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

• 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
• 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

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.

项目摘要/摘要 微管细胞骨架是细胞分化的关键调节剂，必须在空间上组织 为了实现其细胞功能。尽管微管由特定位点组织的概念 称为微管组织中心（MTOC）已被赞赏超过50年，绝大多数 MTOC的研究集中在中心体上。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重新分配到非中心位点。例如， 非中心MTOC（NCMTOC）在上皮细胞的顶膜上形成，向下轴突长度 神经元中的树突以及在肌管和微管中的核包膜上对 神经发生，肌肉功能以及大多数组织的形态发生和极化，包括心脏， 大脑和肠。尽管在体内分化细胞中它们的无处不在和重要性，但对 NCMTOC建立的机制或生物体中NCMTOC组件的身份，部分原因是 缺乏适当的遗传模型。该提案检验了NCMTOC是 由位点特异性适配器和微管负端蛋白组成，并带有链接微管的适配器 通过它们的负末端到标记细胞位置的极性复合物。我们的目标将解决 NCMTOC建立的组成和机制，该领域的两个重要知识差距 在模型有机体中使用尖端遗传和蛋白质组学工具。秀丽隐杆线虫。我们确定了交互者 使用独家NCMTOC组件PTRN-1/Patronin，将发现这些保守的作用 NCMTOC机构中的互动者（AIM 1）。我们将使用最近的 适应的接近标签技术涡轮增压技术，首次在秀丽隐杆线虫中应用，高吞吐量 组织和途径特异性的正向遗传筛查策略（AIM 2）。最后，我们将测试特定模型的 使用我们已经优化的组织特异性降解策略的NCMTOC建立（AIM 3）。恰当的 微管组织对于正常发育和细胞功能和多动MTOC功能至关重要 在中心体是一些癌症的标志。因此，在这些研究中发现的分子可以 提供潜在的治疗靶标，并阐明细胞中重要但研究的主题 发展生物学。