Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan.
Graduate School of Information Science and Technology, Hokkaido University, Sapporo, Japan.
Genes Cells. 2019 Mar;24(3):202-213. doi: 10.1111/gtc.12671.
Fascin, an actin-bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super-resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well-known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.
Fascin 是一种肌动蛋白成束蛋白，存在于生长锥的丝状伪足和片状伪足中。然而，很少有研究检查片状伪足中的 fascin，因为它很难观察。在这项研究中，我们评估了片状伪足中的 fascin。我们通过超分辨率显微镜可视化了活生长锥中片状伪足的肌动蛋白网格。Fascin 与片状伪足中的肌动蛋白网格共定位。Fascin 的 Ser39 是一个众所周知的磷酸化位点，控制 fascin 与肌动蛋白丝的结合。共聚焦显微镜的光漂白后荧光恢复实验表明，Ser39 的磷酸化控制了 fascin 在片状伪足中的结合。此外，TPA，蛋白激酶 C 的激动剂，诱导 fascin 在片状伪足中的磷酸化和与肌动蛋白丝的解离。时间序列图像显示，TPA 诱导 fascin 从肌动蛋白网格解离。随着 fascin 从肌动蛋白丝解离，肌动蛋白丝的方向变得与前缘平行。肌动蛋白丝与前缘的夹角从 73°变为 15°。这通过原子力显微镜测量，使片状伪足的弹性降低了 40%。这些数据表明 fascin 形成的肌动蛋白束有助于生长锥的弹性。
