Moseley James B, Okada Kyoko, Balcer Heath I, Kovar David R, Pollard Thomas D, Goode Bruce L
Department of Biology and The Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454, USA.
J Cell Sci. 2006 Apr 15;119(Pt 8):1547-57. doi: 10.1242/jcs.02860. Epub 2006 Mar 28.
Working in concert, multiple actin-binding proteins regulate the dynamic turnover of actin networks. Here, we define a novel function for the conserved actin-binding protein twinfilin, which until now was thought to function primarily as a monomer-sequestering protein. We show that purified budding yeast twinfilin (Twf1) binds to and severs actin filaments in vitro at pH below 6.0 in bulk kinetic and fluorescence microscopy assays. Further, we use total internal reflection fluorescence (TIRF) microscopy to demonstrate that Twf1 severs individual actin filaments in real time. It has been shown that capping protein directly binds to Twf1 and is required for Twf1 localization to cortical actin patches in vivo. We demonstrate that capping protein directly inhibits the severing activity of Twf1, the first biochemical function ascribed to this interaction. In addition, phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] inhibits Twf1 filament-severing activity. Consistent with these biochemical activities, a twf1Delta mutation causes reduced rates of cortical actin patch turnover in living cells. Together, our data suggest that twinfilin coordinates filament severing and monomer sequestering at sites of rapid actin turnover and is controlled by multiple regulatory inputs.
多种肌动蛋白结合蛋白协同作用，调节肌动蛋白网络的动态更新。在此，我们定义了保守的肌动蛋白结合蛋白双肌动蛋白的一种新功能，此前人们一直认为它主要作为一种单体隔离蛋白发挥作用。我们发现在体外，在pH低于6.0的条件下，通过大量动力学和荧光显微镜检测，纯化的出芽酵母双肌动蛋白（Twf1）能结合并切断肌动蛋白丝。此外，我们使用全内反射荧光（TIRF）显微镜来证明Twf1能实时切断单个肌动蛋白丝。研究表明，封端蛋白直接与Twf1结合，并且是Twf1在体内定位于皮质肌动蛋白斑所必需的。我们证明封端蛋白直接抑制Twf1的切断活性，这是首次将这种相互作用归因于生化功能。此外，磷脂酰肌醇（4,5）-二磷酸[PtdIns(4,5)P2]抑制Twf1的丝切断活性。与这些生化活性一致，twf1Delta突变导致活细胞中皮质肌动蛋白斑更新速率降低。总之我们的数据表明，双肌动蛋白在肌动蛋白快速更新位点协调丝切断和单体隔离，并受多种调节输入的控制。
