Actin Cytoskeleton Dynamics Through the Lens of Disassembly
Creators: name: Alimov, Nikita
Publisher: name: Brandeis University
Year: 2023
Subjects: Actin disassembly
Actin Dynamics
Cyclace Associated Protein
Depolymerization
Treadmilling
Turnover

description: Actin is an essential protein that drives many cellular processes, including muscle contraction, cell motility, polarized growth, cytokinesis, intracellular transport, and endocytosis. Actin structures in these processes are found in distinct networks that depend on spatiotemporal rearrangements of the underlying filaments. This reorganization is accomplished by dozens of actin binding proteins, which work in concert to control actin filament nucleation, growth, disassembly, and spatial organization. The combined actions of these proteins tune the shape and dynamics of actin networks for their specific cellular functions. The different actin structures found in cells have characteristic sizes and filamentous architectures which must not only be assembled with spatiotemporal precision, but also maintained in different states of flux (or subunit turnover) to retain their plasticity and to provide sustained force to drive membrane remodeling and other functions. Turnover is achieved through a delicate balance of filament assembly, disassembly, and subunit recycling. In Chapter 2, I describe a new activity for one of these factors, mouse cyclase-associated protein 1 (CAP1), in regulating dynamics at the barbed ends of actin filaments alone and together with two other actin binding proteins, profilin and formins. I demonstrate that in the absence of actin monomers full-length CAP1 and the C-terminal half of CAP1 (C-CAP1) both accelerate barbed end depolymerization, and that they do so independent of the nucleotide state of the actin filament. Through mutagenesis, I show that the actin-binding CARP and WH2 domains in C-CAP1 are both required for this activity, and using structural modeling I propose a working model for the underlying molecular mechanism by which CAP promotes barbed end depolymerization. I also show that CAP1 directly collaborates with profilin to accelerate barbed end depolymerization, and that their additive effects at the barbed end depend on CAP1-profilin interactions. Lastly, in the presence of actin monomers, I show that CAP1 attenuates barbed end growth, and promotes dissociation of formins from barbed ends, solidifying CAP’s role as a barbed end-associated actin regulator.
description: In Chapter 3, in a separate but thematically related set of experiments I probe the molecular mechanism by which three other actin-binding proteins (cofilin, coronin, and AIP1, which we refer to as ‘CCA’), promote actin disassembly. I present preliminary evidence that AIP1 promotes depolymerization of pointed ends of filaments in addition to its known functions in promoting cofilin-mediated severing and barbed end capping. Further, I demonstrate that intact, oligomeric coronin is more effective in recruiting cofilin to the sides of actin filaments than monomeric coronin, and I define the dwell time of full-length coronin single molecules on the sides of actin filaments. Lastly, I address how the nucleotide state of actin filaments affects the CCA mechanism.
description: Collectively, these results expand our mechanistic understanding of how filamentous actin networks are turned over in cells through the combined activities of actin binding proteins such as CAP1, cofilin, coronin and AIP1.