Investigating the Paxillin and Hic-5 Interactomes

Abstract

Focal adhesions are macromolecular structures that connect the cellular actin cytoskeleton to the surrounding extracellular matrix through transmembrane integrin receptors and the action of numerous proteins, including enzymes, signaling proteins, and scaffolding proteins. Paxillin and Hic-5 are two scaffolding adapter proteins that primarily localize to focal adhesions, as well as other intracellular regions including the nucleus and centrosome. Their signaling partners at focal adhesions and effects on the actin cytoskeleton have been well-characterized over the course of decades through rigorous biochemical studies, but broader analysis and comparison of the interactomes of these two closely related proteins has not yet been thoroughly pursued. In the introduction, recently described roles for paxillin and Hic-5 in regulating cell shape and invadopodia through actin dynamics will be described, in addition to recent discoveries regarding their interaction with the microtubule and intermediate filament cytoskeletons. This background will provide context to data presented in chapter two, in which paxillin and Hic-5 were used as baits for proximity labeling to compare their interactomes, to identify numerous potentially novel interactors for both proteins, and to confirm many previously known partners. Two interesting results of this analysis include a possible proximity interaction between both paxillin and Hic-5 with septin-7, and confirmation of a robust proximity reaction between paxillin and ponsin. In chapter three, another newly discovered interactor with paxillin will be characterized: the formin mDia1. These proteins are shown to bind in vitro and co-localize in vivo. Most interestingly, paxillin relieves mDia1 auto-inhibition to accelerate actin polymerization in in vitro TIRF microscopy assays. Finally, the significance of these results and avenues for future investigation will be discussed, including further confirmation of proximity interactors and experiments to better understand the mechanism by which paxillin interacts with mDia1.