Early-peaking caspase-7 activity at the plasma membrane drives apoptotic phosphatidylserine exposure

Apoptosis is an immunologically silent form of regulated cell death executed by caspase ¹ . Caspase cleaves hundreds of substrates throughout the cell to regulate apoptotic processes ² , including phosphatidylserine (PS) externalization ^3–5 . However, spatio-temporal regulation of caspase activity in dying cells remains unclear. Here, we show that caspase activity peaks earlier at the plasma membrane (PM) during apoptosis by establishing a Dual Förster resonance energy transfer (Dual FRET) imaging system that combines subcellularly targeted FRET-based caspase biosensors with a cytosolic reference counterpart ^6,7 . Genetic analysis identified caspase-7, an executioner caspase considered an inefficient backup for caspase-3, the major executioner caspase ^8,9 , as a caspase responsible for the E arly- P eaking C aspase A ctivity at the P M (EP-CAP). Mechanistically, EP-CAP is mediated via electrostatic interactions between PS in the inner leaflet of the PM and polybasic residues in the N-terminal intrinsically disordered region (IDR) of caspase-7, which are liberated by the caspase-mediated removal of polyacidic residues. Physiologically, EP-CAP facilitates the efficient cleavage of phospholipid scramblases for the rapid externalization of PS and subsequent efferocytosis. Accordingly, we propose that caspase-7, but not caspase-3, is a bona fide immunologically silent death caspase reinforcing the non-inflammatory nature of apoptosis via EP-CAP.