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 caspase1. Caspase cleaves hundreds of substrates throughout the cell to regulate apoptotic processes2, including phosphatidylserine (PS) externalization3-5. 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 Forster resonance energy transfer (Dual FRET) imaging system that combines subcellularly targeted FRET-based caspase biosensors with a cytosolic reference counterpart6,7. Genetic analysis identified caspase-7, an executioner caspase considered an inefficient backup for caspase-3, the major executioner caspase8,9, as a caspase responsible for the Early-Peaking Caspase Activity at the PM (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.