa488/Rhodamin, Calcofluor White, Luciferase Yellow
and DiOC6. Either single images of one plane were taken or a Z-stack generated from
20-30 planes. Images were acquired on a MicroMax1024 CCD-camera (Princeton
Instruments, Trenton NJ, USA). O/n cultures of GFP-tagged C. albicans strains where diluted
in YPD and grown to exponential phase in 30 C before microscopy. Hyphal formation was
induced as previously described. A. gossypii strains were usually taken from an exponentially
growing AFM culture. Calcofluor White staining and actin staining with Alexa488-Phallodin
and Rhodamine-Phallodin was performed as described (Wendland & Philippsen, 2000).
Visualization with FM4-64 and mitochondrial staining with DiOC6 was done according to
(Walther and Wendland 2004a). Luciferase Yellow was added to a fresh AFM culture
(5-10 mg/ml LY) and samples were taken out at different time points and washed with water
before microscopy.
37
7 Results
Several of the proteins involved in the conserved pathways of endocytosis contain
SH3 domains. A set of SH3 domain-containing genes of C. albicans was fused to GFP for
localization studies. Three C. albicans proteins were successfully tagged and their localization
was compared to the S. cerevisiae homologs. The isolated SH3 domains of C. albicans were
also tested in a two-hybrid assay for their interaction with Vrp1, a protein very rich in prolines
and therefore a likely target for SH3 domain-binding. Several truncated versions of Wal1, the
WASP homolog in C. albicans, were used in the two-hybrid experiment with Vrp1 since the
two homologs of S. cerevisiae are known to interact. Sla2 is an important endocytosis factor
in S. cerevisiae but was found to be lacking in the genome of A. gossypii. To investigate this
further, SAC6 of A. gossypii was deleted, a gene that shows synthetic lethality with SLA2 in
S. cerevisiae. Eisosomes have been implicated to be involved in endocytosis in S. cerevisiae
and to colocalize to actin. The A. gossypii homologs were deleted in order to investigate their
importance for endocytosis and localization of eisosomes was compared to the actin patch
distribution.
7.1 Generation of heterozygous C. albicans strains
Heterozygous strains for a set of genes encoding SH3 domain proteins were produced by
deleting one of the two alleles by PCR based gene targeting. The CdHIS1 marker was
amplified with a specific primer pair for each gene producing a deletion cassette with long
homology regions to both flanks of the gene, ensuring efficient integration into the target
locus. Heterozygous C. albicans mutants were verified for eight genes, BBC1, BUD14, CYK3,
PIN3, NBP2, RVS167-2, SHO1 and SLA1. Integration of the CdHIS1 marker into the right
locus of all strains was confirmed at both flanks by PCR (App. I). Fusing of GFP to the
second allele will eliminate expression of any wild type copies of the gene. To this end GFP
fusion constructs on a pRS417 plasmid were first generated for all genes. A fragment with
the last 4-500 bases of each ORF and 4-500 bases of the downstream region was isolated and
fused to a GFP cassette via in vivo recombination in S. cerevisiae. Primers for the GFP fusion
cassette were designed to exclude the stop codon of the target gene and introduce the GFP
ORF in the same reading frame. This strategy allows for sequencing of the fusion point, and
confirmation that the GFP has integrated in the correct reading frame on the plasmid. The
fusion cassette was cut out of the plasmid and transformed into the respective heterozygous
38
strain tagging the remaining allele with GFP. Genomic integration of the GFP downstream of
the target gene was confirmed by PCR in all cases (App. I).
7.2 Localization of C. albicans SH3 domain proteins
Out of eight C-terminally GFP-tagged SH3-domain proteins three produced a convincing GFP
signal in C. albicans cells, namely Cyk3, Sla1 and Bbc1. In all cases the localizations of the
fusion proteins were corresponding to the localization of their homologs in S. cerevisiae
(Fig. 18) (Korinek et al. 2000; Mochida et al. 2002; Warren et al. 2002). Sla1-GFP and
Bbc1-GFP can be seen in cortical patches in both yeast and hyphal cells. Cyk3-GFP shows up
at septal sites, in yeast cells with large buds and in the distal septa of hyphae. Cyk3 in
S. cerevisiae localizes to the bud neck only in the short cytokinesis phase and this would
explain why the GFP-signal of CaCyk3 could only be seen in a small population of the cells
(~30 % of large budded cells, n=82).
B
SLA1-GFP BBC1-GFP CYK3-GFP
A
30 C
37 C
SLA1-GFP BBC1-GFP CYK3-GFP
A
30 C
37 C
bud neck, septabud neckCyk3
cortical actin
patches
cortical actin
patchesBbc1
cortical actin
patches
nucleus, cortical
actin patchesSla1
C. albicansS. cerevisiaeprotein
bud neck, septabud neckCyk3
cortical actin
patches
cortical actin
patchesBbc1
cortical actin
patches
nucleus, cortical
actin patchesSla1
C. albicansS. cerevisiaeprotein
Figure 18. Localization of three SH3 domain proteins in C. albicans. A GFP signals in yeast and hyphal cells of C. albicans. Sla1-GFP and Bbc1-GFP localizes in cortical patches in both daughter and mother cells. Cyk3-GFP localizes to bud necks of yeast cells with a large bud and in the most distal septa of hyphae. Scale bar is 5 µm. B Localizations of homologs in C. albicans and S. cerevisiae.
39
7.3 Yeast two hybrid assay
In S. cerevisiae Vrp1 interacts with Las17 and the SH3 domain of the type I myosin Myo5. To
see whether the same complex also exists among the homologs in C. albicans, Vrp1-Wal1-
Myo5, a yeast two-hybrid analysis was performed (Borth et al 2010) (Fig. 19). SH3 domains
have a preference for proline rich sequences and Vrp1 which is very rich in prolines was
divided in two parts. Both the C-terminal and N-terminal parts were fused to the GAL4 DBD
on plasmid pGBT9. The C-terminus of Vrp1 was also fused to the AD on pGAD424 to be
assayed against its N-terminus. Full length and truncated versions of Wal1 were fused to the
DBD and interactions against the C-terminus of Vrp1 were investigated. A β-galactosidase
assay exploiting the reporter gene lacZ showed that the WH1 domain of CaWal1 interacts
with the C-terminal of CaVrp1 and a stronger interaction was seen when the central proline
rich region of Wal1 was removed. The central proline rich and C-terminal regions of Wal1
and the N-terminal of Vrp1 do not show any interaction with Vrp1-C-terminal. The results
from the ONPG test agreed with the spot assays; the strongest interaction between Wal1 with
out its proline rich region and Vrp1-C-terminus produced the bluest spot on X-gal media.
31
24
115
20
15
21
80
21
β-galactosidase assay [Miller
units]
X-
gal
-trp
-leu
-adeAD-pGAD424BD-pGBT9 β-galactosidase assay [Miller
units]
X-
gal
-trp
-leu
-adeAD-pGAD424BD-pGBT9
LBDWH2 HOTWH2 LBDWH2 HOTWH2
BWH1 P1 VCAP2 P3 P4
VCAP3 P4 HOT
WH1
LBD
WH2 HOT LBDWH2
LBD
WH2WH2
WH2 HOTWH2 SH3
WH2 LBDWH2
WH2 HOTWH2
BWH1 P1 VCAP2 P3 P4BWH1 P1 VCAP2 P3 P4
VCAP3 P4 VCAP3 P4 HOT
WH1
LBD
WH2 HOT LBDWH2
LBD
WH2WH2
WH2 HOTWH2 SH3
WH2 LBDWH2
WH2 HOTWH2
WH1WH1
LBD
WH2 HOT LBDWH2WH2 HOT LBDWH2
LBD
WH2WH2
WH2 HOTWH2 SH3
WH2 LBDWH2
WH2 HOTWH2WH2 HOTWH2
BWH1 P1 VCAP2 P3 P4BWH1 P1 VCAP2 P3 P4 SH3Wal1 Vrp1 SH3-Myo5
Figure 19. Two hybrid analyses of fragments of Wal1, Vrp1 and Myo5, visualised qualitatively on plates and quantitatively in an ONPG assay. Fragments are fused to the binding domain (BD) or the activation domain (AD) of Gal4 on plasmids pGBT9 and pGAD424. When fragments interact the AD and BD forms an active Gal4 and reporter genes ADE2 and lacZ becomes active. Growth on –ade plates (left row of spot assay) indicates that the reporter gene ADE2 is active, colonies turn red on low adenine media (middle row) when ADE2 is off. Blue colour (right row) and Miller units in the ONPG assay is a result of an active lacZ reporter gene. Negative control constitutes the empty plasmids pGBT9/pGAD424.
40
Also, interactions resulting in higher Miller units than the negative control grew on media
lacking adenine, showing that the reporter gene ADE2 was activated. Red colonies on low
adenine media (CSM-Trp-Leu) indicate lack of interactions between the expressed fragments,
as shown for Vrp1-C-terminal against its N-terminal and the proline rich and C-terminus of
Wal1.The SH3 domains of C. albicans were isolated and cloned in the pGAD424 plasmid, as
single domains or, when two SH3 domains resided closely together, as double domains.
Interactions of the C- and N-termini of CaVrp1 were assayed against all the SH3 domains, but
only the SH3 domain of Myo5 would test positive (Fig. 20 and 21).
Growth on plates lacking adenine and a high β-galactosidase activity indicated a strong
interaction of Myo5-SH3 against the C-terminal part of Vrp1. Two hybrid strains with Myo5-
SH3 and the N-terminus of Vrp1 showed some growth on plates lacking adenine but a very
poor result in the β-galactosidase assay. Together, these results show that Vrp1 binds directly
Figure 20. ONPG-assay with yeast two hybrid strains. The DBD of GAL4 was fused to the CaVrp1 N- or C-terminus, A and B. A single or double SH3 domain of each gene was cloned to the AD of GAL4 as described. The dotted line corresponds to the negative control, a strain containing empty plasmids The value of the positive control, containing pGBT-ScBUD3 and pGAD-AgCDC3 is very high (700-1100) and cut off in the graph.
CaVRP1 C-terminus
0
20
40
60
80
100
pos
neg
CaA
BP
1
CaS
LA
1-1
CaS
LA
1-2
CaB
OI1
CaC
YK
3
CaH
SE
1
CaB
BC
1
CaB
EM
1
CaB
EM
1L
CaF
US
1
CaH
OF
1
CaP
EX
13
CaP
IN3
CaB
UD
14
CaR
VS
167-1
CaLS
B3
CaS
HO
1
CaC
DC
25
CaR
VS
167-2
CaQ
59U
90
CaN
BP
2
CaQ
5A
AN
3
CaM
YO
5
CaC
DC
25L
Mill
er
units
CaVRP1 N-terminus
0
20
40
60
80
100
pos
neg
CaA
BP
1
CaS
LA
1-1
CaS
LA
1-2
CaB
OI2
CaC
YK
3
CaH
SE
1
CaB
BC
1
CaB
EM
1
CaB
EM
1L
CaF
US
1
CaH
OF
1
CaP
EX
13
CaP
IN3
CaB
UD
14
CaR
VS
167-1
CaLS
B3
CaS
HO
1
CaC
DC
25
CaR
VS
167-2
CaQ
59U
90
CaN
BP
2
CaQ
5A
AN
3
CaM
YO
5
CaC
DC
25L
Mill
er
units
A
B CaVRP1 C-terminus
0
20
40
60
80
100
pos
neg
CaA
BP
1
CaS
LA
1-1
CaS
LA
1-2
CaB
OI1
CaC
YK
3
CaH
SE
1
CaB
BC
1
CaB
EM
1
CaB
EM
1L
CaF
US
1
CaH
OF
1
CaP
EX
13
CaP
IN3
CaB
UD
14
CaR
VS
167-1
CaLS
B3
CaS
HO
1
CaC
DC
25
CaR
VS
167-2
CaQ
59U
90
CaN
BP
2
CaQ
5A
AN
3
CaM
YO
5
CaC
DC
25L
Mill
er
units
CaVRP1 N-terminus
0
20
40
60
80
100
pos
neg
CaA
BP
1
CaS
LA
1-1
CaS
LA
1-2
CaB
OI2
CaC
YK
3
CaH
SE
1
CaB
BC
1
CaB
EM
1
CaB
EM
1L
CaF
US
1
CaH
OF
1
CaP
EX
13
CaP
IN3
CaB
UD
14
CaR
VS
167-1
CaLS
B3
CaS
HO
1
CaC
DC
25
CaR
VS
167-2
CaQ
59U
90
CaN
BP
2
CaQ
5A
AN
3
CaM
YO
5
CaC
DC
25L
Mill
er
units
CaVRP1 C-terminus
0
20
40
60
80
100
pos
neg
CaA
BP
1
CaS
LA
1-1
CaS
LA
1-2
CaB
OI1
CaC
YK
3
CaH
SE
1
CaB
BC
1
CaB
EM
1
CaB
EM
1L
CaF
US
1
CaH
OF
1
CaP
EX
13
CaP
IN3
CaB
UD
14
CaR
VS
167-1
CaLS
B3
CaS
HO
1
CaC
DC
25
CaR
VS
167-2
CaQ
59U
90
CaN
BP
2
CaQ
5A
AN
3
CaM
YO
5
CaC
DC
25L
Mill
er
units
CaVRP1 N-terminus
0
20
40
60
80
100
pos
neg
CaA
BP
1
CaS
LA
1-1
CaS
LA
1-2
CaB
OI2
CaC
YK
3
CaH
SE
1
CaB
BC
1
CaB
EM
1
CaB
EM
1L
CaF
US
1
CaH
OF
1
CaP
EX
13
CaP
IN3
CaB
UD
14
CaR
VS
167-1
CaLS
B3
CaS
HO
1
CaC
DC
25
CaR
VS
167-2
CaQ
59U
90
CaN
BP
2
CaQ
5A
AN
3
CaM
YO
5
CaC
DC
25L
Mill
er
units
A
B
41
to both Wal1 and Myo5 in vivo suggesting a complex similar to the Vrp1-Las17-Myo5
complex in S. cerevisiae.
7.4 SLA2 is ascent from the A. gossypii genome
Sla2 is an essential part of the endocytic machinery in S. cerevisiae but the SLA2 gene can not
be found in the A. gossypii genome. A search using NCBI pBLAST with the S. cerevisiae
Sla2 sequence generates no hits in A. gossypii and further investigation indicates that the
SLA2 gene has been lost during evolution, possibly in the development of a mating type locus.
The synteny around SLA2 in S. cerevisiae has not been conserved, the gene is found on the
third chromosome next to SUI1. In A. gossypii SUI1 is connected to of all of the three mating
type loci on chromosomes 4, 5 and 6 (Fig. 22). In other yeasts SLA2 is coupled to the mating
type loci as in Klyveromyces lactis where SLA2 is located between SUI1 and the mating type
locus.
+ -
A
B 1 ABP1 13 PIN3
2 SLA1-1 14 BUD14
3 SLA1-2 15 RVS167-1
4 BOI1 16 LSB3
5 CYK3 17 SHO1
6 HSE1 18 CDC25
7 BBC1 19 RVS167-2
8 BEM1 20 Q59U90
9 BEM1L 21 NBP2
10 FUS1 22 Q5AAN3
11 HOF1 23 MYO5
12 PEX13 24 CDC25L
Vrp1-Nt Vrp1-Ct1-12 13-24 1-12 13-24
Vrp1-Nt Vrp1-Ct1-12 13-24 1-12 13-24
+ - + - + -
1 2 3 4
5 6 7 8
9 10 11 12
Figure 21. A yeast two hybrid screen of all SH3 domains from C. albicans against CaVrp1 N-terminus and C-terminus. SH3 domains were fused to the AD of Gal4 on pGAD424 and Vrp1 fragments to the DBD of Gal4 on pGBD9. Physical interaction of AD and DBD in the yeast strains will produce the active transcription factor Gal4 and induce expression of reporter genes, ADE2 and lacZ. Positive control is a strain with pGBT-ScBUD3 and pGAD-AgCDC3, the negative control strain contains empty plasmids. A Strains with the SH3 domain of Myo5 and
either N- or C‑terminus of Vrp1 where able to grow on
selective plates lacking adenine (circles). B All genes providing the SH3 domains are numbered according to their position in the spot grid, from left to right, top to bottom. SLA1-1 contains the two first SH3 domains of SLA1, SLA1-2 contains the third domain.
42
7.5 Generation of Agsac6
SLA2 and SAC6 were found to be essential in abp1 strains of S. cerevisiae and showed
synthetic lethality with each other. SLA2 is not present in the genome of A. gossypii so we
wanted to investigate the impact of a SAC6 deletion in this fungus. To this end, two
independent homokaryotic sac6 strains where generated by replacing the whole SAC6 ORF
with a deletion cassette. The cassette was generated in a two step PCR (Fig. 16). First, 500
base pair long sequences of upstream and downstream regions of the SAC6 ORF and a marker
cassette were amplified by PCR. The primers used in these reactions would add short
sequences to the flank fragments and the marker that overlap. In a second PCR, the three
overlapping fragments were used as a template with primers defining the very ends of the
cassette. Accordingly, a deletion cassette with long homologous regions to the SAC6 locus
was produced. Transformation of the deletion cassette produced heterokaryotic strains;
integration of both ends of the cassette was confirmed by PCR (App. II). Sporulation and
micromanipulation of germinated spores enabled isolation of two homokaryotic strains. No
internal PCR fragments of SAC6 could be amplified from these strains confirming that they
were null mutants (App. II).
Figure 22. Syntenic position of SLA2 in yeast. SLA2 is located next to SUI1 in the S. cerevisiae genome but can not be found in A. gossypii. SUI1 in A. gossypii is in synteny with the mating type locus on all three chromosomes containing the mating type information (in this picture a MATa-strain). In Klyveromyces lactis SLA2 is positioned between SUI1 and the mating type locus.
Chr XIV
S. cerevisiae
Chr IV
Chr V
Chr VI
A. gossypii
K. lactis
CWC25
SUI1 A2A1
SUI1 A2A1
SUI1 A2A1
SUI1 ATG2SLA2
SUI1 A2A1SLA2
YNL246W YNL245C YNL244C YNL243W YNL242W
YBR072W YNL246W YNL244C YHR202W
YBR072W YNL246W YNL244C YHR202W
VPS75
YNL247W YNL246W YNL244C YJL204C
YNL245C YNL244C YNL243W YJL207C
Chr XIV
S. cerevisiae
Chr IV
Chr V
Chr VI
A. gossypii
K. lactis
CWC25
SUI1 A2A1
SUI1 A2A1
SUI1 A2A1
SUI1 ATG2SLA2
SUI1 A2A1SLA2
YNL246W YNL245C YNL244C YNL243W YNL242W
YBR072W YNL246W YNL244C YHR202W
YBR072W YNL246W YNL244C YHR202W
VPS75
YNL247W YNL246W YNL244C YJL204C
YNL245C YNL244C YNL243W YJL207C
43
7.6 Characterization of Agsac6
Both sac6 strains generated by PCR based gene targeting displayed the same features and
mutant phenotypes were restored by introducing a plasmid containing the whole SAC6 gene
(Fig. 23B). This confirms that the observed phenotypes have arisen due to deletion of the
SAC6 ORF. The sac6 strains are temperature sensitive with reduced growth rate on full media
(Fig. 23A). Spores are unable to form visible colonies in 37 C and germination frequency is
20-30 folds lower at 30 C and room temperature in sac6 than in leu2 (Fig. 24).
x100
leu2
sac6
300 180 186
700 900 0
RT 30°C 37°C
x100
leu2
sac6
300 180 186
700 900 0
RT 30°C 37°C
leu2
sac6
300 180 186
700 900 0
RT 30°C 37°C
Figure 24. Germinated spores on AFM plates after three days. leu2 spores were diluted another 100x. Numbers show approximation of germinated spores on the plates. Scale bar is 2 cm.
leu2 sac6 sac6 + p-SAC6
SA
C6p S
AC
6t
NAT5
SAC6
amp
CEN/ARS
pRS418-SAC6
7,9 kb
SA
C6p S
AC
6t
NAT5
SAC6
amp
CEN/ARS
pRS418-SAC6
7,9 kb
B
leu2 sac6
37 ºC
30 ºC
A
leu2 sac6
37 ºC
30 ºC
A
leu2 sac6
37 ºC
30 ºC
A
Figure 23. Growth of sac6 and complementation. A A small mycelial inoculum of leu2 and sac6 on AFM plates after 7 days. B A plasmid bearing the whole gene of SAC6 and the resistance gene for ClonNat is able to restore the growth rate of the mutant. Scale bar is 2 cm.
44
When the mycelia of sac6 were grown on plates in 37 C the hyphae often swelled and burst
close to the tips, a feature similar to the wal1 mutant (Fig. 25). An actin stain revealed that the
actin patches of sac6 are mislocalized and not polarized to the tip as in wild type (Fig. 26).
wal1 shows a similar subapical localization of actin but with fewer patches.
A B C D E F
leu2 sac6sac6+
p-SAC6 wal1
30°C 37°C 30°C 37°C 30°C 30°C
leu2 sac6sac6+
p-SAC6 wal1
30°C 37°C 30°C 37°C 30°C 30°C
Figure 26. Actin stain with Alexa488. Actin patches are subapically localized in sac6, C-D, and not polarized to the very tip as in leu2, A-B. p-SAC6 fully complements the phenotype, E. The actin mislocalization is also pronounced in wal1, F. Scale bar is 10 µm.
Figure 25. Bursting hyphae of sac6 and wal1 on microscope slides. A sac6 spores where germinated in 30 °C o/n and then transferred to 37 °C o/n. Arrows indicate a few of the parts of hyphae that has blown up or burst, two areas are magnified, B and C. For comparison, a germinated spore, D, and part of a hypha, E, from a wal1 strain grown in 30 °C.
C
A
100µM
B
E
D
20µM
C
A
100µM
B
E
D
20µM
45
To see whether the endocytosis was affected in these two mutants, both sac6 and wal1 were
stained with the lipophilic dye FM4-64 and the fluid phase endocytosis marker Luciferase
Yellow, LY. Both assays indicate that sac6 has a slightly reduced endocytosis rate compared
to wild type and that wal1 is even more affected (Fig. 27 and 28). The endosomes become
stained with FM4-64 very fast in all strains but the dye remains in the cell membrane for a
longer time in sac6 and wal1 compared to leu2. After 15 minutes vacuoles are seen in leu2
but they are still only faintly stained in the mutants even after 30 minutes. The FM4-64 dye is
finally delivered to elongated vacuoles in sac6, indicating movement of the vacuolar
membranes. In wal1 the vacuoles are spherical, filling up the diameter of the hyphae but do
not elongate. It takes longer for the LY to reach the vacuoles in sac6 and wal1 than in leu2.
Within 15 minutes the dye lights up vacuoles in leu2 but it takes 30-60 minutes for vacuoles
in sac6 to be readily visible and even longer in wal1.
Figure 27. FM4-64 uptake in sac6 and wal1. Endosomes in the tips are readily visualized by the dye. Eventually the dye ends up in larger fused vacuoles in the proximal areas of hyphae. Scale bar is 5µm.
15min 30min 120min
sac6leu2 wal1
15min 30min 120min
sac6leu2 wal1
46
7.7 Deletion of LSP1, SUR7, PKH1 and YPK1
Null mutant strains of lsp1 and sur7 where generated by PCR based gene targeting as
described for the sac6 strains (Fig. 16). Long flanking deletion cassettes generated in a two
step PCR replaced the whole ORF of the two genes. PCR confirmed integration of the
cassette into the target locus at both ends in the heterokaryons and no internal PCR products
could be amplified from purified DNA from micromanipulated strains. Heterokaryotic strains
of pkh1 and ypk1 were generated using long flanking deletion cassettes and verified by PCR.
All verification PCRs are shown in Appendix II. No homokaryotic mycelia with PHK1 or
YPK1 deletions could be isolated, spores would germinate in an o/n culture at 30 C but after
micromanipulation they seized to grow. Approximately 30 spores that had germinated and
produced one or two germ tubes were selected for each strain but none of them grew further
after isolation. The pkh1 and ypk1 strains where not investigated further.
7.8 Characterization of lsp1 and sur7
Null mutants of lsp1 and sur7 where investigated on the aspects of growth rate on full media
plates, actin distribution and endocytosis visualized by uptake of FM4-64. Aglsp1 displays no
phenotypes compared to the precursor strain leu2 in all these cases. Actin patches are
polarized in growing hyphal tips and actin rings marks septa (Fig. 30). FM4-64 stains
endosomes and is delivered to large vacuoles at a normal rate (Fig. 31)and the size and
morphology of lsp1 colonies after a week on AFM plates are similar to leu2 (Fig. 29).
15min 30min 60min
sac6leu2 wal1
15min 30min 60min
sac6leu2 wal1
Figure 28. Luciferase Yellow is taken up by fluid phase endocytosis and stains the inside of the vacuoles. Scale bar is 5 µm.
47
Deletion of SUR7 results in a weak phenotype and the sur7 colonies are somewhat smaller
than leu2 and lsp1 on full media plates after a week (Fig. 29). At this time point both leu2 and
lsp1 are yellow in the centre of the colonies, due to riboflavin production, but sur7 is still pale
in colour. All of the colonies have produced spores in the central zone and sur7 sporulates
efficiently after a few days in CSM media. Actin is normally distributed in rings at septal sites
and in patches polarized to sites of growth (Fig. 30). Uptake of FM4-64 in hyphal tips of sur7
is fast, endosomes are stained very soon, indicating normal endocytosis (Fig. 31). However,
sur7 is affected in the fusion of vacuoles; it takes up to three hrs before the larger vacuoles are
visualized with the dye, in leu2 it takes less than 30 minutes. Chitin deposition is affected in
sur7, calcofluor staining showed irregularly shaped septa and sometimes clumps of chitin
aggregated on the inside of the cell walls (Fig. 32). Sur7 is a membrane protein and to test
whether the plasma membrane was affected in the mutant sur7 was grown on full media
plates containing SDS or Calcofluor White, CWF. The hyphae of sur7 were indistinguishable
from leu2 under a microscope on any of these plates but after a week the colony diameters of
sur7 were 68 % and 73 %, respectively, compared to leu2 grown under the same conditions
(n=4-5) (Fig. 33). On full media plates the colony size of sur7 was 86 % of leu2 after a week.
leu2 lsp1 sur7
37ºC
30 ºC
leu2 lsp1 sur7
37ºC
30 ºC
Figure 29. Growth of lsp1 and sur7 on AFM after seven
days at 30 C. Scale bar is 2 cm.
48
leu2 sur7leu2 sur7
Figure 32. Calcofluor stain showing chitin depositions at septal sites. Scale bar is 10 µm.
Figure 31. FM4-64 staining of eisosome mutants. Endosomes become visible in the tips very fast and the dye is delivered to the large tubular vacuoles eventually. Scale bar is 5 µm.
15min 30min 120min
lsp1leu2 sur7
180min15min 30min 120min
lsp1leu2 sur7
180min
leu2 lsp1 sur7 leu2 lsp1 sur7leu2 lsp1 sur7 leu2 lsp1 sur7
Figure 30. Alexa488-Phalloidin stain in eisosome mutants showing polarized actin patches and acting rings. Scale bar is 10 µm.
49
7.9 Deletion of PIL1
Deletion cassettes with long flanks could not be generated for PIL1. The upstream region of
the PIL1 start codon could not be amplified by PCR even though different primers were
designed for the purpose (Fig. 34). One primer was placed in the upstream gene ISR1 but the
region between the genes could not be amplified in order to clone and sequenced it.
Mutant strains of PIL1 were instead generated by deletion of almost the complete ORF and by
insertion of a cassette into the ORF. Homokaryotic pil1 mycelia could not be produced,
although independent heterokaryons where confirmed by PCR in both disrupted and ORF
deleted versions. The heterokaryotic strains where sporulated and micromanipulated in order
to isolate homokaryotic strains. Heterokaryotic mycelium contains both transformed and wild
ISR1_AEL330C PIL1
500 bp
426
4
426
0
410
7
410
3
410
4
424
1
410
9
425
6
419
9
411
0
410
5
424
2
410
6
410
8ISR1_AEL330C PIL1
500 bp
426
4
426
0
410
7
410
3
410
4
424
1
410
9
425
6
419
9
411
0
410
5
424
2
410
6
410
8
Figure 34. The PIL1 locus and the neighbouring gene ISR1 according to the annotated sequence in AGD. Lines represent PCR products defined by nine different primer pair combinations, primer numbers below. Whole lines correspond to successfully amplified regions and dotted lines to regions that could not be amplified with the defined primers.
leu2 sur7A
0,0
05%
SD
S 1
00µ
g/m
l C
FW
A
FM
leu2 sur7A
0,0
05%
SD
S 1
00µ
g/m
l C
FW
A
FM
Figure 33. A Growth of leu2 and sur7 on plates containing 100 µg/ml CFW or
0,005 % SDS after one week at 30 C. Scale bar is 2 cm. B Graph showing sizes of the colonies, 4-5 colonies each where measured.
