Cell
Volume 116, Issue 4, 20 February 2004, Pages 577-589
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Article
Nanoscale Organization of Multiple GPI-Anchored Proteins in Living Cell Membranes

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Abstract

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.

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4

These authors contributed equally to this work.

5

Present address: Program in Molecular Pathogenesis, Skirball Institute for Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, New York 10016.

6

Present address: Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218.