Nanoscale organization of multiple GPI-anchored proteins in living cell membranes.
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.
National Centre for Biological Science (TIFR), UAS-GKVK Campus, GKVK PO, Bangalore 560 065, India.
Pranav
P
Sharma
Rajat
R
Varma
R C
RC
Sarasij
Ira
Karine
K
Gousset
G
G
Krishnamoorthy
Madan
M
Rao
Satyajit
S
Mayor
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology* / Q000648:ultrastructure*; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D008027:Light; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D008164:Luminescent Proteins / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry; D008954:Models, Biological; D013997:Time Factors
D003432:Cross-Linking Reagents; D008055:Lipids; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D002784:Cholesterol