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）：负责正确细胞分裂的时间和空间事件的编排需要大量蛋白质协同作用，以确保准确的有丝分裂进展可重复地发生。该提案的焦点是一种功能未知的进化保守蛋白，称为 NUD-1，它是线虫模型生物体秀丽隐杆线虫胚胎发生所必需的基因产物。 NUD-1 蛋白质水平的变化导致细胞分裂异常，包括有丝分裂后期中体微管的明显缺失以及中心体处伽马微管蛋白的丢失。在这里，我们建议进一步辨别中心体 NUD-1 和 γ-微管蛋白之间的功能关系。此外，我们将研究其他中心体相关蛋白对体内 NUD-1 活性的依赖性。先前的工作已经确定 NudC（NUD-1 的哺乳动物同源物）被 polo 样激酶 Plk1 磷酸化，并且这种相互作用对于 Plk1 的正确定位和胞质分裂的进展是必要的。进一步证明，Hsp90 是一种普遍存在的分子伴侣，在有丝分裂过程中调节 Plk1，并且是 Plk1 定位到中心体所必需的。重要的是，Hsp90 活性依赖于称为 p23 的共伴侣蛋白，p23 是一种与 NUD-1/NudC 具有显着结构同源性的蛋白质。我们建议研究 Plk1-NudC 相互作用是否需要涉及 Hsp90 的调节机制，以促进有丝分裂中中心体功能所必需的事件。我们的方法结合了 RNA 干扰 (RNAi)、生物化学、免疫细胞化学和荧光显微镜，使用秀丽隐杆线虫单细胞胚胎，更好地了解 NUD-1 及其伙伴蛋白在细胞分裂中发挥作用的分子机制作为研究中心体异常基因活动的详细后果的模型系统。与公共健康的相关性：研究中提出的蛋白质（NudC、Hsp90 和 Plk1）对于各种癌症（包括白血病、乳腺癌、前列腺癌和皮肤癌）具有重要意义。此外，体外前列腺癌细胞中 NudC 的过量产生可以抑制前列腺肿瘤的生长。阐明这些蛋白质在细胞分裂中介导的功能机制可以更好地了解癌变过程，从而有助于开发更明确的治疗干预途径。