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

项目摘要/摘要 微管细胞骨架是细胞分化的关键调节因子，必须在空间上组织成 才能完成其细胞功能。尽管微管是由特定部位组织起来的概念 被称为微管组织中心(MTOCs)的研究已经有50多年的历史了，其中绝大多数 对MTOCs的研究主要集中在中心体上。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被重新分配到非中心体位置。例如, 非中心体MTOCs(NcMTOCs)形成于上皮细胞顶膜，向下延伸至轴突。 神经元中的树突，以及肌管和微管中的核膜上的树突对 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， 大脑和肠道。尽管它们在体内的分化细胞中普遍存在并具有重要意义，但人们对它们知之甚少 生物体中ncMTOC的建立机制或ncMTOC组分的识别，部分原因是 缺乏合适的遗传模型。这一提议检验了ncMTOCs是 由位点特异性接头和微管负端蛋白组成，接头连接微管 通过它们的负端到达标记细胞位置的极性复合体。我们的目标将解决 NcMTOCs的组成和建立机制，这是该领域的两个重大知识空白 在模式生物秀丽线虫中使用尖端的遗传和蛋白质组学工具。我们确定了相互作用者 与独有的ncMTOC成分PTRN-1/Patronin结合，将揭示这些保守的作用 国家技术合作组织建立中的相互作用者(目标1)。我们将使用我们最近的产品来确定新的ncMTOC组件 改进的邻近标记技术TurboID，首次应用于线虫，并高通量 组织和途径特定的正向遗传筛选策略(目标2)。最后，我们将测试特定型号的 使用我们优化的组织特定降解策略建立ncMTOC(目标3)。恰如其分 微管组织是正常发育和细胞功能以及MTOC功能亢进所必需的 中心体是某些癌症的标志。因此，这些研究中发现的分子可能 提供潜在的治疗靶点，并阐明一个重要的，但在细胞和 发育生物学。

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.