1. SIGNAL PATHWAYS IN CELL MIGRATION AND ADHESION Sam Polak 28 April 2008

2. 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

3. Overview  Migration Cycle  Components of migration  Polarization  Integrins (yes, again)  Summary

4. Migration Cycle Initial Response: Polarization Extend lamellipodia or filopodia Disassemble at back FrontBack

5. Components of Migration Lamellipodia Actin  Barbed and pointed ends  Dendritic vs Parallel  Proteins

6. Components of Migration

8. Filopodial Actin  Treadmilling  Ena/VASP  Fascin http://www.biol.vt.edu/faculty/kuhn/images/TI RFpoly01.gif

9. 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

10. Polarization  Cdc 42  PI3Ks and PTEN  Rac activation  Defining the tail

11. Polarization Cdc42  Located in front of cell  Localizes microtubule- organizing center (MTOC) and Golgi apparatus  Positive feedback loop with target PAK1

12. Polarization PI3Ks and PTEN  Gradient amplifiers via PIP 3 and PI(3,4)P 2  Off-set each other  Feedback loops between PI3K, PTEN, and Cdc42

13. Polarization Rac Activation  Stimulate recruitment/activation of PI3Ks  Microtubules and Rac form activation/stability loop  Integrins and Rac form activation/recruitment loop

14. 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

15. Integrins  Integrin affinity  Formation of adhesions  Tractional forces  Adhesion disassembly in front  Adhesion disassembly in rear

16. Integrins Integrin Affinity  Preferentially localize to leading edge  Binding of ligands leads to conformational changes  Posttranslational modification

17. Integrins Formation of Adhesions  Migration rate influences integrin clusters  Focal complexes and focal adhesions  Rac and Cdc42  Component kinetics

18. 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

19. Integrins Tractional Forces MLC MLCK ROCK MLC Phosphatase Rho-GTP [Ca 2+ ] Phosphorlyation Myosin II Phosphorlyation

20. Integrins Adhesion disassembly at the front  Disassembly vs maturation  Targeting and microtubules  Kinases and phosphatases  FAK and Src/Cas and Crk/Rac-GEFs

21. Integrins Adhesion disassembly at the back  Tethering  Myosin II and retraction  FAK, Src, Calcium

22. Summary

25. 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

26. 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

27. Lipid Rafts and Markers  Modulate signalling pathways  Endocytosed via caveolae  GTPase Arf6 as a regulator  Raft marker CTxB

28. 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

29. Raft Localization after Endocytosis Golgi Investigation  GM130 colocalization  Befeldin A (BFA) – dispersion inducer  Protein kinase D mutant – protein movement blocker

30. Raft Localization after Endocytosis Golgi Investigation – GM130

31. Raft Localization after Endocytosis Golgi Investigation – Brefeldin A LocalizationSpreading

32. Raft Localization after Endocytosis Golgi Investigation – Protein Kinase D Overlap of VSV and CTxB in Golgi

33. Raft Localization after Endocytosis SER Investigation

34. Raft Localization after Endocytosis Recycling Endosome Investigation – Rab11

35. Raft Localization after Endocytosis Recycling Endosome Investigation – Tf

36. Raft Localization after Endocytosis Recycling Endosome Investigation – Rab11

37. Arf6 and Raft Trafficking  Arf6 regulates vesicle trafficking and Rac1 movement  Recycling endosomes and in lamellipodia Recycling EndosomesLamellipodia

38. Arf6 and Raft Trafficking  Arf6 and cell spreading  WT and caveolin -/-

39. Arf6 and Raft Trafficking  Arf6 only involved in raft exocytosis  Cav -/- control

40. Arf6 and Rac1  Compare WT and Cav1 -/-  Suspension and replating

41. Arf6 and Adhesion Adhesion regulation of Arf6

42. Arf6 and Adhesion Arf6 recycling power

43. Arf6 and Adhesion Arf6 recycling power

44. Microtubules and Raft Trafficking MTs and raft components colocalize

45. 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

46. Microtubules and Raft Trafficking WT Nocodazole spreading

47. Microtubules and Raft Trafficking WT Nocodazole CTxB

48. Microtubules and Raft Trafficking  Cav1 -/- Nocodazole

49. Microtubules and Raft Trafficking Cav1 -/- Nocodazole CTxB

50. 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

51. 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

52. 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