50
type nuclei and upon sporulation they will separate into individual spores. The wild type
spores will not germinate in selective media and every spore that germinates should therefore
constitute a null mutant genotype. Spores from pil1 (both deletion- and disruption mutants)
where incubated o/n in selective full media at both 30 C and room temperature. A large
portion of the spores germinated (Fig. 35) and in total 50-60 germlings at different sizes were
selected, from spores with a germ cell to small mycelia with a few lateral branches. None of
them continued to grow after they where transferred to new spots on the plate and placed at
30 C or room temperature.
However, a small population of the pil1 germlings grew long enough in liquid culture to allow
for characterization of several features. The germlings eventually ceased to grow and they
never reached the tip splitting stage. For this reason, no mycelia could be collected to verify
the disruption of the PIL1 gene by PCR. However, both pil1 strains generated by disruption
and by ORF deletion displays exactly the same properties and a plasmid bearing an
AgTEF1p driven PIL1-GFP could rescue the strains. Since all mutants of pil1 show the same
phenotype in all regards they are not distinguished between in the characterization.
7.10 Characterization of the pil1 phenotype
A small population of the germinated pil1 spores continued to grow and generated germ tubes
and lateral branches. Eventually the cells went into a stationary phase where the surface
became full of kernels and they hyphae blew up, giving them features that easily distinguished
them from the still vital germlings. These two sets of pil1 species were been investigated and
compared on several characteristics. Approximately ten germlings of each genotype (deletion
or disruption mutant) reaching at least the state of two germ tubes have been used to define
the typical characteristics of the two phenotypes (vital and stationary) in each test. The vital
Figure 35. Germinated spores of pil1 grown o/n in AFM-G418 at 30 C. A heterokaryotic mycelium was sporulated producing spores with either a wild type or pil1 nucleus. The germ cells that have formed should all be pil1 null mutants since wild type does not germinate in selective media. Scale bar is 10 µm.
51
subset of pil1 mutants has polarized actin patches (Fig. 36), normal septa (Fig. 38) and
FM4-64 uptake after 45 minutes is comparable to leu2 germlings of the same size (Fig. 41).
Luciferase Yellow is taken up by fluid phase endocytosis in pil1 and ends up in large vacuoles
in the tip (Fig. 39). In leu2 only small vacuoles are seen in the tip as the larger vacuoles are
located further away from the tip (not shown). The pil1 germlings cease to grow at different
stages of lateral branching, usually within 10 hrs post germination. None of the hyphae were
observed to reach maturation where they would do dichotomous branching and the growth
speed would increase remarkably. Fig. 40 shows a rather large pil1 germling that branches
laterally from several hyphae, all the tips have polarized actin patches. When the germlings
have ceased to grow the actin is no longer polarized to the tips (Fig. 36) and mitochondria
have been fragmented (Fig. 37). Endocytosis is halted in this stage, fluid phase uptake of LY
is blocked (Fig. 39) and FM4-64 is not seen in small vacuoles as in the vital pil1 germlings
(Fig. 41).
leu2 pil1 pil1
A B C
leu2 pil1 pil1
A B C
Figure 37. Mitokondrial staining with DiOC6 showing long and tubular mitokondria in leu2, A, and in the vital pil1 hyphae, B. Mitochondria have become defragmented in C. Scale bar is 10µm.
leu2 pil1 pil1
A B C
leu2 pil1 pil1
A B C
Figure 36. F-actin staining of pil1. A, B Germlings that still grow have polarized and wild type like distribution of actin patches. C The germlings with a dotty structure and swollen hyphae have depolarized actin. Calibration bar is 10µm.
52
10µM20µM
A B
10µM20µM
A B
Figure 40. A Extensive lateral branching in a small pil1 mycelium. B Actin stain of one hyphae showing polarized patches in the leading tip, arrow.
leu2 pil1 pil1
A B C
leu2 pil1 pil1
A B C
Figure 39. Fluid phase uptake of LY in young germlings after 2 hours. The dye lights up small vacuoles in the tips of leu2, A (arrows), and large vacuoles in the viable pil1 hyphae B. LY has not been internalized in pil1 hyphae in C. Scale bar is 5 µm.
leu2 pil1 pil1
A B C
leu2 pil1 pil1
A B C
Figure 38. Calcofluor staining of pil1. Scale bar is 10µm.
53
Figure 41. FM4-64 uptake of pil1 after 45 minutes, parts of the hyphae are enlarged for greater details. They dye is normally delivered to small vacuoles in the germlings, A and B. In C no vacuoles can be seen, the dye is dispersed in dots all over the hyphae. Scale bar is 10µm.
leu2
pil1
pil1
A
B
C
leu2
pil1
pil1
A
B
C
54
7.11 Localization of AgPIL1-GFP
Overexpression of a PIL1-GFP construct could rescue the pil1 phenotype. Heterokaryotic
mycelia with the complementing plasmid were sporulated and the growth rate of the resulting
homokaryotic strains bearing the plasmid was restored to growth rate of leu2 in 30 C
(Fig. 42). PCR products from a wild type PIL1 copy could not be amplified from the
complemented strain, only the GFP-tagged version was found to be present. The GFP
localized in a punctate cortical pattern (Fig. 43), representing the eisosomes, often leaving the
very tip of hyphae devoid of spots (Fig. 45). Eisosomes formed already in the germ cells
(Fig. 44). The PIL1-GFP plasmid was transformed into leu2 and the other eisosome mutants,
lsp1, sur7, sac6 and wal1. Distribution of eisosomes was the same in these strains even
though their genomes still contain untagged PIL1 (Fig. 45 and 46).
Figure 43 . Pil1-GFP in leu2. Image of one plane show the cortical localisation of eisosomes. Scale bar is 10 µm.
pil1+p-PIL1-GFPleu230ºC
37ºC
BA
AgTEF1pN
AT
5PIL1
ampCEN/ARSpRS418-AgTEF1p-PIL1
8,9 kb
GFP
kanM
X
pil1+p-PIL1-GFPleu230ºC
37ºC
BA
AgTEF1pN
AT
5PIL1
ampCEN/ARSpRS418-AgTEF1p-PIL1
8,9 kb
GFP
kanM
X
AgTEF1pN
AT
5PIL1
ampCEN/ARSpRS418-AgTEF1p-PIL1
8,9 kb
GFP
kanM
X
Figure 42. Complementation of pil1. A Colony morphology on AFM plates after six days. B. A plasmid with an AgTEF1p-driven PIL1-GFP construct was used for complementation of pil1. Scale bar is 2 cm.
55
7.12 PIL1-GFP does not colocalize with actin patches
An overlay of the GFP signal with an actin staining in leu2 shows that actin patches and
eisosomes never colocalize, any overlap would be seen as yellow in Fig. 46. The same is true
for all the eisosome mutants as well for the sac6 and wal1 strains. The two latter strains are
deficient in endocytosis and have mislocalized actin but yet a normal eisosome distribution.
PIL1-GFP was integrated into the genome of A. gossypii, thereby leaving the expression of
the fusion protein to be regulated by its own promoter. Localization of the integrated
PIL1-GFP is the same as of the overexpressed construct. The tips of growing hyphae lack
eisosomes but when the growth slows down or stops the eisosomes catch up and can be seen
all over the tip (Fig 47).
leu2 lsp1 sur7 pil1leu2 lsp1 sur7 pil1
Figure 45. PIL1-GFP overexpressed from a plasmid represents eisosomes in leu2 and the lsp1 and sur7 mutants and rescues pil1, merged Z stacks. Scale bar is 10 µm.
Figure 44. PIL1-GFP representing the eisosomes appears already in the germcells.
56
7.13 Heterologous expression of AgPIL1-GFP in S. cerevisiae
The four proteins Pil1 and Lsp1 of A. gossypii and S. cerevisiae are very similar at the amino
acid level (Fig. 48). Comparison of Lsp1 and Pil1 in A. gossypii shows that the very
C-terminal part of the proteins differs most in sequence (Fig. 49). The AgTEF1p driven
AgPIL1-GFP construct localizes to cortical patches in the S. cerevisiae strain BY4741
(Fig. 50).
exponential growth o/n culture
PIL1-GFP actin merge
A B
exponential growth o/n culture
PIL1-GFP actin merge
A B
Figure 47. Z-stacks of actin and PIL1-GFP, colocalization is shown in yellow. PIL1-GFP was integrated into the genome of leu2 and is driven by its endogenous promoter. A In a fast growing hypha actin is polarized to the tip but depraved of eisosomes. B PIL1-GFP covers the tip when the hypha have stopped growing and actin patches are depolarized. Scale bar is 10 µm.
lsp1 sur7 pil1 sac6 wal1PIL1-GFP actin merge
A B
lsp1 sur7 pil1 sac6 wal1PIL1-GFP actin merge
A B
Figure 46. Actin stains and PIL1-GFP overexpressed from the pRS418-AgTEF1p-PIL1 plasmid, Merged Z-stack pictures of eisosome mutants and two mutants deficient in endocytosis, sac6 and wal1. Co-localisation is visualised in yellow. Scale bar is 10 µm.
57
Figure 50. Heterologous expression of AgPIL1-GFP in S. cerevisiae, Z-stacks. The AgTEF1p driven construct localizes in a patch like pattern in S. cerevisiae. Scale bar is 5 µm.
MHRTYSLRNQKAPTASDLQSPPPPPSSTRSKFFGRAGIASSFRKNAAGNFGPELARKLSSFVKTEKGVLRALEVVANERR 80AgLsp1.pro
MHRTYSLRNSRAPTASQLQNPPPPPSTTKNRFFGKGGLANTFRKNTAGAFGPELSRKLSQLVKIEKNVLRAIEVAANERR 80AgPil1.pro
AAARQLSMWGMDNDDDVSDVTDKLGVLIYELGELQDQFIDKYDQYRVTVKSIRNIEASVQPSRDRKQKITDQIAHLKYKE 160AgLsp1.pro
DAAKQLSLWGLENDDDVSDITDKLGVLIYETSELDDQFIDRYDQYRLTLKSIRDIEGSIQPSRDRKAKITDKIAYLKYKD 160AgPil1.pro
PQSPKIPVLEQELVRAEAESLVAEAQLSNITREKLKAAFNYQFDAIRELSEKFALIAGYGKALLELLDDSPVTPGETRPA 240AgLsp1.pro
PQSPKIEVLEQELVRAEAESLVAEAQLSNITRSKLKAAFNYQFDSLIEHSEKLALIAGYGKALLELLDDSPVTPGETRPA 240AgPil1.pro
YDGYEASRQIIMDAEQALEEWTLDAAAVKPNLSFHQTVDDVYDGEDGGEEHDWEGTQDETEQATK 305AgLsp1.pro
YDGYEASKQIIIDAEAALNDWTLDTAAVKPSLSIRRDYDEEFEEGDDGEQWEQDATEEQVAA 302AgPil1.pro
MHRTYSLRNQKAPTASDLQSPPPPPSSTRSKFFGRAGIASSFRKNAAGNFGPELARKLSSFMHRTYSLRNQKAPTASDLQSPPPPPSSTRSKFFGRAGIASSFRKNAAGNFGPELARKLSSFVKTEKGVLRALEVVANERR 80AgLsp1.pro
MHRTYSLRNSRAPTASQLQNPPPPPSTTKNRFFGKGGLANTFRKNTAGAFGPELSRKLSQLVKIEKNVLRAIEVAANERR 80AgPil1.pro
VKTEKGVLRALEVVANERR 80AgLsp1.pro
MHRTYSLRNSRAPTASQLQNPPPPPSTTKNRFFGKGGLANTFRKNTAGAFGPELSRKLSQLVKIEKNVLRAIEVAANERR 80AgPil1.pro
AAARQLSMWGMDNDDDVSDVTDKLGVLIYELGELQDQFIDKYDQYRVTVKSIRNIEASVQPSRDRKQKITDQIAHLKYKE 160AgLsp1.pro
DAAKQLSLWGLENDDDVSDITDKLGVLIYETSELDDQFIDRYDQYRLTLKSIRDIEGSIQPSRDRKAKITD
AAARQLSMWGMDNDDDVSDVTDKLGVLIYELGELQDQFIDKYDQYRVTVKSIRNIEASVQPSRDRKQKITDQIAHLKYKE 160AgLsp1.pro
DAAKQLSLWGLENDDDVSDITDKLGVLIYETSELDDQFIDRYDQYRLTLKSIRDIEGSIQPSRDRKAKITDKIAYLKYKD 160AgPil1.pro
PQSPKIPVLEQELVRAEAESLVAEAQLSNITREKL
KIAYLKYKD 160AgPil1.pro
PQSPKIPVLEQELVRAEAESLVAEAQLSNITREKLKAAFNYQFDAIRELSEKFALIAGYGKALLELLDDSPVTPGETRPA 240AgLsp1.pro
PQSPKIEVLEQELVRAEAESLVAEAQLSNITRSKLKAAFNYQFDSLIEHSEKLALIAGYGKALLELLDDSPVTPGETRPA 240AgPil1.pro
KAAFNYQFDAIRELSEKFALIAGYGKALLELLDDSPVTPGETRPA 240AgLsp1.pro
PQSPKIEVLEQELVRAEAESLVAEAQLSNITRSKLKAAFNYQFDSLIEHSEKLALIAGYGKALLELLDDSPVTPGETRPA 240AgPil1.pro
YDGYEASRQIIMDAEQALEEWTLDAAAVKPNLSFHQTVDDVYDGEDGGEEHDWEGTQDETEQATK 305AgLsp1.pro
YDGYEASKQIIIDAEAALNDWTLDTAAVKPSLSIRRDYDEEFEEGDDGEQWEQDATEEQVAA 302AgPil1.pro
Figure 49. Sequence alignment of AgLsp1 and AgPil1 with MegAlign.
AgPil1
ScPil1 ScLsp1
AgLsp1
85 %88 %
71 %
75 %
76 %
75 %
AgPil1
ScPil1 ScLsp1
AgLsp1
85 %88 %
71 %
75 %
76 %
75 %
Figure 48. Pil1 and Lsp1 of S. cerevisiae and A. gossypii. Amino acid identity calculated with MegAlign.
58
8 Discussion
Endocytosis and the elaborate network of pathways during this process are well known in the
budding yeast model Saccharomyces cerevisiae. Here, the related ascomycetous fungi
Candida albicans and Ashbya gossypii were used to study some of the key proteins involved
in actin organization that confer endocytosis and growth. SH3 domain proteins promote
protein-protein interactions and are frequently involved in processes like endocytosis and
some of these proteins were studied in C. albicans. This human pathogen displays a wide
array of morphological states and specifically the switch between yeast and hyphal growth is
pivotal for its pathogenicity. C. albicans has evolved pathways that differ from S. cerevisiae
but core mechanisms of the actin polymerization machinery and localization of several
SH3 domain-containing proteins are conserved. In the yeast model, components such as the
WASP homolog Las17, Vrp1 and the SH3 domain myosin I proteins Myo3 and Myo5 are
crucial for proper actin organization, polarity establishment and vacuolar morphology. Strains
of S. cerevisiae and C. albicans with deletions in the genes corresponding to these proteins
have similar phenotypes suggesting that they have related functions in the actin filament
nucleation. A. gossypii is very closely related to the model yeast but is strictly filamentous.
Deletion of the WASP homolog WAL1 affects actin polarization, vacuolar fusion and
septation. A. gossypii sustains unidirectional growth at a high rate even though SLA2 was
found to be absent from its genome. SAC6, which is synthetic lethal with SLA2 in
S. cerevisiae, displays genetic interaction with WAL1 in A. gossypii placing SAC6 and WAL1
in related pathways. Eisosomes, fungal specific structures implicated in endocytosis, are
present in A. gossypii and are distributed in a punctate pattern beneath the cell membrane as in
S. cerevisiae but do not colocalize with actin.
8.1 Localization of C. albicans SH3 domain proteins
C. albicans is a complex organism and many rearrangements in its genome have resulted in
different lifestyles and new functions compared to S. cerevisiae. Genetics are more tedious
due to its diploidy and less efficient homologous recombination machinery. Out of eight GFP
constructs in this study only three produced a convincing fluorescent signal when the GFP
was attached to the 3’ end of the gene. The localization of these C. albicans proteins, Bbc1,
Cyk3 and Sla1, is the same as for their homologs in S. cerevisiae, even though the hyphal
cells of C. albicans adds an extra dimension to the picture. Cyk3 localizes, in addition to bud
necks in yeast cells and pseudohypae, to septal sites in C. albicans hyphae, a structure that is
59
absent from S. cerevisiae. Correct fusion of GFP to the 3’ end of all eight genes on plasmids
was confirmed and incorporation into the genome was proven successful in all cases.
Addition of the bulky GFP might have disturbed the folding or the 3D-structure of the native
protein, abolishing its localization. Fusing the GFP to the N-terminal of the remaining seven
proteins might have been more successful in some of the cases but that was not attempted in
this study. The fluorescence signal might have been too weak to observe if expression of the
GFP fused gene was driven by a weak promoter, a double or triple GFP could have enhanced
the fluorescence. It is even possible that the gene was turned off under given circumstances.
C. albicans can not propagate plasmids so an overexpression construct would have to be
genomically integrated.
8.2 Functional relation between genes regulating actin filamentation in S. cerevisiae,
C. albicans and A. gossypii
Several studies have demonstrated that WASP family proteins, type I myosins and the Arp 2/3
complex are key factors in the nucleation of actin filaments in diverse eukaryotic organisms.
Defects in this pathway often lead to compromised endocytosis and problems to initiate or
sustain polarized growth, resulting in round and swelling cells and slow growth (Holtzman et
al. 1993; Walther and Wendland 2004b). Vacuolar fusion is altered by mutations affecting
actin regulatory factors such as Vrp1, type I myosins, components of the Arp2/3 complex and
the fimbrin Sac6. Deletion of the acidic domain of S. cerevisiae Las17 that interacts with
Arp2/3 complex also results in fragmented vacuoles (Eitzen et al. 2002). Processes involving
cytoskeleton organization are highly conserved and due to the relatively close relationship
between A. gossypii and C. albicans homologs to the key proteins involved could be expected
to be functional equivalents. The S. cerevisiae type I myosins Myo3 and Myo5 have a high
similarity and display functional redundancy, C. albicans has only one homolog, Myo5. The
yeast double myo3/myo5 mutant and the C. albicans myo5 have severe defects in actin
polarization, impairing hyphal growth in C. albicans (Goodson et al. 1996; Oberholzer et al.
2002). Deletion of the WASP homolog in S. cerevisiae, C. albicans and A. gossypii disturbs
actin patch formation, generating temperature sensitive strains (Li 1997; Walther and
Wendland 2004a; Walther and Wendland 2004b). Defects in polarized growth in Cawal1
causes pseudohyphal growth under hypha-inducing conditions and the mislocalized cortical
actin in Agwal1 is a likely reason for swellings of hyphae in subapical regions. These mutants
also present defects in endocytosis of FM4-64 and in vacuolar morphology. This study adds
the notion that deletion of VRP1 in C. albicans produces defects in vacuolar fusion, actin
60
polarization and endocytosis (Borth et al. 2010), thought the defects are not as severe as in
Cawal1.
8.3 Vrp1-Wal1-Myo5 complex in C. albicans
The type I myosins, Myo3 and Myo5, in S. cerevisiae form a complex with Las17 and Vrp1
that controls Arp2/3 mediated actin assembly (Evangelista et al. 2000) (Fig. 51). Part of this
study was assigned to investigate whether this complex also is formed in C. albicans. A two
hybrid assay shows that Vrp1 of C. albicans binds strongly to the WH1 (WASP homology 1)
domain in the N-terminus of Wal1 and the binding is more efficient when the downstream
proline rich stretch is removed (Borth et al. 2010). The assay also indicates a direct binding of
the SH3 domain of Myo5 to Vrp1. The interaction with the C-terminal part of Vrp1 is strong
but, being proline-rich, Vrp1 probably provides multiple docking sites for SH3 domain
binding, as in S. cerevisiae (Anderson et al. 1998). Vrp1, Wal1 and Myo5 in C. albicans are
thus likely to execute the same functions as their homologs in S. cerevisiae, forming an actin
filament nucleating complex in both yeasts. This could provide an explanation for the
observed similarities of phenotypes in the wal1 and myo5 mutants and the less severe
phenotype of vrp1. Both Wal1 and Myo5 are activators of the Arp2/3 complex, and loss of
either of the corresponding these genes may therefore be more detrimental to cells than loss of
VRP1.
Figure 51. Model of the myosin-I-Vrp1-Las17 complex in Arp2/3 mediated actin assembly. The SH3 domain of Myo3/5p (circle) binds to Vrp1p and Las17. Myo3/5p and Las17 bind and activate the Arp2/3 complex through their C-terminal acidic domains (arrow). The WH2 domains of Las17 and Vrp1 (dashed box) bind and couple actin monomers to the Arp2/3 complex for efficient actin nucleation. Activation of the Arp2/3 complex leads to its incorporation into the actin cytoskeleton, either as a cap on the pointed end of an actin filament or attached to the side of a filament, creating cross-links and branches. Modified picture from Evangelista et al. 2000.
61
8.4 Ligand binding of SH3 domains is weak
Other SH3 domains where expected to interact with Vrp1 in the two hybrid experiment in this
assay. The SH3 binding motif PXXP is abundant in verproline but interactions are potentially
too weak for this experiment to discover additional interaction partners. The SH3 domain of
Hof1 in S. cerevisiae binds to the HOT (Hof one trap) domain of ScVrp1 (Ren et al. 2005).
However, the HOT domain is missing in CaVrp1 and the two-hybrid assay did not indicate
any interaction between the Hof1-SH3 and the Vrp1 in C. albicans. In general the binding
properties of SH3 domains are promiscuous and weak and other attempts to fish out ligands
with e.g. Co-IP have proven tedious and hard to interpret (unpublished results within the
Penelope group). Other reasons for low affinity could be that the cloned fragment is too small
or not flexible enough to fold properly, or that the cloned fragment excludes part of the
protein that confers stability and specificity to the ligand binding of the SH3 domain.
8.5 SLA2 is absent from the A. gossypii genome
When searching the A. gossypii genome database for homologs to endocytic factors it
becomes evident that A. gossypii lacks a homolog to Sla2/Hip1, Huntingtin Interacting
Protein 1. Sla2 is one of the earliest factors to arrive at the endocytic site and provides an
essential function in all investigated fungi. The N-terminal ANTH domain targets Sla2 to the
plasma membrane, the central coiled-coil interacts with clathrin and the C-terminal talin-like
domain binds actin filaments. Localization of Sla2 to the marked endocytic site is actin
independent but the protein has a possible role to physically link the clathrin coated pit to the
forces of actin polymerization that will push the invaginating vesicle inwards. S. cerevisiae
sla2 displays stalled actin patches and a severe temperature sensitive and slow growing
phenotype (Holtzman et al. 1993). Sla2 shows redundancy as a clathrin adaptor with other
ANTH/ENTH containing proteins but has additional roles in endocytosis (Baggett et al,
2003). The ENTH domain of the epsins, Ent1/2, is about half the size of the ANTH domain
found in Ap1801/2 and Sla2. The two domains bind to phosphatidyl inositol phosphates but
using different mechanisms. The binding of phosphatidyl inositol phosphates to ENTH
domains causes the folding of a helix that can insert into the membrane and drive membrane
curvature; this does not happen in ANTH domains (Stahelin et al. 2003). Sla2 might
consequently not be critically for induction of membrane curvature but uses its ANTH domain
for membrane targeting. Sla2p is required for viability in abp1 and sac6 mutants and the
N-terminal and central domains of Sla2 where shown to be indispensable in these strains
62
(Holtzman et al. 1993). Sla2 is essential for hyphal growth in C. albicans (Asleson et al.
2001).
In S. cerevisiae SLA2 is genetically linked to SUI1 but this gene appears besides all three
mating type loci in A. gossypii. SLA2 is positioned next to the MAT locus in many yeasts, a
location that often is subjected to rearrangements during mating type switching. It is possible
that SLA2 in A. gossypii was lost during the evolution of its mating-type locus. Sla2 binds to
the N-terminal part of Clc1 in yeast and a closer look at AgClc1 shows that the Sla2
interacting domain is missing in the A. gossypii homolog (Fig. 52). The missing 5’ part of
AgCLC1 is not due to a sequence mistake or a minor insertion/deletion in the genome; the
upstream UTR of AgCLC1 shows no resemblance to the beginning of the ScCLC1 ORF. This,
together with the noted absence of SLA2 strongly indicates a directed evolution against the
need for SLA2.
Efficient endocytosis is a requirement for fast growing filamentous fungi such as A. gossypii.
Though SLA2 is found in all studied ascomycetes and performs an essential task in
endocytosis A. gossypii has found a way to bypass the need for the protein or developed
another pathway that performs the same task. SLA2 was found in a synthetic lethal screen
with ABP1 in yeast and SAC6 was found to be a requirement in abp1 strains as well. In
addition, SLA2 and SAC6 showed synthetic lethality interactions with each other (Holtzman et
al. 1993). This prompted for functional analysis of a SAC6 deletion in A. gossypii.
-----------------------------------------------------------------------MANDYSSSD 9AgClc1.pro
MADKFPELEDDLVQDGFVTGDGDETEFLRREAEILGDEFKTEQDSELLSKDD---SDSKTLGTSVNEADAIAAASYQPQD 77KlClc1.pro
MSEKFPPLEDQNIDFTPNDKKDDDTDFLKREAEILGDEFKTEQDDILETEASPAKDDDEIRDFEEQFPDINSANGAVSSD 80ScClc1.pro
MADKFPEIDTPAAGG----DDDYEGDFLSREKELVGDEFTTDQDKQVFQDDE----DEEINEFKEQFPEVDTKAQPSGIS 72CaClc1.pro
E--LSDRQSSGAERSHAG-------GAHRTGAGATSDSSEPIRKWQERREAEIAERDESEAAATQRLQAEAIKHIDDFYE 80AgClc1.pro
VEGVSNPVEEEEDDDEFG------EPQSSSAEPVVRGKSEALENWKARRELEISERDQAEDKAKADLQEEAAKHIDDFYE 151KlClc1.pro
QN-GSATVSSGNDNGEADDDFSTFEGANQSTESVKEDRSEVVDQWKQRRAVEIHEKDLKDEELKKELQDEAIKHIDDFYD 159ScClc1.pro
VTKGADKYDDDDDEFEGFE------SSNGAAKELNLSESQAIKEWKQRRDLEIEEREKLNSKKKEEIIEKAKSTIDDFYE 146CaClc1.pro
VYSKKKQQQVEQARREAEEFLQQRDTFFDQDNTVWDRVLQLINTE-DADVLGDRDRSKFKDILLRLKGQEHVPGAARG 157AgClc1.pro
NYNIKKQQGIDQTQKEAEEFLAKTHAFASQDLTVWDKALQLINLE-DADIVDGRDRSKFKEILQRLKGNGSAPGATGQK 229KlClc1.pro
SYNKKKEQQLEDAAKEAEAFLKKRDEFFGQDNTTWDRALQLINQD-DADIIGGRDRSKLKEILLRLKGNAKAPGA 233ScClc1.pro
NYNSKRDNHQKEILSEQEKFISKRDDFLKRG-TLWDRVNELVTEVGELPGDESRDKTRFKELLTKLKGKENVPGAGGYQE 225CaClc1.pro
-----------------------------------------------------------------------MANDYSS-----------------------------------------------------------------------MANDYSSSD 9AgClc1.pro
MADKFPELEDDLVQDGFVTGDGDETEFLRREAEILGDEFKTEQDSELLSKDD---SDSKTLGTSVNEADAIAAASYQPQD 77KlClc1.pro
MSEKFPPLEDQNIDFTPNDKKDDDTDFLKREAEILGDEFKTEQDDILETEASPAKDDDEIRDFEEQFPDINSANGAVSSD 80ScClc1.pro
MADKFPEIDTPAAGG----DDDYEGDFLS
SD 9AgClc1.pro
MADKFPELEDDLVQDGFVTGDGDETEFLRREAEILGDEFKTEQDSELLSKDD---SDSKTLGTSVNEADAIAAASYQPQD 77KlClc1.pro
MSEKFPPLEDQNIDFTPNDKKDDDTDFLKREAEILGDEFKTEQDDILETEASPAKDDDEIRDFEEQFPDINSANGAVSSD 80ScClc1.pro
MADKFPEIDTPAAGG----DDDYEGDFLSREKELVGDEFTTDQDKQVFQDDE----DEEINEFKEQFPEVDTKAQPSGIS 72CaClc1.pro REKELVGDEFTTDQDKQVFQDDE----DEEINEFKEQFPEVDTKAQPSGIS 72CaClc1.pro
E--LSDRQSSGAERSHAG-------GAHRTGAGATSDSSEPIRKWQERREAEIAERDESEAAATQRLQAEAIKHIDDFYE 80AgClc1.pro
VEGVSNPVEEEEDDDEFG------EPQSSSAEPVVRGKSEALENWKARRELEISERDQAEDKAKADLQEEAAKHIDDFYE 151KlClc1.pro
QN-GSATVSSGNDNGEADDDFSTFEGAN
E--LSDRQSSGAERSHAG-------GAHRTGAGATSDSSEPIRKWQERREAEIAERDESEAAATQRLQAEAIKHIDDFYE 80AgClc1.pro
VEGVSNPVEEEEDDDEFG------EPQSSSAEPVVRGKSEALENWKARRELEISERDQAEDKAKADLQEEAAKHIDDFYE 151KlClc1.pro
QN-GSATVSSGNDNGEADDDFSTFEGANQSTESVKEDRSEVVDQWKQRRAVEIHEKDLKDEELKKELQDEAIKHIDDFYD 159ScClc1.pro
VTKGADKYDDDDDEFEGFE------SSNGAAKELNLSESQAIKEWKQRRDLEIEEREKLNSKKKEEIIEKAKSTIDDFYE 146CaClc1.pro
QSTESVKEDRSEVVDQWKQRRAVEIHEKDLKDEELKKELQDEAIKHIDDFYD 159ScClc1.pro
VTKGADKYDDDDDEFEGFE------SSNGAAKELNLSESQAIKEWKQRRDLEIEEREKLNSKKKEEIIEKAKSTIDDFYE 146CaClc1.pro
VYSKKKQQQVEQARREAEEFLQQRDTFFDQDNTVWDRVLQLINTE-DADVLGDRDRSKFKDILVYSKKKQQQVEQARREAEEFLQQRDTFFDQDNTVWDRVLQLINTE-DADVLGDRDRSKFKDILLRLKGQEHVPGAARG 157AgClc1.pro
NYNIKKQQGIDQTQKEAEEFLAKTHAFASQDLTVWDKALQLINLE-DADIVDGRDRSKFKEILQRLKGNGSAPGATGQK 229KlClc1.pro
SYNKKKEQQLEDAAKEAEAFLKKRDEFFGQDNTTWDRALQLINQD-DADIIGGRDRSKLKEILLRLKGNAKAPGA 233ScClc1.pro
NYNSKRDNHQKEIL
LRLKGQEHVPGAARG 157AgClc1.pro
NYNIKKQQGIDQTQKEAEEFLAKTHAFASQDLTVWDKALQLINLE-DADIVDGRDRSKFKEILQRLKGNGSAPGATGQK 229KlClc1.pro
SYNKKKEQQLEDAAKEAEAFLKKRDEFFGQDNTTWDRALQLINQD-DADIIGGRDRSKLKEILLRLKGNAKAPGA 233ScClc1.pro
NYNSKRDNHQKEILSEQEKFISKRDDFLKRG-TLWDRVNELVTEVGELPGDESRDKTRFKELLTKLKGKENVPGAGGYQE 225CaClc1.pro
Figure 52. Sequence alignment of Clc1 from A. gossypii, C. albicans, S. cerevisiae and Klyveromyces lactis. A. gossypii is the only fungus among these closely related fungi that lacks the N-terminal Sla2 binding domain. Alignment made with MegAlign.
