Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
J Cell Sci. 2020 Apr 24;133(8):jcs232983. doi: 10.1242/jcs.232983.
Previously, we have shown that bulk microtubule (MT) movement correlates with neurite elongation, and blocking either dynein activity or MT assembly inhibits both processes. However, whether the contributions of MT dynamics and dynein activity to neurite elongation are separate or interdependent is unclear. Here, we investigated the underlying mechanism by testing the roles of dynein and MT assembly in neurite elongation of and chick neurites using time-lapse imaging, fluorescent speckle microscopy, super-resolution imaging and biophysical analysis. Pharmacologically inhibiting either dynein activity or MT assembly reduced neurite elongation rates as well as bulk and individual MT anterograde translocation. Simultaneously suppressing both processes did not have additive effects, suggesting a shared mechanism of action. Single-molecule switching nanoscopy revealed that inhibition of MT assembly decreased the association of dynein with MTs. Finally, inhibiting MT assembly prevented the rise in tension induced by dynein inhibition. Taken together, our results suggest that MT assembly is required for dynein-driven MT translocation and neurite outgrowth.
先前，我们已经表明，大量微管 (MT) 运动与轴突伸长相关，阻断动力蛋白活性或 MT 组装均会抑制这两个过程。然而，MT 动力学和动力蛋白活性对轴突伸长的贡献是独立的还是相互依赖的尚不清楚。在这里，我们通过使用延时成像、荧光斑点显微镜、超分辨率成像和生物物理分析，研究了动力蛋白和 MT 组装在 和鸡神经元突起伸长中的作用机制。用药理学方法抑制动力蛋白活性或 MT 组装均降低了轴突伸长率以及整体和单个 MT 顺向转运。同时抑制这两个过程没有累加效应，表明它们作用机制相似。单分子开关纳米显微镜显示，抑制 MT 组装会减少动力蛋白与 MT 的结合。最后，抑制 MT 组装会阻止因动力蛋白抑制而引起的张力增加。总之，我们的结果表明，MT 组装对于动力蛋白驱动的 MT 转运和轴突生长是必需的。
