The role of the microtubule-binding protein WDR73 in mitotic and post-mitotic cells

对细胞周期有深入的了解，即细胞以受控方式生长和分裂的过程 促进有机体的生长，对于我们理解人类的健康发育、发展至关重要 疾病和癌症。我们最近描述了一种新的蛋白质WDR73，它是一种微管相互作用的蛋白质 在有丝分裂(细胞分裂)过程中很重要。WDR73基因突变与加洛韦-莫瓦特综合征相关 (Gamos)是一种罕见的隐性发育障碍，以小头畸形为特征，导致儿童生长发育减慢。 小脑、智力残疾、发育迟缓、失明、肾脏疾病和过早死亡。我们的 研究确定，WDR73是细胞周期正常进行所必需的，尤其是有丝分裂。在.期间 有丝分裂，WDR73在微管形成有丝分裂纺锤体时定位于微管，并在它们生长过程中与微管保持在一起 捕捉前中期和中期的染色体。WDR73保持在主轴上 后期(染色体的分离)，但也延伸到极间和动粒微管，直到 终末期，当它集中在中体微管时，子细胞在那里最终分离 (细胞质分裂)是协调的。这项建议的目标是研究WDR73在所有阶段的功能 细胞周期和神经元分化。基于WDR73‘S与其他蛋白质相互作用的初步数据， 我们假设WDR73支架在细胞周期的多个阶段和亚细胞周期中动态的蛋白质相互作用. 有丝分裂的阶段。在有丝分裂过程中，初步数据表明WDR73可能在核形成和 有丝分裂微管的稳定。我们对Gamos患者细胞的研究进一步表明，WDR73可能是 细胞存活、退出细胞周期的正常时间和细胞分化所必需的。我们将使用细胞生物学 和生物化学方法通过三个具体目标来研究这些假说：(1)确定 WDR73在有丝分裂和间期微管网络组装和重塑中的作用。现场直播 固定细胞成像将用于Gamos患者的细胞和WDR73将被RNA耗尽的细胞系 干扰(基因沉默)以确定WDR73功能丧失如何损害微管网络和延迟 细胞周期。(2)WDR73蛋白在增殖期和非增殖期的相互作用特征 (分化)细胞。在每个阶段将使用无偏免疫共沉淀和质谱分析 在细胞周期和有丝分裂亚阶段，以及在有丝分裂后神经元中鉴定新的WDR73相互作用蛋白。 (3)探讨WDR73在神经元分化和存活中的作用。RNA干扰将用于 评估WDR73蛋白缺失对神经细胞增殖、存活和分化的影响。这 这项研究具有创新性，因为它的目的是表征一种新的蛋白质的功能，这是 通过细胞周期的进展。它具有重要的意义，因为它将加深我们对细胞调节的理解 维持正常发育所必需的微管网络的周期和相关的调节 大脑、小脑和肾脏。

A strong understanding of cell cycle, the process through which cells grow and divide in a controlled manner to promote the growth of organisms, is vital to our understanding of healthy human development, developmental disorders, and cancers. We recently described a novel protein, WDR73, as a microtubule-interacting protein important during mitosis (cell division). We associated WDR73 gene mutations with Galloway-Mowat Syndrome (GAMOS), a rare, recessive developmental disorder characterized by microcephaly, reduced growth of the cerebellum, intellectual disability, growth retardation, blindness, kidney disease and untimely death. Our research determined that WDR73 is required for normal progression through cell cycle, especially mitosis. During mitosis, WDR73 localizes to the microtubules as they form the mitotic spindles and stays with them as they grow to capture the chromosomes during prometaphase and metaphase. WDR73 remains at the spindles during anaphase (segregation of chromosomes) but also extends into the interpolar and kinetochore microtubules until telophase when it concentrates at the midbody microtubules, where final separation of the daughter cells (cytokinesis) is coordinated. The goal of this proposal is to investigate the function of WDR73 during all phases of cell cycle and neuronal differentiation. Based on preliminary data on WDR73’s interactions with other proteins, we hypothesize that WDR73 scaffolds dynamic protein interactions during multiple phases of cell cycle and sub- phases of mitosis. During mitosis, preliminary data suggest that WDR73 may play a role in the nucleation and stabilization of mitotic microtubules. Our studies of GAMOS patient cells further suggest that WDR73 may be required for cell survival, normal timing of exit from cell cycle, and cell differentiation. We will use cell biological and biochemical approaches to investigate these hypotheses through three Specific Aims: (1) Determine the role of WDR73 in the assembly and remodeling of mitotic and interphase microtubule networks. Live and fixed cell imaging will be used in GAMOS patient cells and in cell lines in which WDR73 will be depleted by RNA interference (gene silencing) to determine how loss of WDR73 function impairs microtubule networks and delays cell cycle. (2) Characterize the WDR73 protein interactome in proliferating and non-proliferating (differentiated) cells. Unbiased co-immunoprecipitation and mass-spectrometry will be used during each phase of cell cycle and sub-phase of mitosis, and in post-mitotic neurons to identify novel WDR73 interacting proteins. (3) Investigate the role of WDR73 in neuronal differentiation and survival. RNA interference will be used to assess the impact of loss of WDR73 protein on proliferation, survival, and differentiation of neural cells. This research is innovative because it aims to characterize the function of a novel protein required for proper progression through cell cycle. It is significant because it will enhance our understanding of the regulation of cell cycle and the associated regulation of microtubule networks necessary to sustain normal development of the brain, cerebellum, and kidneys.