63
8.6 Agsac6 has a similar phenotype to Agwal1
The yeast fimbrin, SAC6, participates in the endocytosis process, binding to actin in both
patches and cables, aiding in the organization and stabilization of the actin network. Deletion
of this actin bundler in S. cerevisiae disrupts the actin cytoskeleton and null mutants are
defective for internalization of α-factor (Kübler and Riezman 1993). In Aspergillus nidulans
the fimbrin homolog FimA is needed for polarity establishment and endocytosis (Upadhyay
and Shaw 2008). It was suggested that filamentous actin have to be bundled into strong
enough structures to aid the scission of vesicles and drive membrane invaginations from the
surface. Sac6 have a redundant function with Scp1, transgelin, in the organization and
stabilization of actin (Goodman et al. 2003). Scp1 contains one CH domain and produces a
loose meshwork of actin filaments, while Sac6 generates tight bundles. A two- to threefold
overexpression of Scp1 partially suppresses defects in sac6 cells.
The phenotype of Agsac6 shows similarities to Agwal1 suggesting that they take part in the
same pathway. Agsac6 have accumulations of cortical actin subapically and when grown in
37 C the hyphae swell and burst. Vacuolar morphology seems unaffected in Agsac6, in
opposition to Agwal1, the former produces large elongated vacuoles, suggesting vivid
movement. Vacuoles in wal1 are round-shaped and immobile indicating that Wal1 activated
Arp2/3 dependent actin polymerization but not filament bundling by Sac6 is required for
vacuolar movement. Agwal1 is deficient in septation and sporulation but Agsac6 is normal in
those aspects. Deletion of WAL1 in the sac6 background might be possible, even though both
deletions cause severe damage on its own, to further study their involvement in the same
pathway. Additionally, investigation of any phenotypic rescue by overexpression of SAC6 in
the wal1 deletion background, or vice verse, could be valuable for the discussion. SAC6 in
A. gossypii is not necessary for actin patch formation but for their polarization to hyphal tips.
Aggregated actin patches behind the growing tip will lead endocytosis from the tip and slow
down growth. Loss of polarization might also be the cause for swelling of hyphae in the
restrictive temperature 37 C. In S. cerevisiae the combined deletion of SLA2 and SAC6 is
lethal, but A. gossypii manages without Sla2 and is viable in the absence of Sac6. How
A. gossypii compensates for the lack of Sla2 is still unclear.
8.7 A. gossypii pil1 germlings cease to grow before reaching maturation
Eisosomes are recently discovered fungi-specific structures that have been argued to mark
sites of endocytosis in S. cerevisiae. Their main components PIL1 and LSP1 share a high
64
degree of similarity but displays distinct non overlapping functions, with only PIL1 being
necessary for regulation of their size and number. LSP1 is not essential in S. cerevisiae but its
deletion decreases the rate of endocytosis and suppresses the rvs161 endocytosis phenotype
(Walther et al. 2006). Recently, eisosome proteins were described in Aspergillus nidulans
where PilA and PilB are closely related to the Pil1 and Lsp1 proteins in S. cerevisiae and
SurG is the Sur7 homolog (Vangelatos et al. 2010). The distribution and expression pattern of
the A. nidulans eisosomal proteins are different from S. cerevisiae, all components are present
in the periphery of ungerminated conidia but only PilA is localized in patch like structures in
hyphal cells. No phenotypes are generated by deletion of any of the A. nidulans eisosome
proteins. Deletion of LSP1 has no observable effect on A. gossypii, as was shown in
C. albicans too (Reijnst et al, unpublished), whereas disruption of PIL1 leads to severe defects
and is barely viable in this study. The few mutant pil1 spores that do germinate seize to grow
before they reach the fast growing mature state. The young germlings have no obvious
phenotype regarding actin patch polarization, FM4-64 uptake, septation or mitochondrial
morphology until they suddenly go into apoptosis. Perhaps the initial slow growth is
independent of the eisosome function or, residual copies of PIL1 in the spore are substantial
to sustain growth for a while. Endocytosis mutants are in many cases temperature sensitive.
Characterization of pil1 was performed in 30 C in liquid culture but an even lower
temperature might have allowed the mutant germlings to persist for longer. Spores of pil1
were also germinated and grown on plates in room temperature but even so no colonies were
formed.
8.8 PIL1-GFP does not localize with cortical actin patches
Cortical actin is known to associate directly with invaginating endocytic pits, following the
vesicle as it internalizes. This study shows that eisosomes in A. gossypii, defined by
PIL1-GFP, never colocalizes with cortical actin patches. In fast growing hyphae with highly
polarized actin patches the very tip is completely deprived of eisosomes, although this could
be due to a slow folding of GFP. Even mutants with grossly mislocalized actin patches
display a normal PIL1-GFP pattern. However, being associated to the lipid rich MCCs,
eisosomes can still play an important role in the regulation of endocytosis. These lipid rafts
form isolated spots where membrane bound transporters are spared from recycling by
endocytosis and PIL1, but not LSP1, is a core component (Grossmann et al. 2008).
65
8.9 Sur7 is not necessary for eisosome formation but affects vacuolar fusion
The membrane protein Sur7 in S. cerevisiae was previously shown to localize to MCCs and
eisosomes but not actin patches. Sur7 and its paralogs FMP45, YNL194C and YLR414C are
not necessary for actin function but affects sphingolipid content in the plasma membrane and
sporulation (Young et al. 2002). Deletion of SUR7 in A. gossypii leads to minor defects in
growth rate, chitin deposition and vacuolar fusion but PIL1-GFP localization and sporulation
appears normal. Elevated chitin production could be a compensatory response to cell wall
damage as has been reported in other yeasts (Smits et al. 2001). Cell wall disturbing agents
slowed down the growth further but did not result in more frequent cell lysis than in wild
type. The C. albicans sur7 mutant displays an even more pronounced phenotype with defects
in chitin distribution and abnormal actin polarization and cell wall growth (Alvarez et al.
2008). Trafficking of vesicles to the vacuoles was delayed in Agsur7 as has been reported in
C. albicans, but since SUR7 is localized to the membrane this is likely an indirect effect. In
Casur7 this could be due to mislocalized actin but not in A. gossypii since the deletion mutant
have wild type like actin polarization.
8.10 A link between lipid rafts and endocytosis
Both LSP1 and SUR7 of S. cerevisiae are genetically linked to RVS161 and RVS167, actin
patch components with a function in the scission event of internalized clathrin coated pits
(Walther et al. 2006). rvs161 and rvs167 mutants are suppressed by mutations in the
sphingolipid synthesis, e. g. overexpression of the transmembrane protein Sur7 (Desfarges et
al. 1993). Sur7 localizes to MCCs which are involved in the regulation of turn over of several
membrane proteins by endocytosis. Furthermore, deletion of PIL1 disrupts MCCs and its
components Can1, Sur7 and Lsp1 cluster in large chunks (Malinska et al. 2004). The
eisosome components Pil1 and Lsp1 are regulated by the sphingolipid-mediated signalling
pathway via the kinases Pkh1/2 and Ypk1/2 (Luo et al. 2008). In wild type C. albicans hyphal
cells lipid rafts and Rvs167 are polarized to the growing tip. Some Rvs167 patches colocalize
to actin patches but the other subset might interact with lipid rafts. Deletion of SLA2 or MYO5
lead to depolymerized lipid rafts and cytoplasmic Rvs167 (Oberholzer et al. 2006). Deletion
of RVS167 in C. albicans did not affect the distribution of eisosomes (unpublished results by
Reijnst et al). Overall this shows that eisosomes are linked to endocytosis even though they do
not appear at the specific site of endocytosis, the cortical actin patch. Eisosome components
might not take part in the actual internalization process but nonetheless have a regulative
function via the lipid metabolism.
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9 Summary
C. albicans displays a wide array of morphological states and has acquired skills to penetrate
the host immune response. It does so with a genetic setup sharing many treats with the model
system S. cerevisiae. In line with this fact, the GFP-tagged C. albicans proteins Bbc1, Cyk3
and Sla1 localized in a way similar to the S. cerevisiae homologs. The strictly filamentous
A. gossypii is a very close relative to S. cerevisiae and is likely to follow conserved pathways
as well. It was previously known that mutants of the actin filament nucleation machinery in
these species showed equivalent defects in regard to actin polarization, hyphal growth,
endocytosis and vacuolar morphology. This study adds a few more connective lines in the
C. albicans endocytosis pathway map showing that Vrp1, Myo5 and Wal1 physically interact.
The closer to the core machinery a mutation occurs the more sever the defect is; deletion of
WAL1 in C. albicans generates a more pronounced phenotype than deletion of VRP1. In
A. gossypii the sac6 strain is similar to but has a weaker phenotype compared to Agwal1,
indicating that Sac6 participates in the same pathway but has a less important mechanism than
Wal1 in the actin filament organization. Synthetic lethality between SLA2 and SAC6 in
S. cerevisiae indicates a functional redundancy in the pathway. The genome of A. gossypii is
missing any homolog to SLA2 and deletion of SAC6 causes a temperature sensitive and
slow-growing phenotype with gross actin patches anomalies. How or why A. gossypii has
developed a pathway around Sla2 to manage the crucial steps of endocytosis is still to be
unveiled. And finally, eisosomes that were previously said to mark sites of endocytosis were
shown not to localize to actin patches and thereby do not participate in the internalization
process. However, according to many studies they are more likely to regulate endocytosis by
associating to lipid structures in the plasma membrane.
67
10 Prospects
SH3 domain proteins are likely to interact with the proline rich Vrp1 but to find interaction
partners in a two-hybrid assay the whole protein might have to be used and not only the
isolated SH3 domain as was done in this study. Misstranslation of C. albicans proteins in a
yeast system due to the ambiguous codon usage could also present problems. To circumvent
the need for heterologous expression the two-hybrid system recently developed by Stynen et
al. 2010 in C. albicans itself could be a solution.
The fact that SLA2 is absent from A. gossypii is intriguing because it performs a conserved
and important function in the endocytosis process of fungi. The Hip1 and Hip1R homologs in
higher eukaryotes share the yeast SLA2 function to associate with clathrin coated pits and
filamentous actin. The N-terminal part of Clc1 (Clathrin light chain 1) that interacts with Sla2
in other fungi is not present in the A. gossypii homolog of Clc1 agreeing with the idea that
A. gossypii has evolved a pathway around the need for Sla2. It would be interesting to see if
heterologous expression of the S. cerevisiae Sla2 would affect the actin organization and if the
protein would localize to cortical actin patches in A. gossypii. And would the ScClc1 work
properly in A. gossypii if the endogenous CLC1 was deleted? This study shows that
A. gossypii can survive and perform endocytosis when the fimbrin-encoding SAC6 is deleted.
This gene is synthetic lethal with SLA2 and ABP1 in S. cerevisiae. Deletion of ABP1 alone
results in no obvious phenotype in S. cerevisiae but how would A. gossypii cope with the
deletion? If A. gossypii manage without ABP1 as well this fungus might have evolved
redundant pathways which do not exist in S. cerevisiae or it compensates otherwise.
Overexpression of Scp1, transgelin, in S. cerevisiae can partially suppress the disrupted actin
cytoskeleton phenotype of sac6. A higher expression of the homologous gene might do the
same in A. gossypii. Deletion of SAC6 and WAL1 in A. gossypii produces phenotypes that
suggest they participate in the same pathway. Overexpression of one gene in the deletion
background of the other could perhaps partially rescue the deletion phenotype. A double
deletion of these genetically interacting genes would also be interesting to study.
The eisosome proteins Pil1 and Lsp1 in A. gossypii are very similar to each other and to the
homologs in S. cerevisiae, only the last stretch of 30 amino acids is substantially different.
Even so, deletion of the two genes produces very different phenotypes in both fungi. Pil1 is
68
responsible for localization of the eisosome components in S. cerevisiae. Lsp1 and Sur7 needs
to be GFP-tagged and localized in the A. gossypii pil1 mutant to see if the same is true in this
fungi. A more thorough investigation of the Agpil1 mutants should be done to test their
viability under different circumstance, e g lower temperature. The upstream region of the
PIL1 ORF should be sequenced so that the promoter function can be tested. Generation of
chimeras between the closely related Pil1 and Lsp1 could indicate which part of the sequence
is responsible for their non-redundant functions.
69
11 Acknowledgements
During my PhD I have met many wonderful people that I am grateful to and wish to thank.
Jürgen Wendland for providing this opportunity to work in the fine surroundings of
Carlsberg and for guiding me during this whole time.
Andrea who has been a support in every possible way, in the lab, at the microscope, as
a tutor and as an inspiration.
Janine and Alex for being very helpful in the lab at the beginning.
Patrick for being a steady colleague for my whole internship who always took time to
help me out on several matters and with whom I travelled a lot during this time.
Anke who I shared office and many discussions with and who would have been the
ideal German teacher had I only given it some more effort.
Sidsel for helping out in the lab with so many things, saving me a lot of precious time.
Colleagues at Carlsberg for great chit-chat in the hallways and for pulling off many
entertaining parties.
All students, PIs and coordinators in the Penelope consortium that attributed so much
more value to this training period, providing long lasting connections all over the EU.
Steen for helping me fulfil all the requirements for a PhD from Københavns
Universitet.
My furry Tristan, who kept me company for hours at my desk and on my keyboard, I
felt your warmth and support during my writing.
And not least, Glenn, you have been a tremendous support and showed real interest
into my work. I am grateful for the firm but gentle boosts that finally got me to pull
this off.
70
12 References
Adams, A. E. and Pringle, J. R. (1984). "Relationship of actin and tubulin distribution to bud
growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae." J Cell Biol
98(3): 934-945.
Alberti-Segui, C., Dietrich, F., et al. (2001). "Cytoplasmic dynein is required to oppose the
force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya
gossypii." J Cell Sci 114(Pt 5): 975-986.
Alvarez, F. J., Douglas, L. M., et al. (2008). "The Sur7 protein regulates plasma membrane
organization and prevents intracellular cell wall growth in Candida albicans." Mol Biol
Cell 19(12): 5214-5225.
Amberg, D. C. (1998). "Three-dimensional imaging of the yeast actin cytoskeleton through
the budding cell cycle." Mol Biol Cell 9(12): 3259-3262.
Anderson, B. L., Boldogh, I., et al. (1998). "The Src homology domain 3 (SH3) of a yeast
type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin
polarization." J Cell Biol 141(6): 1357-1370.
Ashby, S. F. and Nowell, W. (1926). "The Fungi of Stigmatomycosis." Annals of Botany
40(157).
Asleson, C. M., Bensen, E. S., et al. (2001). "Candida albicans INT1-induced filamentation in
Saccharomyces cerevisiae depends on Sla2p." Mol Cell Biol 21(4): 1272-1284.
Ayscough, K. R., Eby, J. J., et al. (1999). "Sla1p is a functionally modular component of the
yeast cortical actin cytoskeleton required for correct localization of both Rho1p-GTPase
and Sla2p, a protein with talin homology." Mol Biol Cell 10(4): 1061-1075.
Bagnat, M., Keranen, S., et al. (2000). "Lipid rafts function in biosynthetic delivery of
proteins to the cell surface in yeast." Proc Natl Acad Sci U S A 97(7): 3254-3259.
Baillie, G. S. and Douglas, L. J. (2000). "Matrix polymers of Candida biofilms and their
possible role in biofilm resistance to antifungal agents." J Antimicrob Chemother 46(3):
397-403.
Berman, J. and Sudbery, P. E. (2002). "Candida Albicans: a molecular revolution built on
lessons from budding yeast." Nat Rev Genet 3(12): 918-930.
Boczkowska, M., Rebowski, G., et al. (2008). "X-ray scattering study of activated Arp2/3
complex with bound actin-WCA." Structure 16(5): 695-704.
71
Borth, N., Walther, A., et al. (2010). "Candida albicans Vrp1 is required for polarized
morphogenesis and interacts with Wal1 and Myo5." Microbiology.
Butler, G., Rasmussen, M. D., et al. (2009). "Evolution of pathogenicity and sexual
reproduction in eight Candida genomes." Nature 459(7247): 657-662.
Chandra, J., Kuhn, D. M., et al. (2001). "Biofilm formation by the fungal pathogen Candida
albicans: development, architecture, and drug resistance." J Bacteriol 183(18): 5385-5394.
D'Agostino, J. L. and Goode, B. L. (2005). "Dissection of Arp2/3 complex actin nucleation
mechanism and distinct roles for its nucleation-promoting factors in Saccharomyces
cerevisiae." Genetics 171(1): 35-47.
Desfarges, L., Durrens, P., et al. (1993). "Yeast mutants affected in viability upon starvation
have a modified phospholipid composition." Yeast 9(3): 267-277.
Dietrich, F. S., Voegeli, S., et al. (2004). "The Ashbya gossypii genome as a tool for mapping
the ancient Saccharomyces cerevisiae genome." Science 304(5668): 304-307.
Drubin, D. G., Miller, K. G., et al. (1988). "Yeast actin-binding proteins: evidence for a role
in morphogenesis." J Cell Biol 107(6 Pt 2): 2551-2561.
Duff, S. J. and Murray, W. D. (1988). "Production and application of methylotrophic yeast
Pichia pastoris." Biotechnol Bioeng 31(1): 44-49.
Eitzen, G., Wang, L., et al. (2002). "Remodeling of organelle-bound actin is required for yeast
vacuole fusion." J Cell Biol 158(4): 669-679.
Enloe, B., Diamond, A., et al. (2000). "A single-transformation gene function test in diploid
Candida albicans." J Bacteriol 182(20): 5730-5736.
Evangelista, M., Klebl, B. M., et al. (2000). "A role for myosin-I in actin assembly through
interactions with Vrp1p, Bee1p, and the Arp2/3 complex." J Cell Biol 148(2): 353-362.
Field, C., Li, R., et al. (1999). "Cytokinesis in eukaryotes: a mechanistic comparison." Curr
Opin Cell Biol 11(1): 68-80.
Gietz, R. D. and Schiestl, R. H. (2007). "High-efficiency yeast transformation using the
LiAc/SS carrier DNA/PEG method." Nat Protoc 2(1): 31-34.
Gimeno, C. J., Ljungdahl, P. O., et al. (1992). "Unipolar cell divisions in the yeast S.
cerevisiae lead to filamentous growth: regulation by starvation and RAS." Cell 68(6):
1077-1090.
Gomes, A. C., Miranda, I., et al. (2007). "A genetic code alteration generates a proteome of
high diversity in the human pathogen Candida albicans." Genome Biol 8(10): R206.
Goode, B. L., Rodal, A. A., et al. (2001). "Activation of the Arp2/3 complex by the actin
filament binding protein Abp1p." J Cell Biol 153(3): 627-634.
72
Goode, B. L., Wong, J. J., et al. (1999). "Coronin promotes the rapid assembly and cross-
linking of actin filaments and may link the actin and microtubule cytoskeletons in
yeast." J Cell Biol 144(1): 83-98.
Goodman, A., Goode, B. L., et al. (2003). "The Saccharomyces cerevisiae calponin/transgelin
homolog Scp1 functions with fimbrin to regulate stability and organization of the actin
cytoskeleton." Mol Biol Cell 14(7): 2617-2629.
Gordon, J. L., Byrne, K. P., et al. (2009). "Additions, losses, and rearrangements on the
evolutionary route from a reconstructed ancestor to the modern Saccharomyces
cerevisiae genome." PLoS Genet 5(5): e1000485.
Grossmann, G., Malinsky, J., et al. (2008). "Plasma membrane microdomains regulate
turnover of transport proteins in yeast." J Cell Biol 183(6): 1075-1088.
Grossmann, G., Opekarova, M., et al. (2007). "Membrane potential governs lateral
segregation of plasma membrane proteins and lipids in yeast." EMBO J 26(1): 1-8.
Grove, S. N. and Bracker, C. E. (1970). "Protoplasmic organization of hyphal tips among
fungi: vesicles and Spitzenkorper." J Bacteriol 104(2): 989-1009.
Hall, A. (1994). "Small GTP-binding proteins and the regulation of the actin cytoskeleton."
Annu Rev Cell Biol 10: 31-54.
Harris, S. D., Read, N. D., et al. (2005). "Polarisome meets spitzenkorper: microscopy,
genetics, and genomics converge." Eukaryot Cell 4(2): 225-229.
Holtzman, D. A., Yang, S., et al. (1993). "Synthetic-lethal interactions identify two novel
genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in
Saccharomyces cerevisiae." J Cell Biol 122(3): 635-644.
Horio, T. and Oakley, B. R. (2005). "The role of microtubules in rapid hyphal tip growth of
Aspergillus nidulans." Mol Biol Cell 16(2): 918-926.
Hull, C. M. and Johnson, A. D. (1999). "Identification of a mating type-like locus in the
asexual pathogenic yeast Candida albicans." Science 285(5431): 1271-1275.
Hull, C. M., Raisner, R. M., et al. (2000). "Evidence for mating of the "asexual" yeast
Candida albicans in a mammalian host." Science 289(5477): 307-310.
Idrissi, F. Z., Grotsch, H., et al. (2008). "Distinct acto/myosin-I structures associate with
endocytic profiles at the plasma membrane." J Cell Biol 180(6): 1219-1232.
Jonsdottir, G. A. and Li, R. (2004). "Dynamics of yeast Myosin I: evidence for a possible role
in scission of endocytic vesicles." Curr Biol 14(17): 1604-1609.
Kaksonen, M., Sun, Y., et al. (2003). "A pathway for association of receptors, adaptors, and
actin during endocytic internalization." Cell 115(4): 475-487.
73
Kaksonen, M., Toret, C. P., et al. (2005). "A modular design for the clathrin- and actin-
mediated endocytosis machinery." Cell 123(2): 305-320.
Kim, K., Yamashita, A., et al. (2004). "Capping protein binding to actin in yeast: biochemical
mechanism and physiological relevance." J Cell Biol 164(4): 567-580.
Knechtle, P., Dietrich, F., et al. (2003). "Maximal polar growth potential depends on the
polarisome component AgSpa2 in the filamentous fungus Ashbya gossypii." Mol Biol
Cell 14(10): 4140-4154.
Kohno, H., Tanaka, K., et al. (1996). "Bni1p implicated in cytoskeletal control is a putative
target of Rho1p small GTP binding protein in Saccharomyces cerevisiae." EMBO J
15(22): 6060-6068.
Korinek, W. S., Bi, E., et al. (2000). "Cyk3, a novel SH3-domain protein, affects cytokinesis
in yeast." Curr Biol 10(15): 947-950.
Kübler, E. and Riezman, H. (1993). "Actin and fimbrin are required for the internalization
step of endocytosis in yeast." EMBO J 12(7): 2855-2862.
Kärkkäinen, S., Hiipakka, M., et al. (2006). "Identification of preferred protein interactions by
phage-display of the human Src homology-3 proteome." EMBO Rep 7(2): 186-191.
Lee, W. L., Bezanilla, M., et al. (2000). "Fission yeast myosin-I, Myo1p, stimulates actin
assembly by Arp2/3 complex and shares functions with WASp." J Cell Biol 151(4):
789-800.
Li, R. (1997). "Bee1, a yeast protein with homology to Wiscott-Aldrich syndrome protein, is
critical for the assembly of cortical actin cytoskeleton." J Cell Biol 136(3): 649-658.
Lo, H. J., Kohler, J. R., et al. (1997). "Nonfilamentous C. albicans mutants are avirulent." Cell
90(5): 939-949.
Luo, G., Gruhler, A., et al. (2008). "The sphingolipid long-chain base-Pkh1/2-Ypk1/2
signaling pathway regulates eisosome assembly and turnover." J Biol Chem 283(16):
10433-10444.
Machesky, L. M., Atkinson, S. J., et al. (1994). "Purification of a cortical complex containing
two unconventional actins from Acanthamoeba by affinity chromatography on profilin-
agarose." J Cell Biol 127(1): 107-115.
Machesky, L. M. and Insall, R. H. (1998). "Scar1 and the related Wiskott-Aldrich syndrome
protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex." Curr Biol
8(25): 1347-1356.
74
Malinska, K., Malinsky, J., et al. (2004). "Distribution of Can1p into stable domains reflects
lateral protein segregation within the plasma membrane of living S. cerevisiae cells." J
Cell Sci 117(Pt 25): 6031-6041.
Martin, R., Walther, A., et al. (2005). "Ras1-induced hyphal development in Candida albicans
requires the formin Bni1." Eukaryot Cell 4(10): 1712-1724.
Mayer, B. J. and Saksela, K., Eds. (2005). Modular Protein Domains. SH3 Domains,
Wiley-VCH.
McCann, R. O. and Craig, S. W. (1997). "The I/LWEQ module: a conserved sequence that
signifies F-actin binding in functionally diverse proteins from yeast to mammals." Proc
Natl Acad Sci U S A 94(11): 5679-5684.
Miller, M. G. and Johnson, A. D. (2002). "White-opaque switching in Candida albicans is
controlled by mating-type locus homeodomain proteins and allows efficient mating."
Cell 110(3): 293-302.
Mochida, J., Yamamoto, T., et al. (2002). "The novel adaptor protein, Mti1p, and Vrp1p, a
homolog of Wiskott-Aldrich syndrome protein-interacting protein (WIP), may
antagonistically regulate type I myosins in Saccharomyces cerevisiae." Genetics 160(3):
923-934.
Moreira, K. E., Walther, T. C., et al. (2009). "Pil1 controls eisosome biogenesis." Mol Biol
Cell 20(3): 809-818.
Morschhäuser, J., Michel, S., et al. (1998). "Expression of a chromosomally integrated,
single-copy GFP gene in Candida albicans, and its use as a reporter of gene regulation."
Mol Gen Genet 257(4): 412-420.
Naglik, J. R., Challacombe, S. J., et al. (2003). "Candida albicans secreted aspartyl
proteinases in virulence and pathogenesis." Microbiol Mol Biol Rev 67(3): 400-428,
table of contents.
