Question

5. (5 points) Activation of Cdc42, a monomeric GTPase, triggers actin polymerization and bundling to form either filopodia or shorter cell protrusions called microspikes. These effects of Cdc42 could be mediated by N-WASp, which is a multifunctional protein. As shown in panel A, N-WASp contains a plectrin homology (PH) domain, which binds to PIP2 (phosphatidylinositol bisphosphate, PI(4,5)P2); a Cdc42 GTPase-binding domain (G); a verprolin homology domain (V), which binds to actin; a cofilin homology domain (C), which can bind to actin filaments; and a C-terminal acidic domain (A), which binds the Arp2/3 complex. In Xenopus egg extracts, a convenient source of cytoskeletal components, the addition of Cdc42 charged with GTPyS, a nonhydrolyzable analog of GTP, stimulates actin polymerization (panel B). If the extract is depleted of N-WASp using N-WASp-specific antibodies, no actin polymerization is observed when CDC42-GTPyS is added. Actin polymerization can be restored by the addition of purified N-WASp, but not by the addition of either of two mutant forms of N-WASp: one (H208D) that cannot bind to CDc42, and a second (Açof) that eliminated the function of the cofilin domain. (A) DOMAINS OF N-WASp (B) ACTIN POLYMERIZATION untreated pleckstrin GTPase homology binding verprolin 2.4 N-WASp depleted PH Acof 1.2 200 400 600 time (seconds) Do these experiments support a role for N-WASp in the rearrangement of actin filaments in response to Cdc42 activation (1 point, Yes/No)? Explain your reasoning and include a discussion of why the two mutant forms of N-WASp do not restore actin polymerization (4 points, 3-4 sentences).

Answer 1

Yes, the experiments support of a role of N-wasp in rearrangement of actin filament in response to Cdc42 activation. However, mutants like H2080 and delta cod do not restore active polymerisation. Following reasons would explain the same;

1. H2080 cannot bind to cdc42 changed with GTPase hence inhibiting cascade reaction that leads to actin polymerisation.
2. Delta COF eliminates function of cofilin domain which is the nest known regulator of non-equilibrium assembly/disassembly of actin filament