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，这是在早期阶段的基本活动 胚胎发生和神经发育。虽然人们对这一规定有很多了解， 中心体数目的变化，关于决定中心体数目的分子机制知之甚少。 中心体大小、微管成核能力和对力的抵抗力。的目标 这项提议旨在揭示中心体蛋白质之间的分子水平相互作用 确定PCM的活性、紧急物质性质和超微结构， 中心体的实质层。 我假设PCM是一种无定形水凝胶，其材料状态（例如，力量， 弹性）由螺旋线圈支架之间的磷酸可调连接调节 proteins.我进一步假设支架结构和材料特性的微调 调节PCM大小、活性和对微管依赖性拉力的抵抗力。我 我建议使用两种创新技术来测试这些假设，我最近 开发：一个最小的PCM重建系统和光学方法来执行纳米- PCM在活胚胎中的流变学。此外，我建议发展细胞内冷冻电子 X射线断层扫描以使PCM超微结构以低于10 nm的分辨率可视化。这些 实验被设计为1）识别生成所需的最小组件 尺寸一致的全有源PCM，2）发现关键调节器和材料设计 原则，使PCM抵抗微管拉力，和3）产生最高- 迄今为止的天然中心体分辨率结构图谱。这一提议意义重大 因为它将阐明中心体的功能是如何决定和调节的， 分子水平，这将提供机制洞察人类疾病引起的 中心体功能障碍，如小头畸形，原始侏儒症，以及各种 癌的

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.