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

项目总结/摘要 微管细胞骨架是细胞分化的关键调节器，并且必须在空间上组织成 以实现其细胞功能。尽管微管是由特定的位点组织起来的这一概念 被称为微管组织中心（MTOC）已经被认识了50多年，绝大多数 关于MTOC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 在有丝分裂期间，在细胞分化期间，MTOC功能被重新分配到非中心体位点。比如说， 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，以及肌管和微管中的核被膜， 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分化的细胞中普遍存在且重要，但对它们的作用知之甚少。 ncMTOC建立的机制或生物体中ncMTOC组分的识别，部分原因是 缺乏合适的遗传模型。该提案检验了核心假设，即ncMTOC是 由位点特异性接头和微管负端蛋白组成，接头连接微管 通过它们的负端连接到标记细胞位置的极性复合物。我们的目标是解决 国家海洋技术选择委员会的组成和建立机制，这是该领域的两个重大知识空白 在模式生物C中使用最先进的遗传和蛋白质组学工具。优雅我们确定了互动者 与独家ncMTOC组件PTRN-1/Patronin，并将揭示这些保守的作用， 建立ncMTOC的相互作用者（目标1）。我们将确定新的ncMTOC组件使用我们最近 采用邻近标记技术TurboID，首次应用于C. elegans和高通量 组织和途径特异性正向遗传筛选策略（目标2）。最后，我们将测试特定的模型， 使用我们优化的组织特异性降解策略建立ncMTOC（目标3）。适当 微管组织对于正常发育和细胞功能以及过度活跃的MTOC功能是必需的 是某些癌症的标志因此，在这些研究中发现的分子可以 提供了潜在的治疗靶点，并阐明了一个重要的，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.