Noble, S. M. and Johnson, A. D. (2005). "Strains and strategies for large-scale gene deletion
studies of the diploid human fungal pathogen Candida albicans." Eukaryot Cell 4(2):
298-309.
Oberholzer, U., Nantel, A., et al. (2006). "Transcript profiles of Candida albicans cortical
actin patch mutants reflect their cellular defects: contribution of the Hog1p and Mkc1p
signaling pathways." Eukaryot Cell 5(8): 1252-1265.
Odds, F. C., Ed. (1988). Candida and Candidosis: a Review and Bibliography, Bailliere Tindall.
Ohama, T., Suzuki, T., et al. (1993). "Non-universal decoding of the leucine codon CUG in
several Candida species." Nucleic Acids Res 21(17): 4039-4045.
75
Pfaller, M. A. and Diekema, D. J. (2007). "Epidemiology of invasive candidiasis: a persistent
public health problem." Clin Microbiol Rev 20(1): 133-163.
Pruyne, D. and Bretscher, A. (2000). "Polarization of cell growth in yeast." J Cell Sci 113 ( Pt
4): 571-585.
Ren, G., Wang, J., et al. (2005). "Verprolin cytokinesis function mediated by the Hof one trap
domain." Traffic 6(7): 575-593.
Richman, T. J. and Johnson, D. I. (2000). "Saccharomyces cerevisiae cdc42p GTPase is
involved in preventing the recurrence of bud emergence during the cell cycle." Mol Cell
Biol 20(22): 8548-8559.
Rodal, A. A., Manning, A. L., et al. (2003). "Negative regulation of yeast WASp by two SH3
domain-containing proteins." Curr Biol 13(12): 1000-1008.
Santos, M. A. and Tuite, M. F. (1995). "The CUG codon is decoded in vivo as serine and not
leucine in Candida albicans." Nucleic Acids Res 23(9): 1481-1486.
Sharpless, K. E. and Harris, S. D. (2002). "Functional characterization and localization of the
Aspergillus nidulans formin SEPA." Mol Biol Cell 13(2): 469-479.
Sivadon, P., Peypouquet, M. F., et al. (1997). "Cloning of the multicopy suppressor gene
SUR7: evidence for a functional relationship between the yeast actin-binding protein
Rvs167 and a putative membranous protein." Yeast 13(8): 747-761.
Smits, G. J., van den Ende, H., et al. (2001). "Differential regulation of cell wall biogenesis
during growth and development in yeast." Microbiology 147(Pt 4): 781-794.
Stahelin, R. V., Long, F., et al. (2003). "Contrasting membrane interaction mechanisms of
AP180 N-terminal homology (ANTH) and epsin N-terminal homology (ENTH)
domains." J Biol Chem 278(31): 28993-28999.
Steiner, S., Wendland, J., et al. (1995). "Homologous recombination as the main mechanism
for DNA integration and cause of rearrangements in the filamentous ascomycete
Ashbya gossypii." Genetics 140(3): 973-987.
Stimpson, H. E., Toret, C. P., et al. (2009). "Early-arriving Syp1p and Ede1p function in endocytic
site placement and formation in budding yeast." Mol Biol Cell 20(22): 4640-4651.
Stynen, B., Van Dijck, P., et al. (2010). "A CUG codon adapted two-hybrid system for the
pathogenic fungus Candida albicans." Nucleic Acids Res.
Sun, Y., Martin, A. C., et al. (2006). "Endocytic internalization in budding yeast requires
coordinated actin nucleation and myosin motor activity." Dev Cell 11(1): 33-46.
76
Tang, H. Y., Xu, J., et al. (2000). "Pan1p, End3p, and S1a1p, three yeast proteins required for
normal cortical actin cytoskeleton organization, associate with each other and play
essential roles in cell wall morphogenesis." Mol Cell Biol 20(1): 12-25.
Thomson, J. M., Gaucher, E. A., et al. (2005). "Resurrecting ancestral alcohol dehydrogenases
from yeast." Nat Genet 37(6): 630-635.
Toshima, J. Y., Toshima, J., et al. (2006). "Spatial dynamics of receptor-mediated endocytic
trafficking in budding yeast revealed by using fluorescent alpha-factor derivatives."
Proc Natl Acad Sci U S A 103(15): 5793-5798.
Trinci, A. P. (1974). "A study of the kinetics of hyphal extension and branch initiation of
fungal mycelia." J Gen Microbiol 81(1): 225-236.
Ungewickell, E. and Branton, D. (1981). "Assembly units of clathrin coats." Nature
289(5796): 420-422.
Upadhyay, S. and Shaw, B. D. (2008). "The role of actin, fimbrin and endocytosis in growth
of hyphae in Aspergillus nidulans." Mol Microbiol 68(3): 690-705.
Walther, A. and Wendland, J. (2003). "An improved transformation protocol for the human
fungal pathogen Candida albicans." Curr Genet 42(6): 339-343.
Walther, A. and Wendland, J. (2004a). "Apical localization of actin patches and vacuolar
dynamics in Ashbya gossypii depend on the WASP homolog Wal1p." J Cell Sci 117(Pt
21): 4947-4958.
Walther, A. and Wendland, J. (2004b). "Polarized hyphal growth in Candida albicans requires
the Wiskott-Aldrich Syndrome protein homolog Wal1p." Eukaryot Cell 3(2): 471-482.
Walther, A. and Wendland, J. (2008). "PCR-based gene targeting in Candida albicans." Nat
Protoc 3(9): 1414-1421.
Walther, T. C., Brickner, J. H., et al. (2006). "Eisosomes mark static sites of endocytosis."
Nature 439(7079): 998-1003.
Vangelatos, I., Roumelioti, K., et al. (2010). "Eisosome Organization in the Filamentous
AscomyceteAspergillus nidulans." Eukaryot Cell 9(10): 1441-1454.
Warren, D. T., Andrews, P. D., et al. (2002). "Sla1p couples the yeast endocytic machinery to
proteins regulating actin dynamics." J Cell Sci 115(Pt 8): 1703-1715.
Watters, M. K. and Griffiths, A. J. (2001). "Tests of a cellular model for constant branch
distribution in the filamentous fungus Neurospora crassa." Appl Environ Microbiol
67(4): 1788-1792.
Wedlich-Söldner, R., Bolker, M., et al. (2000). "A putative endosomal t-SNARE links exo- and
endocytosis in the phytopathogenic fungus Ustilago maydis." EMBO J 19(9): 1974-1986.
77
Wendland, J. (2001). "Comparison of morphogenetic networks of filamentous fungi and
yeast." Fungal Genet Biol 34(2): 63-82.
Wendland, J., Ayad-Durieux, Y., et al. (2000). "PCR-based gene targeting in the filamentous
fungus Ashbya gossypii." Gene 242(1-2): 381-391.
Wendland, J. and Philippsen, P. (2000). "Determination of cell polarity in germinated spores
and hyphal tips of the filamentous ascomycete Ashbya gossypii requires a rhoGAP
homolog." J Cell Sci 113 ( Pt 9): 1611-1621.
Wesp, A., Hicke, L., et al. (1997). "End4p/Sla2p interacts with actin-associated proteins for
endocytosis in Saccharomyces cerevisiae." Mol Biol Cell 8(11): 2291-2306.
Wickerham, L. J., Flickinger, M. H., et al. (1946). "The production of riboflavin by Ashbya
gossypii." Archives of Biochemistry and Biophysics(9): 95-98.
Winter, D., Lechler, T., et al. (1999). "Activation of the yeast Arp2/3 complex by Bee1p, a
WASP-family protein." Curr Biol 9(9): 501-504.
von Nussbaum, F., Brands, M., et al. (2006). "Antibacterial natural products in medicinal
chemistry--exodus or revival?" Angew Chem Int Ed Engl 45(31): 5072-5129.
Wright, M. C. and Philippsen, P. (1991). "Replicative transformation of the filamentous
fungus Ashbya gossypii with plasmids containing Saccharomyces cerevisiae ARS
elements." Gene 109(1): 99-105.
Young, M. E., Karpova, T. S., et al. (2002). "The Sur7p family defines novel cortical domains
in Saccharomyces cerevisiae, affects sphingolipid metabolism, and is involved in
sporulation." Mol Cell Biol 22(3): 927-934.
Yu, H., Rosen, M. K., et al. (1992). "Solution structure of the SH3 domain of Src and
identification of its ligand-binding site." Science 258(5088): 1665-1668.
Zagulski, M., Herbert, C. J., et al. (1998). "Sequencing and functional analysis of the yeast
genome." Acta Biochim Pol 45(3): 627-643.
Zeng, G., Yu, X., et al. (2001). "Regulation of yeast actin cytoskeleton-regulatory complex
Pan1p/Sla1p/End3p by serine/threonine kinase Prk1p." Mol Biol Cell 12(12): 3759-3772.
Zheng, Y., Cerione, R., et al. (1994). "Control of the yeast bud-site assembly GTPase Cdc42.
Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity
by Bem3." J Biol Chem 269(4): 2369-2372.
Zigmond, S. H., Evangelista, M., et al. (2003). "Formin leaky cap allows elongation in the
presence of tight capping proteins." Curr Biol 13(20): 1820-1823.
78
Appendix I – Verification PCR on C. albicans strains
PCR verifying heterozygous C. albicans strains and C-terminal genomic integration of GFP. G1/G2 cover the 5’ end of integration of CdHIS1 into target locus and G3/G4 the 3’ end. G1-GFP anneals in the target ORF and 392 in the GFP, the primer pair spans the integration point of the GFP-cassette. Arrows mark relevant reference bands.
5’ G1+G2
3’ G3+G4
G1-GFP
+ 392
1000bp
500bp
800bp500bp
bbc1 bud14 cyk3 nbp2
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
pin2 rvs167-2 sho2 sla1
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
BBC1 BUD14 CYK3 NBP2 PIN3 RVS167-2 SHO1 SLA2
Integration of GFP at the C-terminus
Heterozygous strains – Integration of CdHIS1 marker
800bp500bp
5’ G1+G2
3’ G3+G4
G1-GFP
+ 392
1000bp
500bp
800bp500bp
bbc1 bud14 cyk3 nbp2
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
pin2 rvs167-2 sho2 sla1
5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
BBC1 BUD14 CYK3 NBP2 PIN3 RVS167-2 SHO1 SLA2
Integration of GFP at the C-terminus
Heterozygous strains – Integration of CdHIS1 marker
800bp500bp
79
Appendix II – Verification PCR on A. gossypii strains
1. 5’ pil1 I1+G2 0.6 kb
2. 3’ pil1 I2+G3 0.6 kb
3. 3’ pil1 G4+G5 0.5 kb
4. iPIL1 I1+I2 0.7 kb
disruption mutant deletion mutant
pil1/PIL1 pil1/PIL1
1 2 3 4
1. 5’ sur7 G1+G2 0.9 kb
2. 3’ sur7 G3+G4 0.6 kb
3. iSUR7 I1+I2 0.7 kb
4. 5’ lsp1 G1+ G2 1.1 kb
5. 3’ lsp1 G3+ G4 0.9 kb
6. iLSP1 I1+I2 0.6 kb
sur7 lsp1
1 2 3 4 5 6
sac6
1 2 3
1. 5’ sac6 G1+G2 0.7 kb
2. 3’ sac6 G3+G4 0.6 kb
3. iSAC6 I1+I2 0.7 kb
1. 5’ pkh1 G1+G6 1.2 kb
2. 3’ pkh1 G4+G5 1.0 kb
3. iPKH1 I1+I2 0.7 kb
4. 5’ ypk1 G1+G2 0.7 kb
5. 3’ ypk1 G3+G4 0.6 kb
6. iYPK1 I1+I2 0.7 kb
pkh1/PKH1 ypk1/YPK1
1 2 3 4 5 6
1000bp
700bp
500bp
1. 5’ pil1 I1+G2 0.6 kb
2. 3’ pil1 I2+G3 0.6 kb
3. 3’ pil1 G4+G5 0.5 kb
4. iPIL1 I1+I2 0.7 kb
disruption mutant deletion mutant
pil1/PIL1 pil1/PIL1
1 2 3 4
1. 5’ pil1 I1+G2 0.6 kb
2. 3’ pil1 I2+G3 0.6 kb
3. 3’ pil1 G4+G5 0.5 kb
4. iPIL1 I1+I2 0.7 kb
disruption mutant deletion mutant
pil1/PIL1 pil1/PIL1
1 2 3 4
1. 5’ sur7 G1+G2 0.9 kb
2. 3’ sur7 G3+G4 0.6 kb
3. iSUR7 I1+I2 0.7 kb
4. 5’ lsp1 G1+ G2 1.1 kb
5. 3’ lsp1 G3+ G4 0.9 kb
6. iLSP1 I1+I2 0.6 kb
sur7 lsp1
1 2 3 4 5 6
1. 5’ sur7 G1+G2 0.9 kb
2. 3’ sur7 G3+G4 0.6 kb
3. iSUR7 I1+I2 0.7 kb
4. 5’ lsp1 G1+ G2 1.1 kb
5. 3’ lsp1 G3+ G4 0.9 kb
6. iLSP1 I1+I2 0.6 kb
sur7 lsp1
1 2 3 4 5 6
sac6
1 2 3
1. 5’ sac6 G1+G2 0.7 kb
2. 3’ sac6 G3+G4 0.6 kb
3. iSAC6 I1+I2 0.7 kb
sac6
1 2 3
1. 5’ sac6 G1+G2 0.7 kb
2. 3’ sac6 G3+G4 0.6 kb
3. iSAC6 I1+I2 0.7 kb
1. 5’ pkh1 G1+G6 1.2 kb
2. 3’ pkh1 G4+G5 1.0 kb
3. iPKH1 I1+I2 0.7 kb
4. 5’ ypk1 G1+G2 0.7 kb
5. 3’ ypk1 G3+G4 0.6 kb
6. iYPK1 I1+I2 0.7 kb
pkh1/PKH1 ypk1/YPK1
1 2 3 4 5 6
1. 5’ pkh1 G1+G6 1.2 kb
2. 3’ pkh1 G4+G5 1.0 kb
3. iPKH1 I1+I2 0.7 kb
4. 5’ ypk1 G1+G2 0.7 kb
5. 3’ ypk1 G3+G4 0.6 kb
6. iYPK1 I1+I2 0.7 kb
pkh1/PKH1 ypk1/YPK1
1 2 3 4 5 6
1000bp
700bp
500bp
PCR verifying mutant strains, homokaryons of sac6, sur7, lsp1 and heterokaryons of pkh1, ypk1 and disrupted and deleted pil1. Primers pairs amplify regions covering the 5’ and 3’ points of integration of the kanMX marker to confirm targeting into the correct locus. G2/G3/G5 and G6 anneal in the kanMX, G1/G4 anneal in the genome outside the target ORF or next to the point of integration in the case of pil1 disruption. Internal PCR fragments from target ORF can only be amplified from heterokaryotic strains; homokaryotic strains lack the internal band. Arrows mark 1000, 700 and 500 base pairs reference bands.
/
80
Appendix III – Plasmids
Plasmids used in this study source
GFP-tags in C. albicans
627 pFA-CdHIS1 Schaub et al 2006
697 pFA-GFP-CmLEU2 Schaub et al 2006
C177 pRS417, GEN3 This study
CAGQS55 BBC1-UAU1-cassette Mitchell
CAGCJ50 CYK3-UAU1-cassette Mitchell
CAGFY04 SLA1-UAU1-cassette Mitchell
C195 pDRIVE-3'BBC1 This study
C199 pRS417-3'BBC1 This study
C255 pRS417- 3'BBC1-GFP This study
C196 pDRIVE-3'CYK3 This study
C200 pRS417-3'CYK3 This study
C257 pRS417- 3'CYK3-GFP This study
C182 pGEM-3'SLA1 This study
C201 pRS417-3'SLA1 This study
C256 pRS417- 3'SLA1-GFP This study
Yeast Two Hybrid
291 pGAD424 lab collection
292 pGBT9 lab collection
339 pGAD424-AgCDC3 lab collection
423 pGBT9-CaWAL1-ΔN-term Borth et al, 2010
481 pGBT9-CaWAL1 Borth et al, 2010
639 pGBT9-ScBUD4-C-term lab collection
C99 pGBT9-CaWAL1-ΔC-term Borth et al, 2010
C100 pGBT9-CaWAL1-Δpro Borth et al, 2010
C103 pGBT9-CaVRP1-N-Term Borth et al, 2010
C104 pGAD-CaVRP1-C-Term Borth et al, 2010
C113 pGBT9-CaVRP1-C-Term Borth et al, 2010
C464 pGAD-SH3-CaABP1 This study
C476 pGAD-SH3-1-CaSLA1 This study
C477 pGAD-SH3-2-CaSLA1 This study
C491 pGAD-SH3-CaBOI1 This study
C492 pGAD-SH3-CaCYK3 This study
C493 pGAD-SH3-CaHSE1 This study
C494 pGAD-SH3-CaBBC1 This study
C495 pGAD-SH3-CaBEM1 This study
C496 pGAD-SH3-CaBEM1L This study
C497 pGAD-SH3-CaFUS1 This study
C498 pGAD-SH3-CaHOF1 This study
C499 pGAD-SH3-CaPEX13 This study
C500 pGAD-SH3-CaPIN3 This study
C501 pGAD-SH3-CaBUD14 This study
C509 pGAD-SH3-CaRVS167-1 This study
C510 pGAD-SH3-CaLSB3 This study
C511 pGAD-SH3-CaSHO1 This study
C512 pGAD-SH3-CaCDC25 This study
C516 pGAD-SH3-CaRVS167-2 This study
C517 pGAD-SH3-CaQ59U90 This study
C520 pGAD-SH3-CaQ5AAN3 This study
81
C519 pGAD-SH3-CaNBP2 This study
C521 pGAD-SH3-CaMYO5 This study
C522 pGAD-SH3-CaCDC25L This study
SAC6/Eisosomes in A. gossypii
121 pFA-kanMX Philippsen
651 pRS-AgTEF1p-lacZ Dünkler & Wendland, 2007
C136 pFA-NAT5 lab collection
C469 pFA-GFP-kanMX lab collection
C548 pGEM-AgPIL1-A This study
C589 pGEM-AgPIL1-B This study
C595 pRS418-AgTEF1p-AgPIL1 This study
C608 pRS418-AgTEF1p-AgPIL1-GFP This study
82
Appendix IV – Strains
Strains used in this study source
GFP-tags in C. albicans
SN148
arg4/arg4, leu2/leu2, his1/his1, ura3::imm434/ura3::imm434, iro1::imm434/iro1::imm434 Noble & Jonhsson, 2005
CAS001 BBC1/bbc1::CdHIS1, arg4, leu2, ura3 This study
CAS034 BBC1-GFP-CmLEU2/bbc1::CdHIS1, arg4, ura3 This study
CAP038 CYK3/cyk3::CdHIS1, arg4, leu2, ura3 Reijnst, this study
CAS030 CYK3-GFP-CmLEU2/cyk3::CdHIS1, arg4, ura3 This study
CAS024 SLA1/sla1::CdHIS1, arg4, leu2, ura3 This study
CAS027 SLA1-GFP-CmLEU2/sla1::CdHIS1, arg4, ura3 This study
SAC6/Eisosomes in A. gossypii
Agleu2 leu2 Mohr & Philippsen
ASJ18 SAC6/sac6:kanMX, leu2 This study
ASJ22 sac6:kanMX, leu2 This study
ASJ06 lsp1::kanMX/LSP1, leu2 This study
ASJ12 lsp1::kanMX, leu2 This study
ASJ10 sur7::kanMX/SUR7, leu2 This study
ASJ16 sur7::kanMX, leu2 This study
ASJ08 pkh1::kanMX/PKH1, leu2 This study
ASJ11 ypk1::kanMX/YPK1, leu2 This study
ASJ21 PIL1/pil1::kanMX, leu2 This study
ASJ24 PIL1/pil1::kanMX, leu2 This study
ASJ31 PIL1-GFP, leu2 This study
AWE37 wal1::GEN3, leu2 Walther & Wendland, 2004
Cd - Candida dubliniensis, Cm - Candida maltosa
83
Appendix V – Primers
Primers used in this study
GFP-tags in C. albicans
3593 BBC1-S1 CTTTACGTAGTTCTTTTGTTACCCCCAATTGATTGCTCGATTATCCGACACTTCAAAACTCCACAATTATTAATAATTATCTTTTCCTGTTTTCAAATTACgaagcttcgtacgctgcaggtc
3310 BBC1-S1-GFP AGATTAAGAGTATTTAGACCAGTTGGAAGACAATTTGTTGGTTGGggtgctggcgcaggtgcttc
3311 BBC1-S2 GAAATAATAAATGTGGTGATCTTCTTTCTCTCATCACCCCACACACTCAAAGAGTTTAACAATGATGGTTACGTTTAAAACAATACTTCTTCTTCGTTAAtctgatatcatcgatgaattcgag
3561 BBC1-G1 GGTACGTCAATGCCGGTTAG
3259 BBC1-G1-GFP GGTAATTGAAGTTGCTTACGACG
3216 BBC1-A1 CACCATTGGGGACTGGAC
3217 BBC1-A4/G4 ATGGCGAATACTCTGGAC
3548 BUD14-S1 CACACACAGACCACTTATTTTTAACAACACACACTTCAACACAGTACACCCTTCCCCCCTTCCCAATACCAATCTATCCACATCTACCTAATTTGAACAGCgaagcttcgtacgctgcaggtc
3571 BUD14-S1-GFP TTTGATGAACTTGCTGAAAAGTTGGCAGAATTGGATGATATACTTggtgctggcgcaggtgcttc
3549 BUD14-S2 ATTTACACAATTTTACACTACCAAATACTTTCCACTATCATTATTAACAGATTCGTATTATCCTTTATTATAAAATTTATGAATTATTATTAATACAAATtctgatatcatcgatgaattcgag
3553 BUD14-G1 CAACCACTACTACCATTAAC
3271 BUD14-G1-GFP CACAACTGATTGATCAGCACG
3220 BUD14-A1 ACTCCAGAGACTGTTGAG
3221 BUD14-A4/G4 CTTGCGGATCAGGTGTCC
4019 CYK3-S1 CCTTTCATTAATTACAAAGAAAAAAATAAGAACATCAACTATCTTTTCACTCTTTTTGAACAAATTTGTATCATACTAAAAGAATTAAATAATAAATAATgaagcttcgtacgctgcaggtc
3312 CYK3-S1-GFP TATGTTTTCGCTCAGTGGGAGTGCATAGGTAGCACAGTTGCAAATggtgctggcgcaggtgcttc
3313 CYK3-S2 AATGTACAAATGGCAAAAAGAAGTAGTAGCAGAAGAGGTAATCTATAAAGAATTTAAAACTAAATAATACCCACTCTGTTTCCCTCTTTATATATATATAtctgatatcatcgatgaattcgag
4020 CYK3-G1 GCACACTTGATGATTTCATC
3539 CYK3-G1-GFP GACTGCAAGGGCAACCAC
3222 CYK3-A1 GCTAAGATCAAGGCAGTG
3223 CYK3-A4/G4 GCAACTGCTGCAGTAGAC
3589 NBP2-S1 GTCTTGTTTGTCCTGTGTGTGTGTGTGTGTGTGTTGATAAATCACCTGAAACATATACTATTTAATCATTTGTTATTCATCATTATTGTCCATTTTGAATAGgaagcttcgtacgctgcaggtc
3578 NBP2-S1-GFP GAAAAGATCAATGCTATTGAAAAGAAATTAAATGATGTTGAAATAggtgctggcgcaggtgcttc
3579 NBP2-S2 CACATACACTCTGTTGGTATGAAAGTATAAAAACATTTGATAAAATTCGTAATCAACATTAATATAACTTAATTGTCCCTATAAGCTGGCTAATATTGGAtctgatatcatcgatgaattcgag
3557 NBP2-G1 GGTGTTTCACATTATTCTCCG
3245 NBP2-G1-GFP GACAAGTCATTTCCCACC
3230 NBP2-A1 CGTCATGGTCAAGGTTGG
3309 NBP2-A4/G4 TGGCCGAACCCTTCCTGG
3588 PIN3-S1 GAAATTGTGGACTAAGGTCAACGCCAGTGTTTAATAATCGGAATGTTGTAAATCTTTGCCTTGACAACAATTTACCTCTTAACACGCAAGAATTACCGCATTGGgaagcttcgtacgctgcaggtc
3576 PIN3-S1-GFP GGTGCTGGTGCGCTGATAGGTAGTAACATTGTCAATTCTATTTTTggtgctggcgcaggtgcttc
3577 PIN3-S2 CTTAGTAAAAAACTCATTTCATCTCAGATAATTGTACACCAAGAAATTCAAATGCCTTTTGGCTATACAACATTACTCCCATATATATGTATATTAAATTtctgatatcatcgatgaattcgag
3556 PIN3-G1 CGGTGTGTGTGGCCACTAATG
3253 PIN3-G1-GFP GTCAGCTGCCGATGTATTAG
3228 PIN3-A1 AAGGCACAACAGGCTGGC
3229 PIN3-A4/G4 CTTGGCTCCGCGTATGTC
3320 RVS167-2-S1 TACCTGGGTTGCAAAAAGTATAAGATACAACAAATAATTACTCCTCCACAAAACACACAAAAATACTAAATGATCTACTAGTAAAAAGTTACACTTCATCgaagcttcgtacgctgcaggtc
3580 RVS167-2-S1-GFP CTTGTTGGGAATGGAACTGGGTGGGAAAGGCAATTAAACGGAAAAggtgctggcgcaggtgcttc
3321 RVS167-2-S2 AGAGAATCAATATACATATTCATTCTATTTTTCACTCCTGTAGTACTTTTAATGCATTTAACAAACCTGATAAAGAGTGTAAAACAATGGAATATCCTTGtctgatatcatcgatgaattcgag
3322 RVS167-2-G1 GGTTTCTGGTTTATTGACCTG
3543 RVS167-2-G1-GFP AGAGCAAAGCGAGCTCAC
3234 RVS167-2-A1 AGGTAATGGGGTACGTCC
3235 RVS167-2-A4/G4 ATCGGTGGGTGCCATTGG
84
3316 SHO1-S1 CAGTGTATCGATCTCCAATAGATTAGTGTTTATTGATAAACTTCCCAACACTACTACTACTATAGACAGAGATAAACTGTATTAAAATATTAAAGATTGAGgaagcttcgtacgctgcaggtc
3586 SHO1-S1-GFP CAAGTTGGTATTTGTCCTTCAAATTATGTTAAATTATTAGATACTggtgctggcgcaggtgcttc
3317 SHO1-S2 CAAATCAAATTAACTCTTCATTTGGGGAAATATAATAATAGTGATAATAATAGTGATAATAAACAGTAACAAATAACAAATAACATCAAACCAAAATATACtctgatatcatcgatgaattcgag
3318 SHO1-G1 CTTCCTTCCTTCTATATCG
3538 SHO1-G1-GFP ACCAGGTAGTGGAACTGG
3552 SHO1-A1 GAGTTGGTGCCGGAAGAG
3319 SHO1-A4/G4 GAATTCAATCAAGTGGAGG
3594 SLA1-S1 CAACTCCTATGTTAGAGCTAGTCGTGCTCAACACAAAACCTGATGTGAAACAATGAAACTTTCGACGATTCTACAAAAGTGCGGAAATTGCTTGAAATCAAAGgaagcttcgtacgctgcaggtc
3314 SLA1-S1-GFP AGAGCTAATCTACAAGCAGCAACACCAGATAATCCCTTTGGATTCggtgctggcgcaggtgcttc
3315 SLA1-S2 AGCATTACAAACTATGAAAGGAATAAGAAATAATGAATAATATTTTGTTTGATATACAATTATAAAATAAAAGAGTTAATAAAGGTTCAAAATGCACTTTtctgatatcatcgatgaattcgag
3562 SLA1-G1 CGGTAGAGATGATGTTGTG
3243 SLA1-G1-GFP CACAACAACAACCGCCACC
3236 SLA1-A1 TGGTGGAGCACCACAGAC
3237 SLA1-A4/G4 CGGCTTTGCAACATCAAGAC
1432 G2-CdHIS1 TCTAAACTGTATATCGGCACCGCTC
1433 G3-CdHIS1 GCTGGCGCAACAGATATATTGGTGC
392 GFPup CATAACCTTCGGGCATGGCACTC
SAC6/Eisosomes in A. gossypii
3982 SAC6-5'a GAGACGGCTACCGTAGACCG
3983 SAC6-5'b-S1 GACCTGCAGCGTACGAAGCTTCcgaaatgcacgtgaccaacgtg
4076 SAC6-3'a-S2 CGATACTAACGCCGCCATCCAGggaggtatacataccaggcgctg
4077 SAC6-3'b attatttctagaCAGTATGTTACACACCCTGGC
3982 SAC6-G1 GAGACGGCTACCGTAGACCG
3986 SAC6-G4 GGTTGACCTTCTACACTTGGCC
304 SAC6-I1 CGACACCAGAGTGCTCAAC
305 SAC6-I2 CTGATTCAAAAGAATGGTATAG
4111 LSP1-5'a attattctcgagCCTTGCTGTCCCGAAGCACC
4112 LSP1-5'b-S1 GACCTGCAGCGTACGAAGCTTCcgtgcttagactggtctggc
4113 LSP1-3'a-S2 CGATACTAACGCCGCCATCCAGgtcacacgtgtcttgtctatcgc
4114 LSP1-3'b attattgagctcCTCGGGATGATACACGTTGGAC
4115 LSP1-G1 CCCAATAACACAGACGTGCG
4116 LSP1-G4 CACGTCTTCCGCCTGCTGGG
4117 LSP1-I1 CGTTCCGCAAGAATGCAGCG
4118 LSP1-I2 CTGTCTAGACGCCTCGTAGC
4119 YPK1-5'a attattctcgagCTTGATTGGCACCGAGGAGC
4120 YPK1-5'b-S1 GACCTGCAGCGTACGAAGCTTCggcctacaagtagatgctagcg
4121 YPK1-3'a-S2 CGATACTAACGCCGCCATCCAGggtcagagcagcttggaagc
4122 YPK1-3'b attattgagctcCTGGGAGTAACCGAGATGCGC
4123 YPK1-G1 CTGCCTACTACGCGCAGACC
4124 YPK1-G4 GCAGATCCTGGCTAGATGATCG
4125 YPK1-I1 CTTCCGTGGAGCAGGTGACG
4126 YPK1-I2 GCGAGCACAGTGCGTTCAGC
4127 PKH1-5'a attattctcgagCCTGTCTACTACCGTCCTCTGC
4128 PKH1-5'b-S1 GACCTGCAGCGTACGAAGCTTCgttgcacagctagaggctcg
4129 PKH1-3'a-S2 CGATACTAACGCCGCCATCCAGctgaccgttccctgcgtcgg
4130 PKH1-3'b attattgaattcCTGATGACAGTGCCAGTGCC
4131 PKH1-G1 CTGCTTTGTGTCCGCCAGCC
4132 PKH1-G4 CAGCCGAGGACATGCGCAGC
4133 PKH1-I1 GCTCATCGGCAGGGAGGACG
4134 PKH1-I2 GCAGCAGCTGCGGCAGAAGC
4135 SUR7-5'a attattctcgagCGTGACCGCGACCGTGCACC
85
4136 SUR7-5'b-S1 GACCTGCAGCGTACGAAGCTTCgtcccgctactctagccacg
4137 SUR7-3'a-S2 CGATACTAACGCCGCCATCCAGctacggcagattggtcacgc
4138 SUR7-3'b attattgagctcCGTGAACTCTAGCAACCCTGGC
4139 SUR7-G1 GTGTGCTGTGCGACGCCACG
4140 SUR7-G4 GTCTAGTCTCCTGGAGTGCG
4141 SUR7-I1 GGTCGAAGTGGACGTTCTGG
4142 SUR7-I2 CGCTGAGGCTCTGCGACAGG
4199 PIL1-A1 GCTGAACGACTGGACGCTGG
4103 PIL1-5’a attattggtaccCATCTGGTCCTGTCCGGTCC
4104 PIL1-5’b gacctgcagcgtacgaagcttcCACTTGCCAGAGTAGCCTCGC
4105 PIL1-3’a cgatactaacgccgccatccagCTTCGGCCTTGGCCGAGTGC
4106 PIL1-3’b attattgagctcGAAGTCTGAACGGCCTCGTGG
4107 PIL1-G1 CACCGTTTCGGACTCGCTGC
4108 PIL1-G6 GTCGCTGATCGTGTGGAGCG
4109 PIL1-I1 GTCGCAGCTGGTGAAGATCG
4110 PIL1-I2 CATCCTGCTCCCACTGCTCG
4199 PIL1-A1 GCTGAACGACTGGACGCTGG
4200 PIL1-S1-GFP GGCGAGCAGTGGGAGCAGGATGCCACTGAGGAGCAAGTCGCAGCCggtgctggcgcaggtgcttc
4201 PIL1-S2-GFP ACGAATAGAAATTAAAGATAGAAAAAGCAGCACTCGGCCAAGGCCtctgatatcatcgatgaattcgagc
4241 PIL1-I3 GCACCGGACATACTCCCTAAGG
4242 PIL1-G4 GATTCAGACCTGCCTGAAGCC
4256 PIL1-I4 attattggcgcgcCGCTCGTTCGCCGCCACCTCG
4260 PIL1-G CCGGCTCCTGCTGCCGTGCC
4264 ISR1-I2 gttcgcggtcttgatgtccccgtg
4270 PIL1-I3 attattgtttaaacATGCACCGGACATACTCCCTAAGG
4758 PIL1-S1 ATGCACCGGACATACTCCCTAAGGAACTCGAGGGCGCCCACGGCGTCGCAGCTTCgaagcttcgtacgctgcaggtc
4759 PIL1-S2 CACTCGGCCAAGGCCGAAGCGGGGGCGCGCAGGCGCGCCGGCGCTTTCCTCGCGCtctgatatcatcgatgaattcgag
3725 S1 GAAGCTTCGTACGCTGCAGGTC
3726 S2 CTGGATGGCGGCGTTAGTATCG
1202 G2-kanMX GCGTTTCCCTGCTCGCAGGTC
1198 G3-kanMX CGCCTCGACATCATCTGCCC
474 G5-kanMX TCGCAGACCGATACCAGGATC
473 G6-kanMX GTTTAGTCTGACCATCTCATCTG
Primers were ordered from Eurofins MWG, Germany. Capital letters correspond to annealing sites in genes, and lowercase letters to sites on pFA-plasmids and added restriction sites.
