Cholesterol-dependent Syntaxin-4 and SNAP-23 Clustering Regulates Caveolar Fusion with the Endothelial Plasma Membrane

Abstract

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 cellfree system involving endothelial PM sheets and isolated biotinlabeled 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. The endothelial cells (ECs)3 lining the blood vessel walls are differentiated to mediate the rapid exchange of substances between the plasma and interstitial fluid. The process involving the fission of caveolae from the apical plasma membrane (PM) and fusion with the basolateral PM is termed transcytosis. Caveolae "pinch off " from the apical PM through a process requiring the recruitment of the GTPase dynamin to the caveolar necks as regulated by another protein intersectin (1, 2). Upon fission, caveolae form vesicular carriers that shuttle through the cytosol and deliver their cargo to the subendothelium by fusion with the basolateral PM (3). The basis of caveolar fusion may involve the same machinery as other vesicular carriers, i.e. the SNARE proteins of the syntaxin, synaptobrevin/cellubrevin, and SNAP-23/25 families (4 -7). However, the organization of SNARE proteins and their function in the fusion of caveolae in ECs are incompletely understood. It is known that syntaxin, cellubrevin, SNAP-23, and the cytosolic factors N-ethylmaleimide-sensitive factor (NSF) and ␣-and ␥-SNAP are key components of the endothelial multimolecular transcytotic complex, the assembly of which depends on the membrane fusion ATPase, NSF, which can be inhibited by alkylation of NSF by N-ethylmaleimide (7). Studies have also shown that N-ethylmaleimide interferes with transcytosis in ECs (4, 8). Based on the SNARE hypothesis, membrane fusion occurs when SNARE proteins on opposing membranes form four helix bundles, bringing the membranes in close apposition, thus providing the driving force necessary for fusion (9, 10). Ultrastructural studies have shown several states of association between caveolae and their target PM, varying in proximity, stability, and readiness for fusion (11). However, the pre-fusion states of caveolae have not been experimentally clarified, and the sequence of events responsible for caveolar fusion...

Cholesterol transport from late endosomes to the Golgi regulates t-SNARE trafficking, assembly, and function

“…Interestingly, STED microscopy revealed that the individual area of clusters was almost identical in CHOwt and CHO-A6 cells (145 ± 35 nm, syntaxin-4; 136 ± 30 nm, SNAP23). These findings correspond to similar electron microscopy data obtained from endothelial cells (Predescu et al , 2005; Figure 2, E–H). Together, results from TIRF and STED microscopy strongly suggest that CHO-A6 cells not only have fewer cholesterol-dependent SNARE clusters in the PM but also that these clusters appear to contain a lower density of molecules per cluster.…”

“…SNAP23 and syntaxin-4 have been implicated in caveolin transport and/or caveolae functioning (Predescu et al , 2005; Choudhury et al , 2006). As CHO-A6 cells are characterized by 1) reduced cholesterol levels in DRMs (Figure 3C) and the PM (Cubells et al , 2007), 2) cholesterol-dependent caveolin retention, and 3) increased amounts of SNAP23/syntaxin-4 in crude Golgi membranes (Figure 3A), we reasoned that SNAP23 and syntaxin-4 could be accumulating in the Golgi of CHO-A6 cells.…”

“…The formation/stability of t-SNARE–containing membrane clusters (e.g., syntaxin-3, syntaxin-4, SNAP23) at the cell surface seems to possess differential sensitivity toward cholesterol, depending on the cell type analyzed. Certainly, treatment with CD in MDCK, endothelial, or CHO cells reveals similar trends, but slightly different impacts on SNARE cluster formation (Predescu et al , 2005; Low et al , 2006). Importantly, the formation of SNARE clusters at the PM depends on the transport of specific SNARE proteins through the secretory pathway and the concomitant availability of cholesterol, which regulates post-Golgi trafficking in a complex and stepwise manner.…”

“…A number of studies identified a crucial role for cholesterol in PM localization of members of the SNAP receptor (SNARE) superfamily (Chamberlain et al , 2001; Predescu et al , 2005; Lang, 2007). Soluble N -ethylmaleimide-sensitive fusion protein 23 (SNAP23) is a ubiquitously expressed target membrane SNARE (t-SNARE) protein attached to the cytoplasmic leaflet of the PM through five palmitoylation moieties.…”

The regulated exocytosis of enlargeosomes is mediated by a SNARE machinery that includes VAMP4

“…Other SNAREs that might be involved are SNAP23, a ubiquitous Q-SNARE active in a large number of other exocytoses, regulated and constitutive (e.g. Bao et al, 2008;Castle et al, 2002;Feng et al, 2002;Predescu et al, 2005;Puri and Roche, 2006;Reales et al, 2005;Wang et al, 2007), and Stx6, another Q-SNARE, also known to operate in the TGN, in which it appears to interact with VAMP4 (Katsumata et al, 2007;Steegmaier et al, 1999;). In the case of these two Q-SNAREs, however, the mechanism of involvement in enlargeosome exocytosis, whether direct or indirect, is still undefined.…”