ARTICLE |
Correspondence to: Ann M. Dvorak, Dept. of Pathology, East Campus, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. Fax: 617-667-2943
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Summary |
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Vesicle-associated membrane proteins (VAMPs) are important to the trafficking of vesicles between membrane-bound intracytoplasmic organelles, in the facilitation of neurosecretion, and in constitutive and regulated secretion in non-neuronal cells. We used a pre-embedding ultrastructural immunonanogold method to localize VAMPs to subcellular sites in human cells of five lineages known to have cytoplasmic vesicles that may function in vesicular transport. We found VAMPs localized to caveolae in pericytes, vascular smooth muscle cells, and endothelial cells of venules, to the vesiculovacuolar organelle, recently defined in venular endothelial cells, to the vesicle-rich intergranular cytoplasm and secretory granule membranes of neutrophils, and to perigranular cytoplasmic secretory vesicles and secretory granule membranes in eosinophils. These specific localizations in five human vascular and granulocyte lineages support the notion that VAMPs have vesicle-associated functions in these cells. (J Histochem Cytochem 49:293304, 2001)
Key Words: VAMP, pericyte, smooth muscle, endothelium, eosinophil, neutrophil, immunonanogold cytochemistry, vesiculovacuolar organelle, caveolae, vesicles
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Introduction |
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Highly conserved molecular machinery for docking and fusion of vesicles to membranes is central to intracellular membrane fusion. This machinery consists of a core complex of N-ethylmaleimide-sensitive fusion (NSF) protein, soluble NSF attachment proteins (SNAPs), and SNAP receptors (SNAREs). SNAP receptor proteins associated with vesicles ([v]-SNAREs) and targets ([t]-SNAREs) provide a mechanism for the specific docking and fusion of transport vesicles to target membranes (
Definition of the tissue and cellular distribution and subcellular organellar localization of specific proteins or classes of proteins is important for understanding their function(s). To this end, we applied a pre-embedding immunonanogold protocol for ultrastructural detection of VAMPs in human skin tissues and granulocyte preparations. The samples included five cell types known to contain small cytoplasmic vesicles which, in many instances, are believed to have a role in intracytoplasmic trafficking (
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Materials and Methods |
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Tissue and Cells
Two foreskins were immersed in freshly prepared 4% paraformaldehyde in a 0.02 M PBS, pH 7.4, immediately after resection. Tissues were fixed for 4 hr at room temperature (RT), immersed in 30% sucrose in the 0.02 M PBS, pH 7.4, overnight at 4C, embedded in OCT compound (Miles; Elkhart, IN), and stored in -176C liquid nitrogen for subsequent use. Frozen 10-µm sections were cut with a standard cryostat and collected on pre-cleaned glass slides which were air-dried for 20 min before staining.
Citrate anticoagulated blood from normal donors was depleted of erythrocytes and centrifuged on Ficoll-Paque gradients (Amersham Pharmacia; Uppsala, Sweden) as described (
Antibody to VAMP-2
Rabbit VAMP-2 polyclonal antibody (StressGen Biotechnologies; Victoria, BC, Canada) was produced using a 21-residue synthetic peptide based on residues 3656 of rat VAMP-2 (
ImmunonanogoldSilver Staining Protocol and Processing for Electron Microscopy
The following steps were performed at RT on cryostat sections mounted on glass slides: (a) one wash in 0.02 M PBS, pH 7.6, 5 min; (b) immersion in 50 mM glycine in 0.02 M PBS, pH 7.4, 10 min; (c) one wash in 0.02 M PBS, pH 7.4, 5 min; (d) immersion in 5% normal goat serum (NGS) (Vector Laboratories; Burlingame, CA), 20 min; (e) incubation in the primary antibody, an affinity-purified rabbit polyclonal antibody against VAMP-2 (StressGen Biotechnologies) (12.5 µg protein/ml, diluted 1:20, 1:100, or 1:140 in 0.02 M PBS), 60 min; (f) three washes in 0.02 M PBS, pH 7.4, 5 min each; (g) incubation in the secondary antibody (affinity-purified 1.4 nm nanogold-conjugated goat anti-rabbit Fab' (Nanoprobes; Stony Brook, NY), 1:50 in 0.02 M PBS, pH 7.4, 30 min; (h) three washes in 0.02 M PBS, pH 7.4, 5 min each; (i) postfixation in 1% glutaraldehyde in 0.02 M PBS, pH 7.4, 5 min; (j) three washes in distilled water, 5 min each; (k) development with HQ silver enhancement solution (Nanoprobes) for 611 min in the darkroom; (l) two washes in distilled water, 2 min each; (m) fixation in 5% sodium thiosulfate, 1 minute; (n) two washes in distilled water, 2 min each; (o) postfixation in 1% osmium tetroxide in Sym-Collidine buffer, pH 7.4, 10 min, RT; (p) one wash in 0.05 M sodium maleate buffer, pH 5.2, 2 min; (q) staining with 2% uranyl acetate in 0.05 M sodium maleate buffer, pH 6.0, 5 min, RT; (r) one wash in distilled water, 2 min; (s) dehydration in graded ethanols and infiltration with a propylene oxideeponate (Eponate 12 Resin; Ted Pella, Redding, CA) sequence; (t) embedment by inverting eponate-filled plastic capsules over the slide-attached tissue sections; (u) polymerization at 60C for 16 hr; (v) separation of eponate blocks from glass slides by brief immersion in liquid nitrogen; (w) cutting of thin sections with a diamond knife on an ultratome (Reichert; Vienna, Austria) and collection of sections on uncoated 200-mesh copper grids (Ted Pella); (x) viewing of unstained grids with a transmission electron microscope (CM 10; Philips, Eindhoven, The Netherlands).
