the miracle of morphogenesis cell adhesion, polarity and ... · formation of the drosophila...
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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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The Miracle of Morphogenesis
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
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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 2
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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
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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 3
7
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
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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 4
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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
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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 5
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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
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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 6
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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
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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 7
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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
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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 8
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)
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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 9
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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?
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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 10
28
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
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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 11
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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
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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 12
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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
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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 13
37
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?
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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 14
40
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
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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 15
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
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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 16
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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
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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 17
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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
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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 18
52
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
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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 19
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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
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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 20
58
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
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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 21
61+
-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
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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 22
64
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
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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 23
67
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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