Deciphering the molecular principles linking PopZ filament structure to condensate function

About This Grant

PROJECT SUMMARY/ABSTRACT Biomolecular condensates critically regulate cellular processes including chromosome segregation, signaling, and protein homeostasis, yet the structural principles linking their architecture to function remain poorly understood. Filamentous condensates represent an important subclass, as their filament-based ultrastructure directly determines mechanical properties and spatial organization, impacting processes ranging from selective autophagy and transcriptional regulation to cell polarity and disease pathology. Leveraging Polar Organizing Protein Z (PopZ) from Caulobacter crescentus as a powerful model system, our recent findings demonstrate that filamentous ultrastructure underpins PopZ's cellular functions, with filament disruption severely impairing chromosome segregation and asymmetric cell division. Given PopZ's unique combination of structured oligomerization domains and intrinsically disordered regions (IDRs), its tunable material properties, evolutionary conservation, and high engineerability, it represents a powerful platform to elucidate how filamentous condensates translate protein sequence and nanoscale architecture into emergent biological functions. To address these fundamental questions, this R35 proposal will integrate advanced methodologies, including cryo-electron tomography, MINFLUX nanoscopy, single-molecule FRET, proteomics, and integrative modeling, across three interconnected research directions. First, we will determine a multiscale structural and dynamic model of PopZ condensates by resolving filament structures at near-atomic resolution, clarifying how IDR conformational dynamics regulate filament assembly via an autoinhibitory switch, and characterizing intrinsic nanoscale filament movements using MINFLUX microscopy. Second, we will elucidate the molecular logic of client selectivity by examining how PopZ condensates remodel their architecture in response to specific client proteins to temporally coordinate chromosome segregation with asymmetric signaling, while proteomics will map dynamic client interactions throughout the cell cycle. Finally, we will define the molecular mechanisms underlying condensate aging by comparing condensate stiffening observed in vitro with sustained fluidity in cells, utilizing FRAP, cryo-electron tomography, and proteomics to identify structural transitions and key client proteins that influence condensate longevity. Together, these studies will yield a comprehensive molecular blueprint for filament-based condensates, providing broadly applicable principles for cellular organization and enabling rational design of synthetic condensates to manipulate cellular processes and target condensate dysfunction in disease.