Investigation of C. elegans NUD-1 in Centrosome Function and Mitosis

线虫 NUD-1 中心体功能和有丝分裂的研究

• 批准号: 7073269
• 负责人: Kim A Caldwell
• 金额: $ 21.15万
• 依托单位: UNIVERSITY OF ALABAMA IN TUSCALOOSA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7073269
• 关键词: Caenorhabditis elegans; RNA interference; cell component structure /function; cell cycle; centrosome; developmental genetics; embryogenesis; heat shock proteins; helminth genetics; invertebrate embryology; protein kinase; protein localization; protein protein interaction; protein structure function

DESCRIPTION (provided by applicant): The orchestration of the temporal and spatial events responsible for proper cell division requires a host of proteins acting in concert to ensure that accurate mitotic progression occurs reproducibly. The focal point of this proposal is one such evolutionary conserved protein of unknown function termed NUD-1, a gene product that is essential for embryogenesis in the nematode model organism, C. elegans. Changes in protein levels of NUD-1 result in aberrant cell division, including a distinctive absence of midbody microtubules at anaphase during mitosis and loss of gamma-tubulin at centrosomes. Here we propose to further discern the functional relationship between NUD-1 and gamma-tubulin at the centrosome. Additionally, we will investigate the dependence of other centrosome-associated proteins on NUD-1 activity in vivo. Previous work has established that NudC, the mammalian homolog of NUD-1, is phosphorylated by the polo-like kinase Plk1, and that this interaction is necessary for the proper localization of Plk1 and progression through cytokinesis. It has been further demonstrated that Hsp90, a ubiquitous molecular chaperone, regulates Plk1 during mitosis and is required for Plk1 localization to centrosomes. Importantly, Hsp90 activity is dependent upon a co-chaperone termed p23, a protein that shares significant structural homology with NUD-1/NudC. We propose to investigate the possibility that Plk1-NudC interaction entails a regulatory mechanism involving Hsp90 to facilitate events essential to centrosome function in mitosis. Our approach employs a combination of RNA interference (RNAi), biochemistry, immunocytochemistry and fluorescent microscopy to gain a greater understanding of the molecular mechanisms by which NUD-1 and its partner proteins act in cell division, using the C. elegans one-celled embryo as a model system to investigate the detailed consequences of aberrant gene activity at the centrosome. Relevance to Public Health: The proposed proteins of study (NudC, Hsp90, and Plk1) are significant in various cancers including leukemia, breast, prostate and skin cancer. Additionally, prostate tumor growth can be inhibited by overproduction of NudC in prostate cancer cells in vitro. Elucidating the functional mechanisms mediated by these proteins in cell division provides a better understanding of the cancerous process, thereby contributing to the development of more defined avenues of therapeutic intervention.

描述（由申请人提供）：负责适当细胞分裂的时间和空间事件的编排需要协同作用的大量蛋白质，以确保可重复地进行准确的有丝分裂进展。该提案的焦点是一种称为NUD-1的未知功能的进化保守蛋白，这是一种基因产物，对于线虫模型生物体中的胚胎发生至关重要。 NUD-1的蛋白质水平的变化导致细胞分裂异常，包括在有丝分裂和中心体γ-微管蛋白丢失的过程中独特的中体微管缺失。在这里，我们建议进一步辨别中心体的NUD-1和γ-微管蛋白之间的功能关系。此外，我们将研究其他与中心体相关蛋白在体内NUD-1活性的依赖性。先前的工作已经确定，NUDC是NUD-1的哺乳动物同源物，被类似polo样激酶PLK1磷酸化，并且这种相互作用对于PLK1的适当定位和通过细胞因子的进展是必要的。已经进一步证明，一种无处不在的分子伴侣HSP90在有丝分裂过程中调节PLK1，并且PLK1定位到中心体所必需。重要的是，HSP90活性取决于称为p23的伴侣，该蛋白质与NUD-1/NUDC具有重要的结构同源性。我们建议调查PLK1-NUDC相互作用需要一种涉及HSP90的调节机制，以促进有丝分裂中心体功能所必需的事件。我们的方法采用了RNA干扰（RNAI），生物化学，免疫细胞化学和荧光显微镜的组合，以更深入地了解NUD-1及其伴侣蛋白在细胞分裂中起作用的分子机制，使用C. elekans exlemans胚胎单细胞的胚胎作为模型系统作为模型，以研究详细的结果，以调查详细的基因基因效果。与公共卫生的相关性：拟议的研究蛋白（NUDC，HSP90和PLK1）在包括白血病，乳腺癌，前列腺癌和皮肤癌在内的各种癌症中都很重要。另外，前列腺肿瘤生长可以通过体外前列腺癌细胞中NUDC过量生产来抑制。阐明这些蛋白在细胞分裂中介导的功能机制提供了更好地理解癌性过程，从而有助于发展更明确的治疗干预途径。