BASIC CELL BIOLOGICAL MECHANISMS USING THE SQUID NERVOUS SYSTEM MODEL

本项目包含两个具体目标：1)研究细胞生物学 蛋白质；以及2)检验轴突蛋白质合成 发生在鱿鱼的巨型轴突中。对于后一项研究，我们生成了 鱿鱼神经丝蛋白的特异性抗体和cDNA探针 研究星状神经节(胞体)和星状神经节的蛋白质合成 轴突。蛋白质生物合成/免疫沉淀实验证实 鱿鱼星状神经节中NF蛋白的强健生物合成，但我们 在巨型轴突中未检测到任何核因子蛋白的生物合成。这是 面对我们在鱿鱼中发现的显著的核因子蛋白mRNA 轴突。在另一项研究中，我们考察了 多聚体介导的磷酸化/去磷酸化反应 激酶和磷酸酶的复合体，它们的调节剂和抑制剂。 我们认为这些复合体被分割成轴突和细胞体。 并建议它们在组装和运输中发挥重要作用 细胞骨架结构和细胞器。在神经元细胞体中， 在调节细胞活性的因素之间存在着微妙的平衡 调节不完全磷酸化的激酶和磷酸酶 (例如，仅在NFS中的头域)在它们组装之前 并运输到轴突中。在轴突中，神经纤维进一步被磷酸化。 (在尾域中)并组织成晶格，以稳定 上述假设，我们使用P13su1琼脂糖偶联珠提取 使轴浆中的神经细丝磷酸化的激酶来自 乌贼巨型轴突。使用Western blotts和体外激酶分析，我们 证明了活性的cdc2样激酶的存在及其推定的 轴浆中的细胞周期蛋白E、P13同系物和P67等调节因子 (CK)I和II也存在于P13-Ax。蛋白质印迹分析 神经细丝(NF60、70和220)、微管蛋白、肌动蛋白和微管 相关蛋白质。NF220和微管蛋白被磷酸化 P13-Ax中的激酶。确定NFS卡是否直接绑定到P13 珠子，或通过与cdc2激酶结合间接结合，洗涤， 微管蛋白，但没有cdc2样激酶，在 与P13su1共孵育。神经丝的洗涤上清液 然而，含有cdc2样激酶的制剂确实产生了 与P13su1结合的细胞骨架成分。此外，一种细菌- 表达的CDK5与P13珠子相关，能够与 在洗涤的神经丝制剂中选择细胞骨架成分。 这些数据表明P13珠子与类CDC2直接结合 激酶可提取活性多聚体组成的轴突复合体 细胞骨架蛋白和蛋白激酶。

This project contains two specific goals: 1) To study the cell biological proteins; and 2) to test the hypothesis that axonal protein synthesis occurs in the squid giant axon. For the latter study, we generated specific antibodies and cDNA probes for squid neurofilament (NF) proteins to study protein synthesis in stellate ganglia (cell bodies) and in axons. Protein biosynthesis/immunoprecipitation experiments confirmed robust biosynthesis of NF proteins in squid stellate ganglia, but we failed to detect any NF protein biosynthesis in the giant axon. This is in the face of our findings of significant NF protein mRNA in the squid axon. In the other study, we have examined the dynamic equilibrium of phosphorylation/dephosphorylation reactions mediated by multimeric complexes of kinases and phosphatases, their regulators and inhibitors. We believe these complexes are compartmentalized in axons and cell bodies and suggest that they play an important role in assembly and transport of cytoskeletal structures and organelles. In the neuronal cell bodies, there is a delicate balance between factors regulating the activity of kinases and phosphatases that modulate the incomplete phosphorylation (e.g., head domains only in NFs) of NFs and tau prior to their assembly and transport into the axon. In axons, NFs are further phosphorylated (in tail domains) and organize into a lattice that stabilizes the above hypothesis, we used P13suc1 sepharose-conjugated beads to extract the kinases that phosphorylate neurofilaments in the axoplasm from the squid giant axon. Using Western blots and in vitro kinase assays, we demonstrated the presence of an active cdc2-like kinase and its putative regulators such as cyclin E, P13 homologue and P67 in axoplasm and a (CK) I and II were also found in the P13-Ax. Western blot analysis of neurofilaments (NFs; NF 60, 70 and 220), tubulin, actin and microtubule associated proteins. NF 220 and tubulin were phosphorylated by the kinases in the P13-Ax. To determine whether NFs bound directly to the P13 beads, or bound indirectly by association with cdc2 kinase, a washed, tubulin, but no cdc2-like kinase, yielded no bound proteins after incubation with P13suc1. The wash supernatant from the neurofilament preparation, however, containing the cdc2-like kinase, did yield cytoskeletal components that bound to P13suc1. Moreover, a bacterial- expressed cdk5 associated with P13 beads, was able to complex with selected cytoskeletal components in the washed neurofilament preparation. These data indicate that direct binding of P13 beads with a cdc2-like kinase could extract active multimeric complexes composed of axonal cytoskeletal proteins and kinases.