SIGNAL PATHWAYS IN CELL MIGRATION AND ADHESION Sam Polak 28 April 2008.
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Transcript of SIGNAL PATHWAYS IN CELL MIGRATION AND ADHESION Sam Polak 28 April 2008.
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SIGNAL PATHWAYS IN CELL MIGRATION AND ADHESION
Sam Polak 28 April 2008
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Ridley A, Schwartz M, Burridge K, Firtel R, Ginsberg M, Borisy G, Parsons J, Horwitz A
Science (302) 5 December 2003 1704-1709
Cell Migration: Integrating Signals from Front to Back
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Overview
Migration Cycle Components of migration Polarization Integrins (yes, again) Summary
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Migration Cycle
Initial Response: Polarization
Extend lamellipodia or filopodiaDisassemble at back
FrontBack
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Components of Migration
Lamellipodia Actin Barbed and pointed ends Dendritic vs Parallel Proteins
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Components of Migration
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Components of Migration
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Components of Migration
Filopodial Actin Treadmilling Ena/VASP Fascin
http://www.biol.vt.edu/faculty/kuhn/images/TIRFpoly01.gif
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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 2/3Positive or negative feedback to Rho-
GTPases
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Polarization
Cdc 42 PI3Ks and PTEN Rac activation Defining the tail
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Polarization
Cdc42 Located in front of cell Localizes microtubule-
organizing center (MTOC) and Golgi apparatus
Positive feedback loop with target PAK1
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Polarization
PI3Ks and PTEN
Gradient amplifiers via PIP3
and PI(3,4)P2 Off-set each other Feedback loops
between PI3K, PTEN, and Cdc42
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Polarization
Rac Activation Stimulate
recruitment/activation of PI3Ks
Microtubules and Rac form activation/stability loop
Integrins and Rac form activation/recruitment loop
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Polarization
Defining the Tail – Rho and Rac Rho stabilizes microtubules Rho and Rac mutually antagonistic Exceptions
Rac involved in tail detachments Rho involved in Rac activation
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Integrins
Integrin affinity Formation of adhesions Tractional forces Adhesion disassembly in front Adhesion disassembly in rear
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Integrins
Integrin Affinity Preferentially localize
to leading edge Binding of ligands
leads to conformational changes
Posttranslational modification
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Integrins
Formation of Adhesions Migration rate influences integrin
clusters Focal complexes and focal adhesions Rac and Cdc42 Component kinetics
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Integrins
Tractional Forces Traction sites and mechanosensors Adhesion strength determined by
Substrate ligand density Adhesion ligand receptor density Receptor affinity
Migrating cells vs more stationary cells Transmitted force regulated by Myosin II
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Integrins
Tractional Forces
MLC
MLCK
ROCK
MLC Phosphatase
Rho-GTP[Ca2+]
Phosphorlyation
Myosin II
Phosphorlyation
Phosphorlyation
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Integrins
Adhesion disassembly at the front
Disassembly vs maturation Targeting and microtubules Kinases and phosphatases
FAK and Src/Cas and Crk/Rac-GEFs
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Integrins
Adhesion disassembly at the back
Tethering Myosin II and retraction FAK, Src, Calcium
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Summary
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Summary
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Summary
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Balasubramanian N, Scott D, Castle D, Casanova J, and Schwartz M
Nature Cell Biology (9) 18 November 2007 1381-1390
Arf6 and microtubules in adhesion-dependent trafficking of lipid rafts
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Overview
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
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Lipid Rafts and Markers
Modulate signalling pathways Endocytosed via caveolae GTPase Arf6 as a regulator Raft marker CTxB
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Raft Relationship with Cytoskeletan Addition of Latrunculin or Nocodazole Addition of CTxB before or after detachment Gamma-tubulin staining
CTxB labelled while attachedCTxB labelled after detachedCTxB labeled while attached, gamma-tubulin stained
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Raft Localization after Endocytosis
Golgi Investigation GM130 colocalization Befeldin A (BFA) – dispersion inducer Protein kinase D mutant – protein
movement blocker
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Raft Localization after Endocytosis
Golgi Investigation – GM130
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Raft Localization after Endocytosis
Golgi Investigation – Brefeldin A
LocalizationSpreading
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Raft Localization after EndocytosisGolgi Investigation – Protein Kinase D
Overlap of VSV and CTxB in Golgi
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Raft Localization after Endocytosis
SER Investigation
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Raft Localization after Endocytosis
Recycling Endosome Investigation – Rab11
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Raft Localization after EndocytosisRecycling Endosome Investigation – Tf
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Raft Localization after Endocytosis
Recycling Endosome Investigation – Rab11
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Arf6 and Raft Trafficking
Arf6 regulates vesicle trafficking and Rac1 movement
Recycling endosomes and in lamellipodia
Recycling EndosomesLamellipodia
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Arf6 and Raft Trafficking
Arf6 and cell spreading WT and caveolin -/-
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Arf6 and Raft Trafficking
Arf6 only involved in raft exocytosis
Cav -/- control
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Arf6 and Rac1
Compare WT and Cav1 -/- Suspension and replating
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Arf6 and Adhesion
Adhesion regulation of Arf6
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Arf6 and Adhesion
Arf6 recycling power
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Arf6 and Adhesion
Arf6 recycling power
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Microtubules and Raft Trafficking
MTs and raft components colocalize
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Microtubules and Raft Trafficking WT and Cav1 -/- Addition of nocodazole
Attached 90 minutes in suspension
Label with CTxB 0 minute of suspension 90 minute of suspension
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Microtubules and Raft Trafficking
WT Nocodazole spreading
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Microtubules and Raft Trafficking
WT Nocodazole CTxB
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Microtubules and Raft Trafficking Cav1 -/- Nocodazole
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Microtubules and Raft Trafficking
Cav1 -/- Nocodazole CTxB
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Conclusions
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
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Critiques
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
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References
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