Mitotic functions of cilia proteins

描述（申请人提供）：中心体在有丝分裂中参与纺锤体的组织和定位，以及在非分裂细胞中参与初级纤毛的组装。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（纺锤极）的中心体中，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，在非循环细胞的纤毛上下运输物质所需的纤毛蛋白（鞭毛内运输，IFT）也在有丝分裂细胞的纺锤极之间运输物质。这些纤毛蛋白的破坏导致有丝分裂纺锤体取向、星状微管组织、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，如着丝点和中间体，表明这类蛋白具有其他有丝分裂功能。本研究的总体目标是验证IFT复合物在非循环细胞中参与纤毛形成和功能的假设，至少部分地重新定向，以执行先前未预料到的有丝分裂功能。为了验证这一点，我们将讨论IFT蛋白复合物在有丝分裂纺锤体中的分子机制。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成核蛋白运送到纺锤极。具体目的是测试IFT蛋白复合物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运动提供动力。这项工作的新方面包括鉴定主轴极组装和主轴方向的新机制，使用超分辨率显微镜对活的有丝分裂细胞中含有新型IFT蛋白的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-动力蛋白复合物，以及使用体外实验研究基于动力蛋白的运动性。这项工作有可能确定纺锤体极组装的新分子途径，并确定有丝分裂细胞中纤毛蛋白的新功能。

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.