KATANIN MEDIATED MICROTUBULE SEVERING

微管被真核细胞用来分离染色体 有丝分裂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细胞器 微管在相间以特定的极化阵列排列， 而在有丝分裂过程中染色体的准确分离需要 微管的双极阵列，有丝分裂的纺锤体。了解以下内容 负责微管重组的特定蛋白质 细胞周期中的阵列对于完全理解将是必不可少的 细胞分裂和肿瘤发生的机制。《长河》 本项目的学期目标是阐明个人 蛋白质在体内调节微管阵列方面发挥作用。之前的工作由 首席研究人员已经提纯了一种名为katanin的 海胆卵中的微管切断蛋白。初步 实验表明，katanin集中在中心体，即 海胆细胞分裂的微管组织中心。具体的 本项目的目的是确定基于katanin的特定作用 间期微管和有丝分裂微管动力学中的微管切断 通过基于抗体的方法抑制Katanin在小鼠体内的活性 体内提取物和体外提取物。具体地说，抗体Fab片段 将抑制卡塔宁的体外活性显微注射到海胆体内 受精卵对微管阵列结构的影响 活着。纯化的、活性的卡塔宁的存在将使 Katanin与抑制性Fab共注射的互补作用 碎片。还将检查注射过量katanin的影响。 在体外，精子中心粒复合体组装成中心体。 从海胆蛋中提取的精华。它们的卡丹宁免疫耗竭作用 萃取物将允许不含katanin的中心体组装，而 将纯化的katanin添加到这些提取物中可以允许组装 含有过量的katanin的着丝粒。有两个特殊的方面 我们将详细研究微管动力学。第一个是释放 微管附着在中心体上，这是一种现象 在体内和无细胞提取物中观察。第二种是微管蛋白流量，一种 体内和体外观察的微管的过程 在中心体的三个附着点上解体。如果 Katanin的失活会抑制这两个过程中的任何一个 实验将第一次提供一个测试 微管蛋白流量和微管从细胞中心体释放 组织。编码katanin亚基和细菌的cDNA的分离 这些cDNA的表达将促进抗体的产生 是这些实验所必需的。

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.