Assessing Cytoskeletal Destruction During Pyroptosis

Pyroptosis is an inflammatory form of cell death driven by the activation of caspase-1 and/or caspase-11 which cleaves and activates the pore-forming and cell-permeabilizing protein gasdermin-D. Pyroptosis is characterized by cell swelling and release of inflammatory cytosolic content, which were thought to be driven by colloid-osmotic lysis. Instead, we previously demonstrated that in vitro, pyroptotic cells do not in fact lyse. We also demonstrated that calpain cleaves vimentin, leading to loss of intermediate filaments, which in turn makes cells fragile and susceptible to rupture by extrinsic pressure. However, if, as our observations suggest, cells do not swell due to osmotic forces, what then causes cell rupture? Interestingly, in addition to intermediate filament loss, we demonstrated that other cytoskeletal networks, such as microtubules, actin, and nuclear lamina, are similarly lost during pyroptosis; however, the mechanisms driving these cytoskeletal disruptions as well as their functional significance are unclear. To facilitate the study of these processes, we present here the immunocytochemical methods by which we detected and assayed cytoskeletal destruction during pyroptosis.