Cholesterol-dependent syntaxin-4 and SNAP-23 clustering regulates caveolar fusion with the endothelial plasma membrane.
We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis.
Predescu
Sanda A
SA
Department of Pharmacology and the Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.
Predescu
Dan N
DN
Shimizu
Kayo
K
Klein
Irene K
IK
Malik
Asrar B
AB
D001710:Biotin / Q000378:metabolism; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002474:Cell-Free System; D002784:Cholesterol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D050766:Qb-SNARE Proteins / Q000378:metabolism*; D050767:Qc-SNARE Proteins / Q000378:metabolism*; D050600:SNARE Proteins / Q000378:metabolism; D019809:Streptavidin / Q000378:metabolism
C495540:CAV1 protein, human; D051242:Caveolin 1; D050765:Qa-SNARE Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; D001710:Biotin; D019809:Streptavidin; D002784:Cholesterol