SIGNAL PATHWAYS IN CELL MIGRATION AND ADHESION

Sam Polak 28 April 2008

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

Overview

Migration Cycle Components of migration Polarization Integrins (yes, again) Summary

Migration Cycle

Initial Response: Polarization

Extend lamellipodia or filopodiaDisassemble at back

FrontBack

Components of Migration

Lamellipodia Actin Barbed and pointed ends Dendritic vs Parallel Proteins

Components of Migration

Components of Migration

Components of Migration

Filopodial Actin Treadmilling Ena/VASP Fascin

http://www.biol.vt.edu/faculty/kuhn/images/TIRFpoly01.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 2/3Positive or negative feedback to Rho-

GTPases

Polarization

Cdc 42 PI3Ks and PTEN Rac activation Defining the tail

Polarization

Cdc42 Located in front of cell Localizes microtubule-

organizing center (MTOC) and Golgi apparatus

Positive feedback loop with target PAK1

Polarization

PI3Ks and PTEN

Gradient amplifiers via PIP3

and PI(3,4)P2 Off-set each other Feedback loops

between PI3K, PTEN, and Cdc42

Polarization

Rac Activation 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 Rho stabilizes microtubules Rho and Rac mutually antagonistic Exceptions

Rac involved in tail detachments Rho involved in Rac activation

Integrins

Integrin affinity Formation of adhesions Tractional forces Adhesion disassembly in front Adhesion disassembly in rear

Integrins

Integrin Affinity Preferentially localize

to leading edge Binding of ligands

leads to conformational changes

Posttranslational modification

Integrins

Formation of Adhesions Migration rate influences integrin

clusters Focal complexes and focal adhesions Rac and Cdc42 Component kinetics

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

Integrins

Tractional Forces

MLC

MLCK

ROCK

MLC Phosphatase

Rho-GTP[Ca2+]

Phosphorlyation

Myosin II

Phosphorlyation

Phosphorlyation

Integrins

Adhesion disassembly at the front

Disassembly vs maturation Targeting and microtubules Kinases and phosphatases

FAK and Src/Cas and Crk/Rac-GEFs

Integrins

Adhesion disassembly at the back

Tethering Myosin II and retraction FAK, Src, Calcium

Summary

Summary

Summary

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

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

Lipid Rafts and Markers

Modulate signalling pathways Endocytosed via caveolae GTPase Arf6 as a regulator Raft marker CTxB

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

Raft Localization after Endocytosis

Golgi Investigation 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

LocalizationSpreading

Raft Localization after EndocytosisGolgi 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 EndocytosisRecycling Endosome Investigation – Tf

Raft Localization after Endocytosis

Recycling Endosome Investigation – Rab11

Arf6 and Raft Trafficking

Arf6 regulates vesicle trafficking and Rac1 movement

Recycling endosomes and in lamellipodia

Recycling EndosomesLamellipodia

Arf6 and Raft Trafficking

Arf6 and cell spreading WT and caveolin -/-

Arf6 and Raft Trafficking

Arf6 only involved in raft exocytosis

Cav -/- control

Arf6 and Rac1

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 WT and Cav1 -/- Addition of nocodazole

Attached 90 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 Cav1 -/- Nocodazole

Microtubules and Raft Trafficking

Cav1 -/- Nocodazole CTxB

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

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

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