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Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis
08:55

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Published on: August 7, 2018

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Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Dan E McNamara1, Giovanni Quarato2, Cliff S Guy2

  • 1Department of Structural Biology, St. Jude Children's Research Hospital; Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital.

Journal of Visualized Experiments : Jove
|August 28, 2018
PubMed

Insights

Researchers identified phosphatidyl-inositol phosphates (PIPs) as key binders of MLKL, a protein crucial for necroptosis (programmed cell death). This finding explains how MLKL targets the plasma membrane, leading to cell rupture in this inflammatory cell death pathway.

Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Necroptosis is a programmed cell death pathway regulated by RIPK3 and executed by MLKL.
  • This inflammatory cell death is implicated in various diseases, including autoimmune disorders, infections, cardiovascular conditions, neurodegeneration, and cancer.

Purpose of the Study:

  • To develop and present protocols for characterizing MLKL's role in necroptosis-induced plasma membrane rupture.
  • To identify molecular interactions and modulators of MLKL activity during necroptosis.

Main Methods:

  • Live-cell imaging (conventional and confocal fluorescence microscopy) to visualize necroptosis dynamics.
  • Electron microscopy for detailed analysis of fixed cells.
  • In vitro nuclear magnetic resonance (NMR) spectroscopy with lipids to determine MLKL-lipid interactions.

Main Results:

  • Visual evidence showed MLKL redistribution from the cytosol to the plasma membrane preceding membrane permeabilization.
  • NMR analysis identified specific lipid-binding preferences for MLKL.
  • Phosphatidyl-inositol phosphates (PIPs) were identified as critical binders of MLKL, essential for its plasma membrane targeting and subsequent permeabilization.

Conclusions:

  • MLKL acts as the executioner of plasma membrane rupture during necroptosis.
  • PIPs are critical regulators of MLKL localization and function in necroptosis.
  • The developed protocols provide valuable tools for studying necroptosis mechanisms.

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