Nanoscale organization of multiple GPI-anchored proteins in living cell membranes

Cell. 2004 Feb 20;116(4):577-89. doi: 10.1016/s0092-8674(04)00167-9.


Cholesterol and sphingolipid-enriched "rafts" have long been proposed as platforms for the sorting of specific membrane components including glycosyl-phosphatidylinositol-anchored proteins (GPI-APs), however, their existence and physical properties have been controversial. Here, we investigate the size of lipid-dependent organization of GPI-APs in live cells, using homo and hetero-FRET-based experiments, combined with theoretical modeling. These studies reveal an unexpected organization wherein cell surface GPI-APs are present as monomers and a smaller fraction (20%-40%) as nanoscale (<5 nm) cholesterol-sensitive clusters. These clusters are composed of at most four molecules and accommodate diverse GPI-AP species; crosslinking GPI-APs segregates them from preexisting GPI-AP clusters and prevents endocytosis of the crosslinked species via a GPI-AP-selective pinocytic pathway. In conjunction with an analysis of the statistical distribution of the clusters, these observations suggest a mechanism for functional lipid-dependent clustering of GPI-APs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CHO Cells
  • Cell Membrane / metabolism*
  • Cell Membrane / physiology*
  • Cell Membrane / ultrastructure*
  • Cells, Cultured
  • Cholesterol / metabolism
  • Cricetinae
  • Cross-Linking Reagents / pharmacology
  • Endocytosis
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins
  • Image Processing, Computer-Assisted
  • Light
  • Lipid Metabolism
  • Lipids / chemistry
  • Luminescent Proteins / metabolism
  • Membrane Microdomains / chemistry
  • Models, Biological
  • Time Factors


  • Cross-Linking Reagents
  • Lipids
  • Luminescent Proteins
  • Green Fluorescent Proteins
  • Cholesterol