86
Scientific publications
Jorde, S., Walther, A., Wendland, J., (2010). "The Ashbya gossypii fimbrin SAC6 is required
for fast polarized hyphal tip growth and endocytosis.” Microbiological Research.
Borth, N., Walther, A., Reijnst, P., Jorde, S., Schaub, Y., Wendland, J., (2010). “Candida
albicans Vrp1 is required for polarized morphogenesis and interacts with Wal1 and Myo5.”
Microbiology.
Reijnst, P., Jorde, S., Wendland, J.,(2010). “Candida albicans SH3-domain proteins involved
in hyphal growth, cytokinesis, and vacuolar morphology.” Current Genetics.
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
ARTICLE IN PRESS+ModelMICRES 25382 1—9
Microbiological Research xxx (2010) xxx—xxx1
Available online at www.sciencedirect.com
www.elsevier.de/micres
The Ashbya gossypii fimbrin SAC6 is required forfast polarized hyphal tip growth and endocytosis
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Sigyn Jorde, Andrea Walther, Jürgen Wendland !4
Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark5
Received 19 August 2010 ; received in revised form 15 September 2010; accepted 25 September 20106
KEYWORDSEndocytosis;Polarized hyphalgrowth;Morphogensis;Actin cytoskeleton
Abstract1
Ashbya gossypii has been an ideal system to study filamentous hyphal growth.Previously, we identified a link between polarized hyphal growth, the organiza-tion of the actin cytoskeleton and endocytosis with our analysis of the A. gossypiiWiskott—Aldrich Syndrome Protein (WASP)-homolog encoded by the AgWAL1 gene.Here, we studied the role of AgSAC6, encoding a fimbrin in polarized hyphal growthand endocytosis, and based on our functional analysis identified genetic interac-tions between AgSAC6 and AgWAL1. SAC6 mutants show severely reduced polarizedgrowth. This growth phenotype is temperature dependent and sac6 spores do not ger-minate at elevated temperatures. Spores germinated at 30 !C generate slow growingmycelia without displaying polarity establishment defects at the hyphal tip. Severalphenotypic characteristics of sac6 hyphae resemble those found in wal1 mutants.First, tips of sac6 hyphae shifted to 37 !C swell and produce subapical bulges. Second,actin patches are mislocalized subapically. And third, the rate of endocytotic uptakeof the vital dye FM4-64 was reduced. This indicates that actin filament bundling, aconserved function of fimbrins, is required for fast polarized hyphal growth, polaritymaintenance, and endocytosis in filamentous fungi.
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© 2010 Published by Elsevier GmbH.
Introduction1
Endocytosis describes a process enabling the2
remodelling of cell surfaces, in the uptake3
of nutrients, and in cellular signalling (Munn,4
"Corresponding author. Tel.: +45 3327 5230; fax: +45 3327 4708.E-mail address: [email protected] (J. Wendland).
2001). In recent years a strong genetic link 5
between the actin cytoskeleton and endocytosis 6
has been established, e.g. by using the actin- 7
depolymerising drug Latrunculin-A it could be 8
shown that loss of filamentous actin prevents 9
endocytosis (Aghamohammadzadeh and Ayscough, 10
2009). Detailed studies using live cell imaging of 11
fluorescently tagged proteins allowed the study of 12
protein dynamics and thus the timing of events 13
0944-5013/$ – see front matter © 2010 Published by Elsevier GmbH.doi:10.1016/j.micres.2010.09.003
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
2 S. Jorde et al.
at sites of endocytosis (Kaksonen et al., 2005).14
With these studies it has been established that15
actin, the Arp2/3 complex and its regulators as16
well as several endocytic proteins co-localize in a17
dynamic yet sequential manner at sites of clathrin-18
coated pits (Merrifield, 2004; Yarar et al., 2005).19
The role of the actin cytoskeleton in endocytosis20
is found in the requirement of an actin meshwork21
and actin polymerization at sites of endocytosis. As22
a part of this, the Arp2/3 complex is required for23
branched actin filament generation. Actin nucle-24
ators like the Wiskott—Aldrich Syndrome Protein25
(WASP) and the type I myosin Myo5 activate the26
Arp2/3 complex resulting in the generation of actin27
filaments (Machesky and Gould, 1999; Higgs and28
Pollard, 1999; Goley et al., 2010). Defects in pro-29
teins interacting with actin or the Arp2/3 complex30
and its regulators typically impair endocytosis, cell31
polarity or morphogenesis (Pruyne and Bretscher,32
2000). Polarized hyphal growth as well as polarized33
pollen tube growth in plants not only depends on34
the targeted delivery of secretory vesicles to the35
growing hyphal tip but also requires endocytosis,36
e.g. for the compensatory recycling of membranes37
and tip-localized proteins (Voigt et al., 2005; Fuchs38
et al., 2006; Araujo-Bazan et al., 2008; Taheri-39
Talesh et al., 2008).40
Ashbya gossypii is a filamentous ascomycete41
belonging to the pre-whole genome duplication Sac-42
charomycetes (Dietrich et al., 2004). It has become43
an excellent system to study polarized hyphal44
morphogenesis, septation and nuclear division45
based on its ease of molecular genetic manipula-46
tion (Wendland and Walther, 2005). In particular47
Rho-protein modules, polarisome and exocyst com-48
ponents and their role for polarity establishment,49
maintenance and tip-growth speed have been char-50
acterized in more detail (Wendland and Philippsen,51
2001; Schmitz et al., 2006; Kohli et al., 2008).52
These studies indicated a strong dependence on the53
actin cytoskeleton for polarized hyphal growth in54
A. gossypii. A link between endocytosis and polar-55
ized growth was established based on the analysis56
of the A. gossypii WASP-homolog WAL1 (Walther and57
Wendland, 2004).58
AgSAC6 encodes the only Ashbya homolog of59
fimbrin. It belongs to the conserved family of60
actin-bundling proteins whose binding to actin is61
mediated by two pairs of calponin homology (CH)62
domains (Nakano et al., 2001; Klein et al., 2004).63
In Saccharomyces cerevisiae, SAC6 is required for64
the establishment and maintenance of cell polarity,65
colocalizes with actin patches, and bundles actin66
filaments (Bretscher, 1981; Adams et al., 1989).67
Similarly, in Schizosaccharomyces pombe the fim-68
brin Fim1 colocalizes with actin patches and the69
cytokinetic ring (Nakano et al., 2001; Wu et al., 70
2001). Studies in Aspergillus nidulans showed that 71
FimA-GFP localizes in a patch-like pattern at the 72
cell cortex. Deletion of fimA resulted in polarity 73
defects, particularly during spore germination, as 74
well as in endocytosis defects (Upadhyay and Shaw, 75
2008). 76
Here we present the functional analysis of 77
AgSAC6. Our data suggest a genetic link between 78
AgSAC6 and AgWAL1 based on the common mutant 79
phenotypes regarding aberrant hyphal growth and 80
endocytosis, which further underscores the link and 81
the importance of endocytosis for polarized hyphal 82
growth. 83
Materials and methods 84
Strains and media 85
The A. gossypii leu2 and wal1 strains were 86
used in this study. The leu2 mutant is referred 87
to as wild type in the text. Independent sac6 88
transformants were generated using Agleu2 as 89
parental strain. Strains were grown in yeast 90
extract—peptone—dextrose (AFM; 1% yeast extract, 91
1% peptone, 2% dextrose) with the addition of 92
the antibiotic G418 at 200 !g/ml for mutant 93
selection when required. For sporulation an A. 94
gossypii culture grown overnight in AFM was fur- 95
ther incubated in minimal medium (1.7 g/l YNB 96
w/o ammonium sulphate w/o amino acids, 0.69 g/l 97
CSM, 20 g/l glucose, 2 g/l asparagine, and 1 g/l 98
myo-inositol) until the cultures were thoroughly 99
sporulated. Escherichia coli strain DH5" was used 100
for plasmid propagation. 101
Transformation and strain construction 102
A. gossypii was transformed by electroporation as 103
described (Wendland and Philippsen, 2001). A SAC6 104
disruption cassette was cloned and used for trans- 105
formation. Primers were obtained from biomers.net 106
GmbH (Ulm, Germany). Sequences will be made 107
available upon request. 108
Plasmid constructs 109
To generate a SAC6 disruption cassette a fusion 110
PCR approach was utilized as described (Noble 111
and Johnson, 2005). Flanking homology regions 112
upstream and downstream of the SAC6-ORF were 113
amplified using primers #3982 and #3983 (384 bp 114
5#-flank) and primers #4076 and #4077 (392 bp 3#- 115
flank). These PCR products were fused to the 116
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
The Ashbya gossypii fimbrin SAC6 is required for fast polarized hyphal tip growth and endocytosis 3
AgSac6-CH1ScSac6-CH1
AgSac6-CH3ScSac6-CH3
AgSac6-CH2ScSac6-CH2
AgSac6-CH4ScSac6-CH4
572.3
AgBim1-CHScBim1-CH
AgCyk1-CHScCyk1-CH
AgScp1-CHScScp1-CH
AgCdc24-CHScCdc24-CH
Amino Acid Substitutions (x100)0100200300400500
Fig. 1. Tree comparing the relation ship of A. gossypii and S. cerevisiae calponin homology domains. The CH-domainsof the indicated proteins were identified using blastp searches at (http://blast.ncbi.nlm.nih.gov). The individual CH-domains have a length of app 100 aa. Trees were constructed using Clustal W of the Lasergene 8 software package.
kanMX4 selection marker in a second PCR. The dele-117
tion cassettes were cloned into pBluescript using118
the terminal restriction sites XhoI/XbaI provided119
with the primers. Plasmid DNA was amplified in E.120
coli and the transformation cassettes were cut from121
their plasmid backbones prior to transformation122
into A. gossypii. Correct integration of the dele-123
tion cassette into target locus and absence of the124
SAC6 ORF in the homokaryotic sac6 null mutant was125
confirmed by PCR using standard diagnostic primers126
(Walther and Wendland, 2008).127
Microscopy and staining procedures128
Microscopy was performed with an Axio-Imager129
microscope (Zeiss, Jena and Göttingen, Ger-130
many) and images processed with Metamorph 7131
software (Molecular Devices Corp., Downington,132
PA). Fluorescent microscopy was performed with133
the appropriate filter combinations for either134
rhodamine—phalloidin or FM4-64. Either single135
images were taken or a Z-stack generated from136
20 to 30 planes. Images were acquired with137
a MicroMax1024 CCD-camera (Princeton Instru-138
ments, Trenton, NJ). Staining procedures were as139
described (Walther and Wendland, 2004).140
Results141
Analysis of A. gossypii calponin homology142
domain proteins143
Within the A. gossypii proteome five proteins144
can be identified harboring calponin homol-145
ogy (CH)-actin-binding domains. Four of these 146
proteins, AAR024wp/Bim1p; ADR388cp/Cdc24, 147
ADR409wp/Scp1, and AFL150cp/Cyk1 contain 148
only a single CH-domain at their N-termini. 149
AGR069cp/Sac6 on the other hand is composed 150
of 4 CH-domains. CH-domains 1 and 3 as well as 151
CH2 and CH4 are more similar towards each other 152
indicating that AgSac6 also forms two actin-binding 153
domains comprising the CH-domains 1/2 and 3/4. 154
Individual CH-domains of A. gossypii are very simi- 155
lar to the homologous CH-domains of S. cerevisiae 156
proteins ranging from 60% amino acids identity 157
in the Scp1 proteins and 92% identity for CH1 of 158
the Sac6 homologs (Fig. 1). Interestingly, ScSAC6 159
contains an intron that is absent from AgSAC6. 160
Deletion of the A gossypii SAC6 gene and 161
the analysis of growth defects 162
To establish a function of AgSAC6 we generated 163
a deletion cassette with long flanking homology 164
regions positioned such as to result in a com- 165
plete ORF deletion. After transformation of A. 166
gossypii heterokaryotic transformants are obtained 167
that show wild-type phenotype as they carry both 168
wild type and mutant nuclei with hyphal com- 169
partments. Clonal selection starting from spores 170
that are uninucleate yields homokaryotic mutants. 171
Verification of correct gene deletion was done 172
by PCR (Fig. 2A). Phenotypic characterization of 173
the sac6 mutants using plate assays indicated a 174
severe reduction in filamentous growth rate. At 175
30—37 !C sac6 mycelia exhibited slow growth and 176
only formed compact small colonies as compared 177
to the wild type (Fig. 2B). We amplified the AgSAC6 178
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
4 S. Jorde et al.
Fig. 2. Slow growth phenotype of the Agsac6 mutant. (A) Diagnostic PCR on a homokaryotic sac6 strain. The expectedsizes at the novel joints of the 5# and 3# ends generated after integration of the kanMX marker into SAC6 locus wereobtained. Conversely, no internal PCR fragments could be amplified from the SAC6 ORF in the null mutant. (B) Theleu2 (herein referred to as wild type) and sac6 strains were grown on AFM plates for 7 days at 30 !C and 37 !C prior tophotography. Reintegration of a plasmid bearing the SAC6 gene (C) is able to restore the wild type like growth of thesac6 mutant.
from genomic DNA and ligated it into a plasmid179
carrying the clonat resistance marker NAT5, which180
consists of the resistance gene ORF and the S. cere-181
visiae TEF2 promoter and terminator regulatory182
sequences (Fig. 2C). Using this construct we could183
complement the slow growth phenotype of the sac6184
mutant indicating that the observed phenotype was185
due to deletion of SAC6.186
The slow growth phenotype was similar to that187
of the Agwal1 mutant strain. However, wal1 mutant188
hyphae are temperature sensitive and do not grow189
at 37 !C (Walther and Wendland, 2004). To deter-190
mine if there is a temperature sensitive phenotype 191
also with the sac6 mutant we germinated sac6 192
spores at different temperatures and compared 193
the colony forming ability with the parental strain 194
(Fig. 3). Colony forming frequency was found to be 195
two orders of magnitude lower in the sac6 mutant 196
compared to the wild type. This assay also showed 197
that sac6 mutants do not generate mycelia when 198
germinated at 37 !C due to cell lysis after germina- 199
tion (Fig. 3). 200
To monitor this phenotype more closely we ger- 201
minated sac6 spores over night at 30 !C and then 202
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
The Ashbya gossypii fimbrin SAC6 is required for fast polarized hyphal tip growth and endocytosis 5
Fig. 3. Germinated frequency of sac6 spores. Spores ofthe leu2 and sac6 strains were plated on AFM plates andresulting colonies were counted and photographed after 3days. Representative plates are shown. Note that the ger-mination frequency of leu2 spores is app. 100-fold higherthan that of sac6 spores at 30 !C.
transferred the cells to 37 !C. Growth at 37 !C was 203
not completely abolished (see also Fig. 2B), yet 204
under these conditions two distinct mutant pheno- 205
types became apparent. First, hyphal tips began to 206
swell and also lysed and, second, subapical hyphal 207
segments formed bulges and swellings (Fig. 4A—C). 208
Both phenotypes are very similar to those observed 209
with the wal1 mutant strain (Fig. 4D and E). 210
The Agsac6 mutant reveals defects in the 211
organization of the actin cytoskeleton 212
The actin cytoskeleton is polarized towards the 213
hyphal tip in filamentous fungi. Thus in the A. 214
gossypii wild type cortical actin patches are con- 215
centrated at the hyphal tip and defects in this 216
organization will result in isotropic growth lead- 217
ing to swellings of hyphal tips (Wendland and 218
Philippsen, 2001). Therefore, we compared the 219
organization of the cortical actin cytoskeleton of 220
the sac6 mutant with the parental strain and that 221
of wal1 mutant hyphae. Cortical actin patches are 222
found along the hyphae but cluster in the tips of 223
wild type hyphae (Fig. 5A and B). It was previously 224
observed that this clustering does not occur in wal1 225
hyphae (Walther and Wendland, 2004). Here we find 226
that also in the sac6 hyphae intense staining of actin 227
is found at subapical positions, which appears to 228
be more pronounced than in wal1 cells (Fig. 5C, 229
D, and F). This defect in patch localization could 230
be complemented upon reintroduction of AgSAC6 231
(Fig. 5E). 232
Fig. 4. Polar growth defects of sac6 hyphae. Mutant sac6 spores where germinated on microscope slides at 30 !C o/nand then transferred to 37 !C. Growth defects in the sac6 mutant lead to tip cell lysis (A and B). Reinitiation of growthat subapical parts of sac6 hyphae led to bulge formation (C). Similar phenotypes were observed for wal1 germinatedspores and hyphae (D and E). Lysis and bulge formation is marked by arrows. Bar is 100 !m.
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
6 S. Jorde et al.
Fig. 5. Comparison of actin cytoskeletal organization in the leu2, sac6 and wal1 mutants. Representative fluorescentimages are shown of hyphae of the indicated strains stained with rhodamine-phalloidin. Prior to fixation cells weregrown at either 30 !C (A, C, E, and F) or 37 !C (B and D). Bar is 10 !m. Septation was analyzed in leu2 (G and H) andsac6 (I and J) strains using calcofluor white staining of hyphae grown at 30 !C.
Using calcofluor white we stained sac6 hyphae233
for septal sites. Previously we noted a septation234
defect and the absence of chitin rich septa in the235
wal1 strain. Concomitantly to the lack of septation,236
wal1 hyphae show a strong sporulation defect. Both237
of these phenotypes could not be observed in sac6238
hyphae as septation did not seem to be affected in239
the sac6 mutant (Fig. 5G—J).240
Agsac6 hyphae show delayed endocytosis241
To monitor endocytosis we stained hyphae of the242
wild type, the sac6 and wal1 mutants with the243
vital dye FM4-64 and used in vivo fluorescence244
at different time points to monitor the uptake of245
the dye (Fig. 6). In the wild type FM4-64 uptake246
is very rapid and early endosomes became visi-247
ble very shortly after the stain was applied. After248
15 min apical compartments showed a large number249
of endosomes. In subapical compartments larger250
vacuoles are formed and they also became visi-251
ble as dye-containing endosomes were delivered252
to the vacuoles. After 2 h the cell membrane253
had essentially been cleared from the dye and254
FM4-64 had been internalized. At this stage large 255
endosomes in the tip compartment and large vac- 256
uoles in subapical compartments can be found. In 257
wild type the shape of large vacuoles is round or 258
elongated and previously we have shown by time- 259
lapse microscopy how vacuolar movement results 260
in shape changes. In the wal1 mutant endocyto- 261
sis is very much delayed particularly in subapical 262
parts of the hyphae. And, strikingly, vacuoles are 263
not actively moved about due to lack of Wal1 264
(Walther and Wendland, 2004). In the sac6 mutant 265
endocytosis is also very slow in subapical parts, 266
although after a 2 h period FM4-64 was observed 267
in endosomes and large vacuoles. Interestingly, the 268
vacuoles of sac6 hyphae did show an elongated 269
morphology indicating that active movement of 270
vacuoles is not affected by the absence of Sac6 271
(Fig. 6). 272
Discussion 273
Yeast fimbrin was isolated in a screen for suppres- 274
sors of a temperature sensitive mutation in the 275
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The Ashbya gossypii fimbrin SAC6 is required for fast polarized hyphal tip growth and endocytosis 7
Fig. 6. Endocytosis is delayed in the sac6 mutant. Hyphae of the indicated strains were grown in AFM medium andstained with FM4-64. At the indicated time points samples were subjected to fluorescence microscopy. Representativeimages of apical and subapical hyphal segments are shown. Scale bar 5 !m.
