Lamellipodial extension driven by actin assembly Rac-GTPase lamellipodia lamella Difference between the lamellipodia and the lamella Protrusion of the membrane during cell edge extension is driven by actin polymerization and depolymerization. Polymerization at the front Stabilization of the meshwork Depolymerization at the back Relatively constant length and a stationary gradient reflecting a steady-state process of assembly at the front and disassembly at the rear Account for extension, but ECM probing? No myosin Myosin is required for migration and cell spreading The cytoskeletal treadmilling cycle can power actin-based movement of beads or bacteria, without the need for myosin-based contractility Components that will be involved in this talk are LIM kinase, cofilin, Rac GTPase. Svitkina T.M. and Borisy G.G., 1999, J Cell Biol, 145:1009-26 Pollard T.D. and Borisy G.G., 2003, Cell, 112:453-65 actin plolymerization (front) and depolymarization (rear) define the lamellipodial width Forces generated by Myosins? Cell spreading and migration require myosin activity

Extension Attachment Contraction Release Recycling C o n t r a c i x e F Attachment Contraction Release Recycling

Cell migration: a cyclic process of force generation 1. lamellipodial extension: forward force exerted by actin polymerization on the membrane 2. extracellular matrix probing: rearward force exerted by actin flow on integrins Cell migration 5 STEP cycle: describe cycle Focus our attention on the 2 STEP extension and attachement Lamellipodial extension and ECM probing involve forces generated by actin: Forward on the memebrane and rearward on integrins Laser tweezers experiments allow us to explore force exerted by the actin reaward flow on integrins Mike already described the effect of forces on the integrin-cytoskeleton interactions Transition: definition of the lamellipodia How the cell locally transduces the rigidity of the ECM into a contractile signal used to direct lamellipodial extension? Sheetz M.P., Felsenfeld D.P., Galbraith C.G., 1998, Trends Cell Biol, 8:51-4

Neural Crest Migration The peripheral nervous system is created by a spatiotemporally co-ordinated migratory process during which the precursor cells, the neural crest (NC) cells, traverse the embryo to reach distantly located sites.

Homing of peripheral neurons and their supportive cells might be dictated by a delicate equilibrium between the multiple actions of stimulatory and inhibitory molecules, which is modulated further by defined responses of the dispersing cells to these ECM components during their successive phases of phenotypic diversification.

Extracellular Matrix-Integrin Interactions in Motility and Signaling Matrix molecules are typically bound by integrins Integrins are alpha-beta dimeric membrane proteins needed for motility, signal, and force transduction. Antibodies to integrins block cell migration and spreading

Integrin Knockouts have Severe Phenotypes

Further Roles for Integrins

Steps In NC Migration Initiation of migration Migration Homing Differentiation

Rigidity of Matrix Directs Motility

Initiation of Migration Transition from tissue ball to migration Loss of cadherins Acquisition of Integrins Acquisition of motile machinery Stimulus to migrate

Important Aspects of Migration 1. Isovolumic 2. Constant Membrane Area 3. Actin filament assembly and disassembly 4. Actin filament contraction 5. Matrix binding 6. Matrix Rigidity 7. Release of matrix binding

Attachment and Contraction

Matrix Forces Directed Inward And Balanced time 4 c e l l d i r e c t i o n rear ) 3 2 m 2 m n u c l e u s / N 1 f r o n t n ( n o t i c - 1 a r T - 2 - 3 - 4

How does a cell treat the ECM? Movie 2: One cell deforms a collagen fibre

Extension Attachment Contraction Release Recycling C o n t r a c i x e F Attachment Contraction Release Recycling