PROJECT SUMMARY/ABSTRACT The actin and microtubule cytoskeletons are filamentous polymers that regulate vital cell processes, including but not limited to division, morphogenesis, DNA synthesis and repair, phagocytosis, and motility. Exactly how the dynamics of individual actin filaments or microtubules are regulated and coordinated in these cell processes is not fully understood. The goal of this research is to understand actin and microtubule assembly and their coordination by 1) developing new tools to visualize these proteins and their regulators, and 2) examining the emerging convergence between these proteins and membrane-less biomolecular condensates. This project utilizes an advanced in vitro biomimetic microscopy system capable of simultaneously monitoring fluorescent actin filaments, microtubules, and other regulatory proteins to elucidate detailed molecular mechanisms at single- molecule resolution. To address these mechanisms in a more physiological setting, we plan to combine these probes with an opto-genetic system that mimics several neurodegenerative disorders by repositioning condensates from the nucleus to the cytoplasm. This innovative approach imitates the onset of most neurodegenerative disorders and allows us to quantify pre- and post-disease state changes at high-resolution in the same cell. Identifying the molecular mechanisms that underlie these intricate biological systems (e.g., the dynamics of the cytoskeleton and biomolecular condensates) will significantly contribute to our understanding of fundamental biological processes and the onset of most neurodegenerative conditions.