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Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

Abstract

The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin–myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin–myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin–myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.

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Figure 1: Membrane blebbing during apoptosis is Rho-independent but ROCK-dependent.
Figure 2: ROCK I is a substrate for caspase-mediated proteolysis during TNFα-induced apoptosis.
Figure 3: Caspase-cleavage of ROCK I generates a truncated form with higher intrinsic kinase activity.
Figure 4: Formation of actin stress fibres, cell contraction and dynamic membrane blebbing induced by active ROCK I.
Figure 5: Active ROCK I, but not active PAK2 or PRK1, promotes membrane blebbing in the presence of caspase inhibitor.
Figure 6: ROCK activity is dispensable for caspase activation, cytochrome c release and phosphatidylserine externalization.
Figure 7: ROCK-dependent membrane blebbing is required for localization of fragmented DNA to blebs and apoptotic bodies.
Figure 8: Caspase-mediated cleavage of ROCK I during apoptosis leads to loss of its auto-inhibitory domain.

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Acknowledgements

We thank R. Marais and C. Marshall for discussions, H. Patterson, A. Tutt, D. Robertson and I. Titley for technical advice, J. Riedl for recombinant Tat–C3 protein, the Welfide Corporation for Y-27632, R. Treisman for pGEX-KG C3, A. Ridley for pCAN-PAK2 (T403E), and S. Narumiya for pCAG-Myc–ROCK I. This work was supported by a project grant from the Cancer Research Campaign. M.F.O. is a Mr and Mrs John Jaffe Donation University Research Fellow of The Royal Society.

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Correspondence to Michael F. Olson.

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Movie 1

Cell contraction and dynamic membrane blebbing induced by active ROCK I. Serum-starved NIH 3T3 fibroblasts were pretreated with 50 μM z-VAD-fmk for 1 h and then microinjected with plasmids encoding both GFP and truncated ROCK I(G1,114opa). After 2 h, time-lapse images were obtained by visualizing expressed GFP using confocal microscopy over a period of 25 min. (AVI 2616 kb)

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Coleman, M., Sahai, E., Yeo, M. et al. Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I. Nat Cell Biol 3, 339–345 (2001). https://doi.org/10.1038/35070009

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