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J. Biol. Chem., Vol. 280, Issue 44, 37130-37138, November 4, 2005

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

From the Department of Pharmacology and the Center for Lung and Vascular Biology, University of Illinois College of Medicine, Chicago, Illinois 60612

Received for publication, May 24, 2005 , and in revised form, August 19, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 and the spatial distribution of the SNARE proteins on the endothelial PM remains uncertain. Unlike intracellular membrane fusion events or regulated exocytosis for which in vitro assays are available (12, 13), an assay for the in vitro reconstitution of caveolar fusion does not exist. A recently described cell-free preparation for exocytosis in PC12 cells demonstrated that sonication of these cells grown on coverslips results in PM patches capable of fusion with secretory vesicles (14). Based on this approach, we have developed an assay for caveolar fusion involving endothelial PM patches and isolated biotin-labeled caveolae. We monitored membrane interactions during the fusion event via the specific detection of biotin-streptavidin complexes on the PM sheets. We correlated fluorescence microscopy (FM) analysis with gold labeling electron microscopy (EM) on ultrathin sections of PM sheets subjected to the fusion reaction. The results identified (i) the presence on PM sheets with numerous docked and fused streptavidin-labeled caveolae, (ii) cholesterol-dependent clustering of SNAP-23 and syntaxin-4 (Synt-4), and (iii) PM target (t) SNARE clusters as the preferential sites for caveolar fusion. Fluorescent staining of ultrathin sections of PM sheets highly correlated with the ultrastructure within the same area. Thus, the fusion of caveolae occurring at specialized cholesterol-rich t-SNARE clusters on the basolateral PM is responsible for exocytosis in ECs and is thereby integral to the mechanism of transcytosis.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials
Human lung microvascular ECs were obtained from BioWhittaker, Inc. (Walkersville, MD). Reagents were obtained as follows: EZ-Link N-hydroxysulfosuccinimidobiotin from Fisher; methyl--cyclodextrin (MCD) and 8-nm gold-conjugated streptavidin from Sigma; FuGENE 6 from Roche Applied Science; Prolong antifade kit, streptavidin-Texas Red, and Alexa Fluor-conjugated antibodies from Molecular Probes, Inc. (Eugene, OR); and SuperSignal chemiluminescent substrate from Pierce. All EM reagents were from EM Science (Fort Washington, PA). All other reagents were from Sigma if not specified otherwise. Relevant antibodies (Abs) were obtained from the following sources: anti-caveolin-1 (Cav-1) Abs and anti-Synt-4 monoclonal antibody (mAb) from BD Transduction Laboratories, anti-SNAP-23 polyclonal antibody (pAb) from Synaptic Systems (Göttingen, Germany), and Synt-4 pAb from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Preparation of PM Sheets
ECs grown on poly-L-lysine-coated coverslips were briefly sonicated in ice-cold 20 mM HEPES (pH 7.2), 120 mM potassium glutamate, 20 mM potassium acetate, and 2 mM EGTA as described (12, 15). PM patches attached to the coverslips were either fixed and subjected to morphological analysis by FM or EM or used in a cell-free assay for caveolar fusion.

Scanning EM of PM Sheets
PM sheets were fixed in 3% glutaraldehyde in 0.05 M sodium cacodylate (pH 7.1) for 30 min at room temperature, post-fixed in 1% OsO₄ in 0.3 M sodium cacodylate for 15 min, dehydrated through a series of graded ethanol dilutions and hexamethyldisilizane, dried, mounted on stubs, coated with a 2-nm layer of platinum/palladium, and then examined and photographed using a Hitachi field emission scanning electron microscope.

