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

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大量蛋白质协同作用，以确保准确的有丝分裂进程可重复地发生。该建议的焦点是一种功能未知的进化保守蛋白，称为NUD-1，是线虫模式生物C.优雅NUD-1蛋白水平的变化导致细胞分裂异常，包括有丝分裂后期中间体微管的明显缺失和中心体γ-微管蛋白的丢失。在这里，我们建议进一步辨别NUD-1和γ-微管蛋白在中心体之间的功能关系。此外，我们将研究其他中心体相关蛋白对NUD-1活性的依赖性。以前的工作已经确定，NUD-1的哺乳动物同源物NudC被Polo样激酶Plk 1磷酸化，并且这种相互作用对于Plk 1的适当定位和通过胞质分裂的进展是必要的。已经进一步证明，Hsp 90，一种普遍存在的分子伴侣，在有丝分裂过程中调节Plk 1，并且是Plk 1定位于中心体所必需的。重要的是，Hsp 90活性依赖于称为p23的共分子伴侣，该蛋白与NUD-1/NudC具有显著的结构同源性。我们建议调查的可能性，Plk 1-NudC相互作用需要一个监管机制，涉及热休克蛋白90，以促进事件必不可少的中心体功能在有丝分裂。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，以获得更好的了解NUD-1及其伴侣蛋白在细胞分裂中发挥作用的分子机制，使用C。elegans单细胞胚胎作为模型系统，以研究异常基因活性在中心体的详细后果。与公共卫生的相关性：研究中提出的蛋白质（NudC，Hsp 90和Plk 1）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具有重要意义。此外，前列腺肿瘤生长可以通过体外前列腺癌细胞中NudC的过度产生来抑制。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理解癌症过程，从而有助于开发更明确的治疗干预途径。

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.