Caspase-mediated cleavage of a scaffold protein, MPRIP, yields a truncated form that is involved in repetitive bleb formation.

Membrane blebbing is a hallmark of apoptotic cell death. However, the molecular mechanism that regulates this event has not been fully elucidated. To understand this underlying mechanism, we developed visualization systems suitable for spatiotemporal analysis. By monitoring the plasma membrane labeled with a fluorescent protein and reconstructing the image data as three-dimensional (3D) volumes based on the rendering technique, we observed that dying cells exhibit cycles of bleb formation at the same region of the cell surface. In addition, a Förster Resonance Energy Transfer (FRET)-based biosensor incorporating a regulatory myosin light chain (RMLC) displayed phosphorylation at the base of the retracting bleb, and dephosphorylation before re-expansion, implying the involvement of not only a kinase but also a phosphatase in the regulation of RMLC. To extend these observations, we focused on a scaffold protein, myosin phosphatase Rho interacting protein (MPRIP), which interacts with RhoA and myosin phosphatase targeting subunit 1 (MYPT1), involved in activation of Rho-associated coiled-coil kinase-I (ROCK-I) or protein phosphatase 1 (PP1), respectively. We found that MPRIP is cleaved both in dying cells and in an in vitro cleavage assay in a caspase-dependent manner. A cleaved C-terminal peptide fragment maintains the interaction with MYPT1. Cytological analysis showed that this fragment forms a complex with MYPT1 and myosin after translocating to the cytoplasm. These results suggest that this complex formation promotes the dephosphorylation of RMLC. Collectively, our study indicates that repetitive bleb formation, which is unique to apoptosis, is regulated by both phosphorylation and dephosphorylation of RMLC through MPRIP in a coordinated manner.