You have created an in vitro artificial cell system to study the formation of la

Question

You have created an in vitro artificial cell system to study the formation of lamellipodia and cell migration (crawling). You have a membrane-bound structure that contains necessary receptors and several cytoskeletal components. In addition you have an external signaling molecule that promotes lamellipodium formation. You perform the following experiments 2 3 4 Membrane External Signal ATP Actin-ATP F-actin Arp213 complex WAVE Profilin Cofilin Rac Experiment 1 is completely successful in achieving normal lamellipodium formation and normal cell migration A. Do you expect the "cells in experiment 2 to migrate? Describe any differences compared to experiment 1 B. Do you expect the "cells in experiment 3 to migrate? Describe any differences compared to experiment 1 C. Do you expect the "cells in experiment 4 to migrate? Describe any differences compared to experiment 1

Explanation / Answer

A) In experiment 2, F-actin is absent. No, the cells will not migrate.

Compared to proper lamellipodium formation observed in experiment 1, there will be ill-defined protrusions in the cell membrane, which will indicate feeble actin polymerisation; but the cell won't protrude and polarize. Since F-actin is absent, actin polymerization cannot take place by severing existing filamentous actin. But since Arp2/3 is present, new nucleation cores can be made from actin monomers, although this de novo nucleation is not sufficient to form an integrated actin network. Arp2/3 complex is suggested to bind to pre-existing actin filaments (by side binding or barbed end binding modes) to form a functional cytoskeletal framework. In absence of F-actin, NO such integrated network will form.

B. In experiment 3, WAVE is absent. NO, the cells will not migrate.

Compared to experiment 1, there will be no lamellipodia formation (protrusion, polarisation and extension) in response to external stimuli. However the cell shape may vary. The WAVE is activated on perception of external stimuli which then activates the Arp2/3 complex. This complex starts de novo nucleation of actin monomers or builds up on severed actin filaments.

C. In experiment 4, Cofilin is absent. NO, the cells will not migrate.

However , lamellipodia will be formed and protrusions from the cell membrane will be visible, comparable to experiment 1. However there will be no further translocation of the cell, as cofilin is absent. cofilin depolymerizes the actin filament at the - end, generating G-actin:ADP to be exchanged at the +end of the growing filament. Therefore a protrusion will occur but there will be no extension.

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