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CELLULAR MOTILITY Lecture 5 January 20th, 2006

CELLULAR MOTILITY Lecture 5 January 20th, 2006. Cell Motility. Migration/Movement of cells requires: 1- Anchorage 2- Active cytosketelon. Intercellular Motility (movements between cell sheets) Cell movements are important for the morphogenesis of the neural tube.

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CELLULAR MOTILITY Lecture 5 January 20th, 2006

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  1. CELLULAR MOTILITY Lecture 5 January 20th, 2006

  2. Cell Motility • Migration/Movement of cells requires: 1- Anchorage 2- Active cytosketelon

  3. Intercellular Motility (movements between cell sheets) Cell movements are important for the morphogenesis of the neural tube • During the formation of the neural tube, a flat layer of cells must roll into a tube (neural tube). This is possible because of contractile forces generated by actin filaments at the apical ends of the cell.

  4. Dynamics of actin polymerization

  5. Barbed end Pointed end Pi D Css ADP ATP T Treadmilling T D T D D (-) (+) ADP-actin ATP-actin Steady state concentration Css Filament turnover Pure actin: 0.1 µM 3µm / 90 min Lamellipodium:? 3 µm / 1 min 2 µM

  6. Actin Depolymerizing Factor (ADF) / Cofilin: • Ubiquitous, conserved, stimulus-responsive regulators of actin dynamics in motile processes: • Embryonic development • Yeast endocytosis • Cytokinesis • Listeria propulsion • Localized in motile regions of cells (at the leading edge)

  7. x 30 ADF ADF D D T ADP ATP ADF increases the treadmilling of F-actin ADF binds F-ADP-Actin better than F-ATP-Actin T D D D T D Increase inCss => faster + end growth

  8. Profilin Actin filament Profilin increase the rate of actin-based motility

  9. x 30 ADF D ADF ADF T T D x125 ADP ATP D T D P Profilin Profilin Profilin Synergy between Profilin and ADF 2. Profilin transports actin-ATP to leading edge. 1. Profilin binding increases rate of actin-ADP to actin-ATP.

  10. MOLECULAR BASIS OF CELL MOTILITY Contraction Contraction Leading edge Net movement FOCAL ADHESION Actin network Focal adhesions Focal complexes Microtubules Stress fibers Forces on the substrate Forces on the cell cortex Relaxation Others Rho kinase Rho Myosin phosphatase MLCK Ca /Calmodulin PAK Some regulators of Focal adhesion turnover FAK PAK Src Microtubules Calpain Rac/Rho Contraction Rac Cdc 42

  11. Actin Filaments in Migrating Cells The organization of actin fibers varies significantly in different areas of the cell.

  12. Arp 2/3 Branching of actin cytoskeleton at the leading edge requires Arp 2/3 Filament branching array in lamellipodia Branching

  13. Arp3 47 Arp2 42 21 20 34 40 16 The Arp2/3 complex: downstream target of multiple signaling pathways leading to actin assembly ARP: Actin Related Protein. Has similar structure to actin. • Seven conserved subunits including Arp2 and Arp3. • Is activated at the surface of Listeria by the ActA protein to stimulate actin polymerization and bacterial propulsion. • Localizes at motile regions of cell. • Generates new actin filaments in a stimulus-responsive and spatially controlled fashion.

  14. B A WH1 Pr o Bee 1 V C B Pr o A WH1 Scar/WAVE V C WASP B GBD A Pr o WH1 V C N-WASP GBD A WH1 B Pr o VV C PIP2 F-Actin G-Actin Cdc42 SH3 Arp 2/3 X (Grb2, Nck, IcsA Fgr, Fyn) WASP family proteins :activators of Arp2/3 complex (signaling molecules)

  15. + X « closed N-WASP » Plasma membrane GBD GBD WH1 WH1 B B Pro Pro Arp2/3 VV C GBD A WH1 B Pr o G « activated N-WASP » VV VV A A C C X WASP family proteins :activators of Arp2/3 complex + Arp2/3 +G-actin

  16. Dynamics of actin polymerization at the leading edge

  17. Reconstitution of actin-based movement from pure proteins (Loisel et al., Nature 1999) • Proteins required for movement: 1) N-WASP (resp. ActA)-activated Arp2/3: site-directed generation of barbed ends 2) Actin, ADF/cofilin,Capping protein: chemostat maintaining a high steady-state concentration of ATP-G-actin • Not required, but improve movement: • VASP, Profilin, a-actinin.

  18. Role of capping proteins in motility: funnelling the treadmilling • Capping proteins are known to be required for efficient motility in vivo. • Capping proteins block most of the barbed ends, hence they increase the concentration of monomeric ATP-actin.

  19. Making many types of actin fibers:Rho family GTPases Rho Myosin Actin stress fibers Contractility Microtubule stability gene expression Cell cycle Rac Cell migration Actin polymerization Cell spreading Membr. trafficking Gene expression Cell cycle apoptosis Cdc42 Actin polymerization Cell polarity Migration Gene expression Cell cycle apoptosis

  20. Signal-transduction pathways involved in Rho-induced stress-fiber assembly

  21. Signal-transduction pathways induced by Rac and Cdc42 (shown in red), which are thought to contribute to the formation of actin-containing lamellipodia and filopodia respectively

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