SIGNAL PATHWAYS IN CELL
MIGRATION AND ADHESION
Sam Polak 28 April 2008
Cell Migration: Integrating Signals
from Front to Back
Ridley A, Schwartz M, Burridge K, Firtel R, Ginsberg
M, Borisy G, Parsons J, Horwitz A
Science (302) 5 December 2003 1704-1709
Overview
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Migration Cycle
Components of migration
Polarization
Integrins (yes, again)
Summary
Migration Cycle
Initial Response: Polarization
Extend lamellipodia or filopodia
Disassemble at back
Back
Front
Components of Migration
Lamellipodia Actin
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Barbed and pointed ends
Dendritic vs Parallel
Proteins
Components of Migration
Components of Migration
Components of Migration
Filopodial Actin
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Treadmilling
Ena/VASP
Fascin
http://www.biol.vt.edu/faculty/kuhn/images/TI
RFpoly01.gif
Components of Migration
Rho Family Guanosine triphosphate binding proteins
 RhoG activates Rac-GEF activates Rac
 Rac actives WAVE
 Cdc42 activates WASP
End results in activation of Arp 2/3
Positive or negative feedback to Rho-GTPases
Polarization
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Cdc 42
PI3Ks and PTEN
Rac activation
Defining the tail
Polarization
Cdc42
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Located in front of cell
Localizes microtubuleorganizing center (MTOC)
and Golgi apparatus
Positive feedback loop with
target PAK1
Polarization
PI3Ks and PTEN
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Gradient amplifiers
via PIP3 and
PI(3,4)P2
Off-set each other
Feedback loops
between PI3K,
PTEN, and Cdc42
Polarization
Rac Activation
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Stimulate
recruitment/activation of
PI3Ks
Microtubules and Rac
form activation/stability
loop
Integrins and Rac form
activation/recruitment
loop
Polarization
Defining the Tail – Rho and Rac
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Rho stabilizes microtubules
Rho and Rac mutually antagonistic
Exceptions
 Rac
involved in tail detachments
 Rho involved in Rac activation
Integrins
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Integrin affinity
Formation of adhesions
Tractional forces
Adhesion disassembly in front
Adhesion disassembly in rear
Integrins
Integrin Affinity
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Preferentially localize to
leading edge
Binding of ligands leads
to conformational
changes
Posttranslational
modification
Integrins
Formation of Adhesions
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Migration rate influences integrin clusters
Focal complexes and focal adhesions
Rac and Cdc42
Component kinetics
Integrins
Tractional Forces
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Traction sites and mechanosensors
Adhesion strength determined by
 Substrate
ligand density
 Adhesion ligand receptor density
 Receptor affinity
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Migrating cells vs more stationary cells
Transmitted force regulated by Myosin II
Integrins
Tractional Forces
Phosphorlyation
[Ca2+]
Rho-GTP
MLCK
ROCK
MLC
Phosphorlyation
MLC Phosphatase
Myosin II
Phosphorlyation
Integrins
Adhesion disassembly at the front
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Disassembly vs maturation
 Targeting
and microtubules
 Kinases and phosphatases
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FAK and Src/Cas and Crk/Rac-GEFs
Integrins
Adhesion disassembly at the back
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Tethering
Myosin II and retraction
FAK, Src, Calcium
Summary
Summary
Summary
Arf6 and microtubules in adhesiondependent trafficking of lipid rafts
Balasubramanian N, Scott D, Castle D, Casanova J, and
Schwartz M
Nature Cell Biology (9) 18 November 2007 1381-1390
Overview
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Lipid rafts and markers
Raft relationship with cytoskeletan
Raft localization after endocytosis
Arf6 and raft trafficking
Arf6 and Rac1
Arf6 and adhesion
Microtubules and raft trafficking
Discussion/Conclusions
Lipid Rafts and Markers
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Modulate signalling pathways
Endocytosed via caveolae
GTPase Arf6 as a regulator
Raft marker CTxB
Raft Relationship with Cytoskeletan
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Addition of Latrunculin or Nocodazole
Addition of CTxB before or after detachment
Gamma-tubulin staining
CTxB labeled
CTxB
while
labelled
attached,
whilegamma-tubulin
after
detached
attached
stained
Raft Localization after Endocytosis
Golgi Investigation
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GM130 colocalization
Befeldin A (BFA) – dispersion inducer
Protein kinase D mutant – protein movement blocker
Raft Localization after Endocytosis
Golgi Investigation – GM130
Raft Localization after Endocytosis
Golgi Investigation – Brefeldin A
Spreading
Localization
Raft Localization after Endocytosis
Golgi Investigation – Protein Kinase D
Overlap of VSV and CTxB in Golgi
Raft Localization after Endocytosis
SER Investigation
Raft Localization after Endocytosis
Recycling Endosome Investigation – Rab11
Raft Localization after Endocytosis
Recycling Endosome Investigation – Tf
Raft Localization after Endocytosis
Recycling Endosome Investigation – Rab11
Arf6 and Raft Trafficking
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Arf6 regulates vesicle trafficking and Rac1 movement
Recycling endosomes and in lamellipodia
Recycling Endosomes
Lamellipodia
Arf6 and Raft Trafficking
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Arf6 and cell spreading
WT and caveolin -/-
Arf6 and Raft Trafficking
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Arf6 only involved
in raft exocytosis
Cav -/- control
Arf6 and Rac1
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Compare WT and Cav1 -/Suspension and replating
Arf6 and Adhesion
Adhesion regulation of Arf6
Arf6 and Adhesion
Arf6 recycling power
Arf6 and Adhesion
Arf6 recycling power
Microtubules and Raft Trafficking
MTs and raft components colocalize
Microtubules and Raft Trafficking
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WT and Cav1 -/Addition of nocodazole
 Attached
 90
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minutes in suspension
Label with CTxB
0
minute of suspension
 90 minute of suspension
Microtubules and Raft Trafficking
WT Nocodazole spreading
Microtubules and Raft Trafficking
WT Nocodazole CTxB
Microtubules and Raft Trafficking
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Cav1 -/- Nocodazole
Microtubules and Raft Trafficking
Cav1 -/- Nocodazole CTxB
Conclusions
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Adhesion recycling of lipid rafts is Arp6 dependent;
and Rab11, Rab22, and caveolin independent;
microtubules and also involved
Arp6 gets raft to the membrane, but additional
steps are needed to get the raft to the surface
Cell detachment sends rafts to recycling endosomes
Rac1 requires rafts, Arp6, and MTs for localization
and activation
Critiques
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Said that WT cell spreading was only moderately
inhibited by late addition of nocodazole, but that’s
not what the data show
Arp6 does not bring rafts to the surface of the
plasma membrane in adherent cells, but data shows
an increase in CTxB in suspended cells with
overactive Arp6 – why would that be so?
Eliminated Golgi for localization of rafts, but the
figure makes it seems as if there is significant
overlap of Golgi marker and CTxB
References
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Balasubramian, et al. Arf6 and microtubules in
adhesion-dependent trafficking of lipid rafts. Nature
Cell Biology (6) Issue 12, Dec 2007 (1381)
Ridley, et al. Cell Migration: Integrating Signals from
Front to Back. Science (302) 5 Dec 2003 (1704)
Kuhn. Department of Biological Sciences
www.biol.vt.edu/research/molceldevcomp/index.htm
Davis. Inside the Cell, Chapter 2 Cells 101.
www.publications.nigmns.nih.gov