Regulation of axon growth and guidance by lipid rafts

在发育过程中，轴突的尖端，称为生长锥，表达调节轴突伸长和引导的各种功能分子。在细胞膜中，蛋白质和脂质不是均匀分布的，而是形成空间上差异化的微区，例如主要由胆固醇和鞘脂组成的脂筏。据报道，细胞粘附分子L1（一种参与轴突伸长的跨膜蛋白）定位于脂筏，并且生长锥外周中的筏对于轴突伸长是关键的（J Cell Biol 159：1097-1108，2002）。在这个项目中，我们进行了差异蛋白质组学研究，以寻找蛋白质，易位到或出脂筏响应配体结合L1表达的神经元在文化。这些研究确定了两种蛋白质，munc-18和突触素，这两种蛋白质都涉及膜胞吐的调节。因此，我们接下来研究了膜胞吐在轴突生长和引导中的作用。用FM 1 -43标记生长锥中的内吞囊泡，并监测它们的运动。我们发现，生长锥的转向是伴随着选择性运输的内吞囊泡面向新的方向。VAMP 2阳性胞吐感受态囊泡显示出类似的运动模式。我们还发现，运输的小泡在生长锥周边被胞吐。这项工作表明，生长锥转向优先供应膜组件面向新的方向的一面，并建议脂筏可能参与调节膜胞吐。

During development, the tip of axons, called the growth cone, expresses various functional molecules that regulate axon elongation and guidance. In cell membranes, proteins and lipids are not uniformly distributed but form spatially differentiated microdomains, such as lipid rafts composed predominantly of cholesterol and sphingolipids. It has been reported that the cell adhesion molecule L1, a transmembrane protein involved in axon elongation, is localized to lipid rafts and that rafts in the growth cone periphery is critical for axon elongation (J Cell Biol 159 : 1097-1108, 2002). In this project, we have conducted differential proteomic studies to search for proteins that translocate into or out of lipid rafts in response to ligand binding to L1 expressed on neurons in culture. These studies identified two proteins, munc-18 and synaptophysin, both of which are implicated in the regulation of membrane exocytosis. Therefore, we next investigated the role of membrane exocytosis in axon growth and guidance. Endocytic vesicles in growth cones were labeled with FM1-43 and their movement was monitored. We found that the growth cone turning is accompanied by selective transport of endocytic vesicles to the side facing the new direction. VAMP2-positive exocytosis-competent vesicles showed the similar pattern of movement. We also found that the transported vesicles are exocytosed at the growth cone periphery. This work has demonstrated that the growth cone turns by preferentially supplying membrane components to the side facing the new direction and suggested that lipid rafts could be involved in the regulation of membrane exocytosis.