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

基本信息

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
项目类别：
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

项目摘要

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.

对细胞周期有深入的了解，细胞周期是细胞以受控方式生长和分裂的过程促进生物体的生长，对于我们理解人类的健康发展、发展至关重要我们最近描述了一种新型蛋白质 WDR73，它是一种微管相互作用蛋白。在有丝分裂（细胞分裂）过程中很重要，我们将 WDR73 基因突变与加洛韦-莫瓦特综合征联系起来。（GAMOS），一种罕见的隐性发育障碍，其特征是小头畸形、大脑发育迟缓小脑、智力障碍、生长迟缓、失明、肾病和过早死亡。研究确定 WDR73 是细胞周期正常进展所必需的，尤其是有丝分裂期间。有丝分裂时，WDR73 在微管形成有丝分裂纺锤体时定位于微管，并在微管生长时与其保持在一起捕获前中期和中期的染色体，WDR73 保留在纺锤体上。后期（染色体分离），但也延伸到极间和动粒微管，直到末期，当它集中在中体微管时，子细胞最终分离该提案的目标是研究 WDR73 在所有阶段的功能。基于 WDR73 与其他蛋白质相互作用的初步数据，我们发现 WDR73 在细胞周期和亚细胞周期的多个阶段中支架动态蛋白质相互作用在有丝分裂期间，初步数据表明 WDR73 可能在成核和形成过程中发挥作用。我们对 GAMOS 患者细胞的研究进一步表明 WDR73 可能是有丝分裂微管的稳定作用。细胞存活、细胞周期退出的正常时间和细胞分化所需的。和生化方法通过三个具体目标来研究这些假设：（1）确定 WDR73 在有丝分裂和间期微管网络的组装和重塑中的作用。固定细胞成像将用于 GAMOS 患者细胞和 WDR73 将被 RNA 耗尽的细胞系干扰（基因沉默）以确定 WDR73 功能的丧失如何损害微管网络和延迟 (2) 表征增殖和非增殖中的 WDR73 蛋白相互作用组（分化的）细胞将在每个阶段使用。细胞周期和有丝分裂的亚阶段，以及有丝分裂后神经元，以新颖地鉴定 WDR73 相互作用蛋白。 (3) 研究WDR73在神经元分化和存活中的作用，将用于RNA干扰。评估 WDR73 蛋白缺失对神经细胞增殖、存活和分化的影响。研究是创新的，因为它的目的是表征适当的新型蛋白质的功能细胞周期的进展非常重要，因为它将增强我们对细胞调节的理解。周期和微管网络的相关调节是维持正常发育所必需的大脑、小脑和肾脏。