the miracle of morphogenesis cell adhesion, polarity and ... · formation of the drosophila...

The Miracle of Morphogenesis

Cell Adhesion, Polarity and CytoskeletalRegulation

Prof. Mark Peifer

The screen versions of these slides have full details of copyright and acknowledgements 1

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Cell Adhesion, Polarity and Cytoskeletal Regulation

Prof. Mark PeiferDepartment of Biology and Lineberger

Comprehensive Cancer Center

University of North Carolina at Chapel Hill

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I love

developmental

biology!

How did

my cells

self-assemble

Into tissues

and organs?

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We need to understand this process at all levelsfrom the whole animal to the molecular level

Molecular Cell Biology, 4thEd. Lodish et al. Fig. 1-1

1 cm

Dead skin cells

Epidermal cells

Basal laminaLoose connective tissue20 µm

Desmosome

Hemidesmosome

1 µm

Multiadhesive protein

Cell-surface receptors

Cytoskeletal proteins

Cell-cell adhesion protein

Intracellular attachment protein

5 nm



Prof. Mark Peifer


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Disassociate embryonic retina into single cells

Disassociate embryonic liver into single cells

Different cell types have specific cell surface adhesion molecules

Mix well

W ait awhile

The cells sort out (almost) perfectly

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Cell adhesion: a critical part of the molecular toolkit to build an animal

Peifer’s

Cell Glue

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The simplest and most commontissue organization is the epithelial sheet

Adherens junction Apical cell surface

Basal Lamina or extracellular matrix

Basolateral cell surfaces

Apical cell surface



Prof. Mark Peifer


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Adherens junctions mediate cell adhesionand allow cells to coordinate their actions

by coordinating their actin cytoskeletons

Adherens

junctions

Actin

8Plasma membrane

Intracellular

ExtracellularIntracellular

Plasma membrane

Armadilloαααα -catenin

F-actin

DE-cadherin

The classic view suggests that the cadherin-catenin complex glues

cells together and also connects their actin cytoskeletons

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We study the process in the mighty fruit fly



Prof. Mark Peifer


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In Drosophila cell adhesion becomes essential as soon as cells form and the embryo begins

the process of gastrulation

Wild-type embryo Mutant lacking

adherens junctions

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Cell adhesion critically important to maintain tissue integrity

Moesin-GFP revealing f-actin

Gastrulation illustrates the amazing process

of morphogenesis in which cell shape changes

and cell migration reshape the body plan

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In the absence of adherens junctionsepithelia fall apart as gastrulation begins



Prof. Mark Peifer


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Cadherin expression is lost or adhesion is otherwise downregulated when epithelial tumors metastasize

Molecular Biology of the Cell, 4th Ed. Alberts et al. Fig. 23-15

Benign tumor in epithelium

Basal lamina

Connective tissue

Break through basal lamina

Invade capillary

Capillary Travel through bloodstream (less than 1 in 1000 cells

will survive to form metastases)

Adhere to capillary wall in liver Escape from capillary (extravasation)

Proliferate to form metastasis in liver

Cadherin expression is lost or adhesion is otherwise downregulated when epithelial tumors metastasize

14Plasma membrane

Intracellular

Extracellular

DE-cadherin

Armadilloαααα -catenin

F-actin

The classic view of adhesion provides a great way to assemble a “stuffed animal”

Intracellular

Plasma membrane

15Developmental Biology Fig. 12.3 Gilbert 7th ed.

In embryos, however, cells are not simply glued together but instead do amazing things

Chick neural tube

In embryos, however, cells are not simply glued together but instead do amazing things



Prof. Mark Peifer


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Red blood

cells

Hyperprolif ertiv e

lymphoid stem

cells

By asking questions about normal cell behavior, we also gain insights into the underlying causes

of human disease

The Biology of Cancer, Fig. 2.8D, Robert Weinberg, Garland Science

Inappropriate activation of Abl kinase plays a key role in human leukemia, by altering cell adhesion

and migration as well as survival and proliferation

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Abelson tyrosine kinase

• A non-receptor tyrosine kinase

• Drosophila abl mutants have CNS defects

SH3

F G

Src-like N-terminus Actin-binding C-terminus

SH2 KinaseAbelson tyrosine kinase

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Abl modulates axon pathfinding by transmitting signals from cell surface receptors

to the actin cytoskeleton

Dlar

Abl

Actin assembly

Ena

Profilin



Prof. Mark Peifer


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We hypothesized that Abl might play a similar role in epithelial morphogenesis

Dorsal closure

Derived from “Atlas of Drosophila development”, Volker Hartenstein, CSHL Press, 1993

