Laboratory of Systems Neurobiology and Medicine, Division of Biological Science, Nara Institute of Science and Technology;
Laboratory of Data-Driven Biology, Division of Biological Science, Nara Institute of Science and Technology.
J Vis Exp. 2021 Oct 21(176). doi: 10.3791/63227.
To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.
为了建立功能网络，神经元必须迁移到它们的适当目的地，然后将轴突延伸到它们的靶细胞。这些过程依赖于位于神经突尖端的生长锥的推进。轴突生长锥通过感知其局部微环境并调节细胞骨架动力学和肌动蛋白-黏附偶联（离合器偶联）来产生驱动力。几十年来的研究已经导致了识别导向分子、它们的受体和下游信号级联，以调节神经元迁移和轴突导向；然而，用于产生驱动力来驱动生长锥前进和导航的分子机制才刚刚开始被阐明。在神经元生长锥的前缘，肌动蛋白丝经历逆行流动，这是由肌动蛋白聚合和肌球蛋白收缩提供动力的。F-肌动蛋白逆行流动和黏附基质之间的离合器偶联产生了生长锥前进的牵引力。本研究描述了一种通过单斑点成像监测 F-肌动蛋白逆行流动的详细方案。重要的是，当与 F-肌动蛋白标记物 Lifeact 结合使用时，该技术可以定量 1）F-肌动蛋白聚合率和 2）F-肌动蛋白逆行流动和黏附基质之间的离合器偶联效率。这两者都是产生生长锥前进和导航力的关键变量。此外，本研究描述了一种牵引力显微镜的详细方案，该方案可以定量 3）生长锥产生的牵引力。因此，通过将单斑点成像和牵引力显微镜分析相结合，研究人员可以监测生长锥前进和导航的分子力学。
