FUNCTIONAL COMPARTMENTALIZATION OF NEURONS AND GLIA

我们的目标是继续我们的研究：1)识别、分离和 膜结合糖偶联物胞内池的特征 正在经历快速轴突运输，2)表征不同的功能 神经元细胞骨架的隔间，尤其与 细胞骨架组分及相关多肽的磷酸化 细胞器。免疫细胞化学分析将用于研究 糖蛋白抗原在小脑生化反应中的分布 特性模拟特化光滑膜的细胞化学特性 浦肯野细胞轴突和树突中的系统。我们将使用特定的 放射性同位素标记及其药理作用 低聚糖加工和轴突运输以确定来源和 新合成的糖偶联物被指定为快速轴突的命运 视网膜感光细胞和神经节细胞的运输。酶连结 细胞化学、放射自显影和生化分析将用于 开始表征运输的糖结合物的低聚糖链 与到达视网膜突触前终末的神经元相比 神经元。我们将使用免疫细胞化学和免疫印迹分析组织 切片、培养的神经元和细胞骨架的分离部分 细胞器用于研究细胞内的分区化 磷脂/钙依赖的蛋白激酶(PKC)及其底物 轴突。内源性和外源性PKC将在体外使用 与体内标记模式比较的磷酸化实验 阐明细胞骨架中该酶的特定底物 多肽。视网膜组织切片和培养的视网膜和 交感神经节神经元将被用来问这个问题 Not-PKC从胞浆转位到细胞膜 对生理刺激或药物激活的反应 PKC。我们将使用ATP的非水解性类似物来探测微管 粘液漏斗的缺陷轴突存在“动蛋白”型 转位分子。将使用来自大鼠脑内的激动素制剂 检测是否存在与神经节细胞相关的轴突多肽 细胞骨架，特别是tau蛋白和PKC。

The objectives are to continue our studies to: 1) identify, isolate and characterize intracellular pools of membrane bound glycoconjugates undergoing fast axonal transport, 2) to characterize different functional compartments of the neuronal cytoskeleton related particularly to phosphorylation of component and associated polypeptides of cytoskeletal organelles. Immunocytochemical analysis will be used to study the distribution of glycoprotein antigens in cerebellum whose biochemical properties mimic the cytochemical properties of specialized smooth membrane systems in Purkinje cell axons and dendrites. We will use specific radioisotope labeling together with pharmacological manipulations of oligosaccharide processing and axonal transport to identify the origin and fate of newly synthesized glycoconjugates destined for fast axonal transport in retinal photoreceptor and ganglion cells. Enzyme-linked cytochemical, autoradiographic and biochemical analysis will be used to begin to characterize oligosaccharide chains of transported glycoconjugates compared to those that have reached the presynaptic terminal of retinal neurons. We will use immunocytochemical and immuno-blot analysis of tissue sections, cultured neurons and isolated fractions of cytoskeletal organelles to study the intracellular compartmentalization of the phospholipid/Ca2+-dependent protein kinase (PKC) and its substrates in axons. Endogenous and exogenous PKC will be used in in vitro phosphorylation experiments for comparison with in vivo labeling patterns to elucidate specific substrates for the enzyme among the cytoskeletal polypeptides. Retinal tissue slices and cultures of retinal and sympathetic ganglion neurons will be used to ask the question whether or not PKC translocates from the cytosol to the plasma membrane of neurons in response to physiological stimulation or pharmacological activation of PKC. We will use non-hydrolyzable analogs of ATP to probe microtubule deficent axons of Myxicola infundibulum for the presence of "kinesin" type translocator molecules. Kinesin preparations from rat brain will be used to test for the presence of axonal polypeptides associated with the cytoskeleton, particularly the tau proteins and PKC.