GAP-43 FUNCTION BY GENETIC MANIPULATION

神经系统特有的“生长相关”的几个特征 GAP-43蛋白提示其在细胞生物学中起重要作用 轴突发生和突触前功能的共同过程： 在脊椎动物进化过程中高度保守，在所有 发育中的神经元：GAP-43水平被显著诱导 在轴突生长期间，但值得注意的是，在损伤后不会重新诱导 不能成功再生的神经元轴突：高度 富含在与膜结合的生长锥体中 骨骼，负责调节形状变化的结构， 是对细胞外引导信号的关键反应：最后，GAP-43是 几种钙依赖酶的靶标，包括那些对 细胞外信号和磷酸化GAP-43水平与 生长锥体的功能状态。所有这些特征都是一致的 GAP-43在细胞外信号整合中的作用 生长锥体，并表明理解 GAP-43的功能将进一步加深我们对 轴突直接生长。一种研究GAP-43功能的有效方法 分子水平是创造一个纯合子零突变细胞系，在其中 GAP-43转录被阻止，然后比较其表型 进一步转染GAP-43cDNA的细胞发生突变 特定的功能部位。为此，我们孤立了 鉴定了小鼠GAP-43的基因组克隆，并用它构建了 通过以下方式敲除GAP-43基因的替换载体 同源重组。我们将把该构造引入 多能胚胎癌细胞系P19分化为 用维甲酸治疗胆碱能样神经元，并将选择 纯合子FOP对GAP-43转录的干扰 对新霉素类似物G418的耐药性。然后，使用一系列具体的 我们开发的检测方法是为了研究GAP-43在生长锥中的作用 功能，我们将对零突变P19细胞的表型进行鉴定 关于层粘连蛋白的附着，轴突生长的调节和 膜骨架和细胞骨架的组织。最后我们会 用突变的GAP-43cDNA转染P19细胞 磷酸化和钙调蛋白结合将会异常，并使用这些 转染细胞以评估磷酸化的相对贡献 GAP-43和钙调蛋白促进轴突生长。这些项目所产生的结果 实验将提供对我们的 了解轴突发生在发育和发育过程中是如何调节的 再生，并将使我们能够开始解决 轴突在多大程度上需要结构正常才能形成 功能性突触。

Several features of the nervous system specific 'growth-associated' protein GAP-43 suggest that it plays a fundamental role in cell biological processes common to axonogenesis and presynaptic function: It has been highly conserved during vertebrate evolution and is expressed in all neurons during development: Levels of GAP-43 are dramatically induced during axon outgrowth but, significantly, are not re-induced in injured axons of neurons that do not regenerate successfully: It is highly enriched in the growth cone where it associates with the membrane skeleton, the structure responsible for regulating the shape changes that are a crucial response to extracellular guidance cues: Finally, GAP-43 is a target for several Ca2+-dependent enzymes, including those responsive to extracellular signals, and levels of phosphorylated GAP-43 correlate with functional states of the growth cone. All of these features are consistent with a role for GAP-43 in the integration of extracellular signals within the growth cone, and suggest that understanding the molecular basis for GAP-43 function will further our understanding of the processes that direct axon growth. One powerful way to investigate GAP-43 function at the molecular level is to create a homozygous null mutant cell line in which transcription of GAP-43 is prevented, and then to compare its phenotype with cells that have further been transfected with GAP-43 cDNAs mutated at specific functional sites. Toward this end we have isolated and characterized a murine genomic clone for GAP-43 and used it to construct a replacement vector to knock out the GAP-43 gene at its native locus by homologous recombination. We will introduce the construct into the pluripotent embryonal carcinoma cell line P19 which differentiates into cholinergic-like neurons on treatment with retinoic acid, and will select lines homozygous fop disruption of GAP-43 transcription by their resistance to G418, a neomycin analog. Then, using a series of specific assays we have developed to investigate the role of GAP-43 in growth cone function, we will characterize the phenotype of the null mutant P19 cells with respect to attachment to laminin, regulation of neurite outgrowth and organization of the membrane skeleton and cytoskeleton. Finally we will transfect the P19 cells with GAP-43 cDNAs that have been mutated so that phosphorylation and calmodulin binding will be abnormal, and use these transfected cells to assess the relative contributions of phosphorylated GAP-43 and calmodulin to neurite outgrowth. The results generated by these experiments will provide information fundamentally important to our understanding of how axonogenesis is regulated during development and regeneration, and will enable us to begin to address the question of to what extent an axon needs to be structurally normal in order to form functional synapses.