Controls for Immunostaining
Four controls were performed on foreskin samples to ensure the specificity of immunostaining: (a) primary antibody was replaced by an irrelevant rabbit IgG; (b) omission of specific primary antibody; (c) omission of the secondary antibody; (d) omission of the HQ silver enhancement solution. Controls a and b were performed on leukocyte preparations.
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Results |
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Human Foreskin
Circumcision samples from newborn infants were prepared for pre-embedding immunonanogold staining. Cells associated with dermal venules in these samples were positive for VAMP. The most consistently labeled cells were pericytes and vascular smooth muscle cells; endothelial cells also were intermittently labeled.
Pericytes. Venular pericytes (Fig 1) beneath venular endothelium were elongated bipolar cells that sometimes formed contacts with overlying endothelial cells. Their cytoplasmic constituents included actin filaments, intermediate filaments, and focal rows of single caveolae attached primarily to their abluminal plasma membranes. In contrast, the luminally oriented plasma membrane was generally devoid of caveolae in most pericytes (Fig 1A, Fig 1C, Fig 1D, and Fig 1E). Basal lamina was associated primarily with the abluminal pericyte surface. Mitochondria, strands of rough endoplasmic reticulum, and rare coated vesicles were also present. Pericyte caveolae were extensively labeled with antibody to VAMP. Label was associated primarily with the cytoplasmic face of abluminal plasma membrane-attached caveolae (Fig 1A1C). Clusters of silver-enhanced nanogold particles sometimes obscured underlying caveolae. Rarely, VAMP was also localized to pericyte abluminal plasma membranes near caveolar attachments (Fig 1C). Coated vesicles were not labeled for VAMP. Controls for the immunonanogold method (omission of the primary antibody or substitution of an irrelevant rabbit IgG for the primary antibody) revealed no label (Fig 1D and Fig 1E).
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Endothelial Cells.
Venular endothelial cells (Fig 1A, Fig 1D, and Fig 2) contained abundant caveolae, associated with both luminal and abluminal plasma membranes. In addition, focal clusters of vesicles and vacuoles, vesiculo-vacuolar organelles (VVOs) (5%) of VVOs were labeled for VAMP (Fig 2B). As was the case for pericyte caveolae, the VAMP labeling of VVO vesicles and vacuoles was primarily associated with their cytoplasmic faces. One vessel that showed morphological criteria of injury displayed heavily labeled endothelial cells. Controls, omission of the primary antibody or substitution for the primary antibody with irrelevant rabbit IgG (Fig 1D and Fig 2C), were negative.
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Smooth Muscle Cells. Processes extended from the surfaces of smooth muscle cells (Fig 3) to form characteristic "ball and socket-like" contacts with adjacent smooth muscle cells, thus creating a distinctive smooth muscle cell syncytium. Smooth muscle cells were encased by basal lamina and displayed many cytoplasmic thick and thin filaments. These actin and myosin filaments were aggregated as fusiform dense bodies in cytoplasmic filaments, and subplasmalemmal, electron-dense attachment areas for these filaments were prominent. The blunt, rounded tips of cell processes were typically packed with caveolae, several mitochondria, and collections of lipid and glycogen (Fig 3AD and Fig 3F). Coated pits and vesicles were infrequently present in smooth muscle cells (Fig 3H). Nanogold particles labeling VAMP in smooth muscle cells were attached to caveolar clusters (Fig 3A, Fig 3C, Fig 3E, and Fig 3F), which encompassed all surfaces of these cells, in contradistinction to pericytes, in which labeled caveolae were generally not clustered and were largely confined to the abluminal surface. Plasma membranes (Fig 3A, Fig 3E and Fig 3F), coated pits (Fig 3H), and coated vesicles were not labeled; caveolae were labeled on their cytoplasmic surfaces (Fig 3G). Specificity controls were negative (Fig 3B and Fig 3D).
