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Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal

Ronald L Mellgren1

  • 1Department of Physiology and Pharmacology, The University of Toledo College of Medicine, 3000 Arlington Avenue, Toledo, OH 43614-2598, USA. ronald.mellgren@utoledo.edu

Journal of Biochemical and Biophysical Methods
|September 18, 2007
PubMed

Insights

Mechanical cell disruption reveals distinct detergent-resistant membrane (DRM) subfractions. These DRMs are physically linked by the actin cytoskeleton, offering new insights into cell signaling.

Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Biology

Background:

  • Detergent-resistant membranes (DRMs) are essential lipid raft domains involved in cellular signaling.
  • Standard homogenization methods often yield a single DRM population, masking potential heterogeneity.

Purpose of the Study:

  • To investigate the existence and characteristics of distinct DRM subfractions.
  • To determine the cellular components responsible for the association of these DRM subfractions.

Main Methods:

  • HEK293 cells were homogenized using different methods (Teflon pestle vs. ground glass) followed by sucrose density gradient ultracentrifugation.
  • DRM subfractions were analyzed for cholesterol, ganglioside GM1, and protein composition.
  • Cell surface labeling and immunofluorescence were used to identify protein localization.
  • The role of cytoskeletal elements (actin, microtubules, vimentin) was assessed using specific inhibitors and knockout cells.

Main Results:

  • Ground glass homogenization yielded three distinct DRM subfractions (A, B, C) with varying protein compositions, unlike Teflon pestle homogenization.
  • All subfractions contained cholesterol and ganglioside GM1, but differed in flotillin-1 and caveolin-1 enrichment.
  • Subfraction C contained mitochondrial proteins, while subfraction B was enriched in plasma membrane proteins.
  • DRM subfraction separation was dependent on the actin cytoskeleton, as disruption with latrunculin B generated all three subfractions, while microtubule or vimentin disruption did not.

Conclusions:

  • DRM subfractions represent physically associated but dissociable membrane domains.
  • The actin cytoskeleton plays a crucial role in maintaining the structural integrity and association of these DRM subfractions.
  • These distinct DRM subfractions may serve as platforms for specific cellular signaling events.

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