Department of Molecular and Integrative Physiology, University of Illinois, Urbana, Illinois 61801.
Department of Cell and Developmental Biology, University of Illinois, Urbana, Illinois 61801.
J Biol Chem. 2012 Oct 12;287(42):35722-35732. doi: 10.1074/jbc.M112.396051. Epub 2012 Aug 17.
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.
快速肌动蛋白解聚对于细胞快速重组肌动蛋白丝网络是必要的。利用李斯特菌荧光肌动蛋白彗星尾实验来监测肌动蛋白解聚速率，我们观察到，尽管肌动蛋白解聚因子（丝切蛋白、冠状蛋白和肌动蛋白相互作用蛋白 1 的混合物足以在生理 G-肌动蛋白浓度存在的情况下分解肌动蛋白彗星尾，但该混合物不足以在生理 F-肌动蛋白浓度存在的情况下分解肌动蛋白彗星尾。通过生化互补，我们从胸腺提取物中纯化了环化酶相关蛋白 (CAP) 作为一种防止过量 F-肌动蛋白抑制的因子。CAP 已被证明参与肌动蛋白动力学，但被认为是通过将丝切蛋白从 ADP·G-肌动蛋白单体中释放出来恢复丝切蛋白活性来发挥作用的。然而，我们发现 CAP 通过加速丝切蛋白介导的切割来增强丝切蛋白介导的解聚。我们还证明 CAP 直接作用于 F-肌动蛋白，并在酸性但不是中性 pH 下切割肌动蛋白丝。在大多数哺乳动物细胞胞质中具有的中性 pH 条件下，我们证明 CAP 和丝切蛋白都不能切割肌动蛋白丝。然而，CAP 和丝切蛋白的组合在整个生理范围内的所有 pH 值下都能迅速切割肌动蛋白。因此，我们的结果揭示了 CAP 在加速丝切蛋白介导的肌动蛋白丝切割中的新功能，并提供了一种机制，通过该机制，细胞可以维持高肌动蛋白周转率，而不必使胞质碱化，这会影响除肌动蛋白解聚之外的许多生化反应。
