GAP-43 FUNCTION BY GENETIC MANIPULATION

神经系统特异性“生长相关”的几个特征 GAP-43蛋白在细胞生物学中起着重要作用， 轴突发生和突触前功能共同的过程：它已经被 在脊椎动物进化过程中高度保守，并在所有 神经元在发育过程中：GAP-43的水平显着诱导 在轴突生长过程中，但重要的是， 不能成功再生的神经元轴突： 在生长锥中富集， 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Ca 2+依赖性酶的靶点，包括那些对 细胞外信号，磷酸化GAP-43水平与 生长锥的功能状态。所有这些特征都是一致的 GAP-43在细胞内整合细胞外信号中的作用 生长锥，并建议理解的分子基础， GAP-43功能将进一步加深我们对 轴突直接生长研究GAP-43功能的一种有效方法是 在分子水平上的目的是创建纯合无效突变细胞系，其中 GAP-43的转录被阻止，然后比较其表型 用进一步用突变为 具体功能部位。为此，我们隔离了 表征了GAP-43的鼠基因组克隆，并将其用于构建 在GAP-43基因的天然基因座敲除GAP-43基因的替换载体， 同源重组我们将把这个结构引入到 多能胚胎癌细胞系P19，其分化为 胆碱能样神经元的治疗与视黄酸，并将选择 株系纯合，通过它们的基因突变破坏GAP-43转录。 对新霉素类似物G418的抗性。然后，使用一系列特定的 我们已经开发了研究GAP-43在生长锥中的作用的试验， 功能，我们将描述无效突变P19细胞的表型 关于与层粘连蛋白的附着，神经突生长的调节， 膜骨架和细胞骨架的组织。最后我们将 用突变的GAP-43 cDNA转染P19细胞， 磷酸化和钙调蛋白结合将是异常的，并使用这些 转染的细胞，以评估磷酸化的相对贡献， GAP-43和钙调素对神经突生长的影响。这些产生的结果 实验将为我们提供重要的信息， 了解轴突发生在发育过程中是如何调节的， 再生，并将使我们能够开始解决的问题， 一个轴突需要在结构上正常到什么程度才能形成 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.