Molecular, material, and structural design principles of centrosomes

项目摘要 中心体使微管阵列成核，并作为力协调中心 定位细胞核和分离染色体，这是早期的基本活动 胚胎发育和神经发育。虽然人们对这一规定有很多了解 对于中心体数量，关于决定分子机制的了解要少得多 中心体大小、微管成核能力和抗力能力。的目标是 这一提议是为了揭示中心体蛋白之间的分子水平相互作用 测定PCM的活性、新材料性质和超微结构，最 中心体的实质一层。 我假设PCM是一种无定形水凝胶，其材料状态(例如，强度， 弹性)由盘绕式脚手架之间的磷可调连接来调节 蛋白质。我进一步假设，脚手架结构和材料性能的微调 调节PCM的大小、活性和对微管依赖的拉力的抵抗。我 建议使用我最近提出的两种创新技术来验证这些假设 开发了一种最小的PCM重建系统和一种执行纳米级的光学方法 活体胚胎中PCM的流变学。此外，我还建议发展细胞内低温电子。 断层扫描显示PCM超微结构，分辨率低于10 nm。这些 设计实验的目的是1)确定生成 一致的大小、完全有效的PCM，2)发现关键调节器和材料设计 允许PCM抵抗微管拉力的原理，以及3)产生最高的- 迄今为止，天然中心体的分辨率结构图谱。这项建议意义重大 因为它将阐明中心体功能是如何在 分子水平，这将提供对人类疾病的机械性洞察 中心体功能障碍，如小头畸形、原始侏儒症和各种 癌症。

Project Summary Centrosomes nucleate microtubule arrays and act as force-coordinating centers to position nuclei and segregate chromosomes, which are essential activities during early embryogenesis and neural development. While much is understood about the regulation of centrosome number, much less is known about molecular mechanisms determining centrosome size, microtubule nucleation capacity, and resistance to forces. The goal of this proposal is to reveal how molecular-level interactions between centrosome proteins determine the activity, emergent material properties, and ultrastructure of PCM, the most substantial layer of a centrosome. I hypothesize that PCM is an amorphous hydrogel whose material state (e.g., strength, elasticity) is regulated by phospho-tunable connections between coiled-coil scaffolding proteins. I further hypothesize that fine-tuning of scaffold structure and material properties regulates PCM size, activity, and resistance to microtubule-dependent pulling forces. I propose to test these hypotheses using two innovative techniques that I recently developed: a minimal PCM reconstitution system and an optical method to perform nano- rheology of PCM in living embryos. In addition, I propose to develop in-cell cryo-electron tomography to visualize PCM ultrastructure with sub-10 nm resolution. These experiments are designed to 1) identify the minimal components needed to generate consistently sized, fully active PCM, 2) discover key regulators and material design principles that allow PCM to resist microtubule-pulling forces, and 3) generate the highest- resolution structural atlas of native centrosomes to date. This proposal is significant because it will illuminate how centrosome function is determined and regulated at the molecular level, which will provide mechanistic insight into human disorders caused by centrosome dysfunction, such as microcephaly, primordial dwarfism, and various cancers.