BASIC CELL BIOLOGICAL MECHANISMS USING THE SQUID NERVOUS SYSTEM MODEL

神经丝的磷酸化， 微管相关蛋白（MAPs）受拓扑学调控 在神经元内。例如，神经丝（NF）（NF-M和NF-κ B）可以是神经纤维。 NF-H）和tau蛋白在轴突中广泛磷酸化 但不在细胞体中。然而，在某些神经退行性疾病中， 这些蛋白质在细胞中被广泛磷酸化 尸体这种磷酸化的机制尚不清楚。 细胞骨架蛋白磷酸化是一个复杂的相互作用， 激酶、磷酸酶及其调节剂。要理解这一点 拓扑调控的细胞骨架蛋白磷酸化，我们 我用鱿鱼作为模型系统， 从星状核巨纤维叶（GFL）的胞体中 神经节这些不同细胞中的磷酸化机制 域可以进行生物化学研究和比较。以来 p13 suc 1与cdc 2样激酶具有高亲和力，我们使用 P13 suc 1亲和层析，结合免疫印迹分析， 研究与P13 suc 1/cdc 2-like激酶相关的蛋白质 从轴质和GFL分离的复合物。虽然裂解物 从轴浆和GFL都含有定性相似的 通过免疫印迹分析确定的蛋白质谱， 我们从轴浆中提取了一个活性P13多聚体复合物 细胞骨架蛋白（微管蛋白、核因子、肌动蛋白和驱动蛋白）， 磷酸化激酶和调节剂。无此类活性P13 从GFL的胞体中获得了复合物; GFL/P13 复合物缺乏微管蛋白，NF和大多数激酶，虽然cdc 2- 检测到类似的激酶，并表现出低水平的 磷酸化活性我们认为，激酶，细胞骨架， 蛋白质和调节剂，虽然在神经元细胞中合成， 在轴突中组装成多聚体复合物， 调节轴突细胞骨架元素的磷酸化。 目前，我们正在更详细地分析这些复合物， 也研究了这些形成的机制， 在神经元的轴突隔室中的多聚体复合物。

Phosphorylation of neurofilaments and microtubule-associated proteins (MAPs) are topologically regulated within neurons. For example, neurofilaments (NFs) (NF-M and NF-H) and tau proteins are extensively phosphorylated in the axon but not in the cell bodies. However, in certain neurodegenerative diseases these proteins are extensively phosphorylated in the cell bodies. The mechanisms of this phosphorylation is not known. Cytoskeletal protein phosphorylation is a complex interplay among kinases, phosphatases and their regulators. To understand this topographic regulation of cytoskeletal protein phosphorylation, we used squid as a model system where giant axons can be isolated free from their perikarya in the giant fiber lobe (GFL) of the stellate ganglion. The phosphorylation mechanisms in these distinct cellular domains can be studied and compared biochemically. Since P13suc1 binds with high affinity to cdc2-like kinases, we used P13suc1 affinity chromatography, coupled to immunoblot analysis, to study proteins associated with the P13suc1/cdc2-like kinase complex isolated from axoplasm and the GFL. Although lysates from axoplasm and the GFL both contained qualitatively similar protein profiles as determined by immunoblot analyses, it was only from axoplasm that we extracted an active P13 multimeric complex of cytoskeletal proteins, (tubulin, NFs, actin and kinesin), their phosphorylating kinases and regulators. No such active P13 complex was obtained from perikarya of the GFL; the GFL/P13 complex lacked tubulin, NFs and most kinases, although a cdc2- like kinase was detected, and exhibited low levels of phosphorylating activity. We propose that kinases, cytoskeletal proteins and regulators, though synthesized in the neuronal cell body, assemble in the axon as a multimeric complex which actively regulates phosphorylation of elements of the axonal cytoskeleton. Currently, we are analyzing these complexes in more detail and are also studying the mechanisms underlying the formation of these multimeric complexes in the axonal compartments of the neuron.