EM of PM Sheets
PM sheets were fixed in 2.5% glutaraldehyde for 15 min at room temperature, post-fixed in 1% OsO₄ in 0.1 M sodium cacodylate buffer (pH 7.4) for 15 min, dehydrated in ethanol, and embedded in Epon 812. After polymerization for 48 h at 60 °C, ultrathin sections were cut perpendicular to the PM sheets, mounted on EM grids, stained with uranyl acetate and lead citrate, and finally examined using a Joel JEM 1220. For immunogold labeling EM, PM patches were prepared by brief sonication of ECs grown on EM gold grids coated first with Formvar and then with a thin layer of poly-L-lysine. PM sheets attached to the EM grid were fixed in 2% paraformaldehyde for 15 min; quenched with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS); then incubated for 1 h at 4 °C with the primary Abs, followed by 6-nm gold-conjugated reporter Abs; diluted in 0.1% BSA in PBS; and centrifuged for 30 min at 30,000 x g before use. In control experiments, the primary Abs were omitted. After washing by transfer through six successive PBS drops, gold-labeled PM sheets were post-fixed in 2% glutaraldehyde for 15 min and stained with uranyl acetate and lead citrate, followed by EM examination. Randomly chosen areas of PM sheets were photographed at the initial x20 magnification. To quantify the distribution of gold particles and to estimate the size of SNARE clusters, we used three sets of micrographs (36 per set representing 200 µm² of PM surface) from three different experiments for each antigen. The clusters were classified based on their number of gold particles (between three and six or more than six gold particles). The size of the gold clusters was estimated using at least 50 clusters per experimental set.

FM of PM Sheets
In control experiments, PM sheets were fixed in 4% paraformaldehyde in PBS; quenched with 50 mM NH₄Cl in PBS; and then sequentially incubated for 30 min with the primary Abs diluted 1:100 in 1% BSA in PBS, followed by a 45-min incubation with the reporter Abs diluted in 0.1% BSA in PBS as described (13). For cholesterol depletion, PM sheets were treated with 10 mM MCD as described (13) and immunostained for Cav-1 and SNAP-23.

Small Interfering RNA (siRNA) for Cav-1
RNA oligonucleotides designed as described (16) were from Integrated DNA Technologies (Coralville, IA). EC monolayers (70-80% confluent) were transfected with Cav-1 siRNA using FuGENE 6 reagent at an siRNA/lipid ratio of 1:3. A four-nucleotide mismatch was used as control.

Cell-free Assay for Caveolar Fusion
To address caveolar fusion experimentally, we used the basolateral endothelial PM as the target PM and the isolated caveolae.

Isolation of Biotin-labeled Caveolae—Freshly prepared PM sheets bearing attached caveolae were biotinylated as described (2). After extensive washing, the PM sheets were incubated for 2 h at 37°C with 5 mg/ml EC cytosol in the presence of 1 mM GTP and an ATP-regenerating system to allow the GTP-dependent fission of biotinylated caveolae as described (1, 2).

Fusion Reaction—The standard fusion assay consisted of PM sheets present on one coverslip (100-150 patches) and the biotin-labeled caveolae released from PM patches present on three coverslips. To initiate docking and fusion, 900 µl of fusion buffer (20 mM HEPES/KOH (pH 7.4), 100 mM KCl, 2 mM dithiothreitol, 2 mM EDTA, and 2 mM ATP); 100 µl of an ATP-regenerating system; and 5 mg/ml EC cytosol were added over the PM sheets, followed by addition of isolated caveolae and incubation for 30 min at 37 °C. The patches were placed on ice, washed, fixed, and stained for biotin and SNARE proteins.

