Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA.
Biochem Biophys Res Commun. 2013 Sep 6;438(4):728-31. doi: 10.1016/j.bbrc.2013.07.109. Epub 2013 Aug 2.
Cofilin is an essential actin filament severing protein that functions in the dynamic remodeling of the actin cytoskeleton. Filament severing activity is most efficient at sub-stoichiometric cofilin binding densities (i.e. <1 cofilin per actin filament subunit), and peaks when the number density of boundaries (i.e. junctions) between bare and cofilin-decorated segments is maximal. A model in which local topological and mechanical discontinuities lead to preferential fragmentation at boundaries accounts for available experimental data, including direct visualization of cofilin and actin during real-time severing events. The boundary-severing model predicts that ligands (e.g. other actin-binding proteins) that compete with cofilin for actin filament binding and modulate cofilin occupancy on filaments will alter the bare-decorated segment boundary density, and thus, the filament severing activity of cofilin. Here, we directly test this model prediction by evaluating the effects of phalloidin and myosin, two ligands that compete with cofilin for filament binding, on the actin filament binding and severing activities of cofilin. Our experiments demonstrate that competitive displacement of cofilin lowers cofilin occupancy and promotes severing when initial cofilin occupancy is high (i.e. >50%). Even in the presence of competitive ligands, maximum severing activity occurs when cofilin-decorated boundary density is highest, consistent with preferential fragmentation at boundaries. We propose a general "severodyne" framework for the modulation of cofilin-mediated actin filament severing by small molecule or actin-binding protein ligands that compete with cofilin for actin filament binding.
丝切蛋白是一种必需的肌动蛋白丝断裂蛋白，在肌动蛋白细胞骨架的动态重塑中发挥作用。丝断裂活性在亚化学计量丝切蛋白结合密度（即<1 个丝切蛋白/肌动蛋白亚基）下效率最高，并且在裸露和丝切蛋白修饰段之间的边界（即连接）的数量密度最大时达到峰值。一种模型认为，局部拓扑和力学不连续性导致在边界处优先发生断裂，该模型解释了现有的实验数据，包括在实时断裂事件中直接观察丝切蛋白和肌动蛋白。边界断裂模型预测，与丝切蛋白竞争肌动蛋白丝结合并调节丝切蛋白在丝上占据的配体（例如其他肌动蛋白结合蛋白）将改变裸露-修饰段边界密度，从而改变丝切蛋白的丝断裂活性。在这里，我们通过评估与丝切蛋白竞争肌动蛋白丝结合的两种配体鬼笔环肽和肌球蛋白对丝切蛋白的肌动蛋白丝结合和断裂活性的影响，直接验证了该模型预测。我们的实验表明，当初始丝切蛋白占据率较高（即>50%）时，竞争取代丝切蛋白会降低丝切蛋白占据率并促进断裂。即使存在竞争配体，当丝切蛋白修饰的边界密度最高时，也会出现最大的断裂活性，这与在边界处优先断裂一致。我们提出了一种通用的“severodyne”框架，用于小分子或与丝切蛋白竞争肌动蛋白丝结合的肌动蛋白结合蛋白配体调节丝切蛋白介导的肌动蛋白丝断裂。
