Grabham Peter W, Reznik Boris, Goldberg Daniel J
Department of Pharmacology and Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA.
J Cell Sci. 2003 Sep 15;116(Pt 18):3739-48. doi: 10.1242/jcs.00686. Epub 2003 Jul 30.
Extracellular cues control the rate and direction of growth of neuronal processes in large part by regulating the cytoskeleton of the growth cone. The actin filament network of the peripheral region is thought to be the primary target for these cues, with consequences for the advance and organization of microtubules. Binding of laminin to integrin receptors is a cue that accelerates the growth of processes from many types of neurons. It was applied acutely to sympathetic neurons in culture to study its effects on the cytoskeleton of the growth cone. Microtubules advance to the edge of the growth cone and bundle in response to laminin, and it was found that small veils of membrane appear near the ends of some of those microtubules. To examine more clearly the relationship between the microtubules and the appearance of actin-rich structures at the periphery, a low dose of cytochalasin D was used to deplete the peripheral region of the growth cone of pre-existing F-actin. The subsequent addition of laminin resulted in the bundling of ends of dynamic (tyrosinated) microtubules at the distal edge of the growth cone, most of which were associated with foci of F-actin. Observations of labeled actin within living growth cones confirmed that these foci formed in response to laminin. Suppression of microtubule dynamics with drugs eliminated the actin foci; washout of drug restored them. Rac 1 did not co-concentrate with F-actin in the peripheral region of the growth cone in the absence of laminin, but did co-concentrate with the foci of F-actin that formed in response to laminin. Inhibition of Rac 1 functioning prevented the formation of the foci and also inhibited laminin-induced neurite growth with or without cytochalasin. These results indicate that extracellular cues can affect actin in the growth cone via microtubules, as well as affect microtubules via actin. They also point to the mediation of microtubule-dependent accumulation of F-actin at the front of the growth cone as a role of Rac 1 in neurite growth.
细胞外信号在很大程度上通过调节生长锥的细胞骨架来控制神经元突起生长的速率和方向。外周区域的肌动蛋白丝网络被认为是这些信号的主要靶点，其结果会影响微管的推进和组织。层粘连蛋白与整合素受体的结合是一种能加速多种类型神经元突起生长的信号。将其急性应用于培养的交感神经元，以研究其对生长锥细胞骨架的影响。微管会响应层粘连蛋白而推进到生长锥边缘并形成束状，并且发现在其中一些微管的末端附近会出现小的膜状结构。为了更清楚地研究微管与外周富含肌动蛋白结构的出现之间的关系，使用低剂量的细胞松弛素D来耗尽生长锥外周区域预先存在的F-肌动蛋白。随后添加层粘连蛋白导致动态（酪氨酸化）微管的末端在生长锥的远端边缘形成束状，其中大多数与F-肌动蛋白的聚集点相关。对活生长锥内标记肌动蛋白的观察证实，这些聚集点是响应层粘连蛋白而形成的。用药物抑制微管动力学消除了肌动蛋白聚集点；药物洗脱后又恢复了它们。在没有层粘连蛋白的情况下，Rac 1在生长锥外周区域不与F-肌动蛋白共聚集，但与响应层粘连蛋白形成的F-肌动蛋白聚集点共聚集。抑制Rac 1的功能可防止聚集点的形成，并且在有或没有细胞松弛素的情况下也抑制层粘连蛋白诱导的神经突生长。这些结果表明，细胞外信号可以通过微管影响生长锥中的肌动蛋白，也可以通过肌动蛋白影响微管。它们还指出，Rac 1在神经突生长中的作用是介导微管依赖的F-肌动蛋白在生长锥前端的积累。
