Mitotic functions of cilia proteins

描述(申请人提供)：中心体有助于有丝分裂中有丝分裂纺锤体的组织和取向，以及在未分裂的细胞中初级纤毛的组装。中心体异常影响纺锤体和纤毛功能的保真度，并与纤毛疾病、小头畸形、侏儒症、癌症和其他人类疾病有关。纤毛蛋白存在于有丝分裂细胞的中心体(纺锤体极)，一些纤毛蛋白参与细胞分裂的定向。然而，这些纤毛蛋白在有丝分裂细胞中的功能尚不清楚。Doxsey实验室的初步结果表明，在非周期细胞中纤毛上下运输所需的纤毛蛋白(鞭毛内运输，IFT)也将物质输送到有丝分裂细胞的纺锤体极和从有丝分裂细胞的纺锤体极运输出来。这些纤毛蛋白的破坏导致有丝分裂纺锤体取向、星形微管组织、纺锤体极功能和有丝分裂进程的缺陷。其他纤毛蛋白定位于其他有丝分裂结构，如动点和中体，表明这类蛋白质具有额外的有丝分裂功能。这项提议的总体目标是测试这样的假设，即参与非周期细胞纤毛形成和功能的IFT复合体至少部分地被重定向以执行以前未预料到的有丝分裂功能。为了验证这一点，我们将探讨IFT蛋白复合体在有丝分裂纺锤体中作用的分子机制。我们的初步研究表明，IFT88在细胞中形成颗粒，利用动力蛋白马达将微管核蛋白运输到纺锤体极。这些特定的目的是为了测试IFT蛋白复合体是否作为有丝分裂货物的载体，以及动力蛋白是否为它们的移动提供动力。这项工作的新方面包括识别纺锤体极组装和纺锤体定向的新机制，使用超分辨率显微镜对活的有丝分裂细胞中新型IFT蛋白颗粒的动力学进行成像，使用新的亲和系统表征新的有丝分裂IFT蛋白-动力蛋白复合体，以及使用体外分析方法研究基于动力蛋白的运动性。这项工作有可能确定一条新的纺锤体极组装的分子途径，并定义纤毛蛋白在有丝分裂细胞中的新功能。 公共卫生相关性：我们试图揭示纺锤体组织的一种新的分子途径，该途径与许多人类疾病有关，包括癌症、发育异常、纤毛疾病、小头畸形和原始侏儒症。这项工作的一个长期目标是开发新的治疗策略来预防或治疗由纺锤体功能障碍引起的疾病。

DESCRIPTION (provided by applicant): Centrosomes contribute to mitotic spindle organization and orientation in mitosis, and to the assembly of primary cilia in nondividing cells. Centrosome anomalies affect the fidelity of spindle and cilia function and are associated with ciliopathies, microcephaly, dwarfism, cancer and other human disorders. Cilia proteins are found at centrosomes in mitotic cells (spindle poles) and some are involved in the orientation of cell division. However, the function of these cilia proteins in mitotic cells is not known. Preliminary results from the Doxsey laboratory suggest that cilia proteins required for transporting material up and down cilia in noncycling cells (intraflagellar transport, IFT) also transport material to and from spindle poles in mitotic cells. Disruption of these cilia proteins induces defects in mitotic spindle orientation, astral microtubule organization, spindle pole function and mitotic progression. Other cilia proteins localize to additional mitotic structures such as kinetochores and midbodies, suggesting additional mitotic functions of this class of proteins. The overall goal of this proposal is to test the hypothesis that IFT complexes involved in cilia formation and function in noncycling cells, are re-directed, at least in part, to perform previously unanticipated mitotic functions. To test this, we will address the molecular mechanism of IFT protein complex function in mitotic spindles. Our preliminary studies indicate that IFT88 forms particles in cells that transport microtubule-nucleating proteins to spindle poles using the dynein motor. The specific aims are designed to test if IFT protein complexes serve as carriers of mitotic cargoes and if dynein provides the force for their movement. Novel aspects of the work include the identification of a novel mechanism for spindle pole assembly and spindle orientation, the use of super- resolution microscopy to image the dynamics of novel IFT protein-containing particles in living mitotic cells, the characterization of new mitotic IFT protein-dynein complexes using new affinity systems and the use of in vitro assays to study dynein-based motility. This work has the potential to identify a new molecular pathway for spindle pole assembly and to define novel functions of cilia proteins in mitotic cells. PUBLIC HEALTH RELEVANCE: We seek to uncover a novel molecular pathway for spindle organization, which is relevant to a number of human disorders including cancer, developmental abnormalities, ciliopathies, microcephalies and primordial dwarfisms. A long-term goal of this work is to develop new therapeutic strategies for prevention or treatment of disorders arising from spindle dysfunction.