Molecular Architecture and Function of the Cytoskeleton

(1)纯化了在青少年大脑中特异性表达的微管相关蛋白2C，并对其进行了生化和超微结构表征。从大鼠脑CDNA文库中克隆MAP2C CDNA并进行测序。为了分析MAP2在体内的功能，我们进行了转染实验。转染CDNA的A.成纤维细胞系表达MAP2C，形成粗束微管。有趣的是，中间细丝也改变了它们的组织并与微管束共定位。(2)为了阐明控制膜细胞器双向运输的机制，我们检测了激酶磷酸化对其与膜结合的影响；我们发现a -激酶对激酶蛋白的磷酸化显著降低了其结合。因此，a -激酶可能在肌动蛋白与细胞膜、细胞器的分离中起重要作用。轴突。(3)为了研究轴突内微管蛋白的动态，我们将荧光素标记的微管蛋白微注射到培养的神经元中，然后用紫外光激活法分析其周转情况。结果表明，轴突内的大多数聚合物是固定的，而自由的低聚物可能是被运输的形式。(4)建立了从哺乳动物脑中纯化10OKD微管相关蛋白（dynamin）的方法。Dynamin显示了微管激活的GTPase活性。克隆并测序了动力蛋白的CDNA。Dynaim是一种新的GTP结合蛋白，在成熟神经细胞中特异性表达。

(1) We purified the microtubule associated protein 2C, which specifically expressed in juvenile brains, and characterized them biochemically and ultrastructurally. The MAP2C CDNA was cloned from rat brain CDNA library and was sequenced. To analyze functions of MAP2 in vivo, we carried out transfection experiments. A. fibroblast cell line which was transfected with the CDNA expressed MAP2C and formed thick bundles of 'microtubules. Ineterestingly intermediate filaments also changed their organization and colocalized with microtubule bundles.(2) To elucidate the mechamism which control bidirectional transport of membranous organelle we examined the effect of phosphorylation of kinesin on their binding to the membranes ; We found phosphorylation of kinesin by A-kinase considerably decreased the binding. Thus A-kinase probably play an important role on the dissociation of kinesin from the membranous, organelles in the. axon.(3) To study the dynamics of tubulin in axons we microinjected caged fluorescein labeled tubulin into cultured neurons and subsequently analysed its turnover by UV photoactivation method. The results suggested that most of the polymers in the axon are stationary and the free oligomers could be the form to be transported.(4) We established a method to purify 10OKD microtubule Associated idrotein (dynamin) from mammalian brains. Dynamin revealed a potent GTPase activity activated by microtubules. CDNA of dynamin was cloned and sequenced. Dynaim was proved to be a new GTP binding protein which specifically expressed in mature nerve cells.