Question

You screen a chemical library to identify novel chemical regulators of the Arp2/3 complex.You will search for three distinct types of chemical regulators, each with one of the following properties:

a. Class A inhibitor that blocks lamellipodia localization of Arp2/3, but retains activity

b. Class B activator that locks the Arp2/3 complex into the inactive conformation

c. Class C inhibitor prevents Arp2/3 binding to nucleators (eg: WASp, WAVE)

Describe a cell-based approach you could use to identify these compounds, and describe how you predict that actin architectures would change upon treatment with each class of inhibitor.

Answer 1

1. the Arp2/3 complex enables rapid actin polymerization without delay for trimer formation. actin polymerization drives the morphological changes that allow cells to undergo dynamic processes such as division, phagocytosis and migration. Arp2 and Arp 3 are actin related proteins that serve as a nucleus for the new actin filament . the other Arp2/3 complex subunits bind to existing actin filaments to generate branch from mother filament.

2. the dendritic branching pattern formed by Arp 2/3 nucleated fragments is optimal for actin polymerisation to exert a protrusive force which suggest that Arp 2/3 induced filaments contribute to lamelliopodial protrusion. in fibroblasts, null cells and knock down cells lost lamelliopodia but migrated by combining formin based filopodia formation with myosin-2 based contractility . the null cells were unable to migrate directionally during wound healing.

3. the WASP and WAVE family have a domain in their c-termini that binds monomeric actin and the actin monomer then forms growing barbed end. WASP have G-actin binding activity which results in formation of many branched filaments. each branching of an elongating filament increases the number of barbed ends available for elongation, thus enabling the rapid formation of an actin filament network.