We discovered a conserved process that drives most epithelial cell death called ‘cell extrusion’, essential to maintaining correct cell numbers and an intact barrier. Importantly, misregulation of extrusion drives tumor formation and invasion and asthma inflammation and may contribute to lung fibrosis. Mechanical crowding from too many cells activates extrusion via an essential stretch-activated channel, Piezo1, which translates mechanical stress into signaling. Most research has focused on how stretch activates Piezo1. Yet extrusion depends on crowding-dependent activation of Piezo1, the mechanism of which remains mysterious. Interestingly, in crowded cells, Piezo1 localizes to novel organelles, piezosomes, that contain innate immunity factors like muc5A and a dsRNA-binding protein. During extrusion, piezosomes appear to explode into secretory vesicles containing signals critical for extrusion (and likely innate immunity). Preliminary conventional resin-section electron microscopy suggests that piezosomes consist of onion-like lamellar bodies. In this collaboration with the Atherton lab, which uses state-of-the-art correlative light and electron microscopy (CLEM), focused ion-beam milling (FIB), cryo-electron tomography (cryoET) and artificial intelligence-based image processing techniques to study organelles and macromolecules in near-native frozen-hydrated conditions, we will elucidate piezosome structure and function. Understanding how these piezosomes form and signal is central to understanding epithelial cell extrusion and how it becomes misregulated to drive numerous diseases.
1. Detail Piezosome ultrastructure via a CLEM-FIB-cryoET workflow (Years 1+2).
2. Obtain Piezo complex structures by sub-tomogram averaging (Years 2+3).
3. Determine how Piezosomes change in crowded states (Years 2-4).
4. Probe piezosomes structures in the presence of extrusion inhibitors (Years 2-4).