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

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协调需要大量的蛋白质协同作用，以确保准确的有丝分裂进程可重复发生。这一提议的焦点是一种功能未知的进化保守蛋白，称为nud-1，这是一种对线虫模式生物-秀丽线虫的胚胎发生至关重要的基因产物。NUD-1蛋白水平的变化导致细胞分裂异常，包括有丝分裂后期明显缺乏中体微管，中心体丢失伽马微管。在这里，我们建议进一步辨别NUD-1和中心体上的伽马微管蛋白之间的功能关系。此外，我们还将在体内研究其他中心体相关蛋白对nud-1活性的依赖性。以前的工作已经证实，nudC是nud-1的哺乳动物同源物，被Polo样激酶Plk1磷酸化，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。Hsp90是一种普遍存在的分子伴侣，在有丝分裂过程中调节Plk1，是Plk1定位到中心体所必需的。重要的是，Hsp90的活性依赖于被称为p23的辅助伴侣，p23是一种与nud-1/nudC有显著结构同源性的蛋白质。我们建议研究Plk1-NudC相互作用涉及Hsp90的调节机制的可能性，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，以线虫单细胞胚胎为模型系统，研究中心体基因活性异常的详细后果，以更好地了解nud-1及其伙伴蛋白在细胞分裂中的分子机制。与公共健康相关：建议的研究蛋白质(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具有重要意义。此外，在体外，前列腺癌细胞中NudC的过量产生可以抑制前列腺癌的生长。阐明这些蛋白质在细胞分裂中的作用机制有助于更好地理解癌症的过程，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.