Date of Completion

8-10-2015

Embargo Period

8-3-2017

Keywords

Actin, lamellipodium, comet tail, cofilin, Cap, formin, N-WASP, Virtual Cell, nucleation, polymerization, actin dynamics, actin-based motility

Major Advisor

Bruce J. Mayer

Co-Major Advisor

Leslie M. Loew

Associate Advisor

Ann E. Cowan

Associate Advisor

William A. Mohler

Associate Advisor

Yi I. Wu

Field of Study

Biomedical Science

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

Rearrangements of the actin cytoskeleton underlie many physiological processes. I investigated actin dynamics in broad thin protrusions (lamellipodia) at the front of migrating cells, and in locally induced dynamic actin structures (comet tails).

Cofilin is an actin regulatory protein that induces either polymerization or depolymerization in live cells. Through biochemical modeling in Virtual Cell software I examined the function of cofilin in a dynamic branched network at the tip of a lamellipodium. Cofilin and capping protein play synergistic roles in regulating branched actin polymerization. The model predicts that cofilin promotes actin disassembly when capping concentration is low and promotes actin assembly when capping is high and a sufficient actin monomer pool is maintained.

Nck, a SH2/SH3 adaptor protein, facilitates recruitment and increases local concentration of cytosolic effectors that induce formation of pathogenic actin comet tails. I characterized comet tails induced by experimental aggregation of Nck SH3 domains at the membrane. Experimental disruption of the balance between unbranched/branched nucleation alters the morphology and dynamics of Nck SH3 actin comets. Inhibition of linear formin-based nucleation results in formation of predominantly circular-shaped actin structures with decreased mobility. Enhancement of branched nucleation by N-WASP overexpression similarly caused loss of the typical actin comet tail shape. The results indicate that formin-based linear actin polymerization is critical for Nck-dependent actin comets. Consistent with this, aggregation of a branched nucleation promoting factor (VCA domain of N-WASP), with density and turnover similar to that of N-WASP in Nck comets, does not reconstitute dynamic elongated actin comets.

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