Investigating the establishment, structure, and function of microtubule organizing centers in differentiated cells in vivo

研究体内分化细胞微管组织中心的建立、结构和功能

基本信息

批准号：
10159297
负责人：
Jessica Lynn Feldman
金额：
$ 31.54万
依托单位：
STANFORD UNIVERSITY
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：
2020-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10159297
关键词：
Address Animal Model Animals Apical Axon Biochemical Genetics Biology Brain Caenorhabditis elegans Cell Differentiation process Cell Polarity Cell division Cell physiology Cells Cellular Structures Cellular biology Centrosome Complex Congenital Abnormality Cytoskeleton Data Defect Dendrites Development Developmental Biology Disease Embryo Epithelial Cells Genes Genetic Genetic Models Genetic Screening Heart Human Hyperactivity Immunoprecipitation In Vitro Intestines Knowledge Label Lateral Length Light Link Location Malignant Neoplasms Microcephaly Microtubule-Organizing Center Microtubules Minus End of the Microtubule Mitosis Mitotic spindle Modeling Molecular Morphogenesis Muscle Fibers Muscle function Neurons Normal Cell Nuclear Envelope Organism Pathology Pathway interactions Pattern Play Process Protein-Serine-Threonine Kinases Proteins Proteomics Radial Research Role Shapes Site Structure Techniques Testing Therapeutic Time Tissues Tubulin WD Repeat Work absorption apical membrane cell type experimental study genetic approach high throughput screening in vivo insight intestinal epithelium neurogenesis new therapeutic target novel programs spatiotemporal therapeutic target tool

项目摘要

PROJECT SUMMARY/ABSTRACT The microtubule cytoskeleton is a critical regulator of cell differentiation, and must be spatially organized in order to fulfill its cellular functions. Although the concept that microtubules are organized by specific sites called microtubule organizing centers (MTOCs) has been appreciated for more than 50 years, the vast majority of research on MTOCs has focused on the centrosome. While all animal cells use centrosomes as MTOCs during mitosis, MTOC function is reassigned to non-centrosomal sites during cell differentiation. For example, non-centrosomal MTOCs (ncMTOCs) form at the apical membrane of epithelial cells, down the length of axons and dendrites in neurons, and at the nuclear envelope in myotubes and microtubules are critical for neurogenesis, muscle function, and in morphogenesis and polarization of most tissues including the heart, brain, and intestine. Despite their ubiquity and importance in differentiated cells in vivo, little is known about mechanisms of ncMTOC establishment or the identity of ncMTOC components in an organism, in part due to the lack of an appropriate genetic model. This proposal tests the central hypothesis that ncMTOCs are composed of site-specific adapters and microtubule minus end proteins, with adapters linking microtubules through their minus ends to polarity complexes that mark cellular locations. Our aims will address the composition and mechanisms of establishment of ncMTOCs, the two significant knowledge gaps in this field using cutting edge genetic and proteomic tools in the model organism C. elegans. We identified interactors with the exclusive ncMTOC component PTRN-1/Patronin and will uncover the role of these conserved interactors in ncMTOC establishment (Aim 1). We will identify novel ncMTOC components using our recently adapted proximity labeling technique TurboID, applied for the first time in C. elegans, and a high throughput tissue- and pathway-specific forward genetic screening strategy (Aim 2). Finally, we will test specific models for ncMTOC establishment using a tissue specific degradation strategy that we have optimized (Aim 3). Proper microtubule organization is essential for normal development and cell function and hyperactive MTOC function at the centrosome is a hallmark of some cancers. Thus, the molecules uncovered in these studies could provide potential therapeutic targets as well as shed light on an important, but understudied topic in cell and developmental biology.

项目概要/摘要微管细胞骨架是细胞分化的关键调节因子，必须在空间上组织以实现其细胞功能。尽管微管是由特定位点组织的概念被称为微管组织中心 (MTOC) 的人们已经认识了 50 多年，绝大多数 MTOC 的研究主要集中在中心体。虽然所有动物细胞都使用中心体作为 MTOC 在有丝分裂过程中，MTOC 功能在细胞分化过程中被重新分配到非中心体位点。例如，非中心体 MTOC（ncMTOC）形成于上皮细胞的顶膜，沿着轴突的长度神经元中的树突和肌管和微管的核膜对于神经发生、肌肉功能以及包括心脏在内的大多数组织的形态发生和极化，脑、肠。尽管它们在体内分化细胞中普遍存在且很重要，但人们对它们知之甚少。 ncMTOC 建立机制或生物体中 ncMTOC 成分的身份，部分原因是缺乏合适的遗传模型。该提案测试了 ncMTOC 的中心假设由位点特异性接头和微管负端蛋白组成，接头连接微管通过它们的负端到标记细胞位置的极性复合物。我们的目标将解决 ncMTOC的组成和建立机制是该领域的两个重大知识差距在模式生物秀丽隐杆线虫中使用最先进的遗传和蛋白质组学工具。我们确定了互动者与独有的 ncMTOC 成分 PTRN-1/Patronin 一起，将揭示这些保守的作用 ncMTOC 建立中的互动者（目标 1）。我们将使用我们最近的技术来识别新型 ncMTOC 组件采用的邻近标记技术 TurboID 首次应用于秀丽隐杆线虫，并且具有高通量组织和途径特异性正向遗传筛选策略（目标 2）。最后，我们将测试特定模型使用我们优化的组织特异性降解策略建立 ncMTOC（目标 3）。恰当的微管组织对于正常发育和细胞功能以及过度活跃的 MTOC 功能至关重要位于中心体是某些癌症的标志。因此，这些研究中发现的分子可以提供潜在的治疗靶点，并阐明细胞和细胞学领域一个重要但尚未充分研究的主题发育生物学。