Cyclase associated protein (CAP) and the physiological disassembly of actin

Abstract

Actin dynamics are important for both driving cellular movement and maintaining cellular structure in apparently static tissues. Actin assembly and disassembly is an energy driven cycle that is utilized by cells to generate force and organize space. Though studied to some extent by a host of fields, the study of the actin cytoskeleton itself is an area with many questions left to be answered, such as how cells disassemble actin in the presence of large concentrations of both monomeric and polymeric actin, how apparently very stable actin structures can be disassembled, and what role actin plays in the nucleus and how this role differs from cytosolic actin dynamics. Using a biochemical reconstitution approach, we set out to find one or more factors that conferred actin disassembly activity to the known actin disassembly factors. Once identified as cyclase associated protein (CAP), we next studied the properties of this factor focusing on its interactions with actin and other actin disassembly factors including cofilin, coronin, and AIP1. We discovered that CAP has a complimentary yet synergistic relationship with cofilin, a partially redundant relationship with coronin, and that CAP can act as an independent actin disassembly factor at low pH. While CAP was a known actin interacting protein, none of these findings were known to the field before our work was published. After an introduction and summary of the state of the field as it was when we started, Chapter 2 begins with the initial characterization of CAP and our efforts to determine its function. We soon realized that the field was mistaken about the role of CAP as an accessory protein not truly involved in actin disassembly, and we showed that CAP accelerates cofilin, coronin and AIP1-mediated actin depolymerization. We then demonstrated a partial redundancy to coronin but showed that the underlying mechanism of CAP-mediated actin disassembly was distinct from that of coronin. Next we set out to discover what the precise role of CAP was through two similar but distinct lines of experiments. In Chapter 3 we study CAP using similar methodologies but with single actin filaments instead of the branched actin networks in the actin comet tails formed by L monocytogenes. This allowed us to more precisely control the experimental conditions while also giving information of single filament off-rates and allowed us to determine the interaction between CAP, cofilin and pH. In Chapter 4 we continue to study branched actin networks formed by L monocytogenese, but formed under defined conditions without any cell extract. This work was designed to allow us to determine whether there might be activities of CAP which were geometrically dependent, such as any activity confined to branch points. What we found was that actin filaments built with ena/vasp-like protein (EVL) were more susceptible to CAP disassembly. Finally in Chapter 5 we offer a few concluding remarks about the state of the field and the recurrent sense of premature accomplishment that it is prone to

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