Correlative FM and EM of Quetol 651-embedded PM Sheets Subjected to Caveolar Fusion
Plastic coverslips containing PM sheets subjected to the fusion reaction were extensively washed; fixed in 2% paraformaldehyde in PBS (pH 7.5) for 10 min at room temperature; rinsed twice in PBS; blocked with 1% BSA in PBS; and subsequently immunostained for SNAP-23, followed by another quenching step and simultaneous application for 30 min of fluorescein isothiocyanate-conjugated anti-rabbit IgG to detect bound anti-SNAP-23 antibody and streptavidin-Texas Red to visualize biotin-labeled caveolae. The PM patches were then washed with PBS; post-fixed in 2% paraformaldehyde in PBS for 10 min at room temperature; and dehydrated with a series of graded ethanol dilutions, followed by embedding with Quetol 651 (EM Science) following the manufacturer's instructions. Ultrathin sections were cut perpendicular to the PM patches and mounted on Formvar-coated EM grids. The grid to be examined was sited on a glass slide, covered with a coverslip using transparent tape, and imaged by FM. All images were collected with identical acquisition parameters. The same grid was subjected to 8-nm gold-conjugated streptavidin labeling, followed by uranyl acetate and lead citrate staining and examined by transmission EM.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Endothelial SNARE Proteins Are Concentrated in Clusters and Co-localize with Cav-1 on Endothelial PM Sheets—To analyze the distribution of t-SNARE proteins in endothelial membranes, we adapted the recently described PM sheet system (12). Basolateral endothelial PM sheets were prepared as described under "Experimental Procedures" by brief sonication of confluent EC monolayers grown on polylysinecoated coverslips. In confluent ECs, tight junctions are formed, and cell polarity is subsequently established. Under these conditions, the apical PM is oriented away from the substrate, thus facilitating the removal of the apical PM and cytosolic structures, whereas the basolateral PM sheets with their membrane protein complexes and caveolar profiles remain attached to the coverslip. These basolateral PM sheets were fixed in 4% paraformaldehyde and incubated first with anti-Cav-1 pAb and then with fluorescein isothiocyanate-conjugated reporter Ab. Cav-1 is known to decorate caveolar coats in different stages of membrane invagination. Cav-1 immunostaining revealed the characteristic vesicular organization (Fig. 1A). Cav-1 clusters were numerous and non-uniformly scattered on the PM sheet. A high resolution scanning EM approach showed a large number of caveolae (Fig. 1B, panels b1 and b2) and, occasionally, caveolar clusters attached to the PM. Fig. 1B shows highly magnified caveolar profiles (panels b3-b5) and caveolar clusters attached to the PM (panels b6 and b7). We also observed some actin cables (panel b2), but no other cell components were detected, indicating that cytosolic structures were removed during the sonication. We very rarely observed membrane folding, and no apparent membrane damage was produced by the sonication. Further morphological analysis by pre-embedding EM of cross-sections through PM sheets (Fig. 1C and panel c1) showed a well preserved membrane structure suitable for studying caveolar fusion in relation to the organization of SNARE proteins located on the inner leaflet of the PM. Given the lack of knowledge about the localization of SNARE proteins in EC membranes, we analyzed the membrane distribution of SNAP-23 and syntaxin. For syntaxin immunostaining, we used anti-Synt-4 pAb. Synt-4, the syntaxin-1 isoform present in ECs (Fig. 2A), has been shown to be the t-SNARE partner of SNAP-23 and cellubrevin in non-neuronal cells (18). Immunofluorescent staining showed, for both SNAP-23 (Fig. 2B, panel b1) and Synt-4 (panel b2), dense puncta and diffuse staining throughout the ECs with significant PM localization. A detailed analysis of endothelial t-SNARE proteins distribution on the inner leaflet of the PM by FM indicated that there was a wide distribution in a dense punctate staining pattern for both proteins (Fig. 2C, panels a1 and b1). Synt-4 puncta were smaller and more discrete, most likely due to the transmembrane domain of Synt-4 and its ability to homo-oligomerize (13), whereas SNAP-23 puncta were more numerous and less distinct, possibly due to the lack of a transmembrane domain and its higher mobility (17). These findings indicate that the basolateral PM endothelial SNARE proteins associate in small clusters.

