APOPTOTIC MEMBRANE BLEBBING IS REGULATED BY MYOSIN LIGHT-CHAIN PHOSPHORYLATION

The evolutionarily conserved execution phase of apoptosis is defined b y characteristic changes occurring during the final stages of death; s pecifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmar k features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous acros s a population of cells. In the present study, a model system is descr ibed for using the caspase inhibitor, z-VAD-FMK, to block apoptosis an d generate a synchronous population of cells actively extruding and re tracting membrane blebs. This model system allowed us to determine sig naling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cell s indicated that only myosin light chain kinase (MLCK) inhibitors decr eased blebbing, Immunoprecipitation of myosin II demonstrated that myo sin regulatory light chain (MLC) phosphorylation was increased in bleb bing cells and that MLC phosphorylation was prevented by inhibitors of MLCK, MLC phosphorylation is also mediated by the small G protein, Rh o. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process, Finally, blebbing was al so inhibited by disruption of the actin cytoskeleton, Based on these r esults, a working model is proposed for how actin/myosin II interactio ns cause cell contraction and membrane blebbing, Our results provide t he first evidence that MLC phosphorylation is critical for apoptotic m embrane blebbing and also implicate Rho signaling in these active morp hological changes. The model system described here should facilitate f uture studies of MLCK, Rho, and other signal transduction pathways act ivated during the execution phase of apoptosis.