Rapid remodeling of the actin cytoskeleton is essential for many cellular processes including cell growth, differentiation, division, and motility. Actin dynamics is expedited by various actin-binding proteins, with Spire and cofilin being prominent among them. In vitro, Spire and cofilin are able to sever, nucleate, and depolymerize filaments, albeit by different methods. This dissertation focuses on characterizing the mechanisms of Spire and cofilin interactions with actin and studying their roles in actin dynamics.
We first study the complex role of Spire in actin dynamics. We observe that the severing activity of Spir is weak and conclude that rapid actin depolymerization is mainly due to the sequestering activity of Spir. Polymerization assays show that Spir and actin form complexes that accelerate polymerization, when present at low stoichiometries, but suggest their heterogeneity. Notably, Spir does not bind readily four actin monomers in a stable complex. Similarly, depolymerization of actin by Spir leads to the formation of several types of their complexes. Strikingly, as detected by cross-linking experiments, Spir induces the formation of lateral actin-actin complexes in addition to the expected longitudinal complexes. The formation of these structures is not affected by the presence of Cappuccino. Finally, using yeast actin mutants labeled with fluorescent probes, we detected different affinities of Spir domains for actin protomers. Together, our data suggest that Spir-actin interactions are more complex than originally believed.
Next, we examined the effects of cofilin isoforms on the dynamics of actin isoforms. First, we studied the change in the persistence length of both vertebrate and yeast F-actin upon binding of human and yeast cofilin. We observe that human cofilin-1 (hCof1) binds to yeast actin filaments but neither increases their flexibility nor severs them. In contrast to that, yeast cofilin increases the flexibility of skeletal and yeast actin filaments and severs them efficiently, confirming the correlation of severing activity with changes in filament flexibility. We further observe that although yeast cofilin is an effective severer of yeast actin at low ratios of cofilin to actin, hCof1 severs yeast actin only when added in excess, despite changing their twist similarly to yeast cofilin. This contradicts previous observations that severing occurs only, or mainly, under subsaturating conditions. Our results raise questions about the role of actin isoforms in their severing by cofilin and the specific actin-cofilin contacts that contribute to this activity. Lastly, actin cross-linkings with benzophenone-4-maleimide (BPM) reveal two types of structural transitions: one associated with actin polymerization into filaments, and the other coupled to coflin binding to F-actin. We map the intramolecular cross-link in F-actin to Cys374 and Asp11 and the intermolecular one, due to cofilin binding, to Cys374 and Met44. We also report differences in dissociation rates of cofilin from cross-linked and uncross-linked F-actin, showing the role of filament flexibility in cofilin dissociation. Cofilin-induced changes may help elucidate the local and global changes in F-actin that destabilize specific interprotomer contacts.
Together, our work on Spir and cofilin provides insight into the mechanisms of their interactions with actin and the roles they play in actin dynamics.