View larger version (76K): [in this window] [in a new window]   FIGURE 1. Morphological characterization of basolateral endothelial PM sheets. A, endothelial PM sheets were fixed in 4% paraformaldehyde in PBS and immunostained with anti-Cav-1 pAb. Staining for Cav-1 resulted in strong punctate labeling, characteristic of a vesicular protein. B, PM sheets were fixed in 3% glutaraldehyde and processed for scanning EM. Panel b1 shows a fragment of an undisrupted EC and an intact PM sheet with no holes or tears. The endothelial junction (arrowhead) was disrupted as result of sonication (panel b2). PM patches were devoid of all cell constituents except attached caveolae (panel b2). Panels b3-b5 show highly magnified attached caveolar profiles, and panels b6 and b7 show caveolar clusters still attached to the PM after sonication. C, the electron micrograph shows an attached caveolar profile. Panel c1 shows a well preserved morphology. Scale bars = 10 µm (A and panel b1), 100 nm (panels b2, b5-b7, C, and c1), and 50 nm (panels b3 and b4).

View larger version (101K): [in this window] [in a new window]   FIGURE 2. Expression and distribution of Synt-4 and SNAP-23 in endothelial cells and on PM sheets. A, a cultured EC lysate subjected to 5-20% SDS-PAGE and analyzed by Western blotting with anti-Synt-4 mAb shows the expression of Synt-4. MW, molecular weight protein standards. B, cultured ECs fixed by methanol permeabilization and immunostained with anti-SNAP-23 pAb (panel b1) and anti-Synt-4 mAb (panel b2) show a preferential distribution of the two t-SNARE proteins at the PM level. Scale bars = 20 µm. C, fixed endothelial PM sheets immunostained for SNAP-23 show numerous brightly fluorescent dots (panel a1). Synt-4 dots seem to be smaller and more discrete (panel b1). Cav-1 (panels a2 and b2) co-localized to a large extent with both SNAP-23 (panel a3) and Synt-4 (panel b3) clusters. Scale bars = 5 µm.

The spatial distribution of SNAP-23 and Synt-4 on endothelial PM sheets was also investigated by 6-nm immunogold labeling EM. The majority of gold particles detecting SNAP-23 (Fig. 3, panel a) or Synt-4 (panel b) were distributed in clusters of three to six gold particles (solid circles) or in larger clusters of more than six gold particles (dashed circles). Frequently, SNAP-23 (panels a1-a4) and Synt-4 (panels b1-b3) immunogold labeling indicated their association with densely stained vesicle-like structures. Because the number of clathrin-coated vesicles in ECs is low compared with caveolae (3% from the total vesicle population (3))⁴ and the clathrin cage was not detected, these structures are likely caveolae. Quantification of the spatial distribution of gold particles on the PM sheets indicated 3000 units/50 µm² of PM surface (TABLE ONE). About 62% of the total number of gold particles detecting SNAP-23 and 71% of the total number of gold particles detecting Synt-4 consisted of clusters of three to six gold particles. The larger clusters consisting of more than six gold particles represented 29% for SNAP-23 and 18% for Synt-4 (TABLE ONE). The distribution of gold particles per unit surface and within the clustered area showed a high density of the recognized antigen. The size of the clusters ranged between 100 and 150 nm. An average of only 10% of the total number of gold particles counted were found singly or in pairs. Control experiments in which specific Abs were omitted showed low background staining (<2%) on a similar endothelial PM surface, and this was always due to gold particles found singly or in pairs, whereas the gold aggregates represented <0.3%.

View larger version (99K): [in this window] [in a new window]   FIGURE 3. t-SNARE clusters visualized by immunogold labeling EM of PM sheets. PM sheets prepared from ECs grown on poly-L-lysine-coated EM gold grids were immunostained with anti-SNAP-23 pAb (panel a) and anti-Synt-4 mAb (panel b), followed by 6-nm gold-conjugated reporter Abs and standard EM staining. Both proteins were distributed in clusters of three to six gold particles (closed circles) or more than six gold particles (dashed circles) and only occasionally as solitary particles or as pairs. The size of the clusters ranged between 100 and 150 nm, considering a scatter radius of 15 nm from the outermost gold particle in the cluster. Panels a1-a4 and b1-b3 show the association of 6-nm gold particles detecting SNAP-23 and Synt-4 with caveolae, respectively. Scale bars = 100 nm (panels a and b) and 30 nm (panels a1-a4 and b1-b3).