actin gene and named SAC6 (Adams and Botstein,276
1989). SAC6 encodes an actin bundling protein that277
interacts with actin through a pair of actin binding278
domains consisting itself of pairs of CH-domains.279
This organization is unique as other members of280
the calponin protein family only possess a single281
CH-domain. ScSac6 was shown to be involved in282
actin organization, endocytosis, and cell polarity283
(Drubin et al., 1988; Adams et al., 1991; Kubler and284
Riezman, 1993).285
We have analyzed the A. gossypii proteome by286
reciprocal blast searches to identify the set of CH-287
domain proteins and identified four proteins with288
single CH-domains in their N-termini, homologs of289
Bim1, Cdc24, Cyk1, and Scp1, as well as Sac6.290
AgCYK1 and AgCDC24 have been analyzed previ-291
ously. AgCYK1 is essential for actin ring formation at292
septal sites. Deletion of AgCYK1 abolishes septation293
and the accumulation of chitin at septal sites with-294
out inhibiting polarized hyphal growth (Wendland295
and Philippsen, 2002). AgCDC24 encodes the gua-296
nine nucleotide exchange factor for AgCdc42 and297
is essential for polarity establishment. Deletion of298
AgCDC24 blocks germ tube formation at the germ299
cell stage and is thus an essential gene (Wendland300
and Philippsen, 2001). However, for both proteins301
the actual contribution of the CH-domains for their302
function has not been elucidated. The BIM1 and 303
SCP1 genes have not been analyzed in A. gossypii 304
yet. 305
We initiated this study to identify domains that 306
when fused to GFP could be used to visualize 307
actin cables in A. gossypii. However, using SAC6 308
actin-binding domains resulted in unphysiological 309
actin bundling (Hebecker and Wendland, our unpub- 310
lished results). ABP140, which encodes another 311
actin binding protein, has been used to analyze 312
actin cables and movement of mitochondria along 313
actin cables in yeast cells in vivo (Yang and Pon, 314
2002; Fehrenbacher et al., 2004). Based on the 315
a 17 amino acid peptide of this protein, termed 316
lifeact, in vivo imaging of the actin cytoskeleton 317
has overcome several technical limitations and has 318
since been used in fungi and mice (Riedl et al., 319
2008, 2010; Delgado-Alvarez et al., 2010). There- 320
fore, introduction of lifeact also in A. gossypii will 321
further our understanding of transport processes 322
and organelle movement in this filamentous fungus. 323
Deletion of AgSAC6 resulted in defects in endocy- 324
tosis. The sac6 hyphae showed an intense staining of 325
actin patches in subapical hyphal regions. In S. cere- 326
visiae sac6 mutants were shown to abolish the fast 327
phase of Abp1 patch movement (Kaksonen et al., 328
2005). Thus the aberrant actin patch accumulation 329
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
8 S. Jorde et al.
in A. gossypii could indicate delays in endocytosis330
at subapical sites similarly as found in S. cerevisiae.331
Endocytosis, measured by uptake and delivery of332
FM4-64 to endosomes and vacuoles, was delayed333
in sac6 hyphae. This phenotype was less drastic as334
the endocytosis delay observed in the wal1 mutant.335
However, polarized growth was similarly affected336
in both mutants. Agwal1 hyphae are temperature337
sensitive and cease growth at 37 !C. At 30 !C wal1338
hyphae form aberrant bulges in subapical regions339
and show increased cell lysis as could be observed340
for sac6 hyphae at elevated temperatures. It is341
currently unknown what triggers the aberrant cell342
growth at the sites of bulge formation. Lateral343
branching events or remedial cell wall biosynthesis344
could cause initiation of polarized growth at such345
sites.346
Interestingly, there was a striking difference347
in vacuolar morphology when comparing the sac6348
hyphae with the wal1 mutant. In wal1 hyphae349
vacuoles are round shaped and largely immobile350
indicating that force generation via Wal1 activated351
Arp2/3 dependent actin polymerization is required352
for this movement (Walther and Wendland, 2004).353
In sac6 hyphae vacuoles had wild type like tubu-354
lar structures suggesting that Sac6 is not required355
for vacuolar motility in A. gossypii. This suggests356
that actin filament bundling via Sac6 is important357
for polarized hyphal growth and endocytosis but358
is dispensable for septation or vacuolar organelle359
movement.360
Acknowledgement361
This study was co-funded by the EU-Marie Curie362
Research Training Network “Penelope”.363
References364
Adams AE, Botstein D. Dominant suppressors of yeast365
actin mutations that are reciprocally suppressed.366
Genetics 1989;121:675—83.367
Adams AE, Botstein D, Drubin DG. A yeast actin-binding368
protein is encoded by SAC6, a gene found by sup-369
pression of an actin mutation. Science 1989;243:370
231—3.371
Adams AE, Botstein D, Drubin DG. Requirement of yeast372
fimbrin for actin organization and morphogenesis in373
vivo. Nature 1991;354:404—8.374
Aghamohammadzadeh S, Ayscough KR. Differential375
requirements for actin during yeast and mammalian376
endocytosis. Nat Cell Biol 2009;11:1039—42.377
Araujo-Bazan L, Penalva MA, Espeso EA. Preferential378
localization of the endocytic internalization machin-379
ery to hyphal tips underlies polarization of the actin380
cytoskeleton in Aspergillus nidulans. Mol Microbiol 381
2008;67:891—905. 382
Bretscher A. Fimbrin is a cytoskeletal protein that 383
crosslinks F-actin in vitro. Proc Natl Acad Sci U S A 384
1981;78:6849—53. 385
Delgado-Alvarez DL, Callejas-Negrete OA, Gomez N, et 386
al. Visualization of F-actin localization and dynamics 387
with live cell markers in Neurospora crassa. Fungal 388
Genet Biol 2010;47:573—86. 389
Dietrich FS, Voegeli S, Brachat S, et al. The Ash- 390
bya gossypii genome as a tool for mapping the 391
ancient Saccharomyces cerevisiae genome. Science 392
2004;304:304—7. 393
Drubin DG, Miller KG, Botstein D. Yeast actin-binding pro- 394
teins: evidence for a role in morphogenesis. J Cell Biol 395
1988;107:2551—61. 396
Fehrenbacher KL, Yang HC, Gay AC, Huckaba TM, 397
Pon LA. Live cell imaging of mitochondrial move- 398
ment along actin cables in budding yeast. Curr Biol 399
2004;14:1996—2004. 400
Fuchs U, Hause G, Schuchardt I, Steinberg G. Endo- 401
cytosis is essential for pathogenic development in 402
the corn smut fungus Ustilago maydis. Plant Cell 403
2006;18:2066—81. 404
Goley ED, Rammohan A, Znameroski EA, Firat-Karalar 405
EN, Sept D, Welch MD. An actin-filament-binding 406
interface on the Arp2/3 complex is critical for nucle- 407
ation and branch stability. Proc Natl Acad Sci U S A 408
2010;107:8159—64. 409
Higgs HN, Pollard TD. Regulation of actin polymerization 410
by Arp2/3 complex and WASp/Scar proteins. J Biol 411
Chem 1999;274:32531—4. 412
Kaksonen M, Toret CP, Drubin DG. A modular design for the 413
clathrin- and actin-mediated endocytosis machinery. 414
Cell 2005;123:305—20. 415
Klein MG, Shi W, Ramagopal U, et al. Structure of 416
the actin crosslinking core of fimbrin. Structure 417
2004;12:999—1013. 418
Kohli M, Galati V, Boudier K, Roberson RW, Philippsen P. 419
Growth-speed-correlated localization of exocyst and 420
polarisome components in growth zones of Ashbya 421
gossypii hyphal tips. J Cell Sci 2008;121:3878—89. 422
Kubler E, Riezman H. Actin fimbrin are required for the 423
internalization step of endocytosis in yeast. EMBO J 424
1993;12:2855—62. 425
Machesky LM, Gould KL. The Arp2/3 complex: a mul- 426
tifunctional actin organizer. Curr Opin Cell Biol 427
1999;11:117—21. 428
Merrifield CJ. Seeing is believing: imaging actin dynam- 429
ics at single sites of endocytosis. Trends Cell Biol 430
2004;14:352—8. 431
Munn AL. Molecular requirements for the internalisation 432
step of endocytosis: insights from yeast. Biochim Bio- 433
phys Acta 2001;1535:236—57. 434
Nakano K, Satoh K, Morimatsu A, Ohnuma M, Mabuchi 435
I. Interactions among a fimbrin, a capping protein, 436
and an actin-depolymerizing factor in organization 437
of the fission yeast actin cytoskeleton. Mol Biol Cell 438
2001;12:3515—26. 439
Noble SM, Johnson AD. Strains and strategies for large- 440
scale gene deletion studies of the diploid human 441
MICRES 25382 1—9Please cite this article in press as: Jorde S, et al. The Ashbya gossypii fimbrin SAC6 is required for fast polarizedhyphal tip growth and endocytosis. Microbiol Res (2010), doi:10.1016/j.micres.2010.09.003
The Ashbya gossypii fimbrin SAC6 is required for fast polarized hyphal tip growth and endocytosis 9
fungal pathogen Candida albicans. Eukaryot Cell442
2005;4:298—309.443
Pruyne D, Bretscher A. Polarization of cell growth in444
yeast. J Cell Sci 2000;113(Pt 4):571—85.445
Riedl J, Crevenna AH, Kessenbrock K, et al. Lifeact:446
a versatile marker to visualize F-actin. Nat Methods447
2008;5:605—7.448
Riedl J, Flynn KC, Raducanu A, et al. Lifeact449
mice for studying F-actin dynamics. Nat Methods450
2010;7:168—9.451
Schmitz HP, Kaufmann A, Kohli M, Laissue PP, Philippsen452
P. From function to shape: a novel role of a formin in453
morphogenesis of the fungus Ashbya gossypii. Mol Biol454
Cell 2006;17:130—45.455
Taheri-Talesh N, Horio T, Araujo-Bazan L, et al. The tip456
growth apparatus of Aspergillus nidulans. Mol Biol Cell457
2008;19:1439—49.458
Upadhyay S, Shaw BD. The role of actin, fimbrin and endo-459
cytosis in growth of hyphae in Aspergillus nidulans.460
Mol Microbiol 2008;68:690—705.461
Voigt B, Timmers AC, Samaj J, et al. Actin-based motility462
of endosomes is linked to the polar tip growth of root463
hairs. Eur J Cell Biol 2005;84:609—21.464
Walther A, Wendland J. Apical localization of actin465
patches and vacuolar dynamics in Ashbya gossypii
depend on the WASP homolog Wal1p. J Cell Sci 466
2004;117:4947—58. 467
Walther A, Wendland J. PCR-based gene targeting in Can- 468
dida albicans. Nat Protoc 2008;3:1414—21. 469
Wendland J, Philippsen P. Cell polarity and hyphal mor- 470
phogenesis are controlled by multiple rho-protein 471
modules in the filamentous ascomycete Ashbya 472
gossypii. Genetics 2001;157:601—10. 473
Wendland J, Philippsen P. An IQGAP-related protein, 474
encoded by AgCYK1, is required for septation in the 475
filamentous fungus Ashbya gossypii. Fungal Genet Biol 476
2002;37:81—8. 477
Wendland J, Walther A. Ashbya gossypii: a model 478
for fungal developmental biology. Nat Rev Microbiol 479
2005;3:421—9. 480
Wu JQ, Bahler J, Pringle JR. Roles of a fimbrin and an 481
alpha-actinin-like protein in fission yeast cell polar- 482
ization and cytokinesis. Mol Biol Cell 2001;12:1061— 483
77. 484
Yang HC, Pon LA. Actin cable dynamics in budding yeast. 485
Proc Natl Acad Sci U S A 2002;99:751—6. 486
Yarar D, Waterman-Storer CM, Schmid SL. A dynamic actin 487
cytoskeleton functions at multiple stages of clathrin- 488
mediated endocytosis. Mol Biol Cell 2005;16:964— 489
75. 490
Candida albicans Vrp1 is required for polarizedmorphogenesis and interacts with Wal1 and Myo5
Nicole Borth,1,23 Andrea Walther,1,2 Patrick Reijnst,1 Sigyn Jorde,1
Yvonne Schaub24 and Jurgen Wendland1,2
Correspondence
Jurgen Wendland
Received 19 May 2010
Revised 18 July 2010
Accepted 19 July 2010
1Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark2Junior Research Group: Growth Control of Fungal Pathogens, Leibniz Institute for Natural ProductResearch and Infection Biology – Hans Knoll Institute and Department of Microbiology,Friedrich Schiller University, D-07745 Jena, Germany
Recently, a link between endocytosis and hyphal morphogenesis has been identified in Candida
albicans via the Wiskott–Aldrich syndrome gene homologue WAL1. To get a more detailedmechanistic understanding of this link we have investigated a potentially conserved interactionbetween Wal1 and the C. albicans WASP-interacting protein (WIP)-homologue encoded byVRP1. Deletion of both alleles of VRP1 results in strong hyphal growth defects under seruminducing conditions but filamentation can be observed on spider medium. Mutant vrp1 cells showa delay in endocytosis – measured as the uptake and delivery of the lipophilic dye FM4-64 intosmall endocytic vesicles – compared to the wild-type. Vacuolar morphology was found to befragmented in a subset of cells and the cortical actin cytoskeleton was depolarized in vrp1
daughter cells. The morphology of the vrp1 null mutant could be complemented by reintegration ofthe wild-type VRP1 gene at the BUD3 locus. Using the yeast two-hybrid system we coulddemonstrate an interaction between the C-terminal part of Vrp1 and the N-terminal part of Wal1,which contains the WH1 domain. Furthermore, we found that Myo5 has several potentialinteraction sites on Vrp1. This suggests that a Wal1–Vrp1–Myo5 complex plays an important rolein endocytosis and the polarized localization of the cortical actin cytoskeleton to promote polarizedhyphal growth in C. albicans.
INTRODUCTION
Candida albicans is a pathogenic yeast that can respond tocertain environmental cues by forming hyphal filaments.This morphogenetic switch is regarded as one of severalattributes that enable C. albicans to cause disease (Sudberyet al., 2004; Whiteway & Oberholzer, 2004; Whiteway &Bachewich, 2007). Hyphal growth is an extreme form ofpolarized morphogenesis that requires constant delivery ofvesicles to support tip growth and remodelling of the cellwall at the tip. The actin cytoskeleton plays an importantrole by providing tracks for the delivery of vesicles to thetip along actin cables, and actin patches at sites ofendocytosis (Pruyne & Bretscher, 2000; Kaksonen et al.,2005). A balance between secretion and endocytosis is also
important for the maintenance of polarized morphogen-esis, although a mechanistic link has not yet been estab-lished (Aghamohammadzadeh & Ayscough, 2009). Twogenes that play a crucial role in endocytosis in C. albicansare CaMYO5, encoding myosin I, and the Wiskott–Aldrichsyndrome homologue CaWAL1 (Oberholzer et al., 2004;Walther & Wendland, 2004). In Saccharomyces cerevisiae,the corresponding homologues ScMYO3/5 and LAS17 havebeen shown to activate the Arp2/3 complex, promotingactin polarization at sites of endocytosis (Evangelista et al.,2000; Machesky, 2000; D’Agostino & Goode, 2005).Deletion of CaMYO5 leads to viable mutant strains thatcannot undergo hyphal development. Yeast cells of Camyo5mutants show depolarization of the actin cytoskeleton,which also affects their budding pattern (Oberholzer et al.,2002, 2004). Similarly, deletion of CaWAL1 results inmutant strains that are unable to generate hyphal filaments.During yeast growth of these mutants, depolarization ;ofthe actin cytoskeleton leads to the formation of round cellsthat show an increase in random budding. Additionally,loss of CaWAL1 leads to defects in the endocytosis of thelipophilic dye FM4-64 as well as defects in vacuolar fusion.Fragmented vacuoles have been observed in other mutant
%paper no. mic041707 charlesworth ref: mic041707&Cell and Molecular Biology of Microbes
Abbreviations: SH3, Src homology domain 3; WASP, Wiskott–Aldrichsyndrome protein; WH2, WASP homology 2; WIP, WASP-interactingprotein.
3Present address: Cell and Molecular Biology, Leibniz Institute forNatural Product Research and Infection Biology – Hans Knoll Institute,D-07745 Jena, Germany.
4Present address: Leibniz Institute for Age Research – Fritz LipmannInstitute, D-07745 Jena, Germany.
Microbiology (2010), 156, 000–000 DOI 10.1099/mic.0.041707-0
041707 G 2010 SGM Printed in Great Britain 1
strains, e.g. vac1 or vps11. These strains were also shown tobe defective in hyphal morphogenesis (Palmer et al., 2003;Franke et al., 2006). Characteristically, during hyphalgrowth large vacuoles are formed in the germ cell and inthe rear parts of the hyphal filaments. An unequal dis-tribution of vacuoles was also shown to influence thetiming of branch emergence (Veses et al., 2008); however,fragmented vacuoles, per se, do not abolish polarizedmorphogenesis, which was recently also shown in a Caboi2mutant (Reijnst et al., 2010).
The functional overlap of C. albicans Myo5 and Wal1 andtheir rather similar mutant phenotypes suggests that thetwo proteins can function in a complex in C. albicans. Inmammalian cells, Wiskott–Aldrich syndrome protein(WASP) was shown to interact with the WASP-interactingprotein (WIP) (Ramesh et al., 1997; Thrasher & Burns,2010). WIP suppresses growth defects of the S. cerevisiaeend5/vrp1 mutant (Vaduva et al., 1999). ScEnd5/Vrp1 is avery proline-rich protein that is involved in cytoskeletalorganization and can interact with both Las17 and Myo5(Anderson et al., 1998; Evangelista et al., 2000; Munn &Thanabalu, 2009). The temperature sensitivity and loss ofviability of an end5-1/vrp1 mutant can be suppressed by theoverexpression of ScLAS17 (Naqvi et al., 1998). And finally,loss of ScEnd5/Vrp1 results in severe defects in cytokinesisand Hof1 cannot be recruited to the bud neck (Ren et al.,2005).
Here we describe the analysis of the C. albicans VRP1homologue. The mutant strain shows defects in hyphalgrowth, endocytosis, organization of the actin cytoskeletonand budding pattern similar to, but less pronounced than,the wal1 and myo5mutant strains. Two-hybrid studies in S.cerevisiae showed that Vrp1 interacts strongly with the N-terminal domain of Wal1 and also with Myo5. The datasuggest that a Wal1–Vrp1–Myo5 complex is crucial forendocytosis and polarized morphogenesis in C. albicans.
METHODS
Strains and media. C. albicans strain SN148 (Noble & Johnson,2005) was used to generate the vrp1 heterozygous and homozygousmutant strains. For the yeast two-hybrid experiment, the followingstrains were used: PJ69-4a< (MATa trp1-901 leu2-3,112 ura3-52 his3-200 gal4D gal80D LYS2 : :GAL1-HIS3 GAL2p-ADE2 met2 : :GAL7-lacZ) and PJ69-4alpha (MATa trp1-901 leu2-3,112 ura3-52 his3-200gal4D gal80D LYS2 : :GAL1-HIS3 GAL2p-ADE2 met2 : :GAL7-lacZ).Strains were grown either in yeast extract/peptone/dextrose medium[YPD; 1% yeast extract, 2% peptone, 2% dextrose (glucose)], or inminimal synthetic defined media= [6.7 g l21 yeast nitrogen base (YNB)with ammonium sulphate and without amino acids, 20 g l21 glucose]supplemented with 0.69 g l21 complete supplement mixture (CSM),or with the addition of required amino acids and uridine. Hyphalformation was induced with 10% serum at 37 uC or by incubation onSpider medium. Escherichia coli strain DH5a served as a host forplasmid propagation.
Transformation and strain construction. S. cerevisiae and C.albicans were transformed using the lithium acetate procedure(Walther & Wendland, 2003; Gietz & Schiestl, 2007). Independent
homozygous mutant strains were constructed and verified followingstandard PCR-based gene targeting methods based on the use of pFAplasmids for cassette generation (Walther & Wendland, 2008).Deleting both ORFs of VRP1 by sequential transformation ofSN148 with PCR-generated cassettes resulted in the heterozygousstrains CAB9 (VRP1/vrp1 : :ARG4) and CAB10 (VRP1/vrp1 : :URA3),and then the homozygous strains CAB12 (vrp1 : :ARG4/vrp1 : :CdHIS1) and CAB13 (vrp1 : :URA3/vrp1 : :CdHIS1). To complementthe Dvrp1 phenotype, VRP1 was amplified from genomic DNA andligated in to cloning vector pDrive, generating #C597. The insert wascloned in #C873, which contains the BUD3 locus for integration andCmLEU2 as selectable marker, using SalI and BamHI restriction sites.This generated #C598. The Dvrp1 homozygous mutant strain CAB13was transformed with SpeI-linearized #C598, generating CAP225.
Strain CAT41 was generated by targeting a GFP-HIS1 cassette to theC. albicans TEF1 locus. All primers were obtained from biomers.netand their sequences will be made available upon request >.
Plasmid constructs. For the yeast two-hybrid experiments, freelyreplicating plasmids were generated using pGAD424 and pGBT9(Clontech) as backbones. These plasmids contained the Gal4-transcription-factor-activation domain or the Gal4-DNA-bindingdomain, respectively. Restriction fragments of WAL1, VRP1 and theregion encompassing the SH3 domain of MYO5 were amplified fromgenomic DNA or plasmid clones and cloned into the correspondingrestriction sites of pGAD424 or pGBT9. Correct cloning was verifiedby sequencing (Eurofins MWG Operon).
Microscopy and staining procedures. Microscopic analyses weredone with an Axio-Imager microscope (Zeiss) using Metamorph 7software tools (Molecular Devices) to drive the automated image-acquisition procedures. Images were acquired with a MicroMax1024CCD camera (Princeton Instruments). Fluorescence microscopy wasperformed using the appropriate filter combinations for FM4-64imaging and actin staining as described previously (Walther &Wendland, 2004; Martin et al., 2005). Samples were analysed bygenerating either single images or Z-stacks of up to 20 images thatwere processed into single-plane projections using Metamorphsoftware.
Yeast two-hybrid analysis. S. cerevisiae was transformed with twoplasmids expressing constructs fused to either the Gal4-DNA-bindingdomain, based on plasmid pGBT9, or the Gal4-activation domain,based on plasmid pGAD424. Transformants were grown on mediaselecting for the maintenance of both plasmids (2Trp 2Leu). Whitecolonies revealed an interaction of the two expressed fusion proteins,which results in the expression of the ADE2 reporter gene, whereasred colonies appeared when the ADE2-reporter could not beactivated. For quantitative analysis, liquid-culture b-galactosidaseassays were performed. To this end, strains were incubated overnightat 30 uC. Cells were harvested by centrifugation, protein extracts wereprepared using a liquid nitrogen/glass-bead method and theconversion of ONPG (o-nitrophenyl b-D-galactopyranoside) wasmeasured photometrically (Rose & Botstein, 1983).
RESULTS
Sequence comparisons
The C. albicans homologue of S. cerevisiae END5/VRP1 hasbeen identified as orf19.2190. C. albicans VRP1 encodes avery proline-rich protein of 664 aa, of which 154 residuesare proline. Sequence comparisons with other fungalhomologues were done using the CLUSTAL W alignment
%paper no. mic041707 charlesworth ref: mic041707&
N. Borth and others
2 Microbiology 156
tool (Fig. 1). The N-terminal region of CaVrp1p contains aproline stretch present in most fungi and only annotated tobe absent in Ashbya gossypii. Reinspection of the VRP1-locus in A. gossypii, however, indicates that there is apolyproline region upstream of the annotated start codon.Furthermore, a Vrp1 homologue in the closely relatedspecies Eremothecium cymbalariae also contains thispolyproline region at the N terminus of Ecym_Vrp1 (ourunpublished results). Downstream of the polyprolineregion in Vrp1, two putative WASP homology 2 (WH2)domains are located. Here, the filamentous ascomyceteNeurospora crassa lacks the second putative WH2 domain.In S. cerevisiae, a short region after the second WH2domain has been characterized as a docking site for Hof1(Ren et al., 2005). This Hof1-trap (HOT)-domain seems tobe rather specific for S. cerevisiae as it is not found in theother fungal species analysed (Fig. 1a). The central part ofVrp1 orthologues exhibits only a low degree of amino acidsequence conservation, not regarding the many proline-rich stretches, whereas the C-terminal regions in fungalVrp1 proteins show better conservation. This domain hasbeen characterized as the Las17p-binding domain (Naqviet al., 1998; Madania et al., 1999; Fig. 1b).
Generation of C. albicans vrp1 mutant strains
To delete both alleles of C. albicans VRP1, a PCR-basedgene targeting approach was applied (Walther &Wendland, 2008). Initially, independent heterozygousmutant strains were generated in which the ORF of oneallele of VRP1 was deleted by either the C. albicans ARG4or URA3 marker gene. To generate homozygous mutantstrains based on these heterozygous strains, the remainingcopy of VRP1 was deleted using the Candida dubliniensisHIS1 gene. Verification of correct gene targeting and the
absence of the VRP1 ORF in the homozygous mutantstrains CAB11 and CAB13 was done by diagnostic PCR andreintegration of VRP1 at the BUD3 locus was used forcomplementation.
Phenotypic assay of growth morphology ofmutant strains
The heterozygous and homozygous VRP1-deletion strainswere compared to the SC5314 wild-type strain, the SN148strain used as a host for transformation and a wal1 mutantstrain deleted for the C. albicans homologue of the humanWASP, described previously (Walther & Wendland, 2004).Hyphal induction was tested on Spider medium, whichcontains mannitol as the primary carbon source. The wild-type showed strong filamentation at the edge of the colony,whereas the wal1 strain was afilamentous. The SN148precursor strain also showed a strong increase in colonywrinkling, which was also found in the heterozygous VRP1/vrp1 strain. The homozygous vrp1 strain did not show thiscolony-wrinkling phenotype; however, the colony edges ofthe mutant vrp1 strain did show invasive filamentousgrowth (Fig. 2a). Hyphal induction in liquid media wasdone using serum as an inducing cue. Here, the wild-typeshowed abundant filamentation. SN148 showed slightlyless filamentation (due to the ura3 deletion) and wal1 againshowed no hyphal formation. The heterozygous VRP1/vrp1strain showed filamentation similar to that of the wild-type, whereas the vrp1 mutant strain showed a strongreduction in filamentation and produced instead a largenumber of pseudohyphal cells and new yeast cells (Fig. 2a).The distribution of cell types after hyphal induction in thevarious strains used was quantified by counting .100 cellsfor each strain (Fig. 2c ?). These analyses indicated a strongdefect in hyphal formation of the vrp1 mutant, which was,
%paper no. mic041707 charlesworth ref: mic041707&
Fig. 1. Alignment of fungal Vrp1 homologues.Amino acid residues corresponding to themajority of analysed sequences are shaded.(a) The N-terminal actin-binding sequencesare boxed in all Vrp1 proteins. S. cerevisiaeVrp1 harbours non-conserved regions with theconsensus sequence ‘PxPSS’ (boxed) thatwere shown to interact with ScHof1. (b)Alignment of the Vrp1 C-terminal regionsharbouring the conserved Las17-bindingdomain. Protein information was obtained fromthe following sources: A. gossypii, the AshbyaGenome Database (http://agd.vital-it.ch/index.html); C. albicans, the Candida GenomeDatabase (http://www.candidagenome.org) @;Kluyveromyces lactis, XP_451805; Neuro-spora crassa, XP_963859; and S. cerevisiae,AAB67263.
C. albicans VRP1
http://mic.sgmjournals.org 3
however, slightly less severe than in the wal1 mutant. Thisresult is in line with the filamentation assay on Spidermedium. To demonstrate that these filamentation defectsare solely due to the deletion of the VRP1 gene, wereintegrated the VRP1 gene at the BUD3 locus in a vrp1/vrp1 mutant strain (see Methods). This reintegrant wasphenotypically like the wild-type, for example, whenassayed for germ tube production (Fig. 2b).
Analysis of the actin cytoskeleton in the vrp1mutant
Hyphal growth defects may be associated with an alteredorganization of the actin cytoskeleton. Therefore, we usedrhodamine-phalloidin staining of fixed cells to analyse thedistribution and polarization of the cortical actin cytoskele-ton. Wild-type cells show a polarization of cortical actin in
the emerging bud and at the hyphal tip. The actincytoskeleton of the wal1 mutant was shown to be largelydepolarized during all growth stages. In the vrp1 mutantsuch a depolarization could also be observed in yeast andpseudohyphal cells. Remarkably, in both yeast and hyphalstages the apical growth region showed more intensestaining indicating an accumulation of actin at sites ofpolarized growth (Fig. 3a). Analysis of the budding patternvia fluorescence microscopy of the bud scars showed thatthe vrp1 mutant is able to generate a bipolar buddingpattern, as found in the wild-type (Fig. 3b).
VRP1 mutants show defects in vacuolar fusionand endocytosis
Altered polarization of the actin cytoskeleton may also affectendocytosis. To study vacuolar morphology and endocytosis
%paper no. mic041707 charlesworth ref: mic041707&
Fig. 2. Characterization of the growth defectsof the vrp1 mutant SN148. (a) The indicatedstrains were grown on Spider plates for 5 daysat 37 6C prior to photography (top row).Microscopic images of the colony edges showthe degree of filamentation (middle row).Hyphal formation in liquid medium wasinduced by using 10% serum. Images weretaken after 6 h of induction (bottom row). (b)Cells induced by serum were counted after 6 hand classified according to their morphology.(c) Complementation of the vrp1 filamentationdefect by reintegration of VRP1 at the C.albicans BUD3 locus.
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4 Microbiology 156
we employed the lipophilic dye FM4-64. Using time-lapsemicroscopy we compared, at the same time, uptake of FM4-64 between the vrp1 mutant and a wild-type strainexpressing cytoplasmic GFP. Within 20 min the dye hadbeen taken up via endocytosis and delivered to the vacuolein the wild-type. This staining of wild-type vacuoles furtherincreased over time. Compared to this the vrp1 mutantshowed a delayed uptake and only after more than 1 h didsmall endocytic vesicles, and possibly vacuoles, becamestained (Fig. 4). Quantification of the vacuoles showed aslight increase in the vrp1 mutant compared to the wild-type. This vrp1 phenotype is thus somewhat intermediatebetween the wal1 mutant and the wild-type (Fig. 5).
Two-hybrid analyses reveal interaction of Vrp1with Myo5 and Wal1
In S. cerevisiae Vrp1 was shown to interact with Srchomology domain 3 (SH3) of the yeast type I myosinMyo5p and the WASP homologue Las17 (Evangelista et al.,2000). To determine if CaVrp1 interacts with both Las17and Myo5 we performed yeast two-hybrid analyses (Fig. 6).In this assay we found that the C-terminal part of Vrp1interacts strongly with the N-terminal part of Wal1containing the WH1-B domains. Interaction of Vrp1 withthe full-length Wal1 protein was somewhat weaker.Highest b-galactosidase activity was obtained with a Wal1fragment in which the central part of WAL1, encodingseveral proline-rich regions, was removed for the assay. Toassay the interaction of the myosin I SH3 domain withVrp1 we used two fragments containing the N and Ctermini of Vrp1 and a fragment containing only the SH3-domain of Myo5. Here, the Myo5 SH3-domain interactedstrongly with the C-terminal part of Vrp1 (Fig. 6).
DISCUSSION
In this report we have characterized the function of the C.albicans VRP1 gene in the polarized morphogenesis andendocytosis of this dimorphic human pathogen. Cellpolarization in C. albicans is important for budding,filamentation and mating. The establishment of cellpolarity occurs either due to intrinsic factors (duringbudding) or in response to environmental stimuli (duringfilamentation and mating) (Whiteway & Bachewich, 2007).One result of this polarity is the polarized organization ofthe actin cytoskeleton, which results in the apicalpositioning of cortical actin patches and the generationof actin cables emanating from the cell apex (Smith et al.,2001). Generation of actin cables from the emerging bud orthe tip of the hyphae has been fairly well characterized. Acascade from locally activated Rho-type GTPases, mostnotably Cdc42, triggers downstream effector genes, such asthe formin Bni1, which nucleates actin filaments(Evangelista et al., 2002; Sagot et al., 2002). Bni1 is partof a complex termed the polarisome, which in S. cerevisiaealso contains Pea2, Spa2 and Bud6 (Sheu et al., 1998).
