UCLA

Rapid remodeling of the actin cytoskeleton is essential for many cellular processes including cell growth, differentiation, division and motility. The structure and dynamics of the actin cytoskeleton are modulated by various actin-binding proteins. Drebrin, coronin and cofilin are three important players in actin's organization in the cell. This dissertation focuses on characterizing the mechanisms of drebrin, coronin and cofilin interactions with actin and studying their independent and inter-dependent roles in actin structure and dynamics.

First, we investigate DrABD binding interface on actin filaments. Our results reveal polymorphism in DrABD binding to F-actin and suggest the existence of two binding sites. We find that DrABD binding is centered on actin subdomain 2 and that this protein bridges two adjacent actin protomers. We also examine the structural effects of drebrin on F-actin in solution. We use the full length protein and its C-terminal truncated constructs to clarify which domains of drebrin are required for its interactions with actin. We demonstrate that F-actin is stabilized by drebrin binding. Also, in different cases of longitudinal and lateral interprotomer contact perturbations, we observe the rescue of filament formation by drebrin. Overall, our data suggests that drebrin stabilizes actin filaments through its effect on their interstrand and intrastrand contacts.

Next, we examine coronin and study the effects of its specific CrnCC construct (a.a. 1-600) on actin filaments. Using a combination of site-directed mutagenesis, solution biochemistry methods, electron and TIRF microscopy, we analyze the effects of coronin on the structure of actin filaments by studying its effect on inter-protomer contacts in F-actin. We compare coronin's effects on F-actin to the changes in filaments induced by cofilin. We also study how the two proteins act when they are present together, to shed light on the mechanism(s) by which coronin modulates cofilin's effects on actin filaments. Our results provide a plausible mechanism for the synergistic effect CrnCC has on the severing activity of cofilin. We find that coronin increases the binding of cofilin to the actin filament and also causes slight structural changes that promote severing by cofilin. Coronin-induced filament rigidity generates a bigger change in stiffness between cofilin free regions and those to which cofilin is bound. This added mechanical asymmetry causes the increase in cofilin's severing of actin filaments.