View larger version (32K): [in this window] [in a new window]   FIGURE 4. Synt-4 and SNAP-23 co-clusters form ring-shaped microdomains. A, double labeling for Synt-4 (panel a1) and SNAP-23 (panel a2) revealed partial co-clustering (panel a3). Circles indicate regions where the t-SNARE co-clusters were highly concentrated. B, shown are highly magnified concentrated SNAP-23/Synt-4 co-clusters. C, shown are representative images used to rate the association of Cav-1-positive puncta (green) with SNAP-23 (red; panels a and b), Synt-4 (blue; panels c and d), and SNAP-23/Synt-4 (panels e-h) clusters. The primary antibodies (mouse anti-Cav-1 mAb, rabbit anti-SNAP-23 pAb, and goat anti-Synt-4 pAb) were visualized using Alexa Fluor 488-conjugated anti-mouse IgG, Alexa Fluor 546-conjugated anti-rabbit IgG, and Alexa Fluor 350-conjugated anti-goat IgG, respectively. After staining, the PM patches were viewed sequentially through optical filter sets appropriate for the fluorophores used, and the resulting images were superimposed. The overlap between signals was assessed as follows: for Cav-1/SNAP-23 (yellow; panels a, b, and e), for Cav-1/Synt-4 (light blue; panels c, e, and f), for SNAP-23/Synt-4 (purple; panels a and h), and for Cav-1/SNAP-23/Synt-4 (white; panels f-h). Scale bars = 5 µm (A, panel a1) and 2 µm (B). All images used for quantification of the degree of co-localization was acquired using identical parameters per experiment.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. Cholesterol dependence of t-SNARE cluster formation. Unfixed PM sheets were treated for 30 min at 37 °C with 15 mM MCD and then immunostained for SNAP-23 (A, panel a2), Synt-4 (B, panel b2), and Cav-1 (A, panel a3; and B, panel b3). Cav-1 left the PM after treatment with 15 mM MCD. Upon cholesterol depletion, SNAP-23 (A, panel a2), and Synt-4 (B, panel b2) staining was dramatically changed in comparison with control PM patches (no MCD treatment) immunostained for SNAP-23 (A, panel a1) or Synt-4 (B, panel b1). The small SNARE proteins clusters disintegrated. Boxes indicate regions that are magnified in panels a1.1, a2.2, a2.3, b1.1, b2.2, and b2.3. Scale bars = 5 µm (A, panels a1 and a2; B, panels b1 and b2) and 1 µm (panel a1.1). ECs membranes were extracted at 4 °C with 1% Triton X-100 before discontinuous OptiPrep density gradient flotation (C). Fractions were collected from the top, and equal volumes were analyzed by SDS-PAGE, electrotransfer to nitrocellulose, and immunoblotting. A pool of both SNAP-23 and Synt-4 co-floated with Cav-1 in the light density fractions (fractions 1-3) of the gradient, corresponding to DRMs. Large pools of both t-SNARE proteins were present in the heavy fractions (fractions 9-12). Similar results were obtained in three different experiments. Treatment with 10 mM MCD (MCD) significantly affected the buoyant properties of all three proteins (D). These analyses were repeated on four separate gradients with similar results.

Docking and Fusion of Caveolae with the PM in a Cell-free System—We visualized by EM docked caveolae in intact cultured ECs, apparently intermediate states in the fusion reaction. Caveolae were found close to the PM but not yet opened to the subendothelial space, tethered and docked, waiting for fusion of their membrane with the basolateral PM (Fig. 7). The region of close apposition between the two membranes extended over 50 nm, more than half the diameter of a typical caveola (panels a-d). The two membranes were <5 nm apart (panels a1-d1), half the length of the coiled bundle of vesicular (v) and t-SNARE proteins forming during exocytosis (19). These findings suggest that SNARE pairing had occurred.

