MOLECULAR BIOLOGY OF NEURAL FUNCTION

我们对促进突触运输的蛋白质感兴趣 沿着轴突形成小泡，然后在突触前膜融合。 其中包括细胞内的马达、激酶和细胞骨架。 蛋白质。目前在实验室研究的蛋白质包括：Kinesin 轻链(KLC)、NSEC-1和酸性钙蛋白(AC)。此前，我们 从乌贼的神经系统中鉴定出一个KLC家族 努力确定其在动蛋白功能中的作用。目前还不清楚 KLc是否在激动素功能或靶向中发挥调节作用 对膜结合细胞器的作用。我们报告说，至少有18人 不同的异构体作为选择性剪接发生的结果出现 几乎只在分子的羧基末端区域 被认为是动蛋白靶向于膜结合的视线 细胞器。此外，我们还发现KLC转录本数量和 在被检查的不同组织中的水平不同，进一步支持 KLC为不同膜提供靶向功能的假说 在不同细胞类型中结合的细胞器。然而，最近，我们 报道了KLC的鉴定和鉴定 和未鉴定出相应的一种 重链提示KLC可能具有一些与 囊泡运输。酸性钙蛋白是一种肌动蛋白结合蛋白，具有 独特的羧基末端尾部结构域，至少具有三个 调节域：一个共同的酪氨酸磷酸化位点，两个 假定的P-环元件，整个尾部是一个PEST域。在 在过去的一年里，我们集中在C-的生化特性上 终端尾域，以努力确定是否以及如何修改 影响AC功能，以及这又如何与其在 轴突生长。在体外，AC是酪氨酸的底物 Src的磷酸化，已被证明在 轴突生长。Ac免疫沉淀法从神经细胞中提取 NGF也是磷酸化的。在一项确定AC是否 抑制镁依赖的ATPase活性(AMA)类似于特征良好的 基本钙蛋白亚型，我们观察到AC刺激了它。 为解决AC对肌动蛋白的影响而设计的其他实验 聚合研究表明，AC可刺激肌动蛋白聚合和 肌动蛋白束的形成。不包括AC的类似检测，但 包括SRC，已经表明肌动蛋白聚合受阻。这 通过添加AC，SRC的抑制是完全可逆的。这些 结果表明，AC的酪氨酸磷酸化不是必需的 AMA或肌动蛋白聚合的刺激。我们已经观察到 在体外，AC在非常年轻的神经元的生长锥体中含量丰富，并且 随着神经元的终末分化，这一水平会降低。我们还有 研究表明，AC的转换是由泛素蛋白分解途径介导的。 可能AC的酪氨酸磷酸化要么靶向，要么保护 蛋白质降解所产生的蛋白质。无论哪种方式，很明显，AC 是参与肌动蛋白动力学的重要分子。 聚合反应。我们的目标是阐明它在 发育中神经元的生长锥体。

We are interested in proteins that facilitate the transport of synaptic vesicles along axons and subsequent fusion at the pre-synaptic membrane. Included among these are intracellular motors, kinases, and cytoskeletal proteins. Proteins currently being studied in the lab include: kinesin light chains (klc), nsec-1, and acidic calponin (AC). Previously, we identified a family of klc from the nervous system of the squid in an effort to determine its role in the kinesin function. It is unclear whether klc plays a regulatory role in kinesin function or a targeting role to membrane bound organelles. We reported that at least 18 different isoforms arose as a result of alternative splicing occurring almost exclusively in the carboxyl terminal domain of the molecule thought to be the sight for kinesin targeting to membrane bound organelles. Furthermore, we found that klc transcript number and levels differed in various tissues examined further supporting the hypothesis that klc provides a targeting function to different membrane bound organelles in disparate cell types. Recently, however, we reported the identification and characterization of klc in a eubacterium, Plectonema boryanum, and failed to identify a corresponding heavy chain suggesting that klc may have some function unrelated to vesicle transport. Acidic calponin is an actin-binding protein with an unique carboxyl terminal tail domain endowed with at least three regulatory domains: a consensus tyrosine phosphorylation site, two putative P-loop elements, and the entire tail is a PEST domain. In the past year, we have focused on the biochemical characterization of C- terminal tail domain in an effort to determine if and how modification affects AC function and how this, in turn, relates to its role in neurite outgrowth. In vitro, AC is a substrate for tyrosine phosphorylation by src which has been shown to play a crucial role in neurite outgrowth. AC immunoprecipitated from neural cells treated with NGF is also phosphotyrosinated. In an assay to determine if AC inhibited Mg-dependent ATPase activity (AMA) like the well-characterized basic calponin isoform, we observed that AC stimulated it, instead. Additional experiments designed to address the effects of AC on actin polymerization have shown that AC stimulates actin polymerization and the formation of actin bundles. Similar assays that exclude AC but include src have shown that actin polymerization is impeded. This inhibition by src is completely reversible by the addition of AC. These results indicate that tyrosine phosphorylation of AC is not required for the stimulation of AMA or actin polymerization. We have observed in vitro that AC is abundant in the growth cones of very young neurons and that levels diminish as neurons terminally differentiate. We have also shown that AC turnover is mediated by the ubiquitin proteolytic pathway. Perhaps tyrosine phosphorylation of AC either targets or protects the protein from proteolytic degradation. Either way, it is clear that AC is an important molecule involved in the dynamics of actin polymerization. Our goal is to elucidate the role it plays in the growth cones of developing neurons.