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

微管结合蛋白 WDR73 在有丝分裂和有丝分裂后细胞中的作用

• 批准号: 10046529
• 负责人: Robert Nelson Jinks
• 金额: $ 38.68万
• 依托单位: FRANKLIN AND MARSHALL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10046529
• 关键词: Affect; Anaphase; Apoptosis; Back; Binding Proteins; Biochemical; Biological; Biological Assay; Biomedical Research; Blindness; Brain; Businesses; Cell Aging; Cell Cycle; Cell Cycle Regulation; Cell Differentiation process; Cell Line; Cell Proliferation; Cell Proliferation Regulation; Cell Survival; Cell division; Cell physiology; Cells; Centrosome; Cerebellum; Cerebral cortex; Cessation of life; Chromosome Segregation; Co-Immunoprecipitations; Confocal Microscopy; Congenital cerebellar hypoplasia; Cortical Blindness; Coupled; Cytokinesis; Cytoplasmic Granules; Data; Development; Developmental Delay Disorders; Disease; End stage renal failure; Family Caregiver; Functional disorder; Galloway syndrome; Gene Mutation; Gene Silencing; Generations; Goals; Government Officials; Growth; Hippocampus (Brain); Human; Human Development; Immunofluorescence Microscopy; Impairment; In Vitro; Individual; Intellectual functioning disability; Interphase; Interphase Cell; Kidney; Kidney Diseases; Kinetochores; Knowledge; Learning; Malignant Neoplasms; Mass Spectrum Analysis; Medicine; Metaphase; Microcephaly; Microtubule Polymerization; Microtubule-Associated Proteins; Microtubules; Minor; Mitosis; Mitotic; Mitotic spindle; Modeling; Morphogenesis; Morphology; Movement Disorders; Mus; Mutation; Neurites; Neuronal Differentiation; Neurons; Nuclear; Optic Nerve; Organ; Organism; Parents; Patients; Phase; Play; Process; Proliferating; Prometaphase; Proteins; Public Health; Publishing; RNA Interference; Rare Diseases; Regulation; Reporting; Research; Resolution; Role; Scaffolding Protein; Skin; Steroid-resistant idiopathic nephrotic syndrome; Students; Testing; Tissues; Tretinoin; WD Repeat; base; cell immortalization; cell type; cellular imaging; cerebral atrophy; chromosome conformation capture; daughter cell; depolymerization; developmental disease; early childhood; gene replacement therapy; genome editing; in utero; innovation; knock-down; live cell imaging; migration; nephrogenesis; nerve stem cell; neuroblast; neuronal survival; novel; premature; progenitor; public health relevance; recruit; scaffold; science teacher; spatiotemporal; stem cell proliferation; telophase; undergraduate student

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.

对细胞周期的深刻理解，细胞以受控的方式生长和分裂的过程， 促进生物体的生长，对我们理解健康的人类发展至关重要， 疾病和癌症。我们最近描述了一种新的蛋白质，WDR 73，作为微管相互作用蛋白 在有丝分裂（细胞分裂）中起重要作用。我们将WDR 73基因突变与Galloway-Mowat综合征联系起来， （GAMOS），一种罕见的隐性发育障碍，其特征是小头畸形， 小脑、智力残疾、生长迟缓、失明、肾病和过早死亡。我们 研究确定WDR 73是细胞周期，特别是有丝分裂的正常进程所必需的。期间 在有丝分裂中，WDR 73定位于微管，因为它们形成有丝分裂纺锤体，并随着它们的生长而与它们在一起 在前中期和中期捕获染色体。WDR 73在运行期间保持在主轴上 分裂后期（染色体分离），但也延伸到极间和动粒微管， 当它集中在中间体微管，在那里最终分离的子细胞时， （细胞分裂）是协调的。本提案的目标是调查WDR 73在所有阶段的功能 细胞周期和神经元分化。基于WDR 73与其他蛋白质相互作用的初步数据， 我们假设WDR 73支架在细胞周期和亚细胞周期的多个阶段动态蛋白质相互作用， 有丝分裂的阶段。在有丝分裂过程中，初步数据表明WDR 73可能在成核和细胞分裂中起作用。 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，WDR 73可能是 这是细胞存活、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 和生物化学的方法来研究这些假设，通过三个具体的目的：（1）确定 WDR 73在有丝分裂和间期微管网络的组装和重塑中的作用。生活和 固定细胞成像将用于GAMOS患者细胞和WDR 73将被RNA耗尽的细胞系中 干扰（基因沉默），以确定WDR 73功能的丧失如何损害微管网络和延迟 细胞周期(2)在增殖和非增殖中表征WDR 73蛋白相互作用体 （分化的）细胞。在每个阶段将使用无偏免疫共沉淀和质谱法 的细胞周期和有丝分裂的亚阶段，并在有丝分裂后的神经元，以确定新的WDR 73相互作用蛋白。 (3)研究WDR 73在神经元分化和存活中的作用。RNA干扰将用于 评估WDR 73蛋白缺失对神经细胞增殖、存活和分化的影响。这 这项研究是创新的，因为它的目的是表征一种新的蛋白质的功能， 通过细胞周期的进展。它的意义在于它将加深我们对细胞调控的理解， 周期和相关的调控微管网络必要的维持正常发展的 大脑小脑和肾脏