We hypothesized that Abl might play a similar role in epithelial morphogenesis

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Dorsal closure requires tight coordination of adhesion and the cytoskeleton to allow

coordinated cell shape changes and cell migration

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Lizz Grevengoed



Prof. Mark Peifer


22abl mutants

Abl is required for dorsal closure and germband retraction

Wild type

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Abl is required for orchestrated cell shape changes during dorsal closure

ablWild type

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The best known target of Abl kinase is Enabled (Ena)

• Substrate of Abl kinase

• Negatively regulated by Abl

• Known modulator of the actin cytoskeleton

• Lizz found that reduction in the level of Ena rescues

abl’s embryonic lethality

The best known target of Abl kinase is Enabled (Ena)



Prof. Mark Peifer


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Lizz found that Ena and Arm co-localize at adherens junctions throughout development

Ena Arm Merged

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Abl regulates apical actin polymerizationin syncytial/cellularizing Drosophila embryos

Grevengoed, Fox, Gates and Peifer J. Cell Biol 163, 1267- 1280, 2003

ActinAbl regulates apical actin polymerization

in syncytial/cellularizing Drosophila embryos

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But how does Abl function during

the more complex events

of morphogenesis?



Prof. Mark Peifer


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Don Fox

29Developmental Biology Fig. 12.3 Gilbert 7th ed.

Formation of the Drosophila mesoderm and the mammalian neural tube are models

of morphogenesis

Chick neural tubeDrosophila ventral

furrow formation

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Apical cell constriction can rearrange a sheet into a tube

Essential Cell Biology 2nd Ed, Alberts et al. Fig. 21-25

Sheet of epithelial cells

Adhesion belts with associated actin filaments

Invagination of epithelial sheet caused by an organized tightening along the adhesion belts in selected regions of the cell sheet

Epithelial tube pinches off from overlying sheet of cells

Epithelial tube



Prof. Mark Peifer


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Both involve apical cell constriction

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Wild-type

Moesin::GFP

Cell constrict in a highly coordinated fashion to form the ventral furrow

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Abl is required for coordinated apical constriction in the ventral furrow

wt abl

E-Cadherin



Prof. Mark Peifer


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In embryos lacking functional Abl Apical constriction becomes highly uncoordinated

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Don then carried out a set of cell biological

experiments to determine how Abl fit

into the classic model of mesoderm invagination

Our new model for Apical constriction: morphogenesis requires careful

coordination of both actin and myosin

Unknown

effector

Abl

Actin

Ena

Fog

Rho

RhoGEF2

Rho Kinase

Myosin

G-protein coupled receptor?

Concertina

Rho

RhoGEF2

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What other actin regulators are critical during morphogenesis?

Extracellular stimuli

2. WASP/Scar activation

3. Arp2/3 complex activation and

filament nucleation

1. Profilin-bound ATP-actin

5. Growing filaments push membrane forward

Extracellular stimuli

10. ADP-ATP exchange

8. ADF/cofilin severs & depolymerizes ATP-actin filaments

6. Capping limits elongation

9. ADF/cofilin inhibition

70 0

Pollard, Blanchoin and Mullins, J. Cell Sci 114, 3-4, 2001



Prof. Mark Peifer


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Don’s work suggested we should examine Ena/VASP proteins

Sutherland and Way, Dev. Cell 2, 692-694, 2002

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Julie Gates

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Remember dorsal closure?



Prof. Mark Peifer


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During migration leading edge cells modulate their actin cytoskeletons to form lamellipodia

and filopodia

en-Gal4; UAS-GFP-actin

Filopodium

Lamellipodium

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Wild-type leading edge cells produce lamellipodia from which filopodia emerge

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In contrast leading edge cells lacking functional Ena produce lamellipodia without filopodia



Prof. Mark Peifer


43en-Gal4; UAS-GFP-Ena

Consistent with this, GFP-Ena localizes to tips of leading edge filopodia

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Another key aspect of epithelia is their apical-basal polarity

Adherens junction Apical cell surface

Basal Lamina or extracellular matrix

Basolateral cell surfaces

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Tony Harris



Prof. Mark Peifer


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SubsequentElaboration

In general, cells use ‘landmarks’ to establish and elaborate polarity

Primary Landmark

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For example, in yeast the bud scar acts as a primary landmark directing both cell

and cytoskeletal polarity

Bud Scar

CytoskeletalPolarization

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Sperm

Entry

Anterior Posterior

Actomysosin-based cortical flow

In C. elegans, sperm entry provides a landmarkfor polarizing the one-cell embryo

The cytoskeleton helps position these cues asymmetrically

Cytoskeletalflow

These cues then interact to establish full polarity

Anterior

cuesPosterior

cues



Prof. Mark Peifer


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How does apical-basal polarity arise

in epithelial tissues?

