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Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro
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Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing

M Sebbagh1, C Renvoizé, J Hamelin

  • 1INSERM U461, Faculté de Pharmacie, 92296 Châtenay-Malabry, France.

Nature Cell Biology
|April 3, 2001
PubMed

Insights

Caspase-3 activation cleaves ROCK I, a key protein in apoptosis. This cleavage deregulates ROCK I, leading to increased myosin light chain (MLC) phosphorylation and membrane blebbing during programmed cell death.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Apoptosis, or programmed cell death, is characterized by dynamic membrane blebbing.
  • Myosin light chain (MLC) phosphorylation is essential for this blebbing process.

Purpose of the Study:

  • To identify novel substrates of caspases involved in apoptotic membrane blebbing.
  • To elucidate the mechanism linking caspase activity to MLC phosphorylation and bleb formation.

Main Methods:

  • Identification of ROCK I as a caspase-3 substrate.
  • Analysis of ROCK I cleavage site (DETD1113/G) and its effect on kinase activity.
  • Assessment of MLC phosphorylation and membrane blebbing in response to caspase or ROCK inhibition, and expression of truncated ROCK I.

Main Results:

  • Caspase-3 cleaves ROCK I, removing its inhibitory domain and causing constitutive kinase activity.
  • Apoptotic cells show increased phosphorylated MLC correlating with ROCK I cleavage.
  • Inhibition of caspases or ROCK abrogates MLC phosphorylation and blebbing; Rho activity is not required.

Conclusions:

  • Caspase-3 mediated activation of ROCK I is a critical event driving membrane blebbing in apoptosis.
  • This pathway provides a direct link between caspase executioners and the morphological changes of cell death.

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