TRACK Regulates Cytokinesis in Trypanosoma brucei

TRACK 调节布氏锥虫的细胞分裂

• 批准号: 6779261
• 负责人: Lawrence Ruben
• 金额: $ 32.47万
• 依托单位: SOUTHERN METHODIST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779261
• 关键词: A kinase anchoring protein; RNA interference; Trypanosoma brucei; affinity chromatography; biological signal transduction; cell cycle; cell growth regulation; cytoskeletal proteins; flow cytometry; genetic library; genetic screening; laboratory mouse; laboratory rat; nucleic acid sequence; phenotype; phosphorylation; protein binding; protein quantitation /detection; protein signal sequence; protein structure function; site directed mutagenesis; two dimensional gel electrophoresis; yeast two hybrid system

DESCRIPTION (provided by applicant): Cytokinesis is the mechanical process by which a cell divides in two. The process is essential for propagation of unicellular parasites. Little is known about cytokinesis in African trypanosomes, but current information suggests that the process is different from that of the mammalian host. My laboratory has a strong interest in cytokinesis because it is clearly tied to signaling events. In particular, it utilizes temporal information to be triggered only at a specific time in the division cycle; and spatial information to form a cleavage furrow only in the appropriate site. One way to provide spatial and temporal information to the signal process is by means of signal anchor proteins. We have recently identified a putative signal anchor protein called TRACK with strong similarity to mammalian and yeast RACK1/Cpc2. We report that TRACK can correct morphological and growth related defects in cpc2 null mutants of S. pombe. In T. brucei, knock-down of TRACK with RNAi demonstrates that it is essential for progression through a mid-stage of cytokinesis. Cells that become "stuck" in cytokinesis continue through multiple rounds of partial division, and thus accumulate multiple nuclei, and multiple cytoplasmic extensions, each with attached flagellum. The observations are significant since they are the first to demonstrate that once cytokinesis is initiated, it is discontinuous and uses a different set of proteins at mid-stage than at the outset. Since no other RACK1/Cpc2 homologue has been implicated in cytokinesis, these data also underscore the unique aspects of the process in T. brucei. The disruption of TRACK blocks cell division, and indicates that novel components within the cytokinesis pathway may be useful as targets for therapy design. The current project utilizes TRACK as a tool to specifically probe molecular events at the mid-stage of cytokinesis. The project has three specific aims. The first aim examines the role of TRACK in cytokinesis through an examination of its binding partners. The second aim investigates proteins downstream of TRACK whose phosphorylation state changes in TRACKi knock-down cells. The third aim is to screen RNAi libraries for the unique phenotypic changes that accompany defective cytokinesis. In this way TRACK binding partners and other components of this essential process will be identified.

描述（由申请人提供）：细胞分裂是细胞一分为二的机械过程。该过程对于单细胞寄生虫的繁殖至关重要。人们对非洲锥虫的胞质分裂知之甚少，但目前的信息表明该过程与哺乳动物宿主的胞质分裂不同。我的实验室对胞质分裂非常感兴趣，因为它显然与信号事件有关。特别地，它利用仅在划分周期中的特定时间触发的时间信息；和空间信息，仅在适当的位点形成解理沟。向信号过程提供空间和时间信息的一种方法是利用信号锚蛋白。我们最近发现了一种假定的信号锚蛋白，称为 TRACK，与哺乳动物和酵母的 RACK1/Cpc2 非常相似。我们报告TRACK可以纠正粟酒裂殖酵母cpc2无效突变体的形态和生长相关缺陷。在布氏锥虫中，用 RNAi 敲低 TRACK 表明它对于胞质分裂中期的进展至关重要。 “陷入”胞质分裂的细胞继续进行多轮部分分裂，从而积累多个细胞核和多个细胞质延伸，每个延伸都带有鞭毛。这些观察结果很重要，因为它们首次证明，一旦胞质分裂开始，它是不连续的，并且在中期使用一组与开始时不同的蛋白质。由于没有其他 RACK1/Cpc2 同源物与胞质分裂有关，这些数据也强调了 T. brucei 中该过程的独特之处。 TRACK 的破坏阻止了细胞分裂，并表明胞质分裂途径中的新成分可能可用作治疗设计的靶标。当前的项目利用 TRACK 作为工具来专门探测胞质分裂中期的分子事件。该项目有三个具体目标。第一个目标是通过检查 TRACK 的结合伴侣来检查 TRACK 在胞质分裂中的作用。第二个目标是研究 TRACK 下游的蛋白质，其磷酸化状态在 TRACKi 敲低细胞中发生变化。第三个目标是筛选 RNAi 文库，寻找伴随有缺陷的胞质分裂的独特表型变化。通过这种方式，TRACK 结合伙伴和这个重要过程的其他组成部分将被识别。