PCDU – Lecture
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Plant Cell, Development & Ultrastructure
Plant Cell Biology Labs
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Myosins V are Related to Plant VIII & XI Myosins
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Heptad-Repeat: a b c d e f g h - a b c d e f g h - a b c d e f g h
Hydrophobic amino acids at positions „a“ and „d“: I, L, V, M, F
DIL: dilute domain, cargo binding C-terminus. dilute was first discovered as a mutation in mice causing color dilution of the fur.
The affected gene codes for a myosin V which moves melanosomes from the cell center to the periphery in skin melanocytes.
Plant Myosins
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Plant Myosins
Nakano et al. 2012
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Plant Myosins of Class XI
Gallus gallina
Homo sapiens
Saccharomyces cerevisiae
Nicotiana tabacum
Chara corallina
Nakano et al. 2012
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Regulation of Myosin XI
Nakano et al. 2012
no Cargo no Cargo
Calmodulin dissociation
Myosin Cross-Bridge Cycle izmb.uni-bonn.de izmb.uni-bonn.de
H5C2
N-Ethylmaleimide (NEM)
C6H7NO2 Mr 125.1
NEM BDM
2,3-Butanedione Monoxime (BDM)
Sulfhydryl alkylalting agent, blocks
SH-groups, prevents disulfide bridges
Synthetic inhibitor against
myosin ATPase activity in
the head domain of myosin
head swings forward
power stroke
Inhibition of Myosin Cross-Bridge Cycle izmb.uni-bonn.de izmb.uni-bonn.de
BDM
„Rigor“
NEM
BDM Inhibits ATP-Hydrolysis NEM Inhibits Release of Head
HMM/S1-Decoration of Actin Filaments izmb.uni-bonn.de izmb.uni-bonn.de
S1
HMM
*
*
In the Absence of ATP, Myosin is Locked on F-Actin Resulting in the Arrow Head Pattern
HMM - Heavy Meromyosin
Phylogenetic Tree of Myosins
17 classes
35 classes curently recognized
Anireddy et al. 2001
Plasma Membrane
Plasmodesmata
Cortical Cytoplasm
Organellar Motors
Domain Structure of Plant Myosins izmb.uni-bonn.de izmb.uni-bonn.de
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Arabidopsis Myosin XI family
Nakano et al. 2012
13 Members in the Myosin XI Family
(16)
(6 / MYA2)
(5 / MYA1)
(8)
(15)
(17)
Cargo of Plant Class XI Myosins izmb.uni-bonn.de izmb.uni-bonn.de
Reisen & Hanson 2007
*
MYA2: peroxisomes XI-B: unknown organelle
XI-K: peroxisomes +? XI-J: ? XI-I: ? + nucleus
MYA1: peroxisomes
Cargo of Plant Class XI Myosins izmb.uni-bonn.de izmb.uni-bonn.de
YFP-Myosin16-tail overlay
Myosin XI-J (16) co-localizes with mitochondria
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Plant Myosin-Dependent Processes
BDM, 5 mM, 3h Control
Shape and motility of the ER depend on functionally active myosin and
structurally intact actin filament system
Helling & Menzel 2001
Lat B, 10 µM, 1.5h
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Plant Myosin-Dependent Processes
BDM, 5 mM, 3h Control
Shape and motility of the ER depend on functionally active myosin and
structurally intact actin filament system
Helling & Menzel 2001
Lat B, 10 µM, 1.5h
Ueda et al., 2010
triple knock-out
XI-K, XI-1, XI-2
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Arabidopsis Myosin XI family
The DIL-domain binds mostly to peroxisomes, but also to Golgi, mitochondria,
nuclear envelope, plasma membrane, plastids and unidentified vesicles.
DIL: dilute domain, cargo binding C-terminus. dilute was first discovered as a mutation in mice causing color dilution of the fur.
The affected gene codes for a myosin V which moves melanosomes from the cell center to the periphery in skin melanocytes.
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Arabidopsis Myosin XI family
The PAL-domain binds to Golgi, mitochondria, nuclear envelope, peroxisomes
ER, plasma membrane and plastids
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Arabidopsis Myosins XI – Cargo Recognition
Arabidopsis Mya1 yeast Myo2
Redundancy of cargo recognition
Peroxisome XI-F
Golgi XI-1, XI-K, XI-I, XI-H, XI-C, XI-E
Chloroplast (stromules) XI-F
Mitochondria XI-J, XI-K, XI-G, XI-C, XI-E
Nuclear envelope XI-1, XI-I, XI-G, XI-F, XI-D, XI-B
N
C
N
C
µm
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Myosin XI-K is Involved in Tip Growth
Growth of root hairs is reduced in
myosin XI-K knock out mutant
Park et al., 2013
Over-expression of myosin XI-K GTD
inhibits root hair growth
µm
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Correct localization of myosin XI-K
depends on the integrity of the
actin cytoskeleton
Park et al., 2013
Myosin XI-K is Involved in Tip Growth
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Cargo of Plant Class XI Myosins
MyoB links myosin XI-K to the membrane of target organelles
metal-binding
domain transmembrane
domain
myosin-binding
domain
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Plant Myosins of Class VIII
CAT-GFP
CAT-GFP
Head Tail
Neck
4/6 IQ Motifs
CC Whole Molecule
GFP : : Tail Domain
Head Domain : : GFP
putative
targeting site GESGAGKT
CC
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Plant Myosins
Head-GFP GFP-Tail
bean leaf epidermis cell Helling & Menzel 2003
Transient Expression of ATM1-GFP Fusion Proteins
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Plant Myosins of Class VIII
Transient expression of GFP-ATM1-tail
in onion bulb epidermis cell after gold particle bomardment
Helling, Menzel 2003
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Plant Myosins VIII
Arabidopsis root Helling, Beck & Menzel 2003
ATM1-GFP accumulates at a lesion afflicted by a micro-
needle. Callose counterstained with aniline blue
ATM1 Callose
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Plant Myosins VIII
Immunofluorescence co-localization of
GFP::ATM1-tail expression in roots of
transgenic A. thaliana
Helling, Menzel 2003
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Plant Myosins VIII
Co-localization of Callose with GFP::ATM1-tail in
stably transformed tobacco BY2 cells
ATM1-tail Callose
Class VIII Myosins
Co-localization with
Callose in/at Plasmodesmata
of Aspen tree pith parenchyma
Plant Myosins VIII at Plasmodesmata izmb.uni-bonn.de izmb.uni-bonn.de
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ATM1
Sadot et al. 2008
ATM1 involved in endocytosis ATM1 associated with pit fields/plasmodesmata
Plant Myosins VIII at Plasmodesmata
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Plant Myosins VIII
GFP: ATM 2 tail: Transient expression
in onion bulb epidermis cells
DIC
GFP:ATM2
GFP:VIIIb
Diana Helling
D. Menzel 2003
BrdU-Cy3, control BrdU-Cy3,
Myosin siRNA knock down
Philimonenko et al. 2004
nucleoli in HeLa-cells ca. 30% reduction
of rDNA-transcription
BrdU= Brom desoxyuridine
thymidine/uridine analogon
indicator of DNA replication or transcription
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Plant Myosins of Class VIII
Myosin VIII - Endocytic Motor in Plants
Helling, Menzel 2003
Roles of Plant Formins in Cell Polarity izmb.uni-bonn.de izmb.uni-bonn.de
polar localization of plant formins
Baluska & Hlavacka 2005
mouse monoclonal
A. thaliana,
root transition zone
GFP-ABD2 10µm 20µm
Zea mays,
root transition zone