A Cryo-Electron Tomography Workflow Reveals Protrusion-Mediated Shedding On Injured Plasma Membrane

Cryo-electron tomography (cryo-ET) provides structural context to molecular mechanisms underlying biological processes. Although straightforward to implement for studying stable macromolecular complexes, using it to locate short-lived structures and events can be impractical. A combination of live-cell microscopy, correlative light and electron microscopy, and cryo-ET will alleviate this issue. We developed a workflow combining the three to study the ubiquitous and dynamic process of shedding in response to plasma membrane damage in HeLa cells. We found filopodia-like protrusions enriched at damage sites and acting as scaffolds for shedding, which involves F-actin dynamics, myosin-1a, and vacuolar protein sorting 4B (a component of the `endosomal sorting complex required for transport' machinery). Overall, shedding is more complex than current models of vesiculation from flat membranes. Its similarities to constitutive shedding in enterocytes argue for a conserved mechanism. Our workflow can also be adapted to study other damage response pathways and dynamic cellular events.