Molecular Architecture and Function of the Cytoskeleton

(1)我们纯化了微管相关蛋白2C，这在青少年脑中特异性表达，并对其进行了生化和超微结构表征。从大鼠脑cDNA文库中克隆了MAP 2C基因的cDNA，并进行了序列测定。为了分析MAP 2在体内的功能，我们进行了转染实验。A.用cDNA转染的成纤维细胞系表达MAP 2C并形成粗束微管。中间丝也改变了它们的组织，并与微管束共存。(2)为了阐明膜性细胞器双向运输的调控机制，我们研究了磷酸化驱动蛋白对驱动蛋白与细胞膜结合的影响，发现A-激酶磷酸化驱动蛋白显著降低了驱动蛋白与细胞膜的结合。因此，A激酶可能在驱动蛋白从细胞膜细胞器上解离的过程中起重要作用。轴突(3)为了研究微管蛋白在轴突中的动力学，我们将荧光素标记的微管蛋白微注射到培养的神经元中，随后用紫外光活化法分析其周转。结果表明，轴突中的聚合物大多数是固定的，游离的寡聚体可能是被运输的形式。(4)我们建立了一种从哺乳动物脑中纯化10 OKD微管相关蛋白（发动蛋白）的方法。动力蛋白揭示了一个强大的GTdR活性激活的微管。克隆发动蛋白cDNA并测序。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.