Transient volume stabilization reveals the key role a PM permeability in pyroptotic cell swelling.

Abstract

Pyroptosis, an inflammatory form of cell death, is characterized by massive cell swelling and plasma membrane rupture. Although swelling was recently shown to occur in two steps, the molecular and biophysical mechanisms driving this process remained unclear. Using fast quantitative microscopy, we reveal that between the two swelling phases, cell volume transiently stabilizes despite sustained plasma membrane permeability to ions and small molecules. From a biophysical perspective, the existence of such a plateau is puzzling, as ion pumps should not be able to regulate cell volume under these conditions. To address this, we developed a physical model based on an ion pump and leak framework that incorporates the dynamics of non-selective pore formation. Experimentally, we demonstrate that the plateau phase is controlled by the dynamics of the GSDMD pore enlargement, which is modulated by Ninj1 activation, possibly through intracellular calcium. Ninj1-mediated lesions are also required for the second swelling phase. We further show that fully opened GSDMD pores display an effective hydrodynamic radius slightly above 1.9 nm, providing an insitu upper bound for pore size. Together, our findings demonstrate that pyroptotic volume dysregulation emerges from the successive and interdependent actions of GSDMD and Ninj1, each imparting distinct permeability regimes associated with increased water filtration and decreased ion selectivity due to pore opening. These insights bridge molecular and biophysical perspectives on lytic cell death and may inform the broader understanding of membrane rupture in inflammatory and pathological contexts. Significance StatementAmong programmed modes of lytic cell death, pyroptosis mediated by gasdermin D (GSDMD) and ninjurin-1 (Ninj1) involves dramatic changes in cell shape and large volume fluctuations, fundamentally altering the cells physical properties. By combining optogenetics, quantitative microscopy, and modeling, we show that a progressive increase in plasma membrane pore size drives cell swelling and membrane lysis through successive and interdependent actions of GSDMD and Ninj1, each imparting distinct permeability regimes associated with increased water filtration and decreased ion selectivity. A deeper understanding of these dynamic cell modifications will shed light on the molecular and biophysical mechanisms driving different forms of cell death.