Because caveolar fusion is difficult to monitor in whole ECs, its basis remains unclear. Thus, to study caveolar fusion experimentally, we used the basolateral endothelial PM and isolated caveolae and monitored fusion by correlative FM and EM analyses. We took advantage of the purity of the PM preparation and the large number of attached caveolae to isolate a pure caveolar fraction. PM sheets and the attached caveolae were labeled with biotin (2) to assess caveolar fusion via biotin-streptavidin complex formation. Biotin-labeled caveolae were released by the GTP-dependent process (1, 2), and aliquots were analyzed biochemically for their biotin, Cav-1, and cellubrevin contents and morphologically using 8-nm gold-conjugated streptavidin labeling and negative staining EM. The released caveolae were immunoreactive to the caveolar marker Cav-1 (Fig. 8A, panel a) and the v-SNARE protein cellubrevin (panel b). Moreover, significant biotinylation of caveolar proteins was detected using streptavidin reagents by dot blotting (panel c) and EM analyses. The electron micrographs shown in Fig. 8B and in more detail in panels b1-b4 show a relatively homogeneous population of caveolae labeled with 8-nm gold-conjugated streptavidin. The released caveolae and freshly prepared naïve PM sheets were incubated as described under "Experimental Procedures." Upon fusion, the membrane patches were washed, fixed, and stained for biotin and SNAP-23 or Synt-4. Fig. 8C shows numerous streptavidin-Texas Red-positive puncta, indicating caveolar attachment to the membrane. In addition, there was significant co-localization between the SNAP-23 clusters and biotin-streptavidin complexes (Fig. 8C, circles), indicating that fusion of caveolae occurred at the specialized sites containing the SNAP-23 clusters (panels c1-c3). Similar findings were obtained when co-localization between Synt-4 and streptavidin-Texas Red was examined on PM patches subjected to the fusion reaction (data not shown). In control experiments in which unlabeled caveolae were used (data not shown) or the cytosol was omitted from the fusion reaction (Fig. 8D), no streptavidin-Texas Red staining was detected. The extent of co-localization between the streptavidin-Texas Red-positive puncta and two SNARE proteins was analyzed on two sets of PM patches (n = 24 per set) obtained from three different fusion reactions per experimental set. This analysis indicated that 50% of the streptavidin-Texas Red-labeled caveolae corresponded to a SNAP-23 or Synt-4 cluster (TABLE THREE). An average of 22% of the streptavidin-Texas Red-positive puncta did not co-localize with the SNARE clusters, whereas for the remaining 28%, co-localization could not be established with accuracy.

View larger version (46K): [in this window] [in a new window]   FIGURE 6. Cav-1 knockdown fails to alter the distribution of t-SNARE clusters on endothelial PM sheets. A, ECs treated with Cav-1 siRNA were analyzed at different time points for Cav-1 expression levels by immunoblotting (panel a1). An average of 80% suppression was found 48-72 h after siRNA transfection. The graph shows data from three different experiments. Under these conditions, caveolin-2 (Cav-2) expression was not affected (panel a2). A mismatched siRNA sequence did not affect Cav-1 expression levels (panel a3) or Cav-1 membrane distribution (panel a4). Error bars represent S.D. RNAi, RNA interference. B and C, PM sheets prepared from Cav-1 siRNA-transfected ECs were fixed in 4% paraformaldehyde and immunostained for Cav-1 (panels b1 and c1), SNAP-23 (panel b2), and Synt-4 (panels c2). Panels b1 and c1 show PM patches with a variable extent of Cav-1 down-regulation. SNAP-23 distribution was not detectably affected on the same patches. However, Synt-4 clusters became slightly larger and more diffuse. Scale bars = 4 µm (panel a4) and 8 µm (panels b1 and c1).

