Regulation of neuronal motility

描述(摘自申请者摘要)：生长中神经元的指导 发育中的胚胎是建立神经元的关键步骤 连接，这是神经系统功能的要求。 虽然大量的引导分子和它们的 神经元受体已被识别，但在细胞水平上知之甚少。 关于受体激活如何引起细胞形状的变化 引导轴突生长所必需的。神经细胞黏附受体的研究进展 其他迁移细胞表明，细胞牵引力的产生 通过细胞与环境接触的部位的作用力可能起到中心作用 在调节细胞移位中的作用。这项提案的总体目标是界定 生长中的神经元产生牵引力的机制， 以神经元免疫球蛋白L1为黏附模型 受体。我们的初步数据表明，L1家庭成员与 细胞骨架中产生力量的组件。基于这一观察，我们 提出三个主要目标：1)明确监管和结构 L1-细胞骨架相互作用的要求和由此产生的细胞 牵引力。2)量化L1在一个位点的磷酸化动力学。 已知的调节细胞骨架连接物结合的细胞质尾巴 蛋白粘蛋白B。3)在基质上进行轴突生长的体外实验 涂有L1配体以确定结构域如何识别为必需的 L1介导的轴突生长需要上述1和2中的TO L1功能。vbl.使用 高分辨率视频显微镜和光学梯度激光器的组合 捕捉(激光镊子)以拉出绑定在细胞表面L1上的微珠，我们 可以监测L1功能的调节。这些方法允许 L1-细胞分裂素相互作用的高时空检测 准确性，使我们能够直接检查L1函数是如何由 配体活化、交联化和磷酸化。该基因的突变 在人类中编码L1会导致一系列复杂的神经疾病，包括 痉挛性截瘫和智力低下，表明L1起着 在神经发育中起着重要作用。通过刻画 在细胞和生物物理水平上的L1功能，我们可以开始发展一个 详细了解定向神经元生长的潜在机制。 此外，我们从研究黏附受体中获得的信息 神经元可能会帮助我们理解细胞在不同环境中的迁移 其他系统，从免疫系统功能到肿瘤细胞转移。

DESCRIPTION (From the Applicant's Abstract): The guidance of growing neurons in the developing embryo is an essential step in the establishment of neuronal connections, a requirement for nervous system function. Although a large number of guidance molecules and their neuronal receptors have been identified, little is known at the cellular level about how receptor activation gives rise to the changes in cell shape that are required for guided axon growth. The study of adhesion receptors in neurons and other migrating cells has suggested that the generation of cellular traction forces through sites of cell contact with the environment may play a central role in the regulation of cell translocation. The overall aim of this proposal is to define the mechanism underlying the generation of traction forces in the growing neuron, focusing on the neuronal immunoglobulin (Ig) protein L1 as a model adhesion receptor. Our preliminary data suggest that L1 family members interact with force-generating components of the cytoskeleton. Based on this observation, we propose three major aims: 1) To characterize the regulation and structural requirements of L1-cytoskeleton interactions and the resulting cellular traction forces. 2) To quantify the kinetics of L1 phosphorylation at a site in the cytoplasmic tail known to regulate the binding of the cytoskeleton linker protein ankyrinB. 3) To use in vitro neurite outgrowth assays on substrates coated with L1 ligands to determine how domains identified as being essential to L1 function in 1 and 2 above are required for L1-mediated axon growth. Using a combination of high-resolution video microscopy and an optical gradient laser trap (laser tweezers) to pull on microscopic beads bound to cell-surface L1, we can monitor the regulation of L1 function. These approaches permit the detection of L1-cytosksleton interactions with high spatial and temporal accuracy, allowing us to examine directly how L1 function is modulated by ligand activation, crosslinking and phosphorylation. Mutations in the gene encoding L1 in humans leads to a complex of neurological disorders including spastic paraplegia and mental retardation, suggesting that L1 plays an important role in neural development. By characterizing L1 function at a cellular and biophysical level, we can begin to develop a detailed understanding of the mechanisms underlying directed neuronal growth. Moreover, the information that we gain from studying adhesion receptors in neurons is likely to inform our understanding of cell migration in a variety of other systems, ranging from immune system function to tumor cell metastasis.