Abstract
Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.
Highlights
Cytosolic pH blocks actin disassembly in vitro
Coronin, and actin-interacting protein 1 (AIP1) Are Insufficient to Disassemble Actin in the Presence of Excess F-actin—We have previously demonstrated that cofilin, coronin, and AIP1 are each necessary and together are sufficient to disassemble the actin comet tails assembled by L. monocytogenes in our perfusion chamber setup despite the presence of a physiological excess of G-actin [13]
Cofilin is necessary for actin filament disassembly, it is increasingly clear that cofilin alone cannot account for actin disassembly in cells where high concentrations of both polymerizable G-actin and F-actin in addition to neutral pH either favor actin assembly or limit cofilin activity
Summary
Cytosolic pH blocks actin disassembly in vitro. Results: CAP actively participates in actin disassembly, augmenting cofilin activity across the physiological pH range. Our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization. Actin-depolymerizing factor/ cofilin, cofilin [10], has been shown to be necessary to disassemble physiological branched actin arrays in cell extracts [6, 11] and actin filaments in pure solution [12] but is insufficient to reconstitute physiological actin disassembly both qualitatively in terms of the ability to function in the presence of high concentrations of actin and quantitatively in terms of observed disassembly rate. Coronin and actin-interacting protein 1 (AIP1) were identified as factors necessary for actin depolymerization in the presence of physiological concentrations of G-actin [13]. Listeria comet tails offer a physiological actin substrate for the in vitro study of cellular mechanisms of actin
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