View larger version (59K): [in this window] [in a new window]   FIGURE 7. EM morphological evidence for docked caveolae in intermediate states of the fusion reaction. Note the wide region of close apposition between the caveolae and basolateral PM (panels a-d) and the proximity and the staining-dense strands between the two membranes (panels a1-d1), suggestive of v-SNARE/t-SNARE pairing. Scale bars = 35 nm (panel d) and 20 nm (panel d1).

View larger version (53K): [in this window] [in a new window]   FIGURE 8. Biotin-labeled caveolae preferentially dock to sites of PM sheets containing SNAP-23 clusters. A, aliquots of the GTP-dependent released caveolae (100 and 200 µl, comprising nanogram ranges of total protein) were analyzed by immunodot blotting for their Cav-1 (panel a), cellubrevin (panel b), and biotin (panel c) content. B, 8-nm gold-conjugated streptavidin-labeled caveolae released from basolateral endothelial PM sheets subjected to the fission reaction were analyzed by negative staining EM. Panels b1-b4 show magnified caveolar profiles labeled by 8-nm gold-conjugated streptavidin. C, PM sheets subjected to the fusion reaction were fixed and stained for SNAP-23 (green) and biotin (red). Numerous streptavidin-Texas Red-positive spots were detected on the PM patch. Circles and panels c1-c3 show the high concentration of SNAP-23 clusters and their co-localization with Texas Red-positive puncta. D, no streptavidin-Texas Red staining was detected when the fusion reaction was performed in the absence of EC cytosol. Scale bars = 100 nm (B and panels b1-b4) and 2 µm (C and D).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Using these approaches, we carried out a detailed analysis of the distribution of SNAREs on the target endothelial PM. We observed a generalized clustering of t-SNARE proteins as evidenced by both SNAP-23 and Synt-4 forming individual clusters distributed throughout the PM. Immunogold labeling EM showed a high density of t-SNARE proteins on the cytosolic leaflet of the PM. The majority of gold particles detecting SNAP-23 or Synt-4 were found as clusters of 100-150 nm in diameter. Strikingly, SNAP-23 and Synt-4 also co-localized and formed patches of five to seven co-clusters/100 µm² of PM surface. We observed that a total of 61% of the Cav-1-positive puncta co-localized with t-SNARE clusters (either SNAP-23 or Synt-4 clusters), representing 47% of the total, and with SNAP-23/Synt-4 co-clusters, representing 14% of the total. Because the ECs provide a suitable system for studying membrane fusion due to their high transcytotic activity, we were able to visualize with high optical resolution and thereby quantify the existence of binary complexes of SNAP-23 and Synt-4 and their co-localization with Cav-1. An important question is why there is only a 14% co-localization of Cav-1-positive puncta with the SNAP-23/Synt-4 co-clusters. An explanation of this finding may be the fact that, at any given time, approximately one-third of the total caveolar population of a typical EC is open to the basolateral side of the cell. Thus, while attached to the PM, the caveolae are expected to co-localize with the SNAP-23/Synt-4 co-clusters only during the fusion event. As membrane fusion is a very rapid process and Cav-1/t-SNARE interactions are short-lived, at any given time, relatively few caveolae are expected to co-localize with the t-SNARE co-clusters, as evident from our data. We surmise that co-localization of Cav-1-positive puncta with the organized clusters and co-clusters represents the sites of fusion of caveolae with the PM. Interestingly, our in vitro fusion assay data demonstrating that 50% of the fused caveolae co-localized with SNAP-23- or Synt-4-positive puncta are in close agreement with the morphological evidence of 61% co-localization discussed above. Moreover, our results are in the range of values obtained for docking and fusion of secretory granules during regulated exocytosis in PC12 cells (13).