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Lumen or exterior space

Underlying tissue

Apical

domain

Basolateral

domain

AJs

Adherens junctions (AJs) were thought to act as primary landmarks for epithelial polarity

51Without AJs

With AJs

Adherens junctions (AJs) were thought to act as primary landmarks for epithelial polarity



Prof. Mark Peifer


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AJs as landmarks for establishing epithelial polarity

Cadherin

αααα-catArm

Apical

domain

Basolateral

domain

Apical

cues

Basal

cues

aPKC

Baz

PAR-3

Crb

Sdt

PatJ

PAR-6

Are AJs

the primary

landmark?

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AJs Baz?

Baz?

After polarity is established, complex interactions among polarity determinants

complicate answering this question

AJs

AJs are mislocalized

during gastrulation

in baz mutants

Müller and Wieschaus, 1996

Baz is mislocalized

during gastrulation

in arm mutants

Bilder et al., 2003

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Tony went back to the beginning, examining the initial establishment of polarity



Prof. Mark Peifer


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• Polarity can be analyzed from the outset

• Polarity cues can be studied before they affect

epithelial structure

• Polarity can be studied w ith powerful tools of genetics,

microscopy and biochemistry

Drosophila cellularization; a model for studying epithelial polarity establishment

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Lumen or exterior space

Underlying tissue

Apical

domain

Basolateral

domain

AJs

Our hypothesis was that adherens junctions (AJs) were the primary landmarks

for epithelial polarity

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Baz AJs

No!

Baz (Fly Par3) acts upstream of AJs

as epithelial polarity is established in Drosophila



Prof. Mark Peifer


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Baz forms apical complexes in the absence of AJ assembly

Dlg Baz

CadCad Baz

Baz

Baz forms apical complexes in the absence of AJ assembly

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DE-cadherin cannot form apical complexesin the absence of Bazooka

Cad Dlg Cad Cad

baz baz WT

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We hypothesized that Bazooka is initially positioned in response to cytoskeletal

landmarks present prior to cellularization



Prof. Mark Peifer


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-Cad

Cad

Baz

Baz

Baz-bindingscaffold

Dynein may ferry Baz/cadherin complexes to the apical domain

62Baz

Dlg Baz

Baz

WT dhc

Dynein is required for positioning Bazooka

Dlg Baz

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Baz

PAR-6 aPKC

Bazooka is traditionally thought to act in a complex with aPKC and PAR-6

AJs

C. Elegans one-cell embryo Vertebrate epithelia



Prof. Mark Peifer


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However, Baz localizes below aPKC and PAR-6during WT cellularization, gastrulation and later

Cellularization

Gastrulation

Cad aPKCBaz Cad Baz PAR-6

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These data help reshape our view of the establishment and maintenance

of epithelial polarity

However, many challenges remain

in unraveling how tissues assemble

and are maintained from egg to adult

• In Drosophila, AJ do not serve as the primary cue

for the establishment of epithelial polarit y

• Baz/Par-3 plays a key role in polarity establishment

• Is Baz an obligate partner of PAR-6 and aPKC?

• Things may be more complex

in animals other than Drosophila

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I would like to thank members of my lab, past and present,

who have led our exploration of cell adhesion, signaling

and cytoskeletal regulation, especially those whose work

I presented today

Rachel Cox Joe Loureiro

Lizz Grevengoed Don Fox

Julie Gates Tony Harris

Thanks to our collaborators on the Ena project

And to my audience for indulging my enthusiasm

for this exciting field

Frank Gertler Dav ie van Vactor



Prof. Mark Peifer


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You can learn more about the work I presented from our lab in:

• Cox, R., Kirkpatrick, C. and Peifer, M. (1996) Journal of Cell Biology 134: 133-148

• Grevengoed, E.E., Loureiro, J.J., Jesse, T.L. and Peifer M. (2001) Journal of Cell Biology

155, 1185-1197

• Harris, T.J.C. and Peifer, M. (2004) Journal of Cell Biology 165, 135-147

• Fox, D.T. and Peifer, M. (2007) Development 143, 567-578

• Harris, T.J.C. and Peifer, M. (2007) Developmental Cell 12, 727-738

• Gates, J., Mahaffey, J.P., Rogers, S.L., Emerson, M., Rogers, E.M., Sottile, S.L.,

Van Vactor, D., Gertler, F.B. and Peifer, M. (2007) Development 134 2027-2039

or the cytoskeleton or the developmental events of morphogenesis,

these textbooks are an excellent resources:

• Molecular Biology of the Cell, Alberts et al., Garland Science

• Molecular Cell Biology, Lodish et al., WH Freeman and Co.

• Developmental Biology, Scott Gilbert, Sinauer Assoc. Inc.

For more information on the cell biology of adhesion

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