Clustered assembly of actin patches occurs at sites ofpolarized growth. Mutants of S. cerevisiae and C. albicansthat are affected in the position of actin patches, e.g. in theLAS17/WAL1 or MYO3/5 genes, show defects in polarizedgrowth (Li, 1997; Lechler et al., 2000; Oberholzer et al.,2002; Walther & Wendland, 2004). Las17 and Myo3/5 havebeen shown to stimulate actin filament formation via theArp2/3 complex (Lechler et al., 2000). Mutants affected inthese genes show defects in the assembly and organizationof the actin cytoskeleton and since the actin cytoskeleton isessential for endocytosis in S. cerevisiae they also showdefects in clathrin-mediated endocytosis (Munn, 2001;Kaksonen et al., 2003). Most of the proteins known to beinvolved in endocytosis co-localize with actin patches.
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Fig. 3. Analysis of the actin cytoskeleton and budding pattern. (a)Strains were grown to form yeast and hyphal stages, fixed byformaldehyde and stained using rhodamine-phalloidine. Bar,10 mm. (b) Representative images of calcofluor-stained cells.
C. albicans VRP1
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Thus actin patches are sites of endocytosis (Kaksonen et al.,2005).
Deletion of S. cerevisiae VRP1 results in temperaturesensitivity and the depolarization of actin patches. Specifi-cally, actin patches do not cluster in emerging buds(Lambert et al., 2007). We have also observed adepolarization of actin patches in both mother anddaughter cells of the C. albicans vrp1 mutant. Further-more, hyphal morphogenesis in the vrp1 strains wasinhibited – although not abolished as in wal1 cells.Nevertheless, during growth of vrp1 germ tubes, actinappeared to accumulate in a cap-like structure.
Our two-hybrid analysis suggests that a Wal1–Vrp1–Myo5complex may be formed in C. albicans similar to thatidentified in S. cerevisiae (Evangelista et al., 2000). Thiscould provide an explanation of the mechanism responsiblefor the similar phenotypes of the wal1 and myo5 mutantsobserved previously. Both of these genes are activators of theArp2/3 complex, and loss of either of these genes may bemore detrimental to cells than loss of VRP1. Consequently,the observed defects in endocytosis and vacuole formationwere less severe in the vrp1 strains compared to wal1.Interestingly, S. cerevisiae Vrp1 contains a region character-ized as the Hof1-trap domain, which is essential for bindingthe Hof1-SH3 domain (Ren et al., 2005). A Hof1-trapdomain could not be identified in C. albicans Vrp1. Ourattempts to identify a two-hybrid interaction of the C.albicans SH3-domain of Hof1 with Vrp1 were unsuccessful,which may suggest that this interaction is occurring either
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Fig. 4. Time-lapse analysis of endocytosis ofthe lipophilic dye FM4-64. Wild-type cellscarrying a cytoplasmic GFP label (on the rightside of each panel) were mixed with vrp1/vrp1cells (on the left side of each panel).Microscopy slides with wells were filled with0.75 ml 0.5! YPD and 0.75 ml 3.4% agarose.To this mixture 1 ml FM4-64 (200 mg ml”1 inDMSO) was added. Image acquisition started10 min after preparation of the slide for a dura-tion of 3 h with a frequency of 1 image min”1.Selected frames are shown at the indicatedtime points, starting with a GFP imageidentifying the wild-type cells followed by abright-field differential interference contrast(DIC) image Aof all cells. Bar, 10 mm.
Fig. 5. Analysis of vacuolar morphology. Strains were grownovernight and then stained with FM4-64 for 2 h prior tophotography. Cells were counted and characterized according tothe number of vacuoles they contained. Representative images ofcells displaying one, two to three, or more than four vacuoles areshown.
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6 Microbiology 156
with a low affinity or not at all (our unpublished results). Onthe other hand, it was shown that the SH3 domain of S.cerevisiae Hof1 also interacts with formins Bnr1 and Bni1,which could provide an alternative route for the localizationof Vrp1 to sites of polarized growth and septation(Evangelista et al., 2003). Formins and Vrp1 may shareanother feature: the binding of profilin. Bni1 binds via itsFH1 domain to profilin. This domain includes a polyprolinestretch similar to that found at the N terminus of Vrp1homologues, which may explain, mechanistically, how Vrp1contributes to F-actin formation.
Thus, our analysis contributes to our understanding of themechanistic link between Wal1 and Myo5 in C. albicans.With the defects in hyphal morphogenesis and endocytosisof the vrp1 mutant strain we have identified another playerpartaking in the yeast-to-hyphal switch in C. albicans.
ACKNOWLEDGEMENTS
We thank Alexander Johnson and Suzanne Noble for generouslyproviding the reagents used in this study. Parts of this study were
funded by the EU-Marie Curie Research Training Network‘Penelope’.
REFERENCES
Aghamohammadzadeh, S. & Ayscough, K. R. (2009). Differentialrequirements for actin during yeast and mammalian endocytosis. NatCell Biol 11, 1039–1042.
Anderson, B. L., Boldogh, I., Evangelista, M., Boone, C., Greene, L. A.& Pon, L. A. (1998). The Src homology domain 3 (SH3) of a yeast typeI myosin, Myo5p, binds to verprolin and is required for targeting tosites of actin polarization. J Cell Biol 141, 1357–1370.
D’Agostino, J. L. & Goode, B. L. (2005). Dissection of Arp2/3 complexactin nucleation mechanism and distinct roles for its nucleation-promoting factors in Saccharomyces cerevisiae. Genetics 171, 35–47.
Evangelista, M., Klebl, B. M., Tong, A. H., Webb, B. A., Leeuw, T.,Leberer, E., Whiteway, M., Thomas, D. Y. & Boone, C. (2000). A rolefor myosin-I in actin assembly through interactions with Vrp1p,Bee1p, and the Arp2/3 complex. J Cell Biol 148, 353–362.
Evangelista, M., Pruyne, D., Amberg, D. C., Boone, C. & Bretscher, A.(2002). Formins direct Arp2/3-independent actin filament assemblyto polarize cell growth in yeast. Nat Cell Biol 4, 32–41.
%paper no. mic041707 charlesworth ref: mic041707&
Fig. 6. Two-hybrid analysis. PCR fragmentscarrying different domains were combined withthe DNA-binding domain (BD) or the DNA-activation domain (AD) as indicated in theupper panel. For CaMYO5, only the core SH3domain was used (for details see Methods).Domains: WH1, WASP-homology 1; WH2,WASP-homology 2; P1–P4, proline-richregions in CaWal1; VCA, verprolin-central-acidic domain; LBD, conserved Las17-bindingdomain; HOT, Hof1-trap domain. Plasmidscarrying the BD/AD pairs were transformedinto S. cerevisiae and selected on –Trp/–Leuplates (lower panel). Transformant colonieswere tested by X-Gal overlay and the b-galactosidase activity was tested in triplicate.
COLOURFIGURE
C. albicans VRP1
http://mic.sgmjournals.org 7
Evangelista, M., Zigmond, S. & Boone, C. (2003). Formins: signalingeffectors for assembly and polarization of actin filaments. J Cell Sci116, 2603–2611.
Franke, K., Nguyen, M., Hartl, A., Dahse, H. M., Vogl, G., Wurzner, R.,Zipfel, P. F., Kunkel, W. & Eck, R. (2006). The vesicle transport proteinVac1p is required for virulence of Candida albicans. Microbiology 152,3111–3121.
Gietz, R. D. & Schiestl, R. H. (2007). High-efficiency yeasttransformation using the LiAc/SS carrier DNA/PEG method. NatProtoc 2, 31–34.
Kaksonen, M., Sun, Y. & Drubin, D. G. (2003). A pathway forassociation of receptors, adaptors, and actin during endocyticinternalization. Cell 115, 475–487.
Kaksonen, M., Toret, C. P. & Drubin, D. G. (2005). A modular designfor the clathrin- and actin-mediated endocytosis machinery. Cell 123,305–320.
Lambert, A. A., Perron, M. P., Lavoie, E. & Pallotta, D. (2007). TheSaccharomyces cerevisiae Arf3 protein is involved in actin cable andcortical patch formation. FEMS Yeast Res 7, 782–795.
Lechler, T., Shevchenko, A. & Li, R. (2000). Direct involvement ofyeast type I myosins in Cdc42-dependent actin polymerization. J CellBiol 148, 363–373.
Li, R. (1997). Bee1, a yeast protein with homology to Wiscott-Aldrichsyndrome protein, is critical for the assembly of cortical actincytoskeleton. J Cell Biol 136, 649–658.
Machesky, L. M. (2000). The tails of two myosins. J Cell Biol 148,219–221.
Madania, A., Dumoulin, P., Grava, S., Kitamoto, H., Scharer-Brodbeck, C., Soulard, A., Moreau, V. & Winsor, B. (1999). TheSaccharomyces cerevisiae homologue of human Wiskott-Aldrichsyndrome protein Las17p interacts with the Arp2/3 complex. MolBiol Cell 10, 3521–3538.
Martin, R., Walther, A. & Wendland, J. (2005). Ras1-induced hyphaldevelopment in Candida albicans requires the formin Bni1. EukaryotCell 4, 1712–1724.
Munn, A. L. (2001). Molecular requirements for the internalisationstep of endocytosis: insights from yeast. Biochim Biophys Acta 1535,236–257.
Munn, A. L. & Thanabalu, T. (2009). Verprolin: a cool set of actin-binding sites and some very HOT prolines. IUBMB Life 61, 707–712.
Naqvi, S. N., Zahn, R., Mitchell, D. A., Stevenson, B. J. & Munn, A. L.(1998). The WASp homologue Las17p functions with the WIPhomologue End5p/verprolin and is essential for endocytosis in yeast.Curr Biol 8, 959–962.
Noble, S. M. & Johnson, A. D. (2005). Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogenCandida albicans. Eukaryot Cell 4, 298–309.
Oberholzer, U., Marcil, A., Leberer, E., Thomas, D. Y. & Whiteway, M.(2002).Myosin I is required for hypha formation in Candida albicans.Eukaryot Cell 1, 213–228.
Oberholzer, U., Iouk, T. L., Thomas, D. Y. & Whiteway, M. (2004).Functional characterization of myosin I tail regions in Candidaalbicans. Eukaryot Cell 3, 1272–1286.
Palmer, G. E., Cashmore, A. & Sturtevant, J. (2003). Candida albicansVPS11 is required for vacuole biogenesis and germ tube formation.Eukaryot Cell 2, 411–421.
Pruyne, D. & Bretscher, A. (2000). Polarization of cell growth inyeast. J Cell Sci 113, 571–585.
Ramesh, N., Anton, I. M., Hartwig, J. H. & Geha, R. S. (1997). WIP, aprotein associated with Wiskott–Aldrich syndrome protein, inducesactin polymerization and redistribution in lymphoid cells. Proc NatlAcad Sci U S A 94, 14671–14676.
Reijnst, P., Jorde, S. & Wendland, J. (2010). Candida albicans SH3-domain proteins involved in hyphal growth, cytokinesis, and vacuolarmorphology. Curr Genet 56, 309–319.
Ren, G., Wang, J., Brinkworth, R., Winsor, B., Kobe, B. & Munn, A. L.(2005). Verprolin cytokinesis function mediated by the Hof one trapdomain. Traffic 6, 575–593.
Rose, M. & Botstein, D. (1983). Construction and use of gene fusionsto lacZ (beta-galactosidase) that are expressed in yeast. MethodsEnzymol 101, 167–180.
Sagot, I., Rodal, A. A., Moseley, J., Goode, B. L. & Pellman, D. (2002).An actin nucleation mechanism mediated by Bni1 and profilin. NatCell Biol 4, 626–631.
Sheu, Y. J., Santos, B., Fortin, N., Costigan, C. & Snyder, M. (1998).Spa2p interacts with cell polarity proteins and signaling compo-nents involved in yeast cell morphogenesis. Mol Cell Biol 18, 4053–4069.
Smith, M. G., Swamy, S. R. & Pon, L. A. (2001). The life cycle of actinpatches in mating yeast. J Cell Sci 114, 1505–1513.
Sudbery, P., Gow, N. & Berman, J. (2004). The distinct morphogenicstates of Candida albicans. Trends Microbiol 12, 317–324.
Thrasher, A. J. & Burns, S. O. (2010). WASP: a key immunologicalmultitasker. Nat Rev Immunol 10, 182–192.
Vaduva, G., Martinez-Quiles, N., Anton, I. M., Martin, N. C., Geha, R. S.,Hopper, A. K. & Ramesh, N. (1999). The human WASP-interactingprotein, WIP, activates the cell polarity pathway in yeast. J Biol Chem274, 17103–17108.
Veses, V., Richards, A. & Gow, N. A. (2008). Vacuoles and fungalbiology. Curr Opin Microbiol 11, 503–510.
Walther, A. & Wendland, J. (2003). An improved transformationprotocol for the human fungal pathogen Candida albicans. Curr Genet42, 339–343.
Walther, A. & Wendland, J. (2004). Polarized hyphal growth inCandida albicans requires the Wiskott–Aldrich Syndrome proteinhomolog Wal1p. Eukaryot Cell 3, 471–482.
Walther, A. & Wendland, J. (2008). PCR-based gene targeting inCandida albicans. Nat Protoc 3, 1414–1421.
Whiteway, M. & Bachewich, C. (2007). Morphogenesis in Candidaalbicans. Annu Rev Microbiol 61, 529–553.
Whiteway, M. & Oberholzer, U. (2004). Candida morphogenesisand host–pathogen interactions. Curr Opin Microbiol 7, 350–357.
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Curr GenetDOI 10.1007/s00294-010-0301-7
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RESEARCH ARTICLE
Candida albicans SH3-domain proteins involved in hyphal growth, cytokinesis, and vacuolar morphology
Patrick Reijnst · Sigyn Jorde · Jürgen Wendland
Received: 23 December 2009 / Revised: 22 March 2010 / Accepted: 29 March 2010! Springer-Verlag 2010
Abstract This report describes the analyses of threeCandida albicans genes that encode Src Homology 3(SH3)-domain proteins. Homologs in Saccharomyces cere-visiae are encoded by the SLA1, NBP2, and CYK3 genes.Deletion of CYK3 in C. albicans was not feasible, suggest-ing it is essential. Promoter shutdown experiments ofCaCYK3 revealed cytokinesis defects, which are in linewith the localization of GFP-tagged Cyk3 at septal sites.Deletion of SLA1 resulted in strains with decreased abilityto form hyphal Wlaments. The number of cortical actinpatches was strongly reduced in !sla1 strains during allgrowth stages. Sla1-GFP localizes in patches that are foundconcentrated at the hyphal tip. Deletion of the Wrst twoSH3-domains of Sla1 still resulted in cortical localizationof the truncated protein. However, the actin cytoskeleton inthis strain was aberrant like in the !sla1 deletion mutantindicating a function of these SH3 domains to recruit actinnucleation to sites of endocytosis. Deletion of NBP2resulted in a defect in vacuolar fusion in hyphae. Germcells of !nbp2 strains lacked a large vacuole but initiatedseveral germ tubes. The mutant phenotypes of !nbp2 and!sla1 could be corrected by reintegration of the wild-typegenes.
Keywords Candida albicans · PCR-based gene targeting · pFA-plasmids · Actin cytoskeleton · FM4-64
Introduction
Candida albicans is one of the most important human fun-gal pathogens. It occurs as a commensal on epithelial sur-faces in oropharyngeal tissue, the gastro-intestinal tract,and in the vagina. Particularly, vaginitis and urinary tractinfections caused by C. albicans are frequent in otherwisehealthy individuals. Immuno-compromised patients mayadditionally develop life-threatening systemic infections ofinner organs (Odds 1994; Calderone and Fonzi 2001).
The morphological transition of C. albicans from yeastto hyphal growth has been recognized as an important viru-lence attribute amongst others (Sudbery et al. 2004;Kumamoto and Vinces 2005). Filamenting germ cellscharacteristically generate large vacuoles. This compart-mentalizes the germ tube in an apical region that containsendosomes and small vacuoles, and subapical regionswhich harbor large vacuoles at the expense of cytoplasm.Septation in hyphal Wlaments further promotes this com-partmentalization. The unequal distribution of vacuolar vol-ume inXuences the branching frequency during Wlamentousgrowth (Barelle et al. 2006; Veses et al. 2009). The actincytoskeleton is polarized at sites of polarized growth andcortical actin patches cluster in the hyphal tips. Defects inthe polarization of the actin cytoskeleton, e.g. interferingwith the function of several Rho-type GTPases generallylead to growth defects (Wendland 2001; Court and Sudbery2007; Zheng et al. 2007). Actin ring formation promotedvia Iqg1 at sites of septation is required for septum forma-tion (Epp and Chant 1997; Wendland and Philippsen 2002).In S. cerevisiae, CYK3 can act as a multicopy suppressor ofan IQG deletion (Korinek et al. 2000). Processes like polar-ized hyphal growth, endocytosis, and cytokinesis requireprotein networks and timely regulation within the cellcycle. SH3-domain encoding proteins are well suited to
Communicated by C. D'Enfert.
P. Reijnst · S. Jorde · J. Wendland (&)Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, 2500 Valby, Copenhagen, Denmarke-mail: [email protected]
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play important roles in these processes since they can medi-ate protein–protein interactions via their SH3-domains (amBusch et al. 2009). Several C. albicans SH3-protein encod-ing genes have already been characterized including MYO5,BEM1, and CDC25 (Enloe et al. 2000; Michel et al. 2002;Oberholzer et al. 2002; Bassilana et al. 2003). BEM1 wasfound to be essential, while MYO5 plays an important roleduring endocytosis. Both Myo5 and Cdc25 are required forWlamentation under speciWc conditions. Therefore, otherSH3-domain encoding genes may also play important mor-phogenetic roles.
Establishing the genome sequence of C. albicans hasopened the way for the functional analysis of the C. albi-cans gene set as has been elegantly achieved in S. cerevi-siae (Winzeler et al. 1999). PCR-based gene targetingapproaches similar to those used in S. cerevisiae have beenestablished to generate homozygous mutant strains aftertwo successive rounds of transformation (Berman and Sud-bery 2002; Walther and Wendland 2008).
To contribute further to the functional analysis ofC. albicans genes, we have functionally analyzed theC. albicans homologs of the S. cerevisiae SH3-domainencoding genes SLA1, NBP2, and CYK3.
Materials and methods
Strains and media
The C. albicans strains used and generated in this study arelisted in Table 1. Generally, at least two independent
transformants were generated for each desired geneticmanipulation. Strains were grown either in yeast extract–peptone–dextrose (YPD; 1% yeast extract, 2% peptone,2% dextrose) or in deWned minimal media [CSM; com-plete supplement mixture; 6.7 g/l yeast nitrogen base(YNB) with ammonium sulfate and without amino acids;0.69 g/l CSM; 20 g/l glucose] with the addition ofrequired amino acids and uridine. Promoter shut down ofMET3-promoter or MAL2-promoter controlled geneexpression was done as described previously (Bauer andWendland 2007).
Strains were generally grown at 30°C to keep them inthe yeast phase; hyphal induction of C. albicans cells wasdone at 37°C with the addition of 10% serum to the growthmedium. Escherichia coli strain DH5" was used for pFA-plasmid propagation.
Transformation of C. albicans
Completely independent C. albicans homozygous com-plete ORF-deletion strains were constructed startingfrom C. albicans strain SN148 (Noble and Johnson2005). PCR-generated disruption cassettes were used totarget both alleles of a gene, which were deleted bysequential transformation of Wrst SN148 and then theresulting heterozygous strains. PCR-products for trans-formation of C. albicans were ampliWed from pFA-vec-tors (Table 2) using S1- and S2-primers as described(Walther and Wendland 2008). Primers were purchasedfrom biomers.net GmbH (Ulm, Germany). S1- and S2-prim-ers (see Table 3) harbor 100 nt of target homology at their
Table 1 C. albicans strains used in this study
Straina Genotype Source
SC5314 C. albicans wild type Gillum et al. 1984
SN148 arg4/arg4, leu2/leu2, his1/his1ura3::imm434/ura3::imm434, iro1::imm434/iro1::imm434
Noble and Johnson 2005
CAP046 NBP2/nbp2::CdHIS1, leu2, ura3, arg4 This study
CAP015 nbp2::CdHIS1/nbp2::URA3, leu2, arg4 This study
CAP191 nbp2::CdHIS1/nbp2::URA3, BUD3/bud3::NBP2-CmLEU2, arg4 This study
CAP147 CYK3/cyk3::CdHIS1, leu2, ura3, arg4 This study
CAP007 URA3-MET3p-CYK/cyk3::CdHIS1, arg4, leu2 This study
CAP054 SLA1/sla1::CdHIS1, leu2, ura3, arg4 This study
CAP024 sla1::CdHIS1/sla1::URA3, leu2, arg4 This study
CAP204 sla1::CdHIS1/sla1::URA3, BUD3/bud3::SLA1-CmLEU2, arg4 This study
CAP025 sla1::ARG4isla1::URA3, his1, leu2 This study
CAP026 sla1::ARG4/sla1::URA3, his1, leu2 This study
CAS024 SLA1/sla1::CdHIS1, leu2, ura3, arg4 This study
CAP206 URA3-MAL2p-sla1!SH3#1,2/sla1::CdHIS1, leu2, arg4 This study
CAP221 URA3-MAL2p-sla1!SH3#1,2-GFP-CmLEU2/sla1::CdHIS1, arg4 This study
CAS027 SLA1-GFP-CmLEU2/sla1::CdHIS1 This study
CAS030 CYK3-GFP-CmLEU2/cyk3::CdHIS1, ura3, arg4 This study
Cm C. maltosa, Cd C. dubliniensisa All CAxxxx strains are derivates of SN148
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5!-ends. Shorter primers were used for diagnostic PCR toverify the integration of the cassettes and absence of the tar-get gene in homozygous mutants. Transformation was doneas described (Walther and Wendland 2003).
SLA1 was also disrupted using two gene-speciWcUAU1 cassettes kindly provided by Aaron Mitchell.Transformation with the SLA1 cassettes on plasmidsCAGFY04 and CAGO130 required linearization of theplasmid using NotI and transformation of C. albicanswith a Wrst selection on ¡Arg media. Restreaking of theprimary transformants on ¡Arg and ¡Ura media wasdone to select for recombinants in which both alleleshave been disrupted (see Nobile and Mitchell 2009 forfurther details).
Reintegration of SLA1 and NBP2
Reconstitution of the !sla1 and !nbp2 strains was done byreintegration of the wild-type gene at the BUD3 locus. Tothis end, SLA1 was ampliWed using primers #3562 and#3237 and cloned into pDrive (C508). From there SLA1was cloned as an XhoI/BamHI fragment into plasmid #873to yield plasmid C553. The !sla1 strain CAP024 was trans-formed with SpeI-linearized plasmid C553. Similarly,NBP2 was ampliWed using primers #3557 and #4196 andcloned into pDrive generating plasmid C527. NBP2 wasthen cloned as a XhoI/BamHI fragment into #873 generat-
ing plasmid C530. Plasmid C530 was transformed aftercleavage with SpeI to generate strain CAP191.
Construction of SLA1-GFP and CYK3-GFP
To generate chromosomally GFP-tagged strains, the fol-lowing procedure was applied. The 3!-ends of SLA1 andCYK3 were ampliWed using primer pairs #3236/#3237 and#3222/#3223, respectively, and cloned into pDrive (plas-mids C182 and C196). The fragments were recloned intopRS417, which is based on pRS415 but carries a GEN3marker instead of LEU2. This generated plasmids C200 andC201, which were used for in vivo recombination in S.cerevisiae to add the GFP-CmLEU2 cassettes ampliWedusing the primer pairs #3314/#3315 for SLA1 and #3312/#3313 for CYK3, respectively. The resulting plasmids C256and C257 were cleaved by XhoI/BamHI and SacII/XhoI,respectively, to release the targeting cassettes used fortransformation of C. albicans. Correct fusion was veriWedby sequencing and correct integration of the cassettes wasveriWed by diagnostic PCR.
Construction of sla1!SH3#1,2-GFP
To generate a SLA1-allele which is expressed from the reg-ulatable MAL2-promoter and lacks the Wrst two SH3-domains, a PCR-based gene targeting approach was used.The URA3-MAL2p-cassette was ampliWed from a pFAvector using primers #3594 and #4265. This cassette wastransformed into strain CAS023 (SLA1/sla1::CdHIS1).This generated strain CAP206 bearing a deletion of oneSLA1 allele and converting the remaining allele tosla1!SH3#1,2. To be able to record the localization of thetruncated protein in living cells, this SLA1 allele was taggedwith GFP. To this end, the SLA1-GFP-tagging cassette wasused and CAP206 was transformed with SpeI/SacIIdigested C256 (pRS417-3!-SLA1-GFP). This resulted inthe addition of GFP to the C-terminus of sla1!SH3#1,2.
Microscopy and staining procedures
Fluorescence microscopy was done with an Axio-Imagermicroscope (Zeiss, Jena and Göttingen, Germany) usingMetamorph software tools (Molecular Devices Corp.,Downington, PA, USA) and a MicroMax1024 CCD-cam-era (Princeton Instruments, Trenton, NJ, USA). Imagingwas performed using the appropriate Wlter combinations forFM4-64-imaging, GFP-localization, and actin-staining asdescribed (Walther and Wendland 2004a, b). Quinacrinestaining was done according to (Weisman et al. 1987). Tothis end, strains were grown overnight in YPD, and thendiluted in YPD + Serum and grown for an additional 4 h.Quinacrine was added to a Wnal concentration of 200 !M.
Table 2 Plasmids used in this study
Ca C. albicans, Cm C. maltosa, Cd C. dubliniensis
Plasmid Description Source
200 pFA-URA3 Gola et al. 2003
230 pFA-URA3-MAL2p Gola et al. 2003
627 pFA-CdHIS1 Schaub et al. 2006
697 pFA-GFP-CmLEU2 Schaub et al. 2006
873 pRS-CaBUD3-CmLEU2 Wendland
C508 pDrive-SLA1 This study
C553 pRS-BUD3-SLA1-CmLEU2 This study
C527 pDrive-NBP2 This study
C530 pRS-BUD3-NBP2-CmLEU2 This study
C177 pRS417 (GEN3) This study
C196 pDRIVE-3!-CYK3 This study
C200 pRS417-3!-CYK3 This study
C257 pRS417-3!-CYK3-GFP This study
C182 pGEM-3!-SLA1 This study
C201 pRS417-3!-SLA1 This study
C256 pRS417-3!-SLA1-GFP This study
CAGFY04 SLA1-UAU1-cassette Mitchell
CAGO130 SLA1-UAU1-cassette Mitchell
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Cells were incubated at 37°C for 5 min, collected by centri-fugation and resuspended in 200 !l YPD + Serum with50 mM NaH2PO4. Visualization was done with the GFPWlter. Samples were analyzed by generating either singleimages or stacks of 5–20 images that were processed intosingle plane projections using Metamorph software.
Results
Sequence comparisons
The C. albicans Sla1, Nbp2, and Cyk3 proteins share onefeature in the possession of SH3-domains, which were
Table 3 Primers used in this study
Ca C. albicans, Cm C. maltosa, Cd C. dubliniensisa Upper case sequences correspond to C. albicans DNA sequences and lower case sequences correspond to 3!-terminal annealing regions for theampliWcation of pFA-cassettes. All sequences are written from 5! to 3!
