Tseng Y, Fedorov E, McCaffery J M, Almo S C, Wirtz D
Department of Chemical Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
J Mol Biol. 2001 Jul 6;310(2):351-66. doi: 10.1006/jmbi.2001.4716.
Fascin is an actin crosslinking protein that organizes actin filaments into tightly packed bundles believed to mediate the formation of cellular protrusions and to provide mechanical support to stress fibers. Using quantitative rheological methods, we studied the evolution of the mechanical behavior of filamentous actin (F-actin) networks assembled in the presence of human fascin. The mechanical properties of F-actin/fascin networks were directly compared with those formed by alpha-actinin, a prototypical actin filament crosslinking/bundling protein. Gelation of F-actin networks in the presence of fascin (fascin to actin molar ratio >1:50) exhibits a non-monotonic behavior characterized by a burst of elasticity followed by a slow decline over time. Moreover, the rate of gelation shows a non-monotonic dependence on fascin concentration. In contrast, alpha-actinin increased the F-actin network elasticity and the rate of gelation monotonically. Time-resolved multiple-angle light scattering and confocal and electron microscopies suggest that this unique behavior is due to competition between fascin-mediated crosslinking and side-branching of actin filaments and bundles, on the one hand, and delayed actin assembly and enhanced network micro-heterogeneity, on the other hand. The behavior of F-actin/fascin solutions under oscillatory shear of different frequencies, which mimics the cell's response to forces applied at different rates, supports a key role for fascin-mediated F-actin side-branching. F-actin side-branching promotes the formation of interconnected networks, which completely inhibits the motion of actin filaments and bundles. Our results therefore show that despite sharing seemingly similar F-actin crosslinking/bundling activity, alpha-actinin and fascin display completely different mechanical behavior. When viewed in the context of recent microrheological measurements in living cells, these results provide the basis for understanding the synergy between multiple crosslinking proteins, and in particular the complementary mechanical roles of fascin and alpha-actinin in vivo.
Fascin是一种肌动蛋白交联蛋白，它将肌动蛋白丝组织成紧密排列的束状结构，据信这种结构介导细胞突起的形成，并为应力纤维提供机械支持。我们使用定量流变学方法，研究了在人Fascin存在下组装的丝状肌动蛋白（F-肌动蛋白）网络的力学行为演变。将F-肌动蛋白/Fascin网络的力学性能与由典型的肌动蛋白丝交联/成束蛋白α-辅肌动蛋白形成的网络的力学性能进行了直接比较。在Fascin存在下（Fascin与肌动蛋白的摩尔比>1:50），F-肌动蛋白网络的凝胶化表现出非单调行为，其特征是弹性突然增加，随后随时间缓慢下降。此外，凝胶化速率对Fascin浓度表现出非单调依赖性。相比之下，α-辅肌动蛋白单调地增加了F-肌动蛋白网络的弹性和凝胶化速率。时间分辨多角度光散射以及共聚焦和电子显微镜表明，这种独特行为一方面是由于Fascin介导的肌动蛋白丝和成束的交联与侧支化之间的竞争，另一方面是由于肌动蛋白组装延迟和网络微不均匀性增强。F-肌动蛋白/Fascin溶液在不同频率振荡剪切下的行为模拟了细胞对以不同速率施加的力的反应，这支持了Fascin介导的F-肌动蛋白侧支化的关键作用。F-肌动蛋白侧支化促进相互连接网络的形成，这完全抑制了肌动蛋白丝和成束的运动。因此，我们的结果表明，尽管α-辅肌动蛋白和Fascin具有看似相似的F-肌动蛋白交联/成束活性，但它们表现出完全不同的力学行为。从最近在活细胞中进行的微观流变学测量的背景来看，这些结果为理解多种交联蛋白之间的协同作用提供了基础，特别是Fascin和α-辅肌动蛋白在体内的互补力学作用。