View larger version (49K): [in this window] [in a new window]   FIGURE 9. Correlative FM and EM analyses of ultrathin cross-sections of PM sheets subjected to the fusion reaction. Panel A shows ultrathin sections of Quetol 651-embedded PM sheets subjected to the fusion reaction were mounted on EM grids and stained for SNAP-23 and biotin. SNAP-23 immunostaining showed three PM sheets (PM1-PM3) comprising sharp, bright SNAP-23 clusters. High magnification of the boxed area shows in detail well defined SNAP-23 clusters (panel a1) and streptavidin-Texas Red-positive puncta (panel a2). Note the degree of co-localization between SNAP-23 and streptavidin-Texas Red-positive puncta (panel a3, spots *1-*3, 4*, and 5*). Upon EM analysis, the fluorescent spots correlated with individual docked caveolae (panel a4, spots *2-4*). The inset in panel a4 shows a better view of the caveolar profile of spot *3. A gallery of highly magnified 8-nm gold-conjugated streptavidin-labeled caveolae docked (panels a5-a8) or fused (panel a9) to the PM sheets on the same sections is shown. Scale bars = 5 µm (panel A), 1 µm (panel a1), 250 nm (panel 1*), 100 nm (panels a4-a8), and 75 nm (panel a9).

The cholesterol-dependent clustering of t-SNARE proteins and their distribution on flotation gradients demonstrated that a small fraction of t-SNARE proteins was associated with Cav-1, a marker of DRMs, whereas a larger pool of SNARE proteins was localized in the cholesterol-rich microdomains of the PM distinct from DRMs. In other cell types, SNARE proteins display different association with DRMs ranging from 20% in PC12 cells to 70% in adipocytes, suggesting an important role of lipid rafts in membrane fusion and probably in regulating SNARE function (21). The functional significance of these pools of t-SNARE proteins in the mechanism of caveolar fusion to the PM is not clear. As caveolar fusion was shown to occur mainly with t-SNARE clusters in the target PM, a possible function of these two pools may be to hold t-SNARE proteins in reserve in distinct PM microdomains until they are needed for caveolar fusion.

The docking and fusion of caveolae with the target PM and the involvement of caveolae in the transcytotic pathway in ECs were further assessed by correlative FM and EM. Optical imaging with virtually no background by high resolution FM on ultrathin sections of PM sheets correlated with the ultrastructure of the same region, i.e. the streptavidin-Texas Red-positive spots were identified with high fidelity by EM with the individual docked and fused caveolae. These EM results reinforce the observation that the t-SNARE clusters are the preferential sites for caveolar fusion with the target PM. Moreover, the results unequivocally show that caveolar fusion with the PM occurs in our system.

In summary, our results demonstrate that clusters of endothelial t-SNARE proteins (SNAP-23 and Synt-4) form in cholesterol-rich microdomains of the PM and that these clusters are the preferential sites for caveolar fusion with the target PM during exocytosis. These findings support the hypothesis that caveolar interaction with t-SNARE clusters is essential for fusion and, as such, is important in the regulation of transcytosis across the endothelial barrier.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant PO1 HL60678 (to A. B. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Parker B. Francis Fellow in pulmonary research.

² To whom correspondence should be addressed: Dept. of Pharmacology (m/c868), University of Illinois, 835 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312-996-7635; Fax: 312-996-1225; E-mail: abmalik{at}uic.edu.

³ The abbreviations used are: ECs, endothelial cells; PM, plasma membrane; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; t, target; v, vesicular; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; NSF, N-ethylmaleimide-sensitive factor; FM, fluorescence microscopy; EM, electron microscopy; Synt-4, syntaxin-4; MCD, methyl--cyclodextrin; Abs, antibodies; Cav-1, caveolin-1; mAb, monoclonal antibody; pAb, polyclonal antibody; BSA, bovine serum albumin; PBS, phosphate-buffered saline; siRNA, small interfering RNA; DRMs, detergent-resistant membranes.

⁴ D. N. Predescu and S. A. Predescu, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Nicki Watson for help with EM and Dr. T. Lang for technical advice in PM sheet preparation.

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