Molecular Architecture and Function of the Cytoskeleton

细胞骨架的分子结构和功能

基本信息

批准号：
62065007
负责人：
HIROKAWA Nobutaka
金额：
$ 155.52万
依托单位：
University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Specially Promoted Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1991
项目状态：
已结题

项目摘要

(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.

(1)我们纯化了在幼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。 MAP2C CDNA 从大鼠脑 cDNA 文库中克隆并测序。为了分析MAP2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MAP2C并形成粗的微管束。有趣的是，中间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，我们研究了驱动蛋白磷酸化对其与膜结合的影响；我们发现 A-激酶对驱动蛋白的磷酸化显着降低了结合。因此，A-激酶可能在驱动蛋白从膜细胞器解离的过程中发挥重要作用。 (3)为了研究轴突中微管蛋白的动态，我们将笼状荧光素标记的微管蛋白显微注射到培养的神经元中，随后通过紫外光激活方法分析其周转。结果表明，轴突中的大多数聚合物是固定的，游离寡聚体可能是被运输的形式。(4)我们建立了从哺乳动物大脑中纯化10OKD微管相关idrotein (dynamin)的方法。 Dynamin 揭示了微管激活的有效 GTP 酶活性。 Dynamin 的 cDNA 被克隆并测序。 Dynaim被证明是一种新的GTP结合蛋白，在成熟神经细胞中特异性表达。