KATANIN MEDIATED MICROTUBULE SEVERING

微管被真核细胞用来分离染色体， 并将细胞器运输到细胞中的特定位置 在间期。 细胞器运输的准确性要求 微管在相间排列成特定的极化阵列， 而在有丝分裂过程中染色体的精确分离需要 微管的双极排列，有丝分裂纺锤体。 知识 负责微管重组的特异性蛋白质 在细胞周期中的阵列将是必不可少的完整的理解 细胞分裂的机制以及肿瘤发生的机制。长 这个项目的长期目标是阐明个人的角色， 蛋白质在调节体内微管阵列中发挥作用。之前的工作 首席研究员已经导致了katanin的纯化， 微管切断蛋白，来自海胆卵。 初步 实验表明，katanin集中在中心体， 微管组织中心，分裂的海胆细胞。具体 该项目的目的是确定卡他宁的具体作用， 分裂间期和有丝分裂期微管动力学中的微管断裂 使用基于抗体的方法抑制卡他宁的活性 体内和体外提取物。 具体地，抗体Fab片段， 抑制katanin体外活性将被显微注射到海胆中 受精卵的微管阵列结构的影响， vivo. 净化的、活跃的katanin的存在将允许 通过共注射katanin与抑制性FAb的互补 片段注射过量katanin的影响也将被检查。 在体外，精子中心粒复合体在体外组装成中心体， 海胆卵提取物。免疫耗竭的katanin从这些 提取物将允许缺乏katanin的中心体的组装， 向这些提取物中加入纯化的katanin可以允许组装 含有过量katanin的中心体。两个特别的方面 将详细检查微管动力学。一是释放 微管附着在中心体上， 在体内和无细胞提取物中观察到。第二个是微管蛋白通量， 在体内和体外观察到的过程中，微管 在中心体的三个附着位点处被分解。如果 katanin的失活抑制了这些过程中的任何一个， 实验将首次提供一个测试的重要性， 微管蛋白通量和微管从中心体释放 师.分离编码katanin亚基的cDNA和细菌 这些cDNA的表达将促进抗体的产生 这些实验所需要的。

Microtubules are used by eukaryotic cells to segregate chromosomes at mitosis and to transport organelles to specific locations in the cell during interphase. The accuracy of organelle transport requires that the microtubules be arranged in a specific polarized array in interphase, whereas accurate segregation of chromosomes during mitosis requires a bipolar array of microtubules, the mitotic spindle. Knowledge of the specific proteins responsible for the reorganization of the microtubule array during the cell cycle will be essential for a complete understanding of the mechanisms of cell division and therefore of oncogenesis. The long term goal of this project is to elucidate the roles that individual proteins play in regulating microtubule arrays in vivo. Previous work by the principal investigator has resulted in the purification of katanin, a microtubule-severing protein, from sea urchin eggs. Preliminary experiments indicate that katanin is concentrated at the centrosomes, the microtubule organizing centers, of dividing sea urchin cells. The specific aim of this project is to determine the specific role of katanin-based microtubule severing in the dynamics of interphase and mitotic microtubule arrays by using antibody-based methods to inhibit katanin's activity in vivo and in in vitro extracts. Specifically, antibody FAb fragments that inhibit katanin's in vitro activity will be microinjected into sea urchin zygotes to examine the effects on the structure of microtubule arrays in vivo. The existence of purified, active katanin will allow complementation by coinjection of katanin with the inhibitory FAb fragments. The effects of injecting excess katanin will also be examined. In vitro, sperm centriole complexes are assembled into centrosomes in extracts made from sea urchin eggs. Immunodepletion of katanin from these extracts will allow assembly of centrosomes devoid of katanin whereas addition of purified katanin to these extracts may allow assembly of centrosomes containing excess katanin. Two particular aspects of microtubule dynamics will be examined in detail. The first is the release of microtubules from their attachment to the centrosome, a phenomenon observed in vivo and in cell free extracts. The second is tubulin flux, a process observed in vivo and in vitro in which microtubules are disassembled at there sites of attachment at the centrosome. If inactivation of katanin inhibits either of these processes, these experiments will, for the first time, provide a test of the importance of tubulin flux and of microtubule release from the centrosome in cell division. Isolation of cDNAs encoding the katanin subunits and bacterial expression of these cDNAs will facilitate production of the antibodies required for these experiments.