Molecular, material, and structural design principles of centrosomes

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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.