ALIX mediates reversible gasdermin-D pore formation via the endosomal pathway to limit pyroptosis by active membrane repair.

Pyroptosis is a form of regulatory cell death characterized by membrane rupture and release of pro-inflammatory signals. In pyroptosis, Caspase-1 activation leads to the cleavage of gasdermin-D (GSDMD). Upon cleavage, GSDMD's N-terminal (N-GSDMD) fragments insert into the plasma membrane, oligomerize, and form pores. The molecular details that define whether GSDMD pore formation results in cell death or survival are largely unknown. In this study, we show that a shorter duration of membrane N-GSDMD pores (t ≤ 2 h), along with associated membrane permeability does not harm cellular viability. We demonstrate that N-GSDMD is removed, and membrane integrity is restored if the pyroptotic stimulus is washed out within 1 hour. In contrast, longer duration of N-GSDMD pore formation leads to large-scale membrane damage and cell death. Using a selective dynamin inhibitor and confocal microscopy, to co-label N-terminal GSDMD (N-GSDMD) and the early endosomal marker EEA1, we demonstrate that N-GSDMD is cleared from the plasma membrane via the endosomal pathway. Through stable ALIX knockdown and overexpression approaches, we further show that ALIX, a key accessory protein of the ESCRT machinery, regulates N-GSDMD pore dynamics by promoting its removal and facilitating membrane repair via N-GSDMD internalization. In summary, we show that the duration of N-GSDMD membrane pores is a decisive factor and ALIX-dependent mechanism facilitates N-GSDMD removal and restores membrane integrity. The identification of these factors can open the development of new therapeutic strategies in chronic inflammatory conditions by bolstering the cell's inherent self-healing potential.