Genes Primer names and sequencesa
CaCYK3 #4019: S1-CaCYK3: CCTTTCATTAATTACAAAGAAAAAAATAAGAACATCAACTATCTTTTCACTCTTTTT GAACAAATTTGTATCATACTAAAAGAATTAAATAATAAATAATgaagcttcgtacgctgcaggtc
CaCYK3 #3313: S2-CaCYK3: AATGTACAAATGGCAAAAAGAAGTAGTAGCAGAAGAGGTAATCTATAAAGAATTTAAAACTAAATAATACCCACTCTGTTTCCCTCTTTATATATATATAtctgatatcatcgatgaattcgag
CaCYK3 #4020: G1-CaCYK3: GCACACTTGATGATTTCATC
CaCYK3 #3222: G3-CaCYK3: GCTAAGATCAAGGCAGTG
CaCYK3 #3223: G4-CaCYK3: GCAACTGCTGCAGTAGAC
CaCYK3 #3312: S1-GFP-CaCYK3: TATGTTTTCGCTCAGTGGGAGTGCATAGGTAGCACAGTTGCAAATggtgctggcgcaggtgcttc
CaCYK3 #3539: G1-CaCYK3-GFP: GACTGCAAGGGCAACCAC
CaCYK3 #3747: G2-CaCYK3: AGGATTTAaagcttttaCCCAAGTGGGGTTGTTCCAGC
CaNBP2 #3589: S1-CaNBP2: GTCTTGTTTGTCCTGTGTGTGTGTGTGTGTGTGTTGATAAATCACCTGAAACATATACTATTTAATCATTTGTTATTCATCATTATTGTCCATTTTGAATAGgaagcttcgtacgctgcaggtc
CaNBP2 #3579: S2-CaNBP2: CACATACACTCTGTTGGTATGAAAGTATAAAAACATTTGATAAAATTCGTAATCAACATT AATATAACTTAATTGTCCCTATAAGCTGGCTAATATTGGAtctgatatcatcgatgaattcgag
CaNBP2 #3557: G1-CaNBP2: GGTGTTTCACATTATTCTCCG
CaNBP2 #3309: G4-CaNBP2: TGGCCGAACCCTTCCTGG
CaNBP2 #3245: I1-CaNBP2: GACAAGTCATTTCCCACC
CaNBP2 #3246: I2-CaNBP2: CTTCAGCAACTAACCAACCTTG
CaNBP2 #4196: A4-CaNBP2: CACATACACTCTGTTGGTATG
CaSLA1 #3594: S1-CaSLA1: CAACTCCTATGTTAGAGCTAGTCGTGCTCAACACAAAACCTGATGTGAAACAATGAA ACTTTCGACGATTCTACAAAAGTGCGGAAATTGCTTGAAATCAAAGgaagcttcgtacgctgcaggtc
CaSLA1 #3315: S2-CaSLA1: AGCATTACAAACTATGAAAGGAATAAGAAATAATGAATAATATTTTGTTTGATATACAATTA TAAAATAAAAGAGTTAATAAAGGTTCAAAATGCACTTTtctgatatcatcgatgaattcgag
CaSLA1 #3562: G1-CaSLA1: CGGTAGAGATGATGTTGTG
CaSLA1 #3765: G2-SLA1: AGGATTTAaagcttttaAGGTGGTGCAGGGAAATCCG
CaSLA1 #3236: G3-CaSLA1: TGGTGGAGCACCACAGAC
CaSLA1 #3237: G4-CaSLA1: CGGCTTTGCAACATCAAGAC
CaSLA1 #3241: I1-CaSLA1: CATAGGGATAGATCACCAG
CaSLA1 #3242: I2-CaSLA1: CTTCTCTCAAACCATGGGC
CaSLA1 #3243: I3-CaSLA1: CACAACAACAACCGCCACC
CaSLA1 #3244: I4-CaSLA1: CCATACCAGTTGGTTGTGAC
CaSLA1 #3314: S1-GFP-CaSLA1: AGAGCTAATCTACAAGCAGCAACACCAGATAATCCCTTTGGATTCggtgctggcgcaggtgcttc
CaSLA1 #4265: S2-MALp-SLA1"SH3#1-2: GAATCTGAGTCTGTTGCTGTGGAATAGCCTGCTGTTGTTGTGGTGGTGGTTGGAAAACCTGTTGTGGTTGTTGCTGTTGATGCTGTGCTGGCTCTGCTGTcattgtagttgattattagttaaaccac
CaURA3 #600: U2: GTGTTACGAATCAATGGCACTACAGC
CaURA3 #599: U3: GGAGTTGGATTAGATGATAAAGGTGATGG
CdHIS1 #1432: H2: TCTAAACTGTATATCGGCACCGCTC
CdHIS1 #1433: H3: GCTGGCGCAACAGATATATTGGTGC
CmLEU2 #1743: L3: GCTGAAGCTTTAGAAGAAGCCGTG
CaMAL2 #4269: G3-CaMALp: GTACAACTAAACTGGGTGATG
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identiWed using the SMART tool at http://smart.embl.de/.An SH3-domain is composed of app. 70 amino acids andseveral conserved residues can be found (Fig. 1a). Theposition of a SH3-domain within a protein may vary andthere are also proteins like Sla1 that contain more than oneSH3-domain (Fig. 1b). Amino acid sequence identitiesbetween the C. albicans and, for example, Saccharomycescerevisiae proteins are overall not very high and rangebetween 27 and 37%. The C. albicans proteins are largerthan the S. cerevisiae homologs: Sla1 by only 13aa, Cyk3by 135aa, and Nbp2 by one-third (342aa compared to236aa). The characterizations of the yeast genes revealedthat SLA1 plays a role in actin cytoskeleton assembly andendocytosis; Nbp2 is required for mitotic growth at hightemperatures and for cell wall integrity and Cyk3 isinvolved in cytokinesis (Korinek et al. 2000; Warren et al.2002; Ohkuni et al. 2003).
Generation of C. albicans mutant strains
In this report, we have employed several strategies forgene function analysis relying on diVerent pFA-vectorsand also used a single transformation approach relyingon UAU1-cassettes as described below. Initially, we usedthe pFA-series to generate complete ORF-deletion strainsin the three genes. From two heterozygous mutantstrains, we went onto obtain two independent homozy-gous mutant strains thereof using the C. albicans URA3and the Candida dubliniensis HIS1 marker genes. In
order to characterize SLA1 in more detail, we used inser-tional disruption cassettes based on SLA1-UAU1-cas-settes. Since deletion of CYK3 was not feasible, we useda promoter shutdown approach to analyze the conse-quences of Cyk3 depletion. Mutant phenotypes could beobtained with the homozygous mutants of sla1 and nbp2,which in both cases could be complemented by the rein-tegration of the wild-type gene at the BUD3 locus. Local-ization of Sla1 and Cyk3 was done by Xuorescencemicroscopy of GFP-tagged strains. Using this array oftools, we were able to achieve an initial characterizationof gene function for these genes, which will be describedin the following sections.
Deletion of SLA1 and NBP2 results in hyphal growth phenotypes
Homozygous mutant strains of sla1 and nbp2 were charac-terized to reveal their growth potential under diVerentgrowth conditions. When grown on minimal media, nostrong defects during yeast growth were observable.Hyphal growth was monitored by inducing yeast cellseither on solid media or in liquid culture (Fig. 2). The wildtype strongly Wlaments after addition of serum or in spidermedium. Mutants in sla1 or nbp2 were also able to induceWlament formation. Hyphae of these mutant strains, how-ever, were shorter than the wild type after several hours ofinduction. Interestingly, the nbp2 mutant showed frequentreinitiating of germ tube formation from the germ cell.
Fig. 1 Alignment and position of SH3-domains. a The Wve SH3-do-mains of the three genes that were functionally analyzed in this studywere aligned using the MegAlign tool of the DNASTAR software (ht-tp://www.dnastar.com). Consensus sites are shaded in gray while iden-tical sites in all Wve domains are shaded in black. b The positions of theSH3-domain vary within the proteins and are for Nbp2 at amino acids
127–184, for Cyk3 at position 11–68 and for Sla1 at positions 7–73,76–133, and 399–457. For reference, the complete protein length isdrawn to scale. No other domains were found using the SMART toolat (http://smart.embl.de/) also Sla1 has several repeats at its C-termi-nus. Deletion of the N-terminal SH3-domains results in a sla1 alleletermed Sla1!SH3#1,2
A
Cyk3
Nbp2B
CAP026CAP025
Sla1
Sla1 SH3#1-2 MAL2-prom
SH3-domain
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Thus, after 3 h, most of the germ cells had formed two orthree germ tubes (Fig. 2).
SLA1-deletion leads to defects in actin patch assembly and distribution
The actin cytoskeleton plays a major role in polarizedgrowth. During hyphal growth, actin cables in C. albicansare formed by the tip-localized polarisome and actinpatches localize to the hyphal tip at sites of endocytosis (forreview see Pruyne and Bretscher 2000a, b). To analyze thegrowth defects of sla1 in detail, we used Xuorescencemicroscopy of rhodamine stained germ tubes. We analyzedthree diVerent sla1 mutant strains: two bearing UAU1 cas-settes, CAP025 and CAP026, and a complete ORF-deletionstrain. In this way, we could analyze the eVect of truncatingSLA1 at positions that potentially leave all three SH3-domainsintact, truncate the protein after the Wrst two SH3-domainsor entirely delete SLA1 (see also Fig. 1). The two UAU1insertions in SLA1 already showed diVerent phenotypes.The CAP026 strain basically showed no defect, grew likewild type and accumulated actin patches in the hyphal tips.The CAP025 strain in which the UAU1 insertion truncatesSLA1 downstream of the region coding for the second SH3-domain, shows decreased hyphal lengths when comparedwith the wild type. The number of actin patches in thismutant is decreased and the patches do not accumulate inthe tips of hyphae (Fig. 3a). This phenotype is even morepronounced in the complete ORF-deletion strain. Thisstrain has a more drastic growth defect and even fewer cor-tical actin patches. This demonstrates that Sla1 is involvedin the assembly and polarization of cortical actin patches inC. albicans. The assembly of actin cables in the hyphal Wla-ments was not impaired (Fig. 3b). The growth defect of thenull mutant could be complemented by reintegration of thewild-type SLA1 gene at the BUD3 locus. The assembly ofcortical actin patches and their polarized localization wasalso restored in both yeast and hyphal cells (Fig. 3b).
To visualize the localization of Sla1 in vivo, a chromo-somally tagged SLA1-GFP strain was constructed based ona heterozygous mutant. Sla1 shows a patch-like localizationin yeast cells and hyphae (Fig. 3b). An increased number ofSla1-GFP patches can be found at sites of polarized growth,
Fig. 2 Characterization of growth phenotypes of the sla1 and nbp2mutants. The strains were grown overnight in liquid culture and theninoculated on minimal medium (a), on serum containing plates or inliquid medium supplemented with 10% serum (b), or on spider platesand in spider liquid medium (c). Plates were incubated for 3 days at
30°C (a) or 37°C (b, c) prior to photography. Hyphal induction in liq-uid media was done for 3 h prior to microscopy. Bar 10 !m. Note theshort germ tube in the sla1 strain (middle row) and the multiple germtubes in the nbp2 mutant (bottom row)
sla1
nbp2
A B C
WT
Fig. 3 Deletion of SLA1 leads to defects in cortical actin patch assem-bly. Strains were grown overnight in YPD, diluted in new medium andgrown for 4 h at 30°C for yeast cell growth or at 37°C in the presenceof 10% serum to induce Wlament formation. Cells were then Wxed andstained with rhodamine-phalloidin. Bright-Weld and Xuorescence im-ages showing the actin cytoskeleton of the indicated strains are shown.a DiVerential eVect on cortical actin patch assembly of SLA1-UAU1insertions compared to the wild type. b Reintegration of SLA1 comple-ments the actin patch defect of the sla1 complete ORF-deletion mutant.In vivo localization of Sla1-GFP was done without Wxation. Bars 5 !m
SC5314 sla1::UAU1-CAP025 sla1::UAU1-CAP026 A
B SC5314 sla1 sla1,
BUD3/bud3::SLA1 SLA1-GFP/sla1
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e.g. the hyphal tip. This localization pattern resembles, forexample, that of C. albicans Abp1 and other proteinsinvolved in actin patch assembly or endocytosis (Martinet al. 2007).
The N-terminal SH3-domains of Sla1 are important for actin cytoskeleton assembly but not for localization of Sla1
To demonstrate that the two N-terminally located SH3-domains of Sla1 contribute to the function of Sla1, we gen-erated a truncated allele, sla1!SH3#1,2. This allele was placedunder control of the regulatable MAL2-promoter using aPCR-based gene targeting approach, which at the sametime eliminated the Wrst two SH3-domains. Furthermore, tobe able to localize the truncated protein, a C-terminal tagwas added to sla1!SH3#1,2. This strain was then used to visu-alize both the localization of Sla1!SH3#1,2-GFP and the orga-nization of actin cytoskeleton in yeast and hyphal cells(Fig. 4). When grown in glucose, sla1!SH3#1,2 expressionwas turned down and the protein could not be detected.Actin organization resembled that of a sla1 mutant strain(compare Figs. 3, 4). Growth in maltose medium inducedthe expression of sla1!SH3#1,2-GFP. Hence Sla1!SH3#1,2-GFP could be detected as cortical patches in yeast andhyphal cells. Sla1!SH3#1,2-GFP was also found enriched inthe hyphal tips. Thus, Sla1!SH3#1,2-GFP localizes in a similarmanner as full length Sla1-GFP indicating that the N-terminalSH3 domains do not play a role in Sla1-targeting to thecortex. However, under inducing conditions the actin cyto-skeleton assembly in the strain expressing Sla1!SH3#1,2-GFPwas still aberrant. Cortical actin patches that did not
localize to the hyphal tip were reduced in number and thusresembled the situation in the sla1 deletion strain. Thisindicates that the N-terminal SH3 domains of Sla1 do playan important role in organization of the actin cytoskeletonat sites of endocytosis (Fig. 4).
NBP2-deletion leads to multiple germ tube formation
Deletion of NBP2 did not reveal any defects during theyeast growth phase. Yet, under hyphal inducing conditions,we observed that all germ cells developed multiple germtubes after 4 h and the length of the primary germ tube wasdecreased in nbp2 compared to that of the wild type(Figs. 2, 5). Previously, it was shown in C. albicans andAshbya gossypii that in hyphae large vacuoles are formed insubapical compartments (Walther and Wendland 2004a;Veses and Gow 2008). This inXuences the ratio of cyto-plasm versus vacuole and inXuences the branching fre-quency (Veses et al. 2009). Therefore, we analyzed thevacuolar compartments in FM4-64 stained yeast cells andhyphae of the wild type and the nbp2 mutant (Fig. 5). Dur-ing yeast growth, both strains accumulated a larger vacuolein mother cells and showed no observable diVerence. How-ever, staining of germlings revealed the inability of nbp2germ cells to generate large vacuolar compartments. Frag-mented vacuoles were found throughout nbp2 hyphae.Thus, the altered ratio of cytoplasm versus vacuolar spacemay be the causal link to increased branching of germ cellsin the nbp2 mutant. To corroborate that the defect in vacuo-lar fusion was speciWc for the nbp2 deletion strain, we rein-tegrated the NBP2 gene at the BUD3-locus. As expected,
Fig. 4 The two amino terminal SH3-domains of Sla1 are re-quired for the organization of the actin cytoskeleton. CAP221 was grown overnight in YPD for a full SLA1 shut-down. Then cells were diluted in either YPD (re-pressed) or YPM (induced) with or without serum and grown for 4 h. Afterwards, cells were Wxed and stained with rhodamine-phalloidin prior to GFP and actin Xuorescence microscopy. Bar 5 !m
DIC DIC DIC DICGFP actin GFP actin
MAL2p-sla1 SH3#1,2-GFP repressed MAL2p-sla1 SH3#1,2-GFP induced
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the reintegrant showed wild type vacuolar phenotype. Wealso generated a chromosomally encoded NBP2-GFP,which, however, did not yield a Xuorescent signal. To ana-lyze vacuolar acidiWcation in the nbp2 strain, we used quin-acrine staining and Xuorescence microscopy (Fig. 6).Quinacrine diVuses through membranes and accumulates inacidic compartments like the vacuole (Weisman et al.1987). The accumulation of the dye and staining of vacu-oles of nbp2 hyphae indicated that vacuolar fusion but notthe function of the vacuoles was aVected in the nbp2 strain(Fig. 6, 7).
Depletion of Cyk3 results in cytokinesis defects
We were unable to generate homozygous cyk3 strains frominitial heterozygous mutants, without also generating sometriplication event that left a wild-type copy of the gene inthe genome. Thus, we conclude that CYK3 is an essentialgene. In S. cerevisiae, CYK3 is involved in cytokinesis andlocalizes to the bud neck in large budded cells (Korineket al. 2000). Cyk3 localization in C. albicans was deter-mined by producing a fusion between the chromosomalCYK3 gene with GFP in a heterozygous mutant. In C. albi-cans, CYK3 was found to localize at the bud neck in largebudded cells similar to Cyk3 in S. cerevisiae (Fig. 5a).
In S. cerevisiae, deletion of CYK3 results in only mildcytokinesis defects, which contrasts the situation inC. albicans. To assess a phenotype upon depletion of CYK3transcript, we produced a strain which expressed CYK3 from
the regulatable C. albicans MET3 promoter (Care et al.1999). Shutdown of CYK3 expression resulted in a severecytokinesis defect. CYK3-depleted cells were elongated ormisshapen and showed abnormal chitin deposition (Fig. 5b).
Discussion
In this study, we have generated C. albicans mutant strainsfor three SH3-domain encoding genes using PCR-basedgene targeting methodologies and a single-step transforma-tion protocol with UAU1 cassettes (Walther and Wendland2008; Nobile and Mitchell 2009). SH3-domains are smallprotein domains that promote protein–protein interactions,particularly by binding to proline-rich ligands with a PxxPmotif (Mayer 2001). The binding aYnity and binding spec-iWcity are inherently rather low. This may pose some diY-culties when trying to establish protein interactions usingthe yeast two-hybrid system. Sla1, on the other hand, con-tains three SH3 domains, which may help to increase spe-ciWc binding of target proteins.
Given the strong potential of SH3-domains to promotesignaling and morphogenesis, a large variety of SH3-domain
Fig. 5 Deletion of NBP2 results in vacuolar fragmentation in hyphae.Strains were grown under yeast or germ tube inducing conditions for4 h. FM4-64 (0.2 !g/ml) was added and samples were processed forbrightWeld and Xuorescence microscopy after 1 h to allow uptake of thedye. Bars 5 !m
SC5314 nbp2nbp2,
BUD3/bud3::NBP2
Fig. 6 Vacuoles of an nbp2 mutant strain are acidic. Strains weregrown under yeast or germ tube inducing conditions for 4 h. Quina-crine staining reveals acidiWed and functional vacuoles
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proteins can be found in eukaryotic genomes ranging from20–30 in yeast-like ascomycetes to over 300 in humans(Karkkainen et al. 2006). In yeast-like ascomycetes, thereseems to be limited evolution of SH3-domain encodinggenes. For example, in S. cerevisiae, Abp1 contains oneSH3-domain, while in C. albicans, the Abp1 homolog hastwo adjacent SH3-domains, yet deletion of CaABP1showed no discernible phenotype (Martin et al. 2007).
Deletion of the C. albicans SLA1 resulted in similar actinassembly defects compared to a SLA1 deletion in S. cerevi-siae. The severe reduction in actin patches, however, didnot abolish the ability to generate germ tubes in the C. albi-cans sla1 mutants although a decreased polarized growthrate could be observed. A similar phenotype was observedin a Camyo5(S366D) allele, which mimics a phosphory-lated serine. A strain bearing this allele was found to Wla-ment, yet shows a largely delocalized actin cytoskeleton(Oberholzer et al. 2002). In this paper, we identiWed theN-terminal region of C. albicans Sla1 containing two SH3-domains to be required for correct organization of the actincytoskeleton. In S. cerevisiae, Sla1 localizes to the cortexvia an interaction of the Sla1 C-terminal repeat region withEnd3 (Tang et al. 2000; Warren et al. 2002). Furthermore,Sla1 interacts with Las17 and Abp1 as shown by immuno-precipitation (Warren et al. 2002). The elimination of twoSH3 domains from Sla1 resulted in profound disorganizationof the actin cytoskeleton indentifying Sla1 as a major
player linking early events of endocytosis with the actincytoskeleton. Nevertheless, sla1 mutants were able to gen-erate, albeit short, hyphae.
Surprisingly, sla1 did not show a defect in the formationof large subapical vacuoles (see also Fig. 2). This, on theother hand, was observed for the nbp2 mutant. In S. cerevi-siae, nbp2 mutants are temperature sensitive and also sensi-tive to cell wall stress (Ohkuni et al. 2003). Our C. albicansnbp2 mutants were not temperature sensitive and grew wellat 40°C even with the addition of 1 M sorbitol or 1.5 MNaCl (data not shown). The transformation frequency,which requires a heat shock, was also not aVected in nbp2cells. Thus, our results indicate some novel vacuolar func-tions for NBP2 which are more pronounced during hyphalgrowth stages and not apparent in yeast cells. Germ tubeformation in the nbp2 strain was altered in a way that germcells quickly generated multiple hyphae rather than onedominant germ tube as in the wild type. Thus, such a phe-notype could be useful in larger scale screenings of aC. albicans mutant collection once available.
SH3-domain proteins in C. albicans are taking part in avariety of processes. In this study, we identiWed a key roleof the Wrst two Sla1 SH3-domains for the polarized assem-bly of the actin cytoskeleton, which had not previouslybeen identiWed in other studies. We also revealed theinvolvement of Nbp2 in vacuolar fusion, and of Cyk3 incytokinesis. The promoter shutdown experiment using
Fig. 7 CYK3 localization and depletion after promoter shut-down. a Cyk3-GFP Xuorescence in large budded yeast cells was observed at the bud neck. b Cells in which CYK3 expression is controlled by the regulatable MET3-promoter were grown overnight in YPD at 30°C with (repressed) or without (induced) the addition of 3.5 mM methio-nine and cysteine. Prior to microscopy, calcoXuor was add-ed to the medium to stain chitin rich regions. Bar 10 !m
A
BMET3p-CYK3
inducedMET3p-CYK3
repressed
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MET-promoter controlled CYK3 did not result in growtharrest. This may be due to the leakiness of the promoter.However, cells were found to be deWcient in cell separationproviding evidence that also in C. albicans Cyk3 isinvolved in this process. Our GFP-localization data ofCyk3-GFP provide further evidence for that. Similar toS. cerevisiae, C. albicans Cyk3 may, therefore, act at thelevel of actin ring formation or constriction.
Due to the diploidy of C. albicans, gene function analy-ses still require much more eVort to produce the correctdeletion strains. Using PCR-based gene targeting methods,detailed structure–function analyses are possible andreduce the time required to construct the desired strains.Thus, larger scale approaches can be undertaken also inC. albicans (Noble and Johnson 2005). Our study of threepreviously uncharacterized C. albicans genes, therefore,adds to the repository of functional analysis information forthis human fungal pathogen.
Acknowledgments We thank Alexander Johnson, Suzanne Noble,and Aaron Mitchell for generously providing reagents used in thisstudy; Sidsel Ehlers for providing technical assistance and AndreaWalther for support on microscopy. This study was funded by the EU-Marie Curie Research Training Network “Penelope” and we thankmembers of this consortium for discussions.
References
am Busch MS, Mignon D, Simonson T (2009) Computational proteindesign as a tool for fold recognition. Proteins 77:139–158
Barelle CJ, Richard ML, Gaillardin C, Gow NA, Brown AJ (2006)Candida albicans VAC8 is required for vacuolar inheritance andnormal hyphal branching. Eukaryot Cell 5:359–367
Bassilana M, Blyth J, Arkowitz RA (2003) Cdc24, the GDP-GTP ex-change factor for Cdc42, is required for invasive hyphal growthof Candida albicans. Eukaryot Cell 2:9–18
Bauer J, Wendland J (2007) Candida albicans SX1 suppresses Xoccu-lation and Wlamentation. Eukaryot Cell 6:1736–1744
Berman J, Sudbery PE (2002) Candida albicans: a molecular revolutionbuilt on lessons from budding yeast. Nat Rev Genet 3:918–930
Calderone RA, Fonzi WA (2001) Virulence factors of Candida albi-cans. Trends Microbiol 9:327–335
Care RS, Trevethick J, Binley KM, Sudbery PE (1999) The MET3 pro-moter: a new tool for Candida albicans molecular genetics. MolMicrobiol 34:792–798
Court H, Sudbery P (2007) Regulation of Cdc42 GTPase activity in theformation of hyphae in Candida albicans. Mol Biol Cell 18:265–281
Enloe B, Diamond A, Mitchell AP (2000) A single-transformationgene function test in diploid Candida albicans. J Bacteriol182:5730–5736
Epp JA, Chant J (1997) An IQGAP-related protein controls actin-ringformation and cytokinesis in yeast. Curr Biol 7:921–929
Gillum AM, Tsay EY, Kirsch DR (1984) Isolation of the Candida albi-cans gene for orotidine-5!-phosphate decarboxylase by comple-mentation of S. cerevisiae ura3 and E. coli pyrF mutations. MolGen Genet 198:179–182
Gola S, Martin R, Walther A, Dunkler A, Wendland J (2003) Newmodules for PCR-based gene targeting in Candida albicans: rapid
and eYcient gene targeting using 100 bp of Xanking homology re-gion. Yeast 20:1339–1347
Karkkainen S, Hiipakka M, Wang JH, Kleino I, Vaha-Jaakkola M,Renkema GH, Liss M, Wagner R, Saksela K (2006) IdentiWcationof preferred protein interactions by phage-display of the humanSrc homology-3 proteome. EMBO Rep 7:186–191
Korinek WS, Bi E, Epp JA, Wang L, Ho J, Chant J (2000) Cyk3, a nov-el SH3-domain protein, aVects cytokinesis in yeast. Curr Biol10:947–950
Kumamoto CA, Vinces MD (2005) Contributions of hyphae and hy-pha-co-regulated genes to Candida albicans virulence. CellMicrobiol 7:1546–1554
Martin R, Hellwig D, Schaub Y, Bauer J, Walther A, Wendland J(2007) Functional analysis of Candida albicans genes whose Sac-charomyces cerevisiae homologues are involved in endocytosis.Yeast 24:511–522
Mayer BJ (2001) SH3 domains: complexity in moderation. J Cell Sci114:1253–1263
Michel S, Ushinsky S, Klebl B, Leberer E, Thomas D, Whiteway M,Morschhauser J (2002) Generation of conditional lethal Candidaalbicans mutants by inducible deletion of essential genes. MolMicrobiol 46:269–280
Nobile CJ, Mitchell AP (2009) Large-scale gene disruption using theUAU1 cassette. Methods Mol Biol 499:175–194
Noble SM, Johnson AD (2005) Strains and strategies for large-scalegene deletion studies of the diploid human fungal pathogen Can-dida albicans. Eukaryot Cell 4:298–309
Oberholzer U, Marcil A, Leberer E, Thomas DY, Whiteway M (2002)Myosin I is required for hypha formation in Candida albicans.Eukaryot Cell 1:213–228
Odds FC (1994) Pathogenesis of Candida infections. J Am Acad Der-matol 31:S2–S5
Ohkuni K, Okuda A, Kikuchi A (2003) Yeast Nap1-binding proteinNbp2p is required for mitotic growth at high temperatures and forcell wall integrity. Genetics 165:517–529
Pruyne D, Bretscher A (2000a) Polarization of cell growth in yeast.J Cell Sci 113:571–585
Pruyne D, Bretscher A (2000b) Polarization of cell growth in yeast. I.Establishment and maintenance of polarity states. J Cell Sci113:365–375
Schaub Y, Dunkler A, Walther A, Wendland J (2006) New pFA-cas-settes for PCR-based gene manipulation in Candida albicans.J Basic Microbiol 46:416–429
Sudbery P, Gow N, Berman J (2004) The distinct morphogenic statesof Candida albicans. Trends Microbiol 12:317–324
Tang HY, Xu J, Cai M (2000) Pan1p, End3p, and S1a1p, three yeastproteins required for normal cortical actin cytoskeleton organiza-tion, associate with each other and play essential roles in cell wallmorphogenesis. Mol Cell Biol 20:12–25
Veses V, Gow NA (2008) Vacuolar dynamics during the morphoge-netic transition in Candida albicans. FEMS Yeast Res 8:1339–1348
Veses V, Richards A, Gow NA (2009) Vacuole inheritance regulatescell size and branching frequency of Candida albicans hyphae.Mol Microbiol 71:505–519
Walther A, Wendland J (2003) An improved transformation protocolfor the human fungal pathogen Candida albicans. Curr Genet42:339–343
Walther A, Wendland J (2004a) Apical localization of actin patchesand vacuolar dynamics in Ashbya gossypii depend on the WASPhomolog Wal1p. J Cell Sci 117:4947–4958
Walther A, Wendland J (2004b) Polarized hyphal growth in Candidaalbicans requires the Wiskott–Aldrich Syndrome protein homo-log Wal1p. Eukaryot Cell 3:471–482
Walther A, Wendland J (2008) PCR-based gene targeting in Candidaalbicans. Nat Protoc 3:1414–1421
Curr Genet
123
Warren DT, Andrews PD, Gourlay CW, Ayscough KR (2002) Sla1pcouples the yeast endocytic machinery to proteins regulating actindynamics. J Cell Sci 115:1703–1715
Weisman LS, Bacallao R, Wickner W (1987) Multiple methods ofvisualizing the yeast vacuole permit evaluation of its morphologyand inheritance during the cell cycle. J Cell Biol 105:1539–1547
Wendland J (2001) Comparison of morphogenetic networks of Wla-mentous fungi and yeast. Fungal Genet Biol 34:63–82
Wendland J, Philippsen P (2002) An IQGAP-related protein, encodedby AgCYK1, is required for septation in the Wlamentous fungusAshbya gossypii. Fungal Genet Biol 37:81–88
Winzeler EA, Shoemaker DD, AstromoV A, Liang H, Anderson K,Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM,
Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M,Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, JonesT, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A,Lussier M, M’Rabet N, Menard P, Mittmann M, Pai C,Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P,Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, VeronneauS, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K,Zimmermann K, Philippsen P, Johnston M, Davis RW (1999)Functional characterization of the S. cerevisiae genome by genedeletion and parallel analysis. Science 285:901–906
Zheng XD, Lee RT, Wang YM, Lin QS, Wang Y (2007) Phosphoryla-tion of Rga2, a Cdc42 GAP, by CDK/Hgc1 is crucial for Candidaalbicans hyphal growth. EMBO J 26:3760–3769