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Human Peripheral Blood
Human peripheral blood samples from normal donors were partially purified to enrich granulocytes (
Eosinophils.
Eosinophils (Fig 4), granulocytes with polylobed nuclei, and typical crystalloid-containing cytoplasmic secretory granules displayed many cytoplasmic vesicles adjacent to granules and beneath the plasma membrane in a relatively granule-free zone. VAMP label was associated with the granule-poor subplasmalemmal, and inter-granule cytoplasm in eosinophils (Fig 4A and Fig 4B). At higher magnifications and in cells showing evidence of secretion (
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Neutrophils. Neutrophils (Fig 5) with polylobed nuclei and a full component of secretory granules were labeled for VAMP (Fig 5A). Although some gold particles were associated with secretory granule membranes on their cytoplasmic surfaces (Fig 5A) and some were found at the interface between condensed and non-condensed nuclear chromatin, in many cells VAMP label was present in a granule-poor, circumferential cytoplasmic band just beneath the plasma membrane. Substitution of irrelevant rabbit IgG for the VAMP-specific antibody gave no labeling (Fig 5B).
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Discussion |
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We used a pre-embedding immunonanogold ultrastructural method (
Caveolae, also called plasmalemmal vesicles, were originally described in capillary endothelial cells, which contain rows of these flask-shaped vesicles individually attached to endothelial cell luminal and abluminal surfaces (
Purified rat lung endothelial cell caveolae fractions have been prepared (
Although capillary endothelial cell caveolae are believed by many to be capable of detachment from the plasma membrane and movement across cells (motile caveolae), cells also display single caveolae and clusters of surface-attached caveolae [capillary endothelial cells (
Smooth muscle cells also displayed surface-attached caveolae but, unlike pericytes, these were distributed on all surfaces (
Vesiculovacuolar organelles (VVOs) were first identified in tumor-associated microvessels and in the endothelia of normal venules of mice and guinea pigs (
Small secretory vesicles have been described in all three granulocyte lineages (
Eosinophils (and basophils) contain small cytoplasmic vesicles in similar locations that have a transport function, shuttling between their major granule populations and the plasma membrane in a secretory response termed piecemeal degranulation (
In summary, we have localized VAMP, an important family of v-SNAREs, to caveolae in pericytes, smooth muscle cells, and endothelial cells of human venules, to VVOs in venular endothelial cells, and to vesicle-rich cytoplasmic areas of human neutrophils and eosinophils. This distribution of label supports a role(s) for VAMP in vesicle transport mechanisms involved in macromolecular extravasation across venular endothelia, through sessile closed VVOs (which open), or in caveolae, which either do or do not (potocytosis) move. Single sessile caveolae in pericytes and clusters of sessile caveolae in vascular smooth muscle also labeled for VAMP, suggesting a role for this family of v-SNAREs in vesicular containers that may exert their function while remaining attached to the plasma membrane, much like VVOs. Circulating granulocytes also displayed label for VAMP in cytoplasmic vesicle-rich sites, lending support for a role(s) for this family of v-SNAREs in the vesicular transport of piecemeal degranulation, a secretory mode demonstrated in eosinophils (
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Acknowledgments |
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Supported by NIH grants AI-44066 and AI-33372 to A.M. Dvorak, HL-46563 and AI-20241 to Peter F. Weller, and HL-63250 to R. Flaumenhaft. R. Flaumenhaft is a Burroughs Wellcome Fund Career Awardee and is a participant in the Clinical Investigator Training Program, Beth Israel Deaconess Medical CenterHarvard/MIT Health Sciences and Technology, in collaboration with Pfizer, Inc.
We thank Peter K. Gardner for editorial assistance in the preparation of the manuscript, Patricia Fox for photographic assistance, and Tracey Sciuto for technical assistance.
Received for publication August 9, 2000; accepted November 15, 2000.
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