RAPID COMMUNICATION |
Correspondence to: Stanley L. Erlandsen, Dept. Genetics, Cell Biology, and Development, 6-160 Jackson Hall, U. of Minnesota School of Medicine, Minneapolis, MN 55455. E-mail: erlan001@umn.edu
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Summary |
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The aim of this study was to develop a model for the detection of individual cell adhesion molecules (CAMs) in the glycocalyx of spread human platelets using high-resolution cryo-field emission scanning electron microscopy (cryoFESEM). Three surface glycoprotein CAMs, P-selectin (CD62P), GPIba in the GPI-IX complex (CD42a/CD42b,bß), and the integrin GPIIbIIIa (CD41/CD61) in the human platelet were selected on the basis of their unique topographic shape. Spread human platelets were indirectly immunolabeled with 10-nm colloidal gold and then cryoimmobilized. After sublimation of water from the cryoimmobilized sample, partially freeze-dried platelets were coated unidirectionally with Pt, stabilized with carbon, and examined in an in-lens cryoFESEM using high-resolution backscattered electron imaging. CAMs were detected by indirect immunogold labeling and the length of each type of CAM was determined using analysis of differences in parallax as measured in the software program Sterecon. Our results demonstrate the efficacy of using high-resolution cryoFESEM to recognize and detect individual CAMs in the glycocalyx. Further advances in production of metal coatings with finer granularity, together with improvements in imaging (tilting and angle of stereo images), may provide better definition of the topography associated with glycosylation and formation of multimeric CAM complexes. (J Histochem Cytochem 49:809819, 2001)
Key Words: cryo-field emission SEM, glycocalyx, cell adhesion molecules, P-selectin, GPI-IX, GPIIbIIIa, immunogold, backscattered electron imaging
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Introduction |
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CELLS INTERACT with neighboring cells and their extracellular matrix by means of cell adhesion molecules (CAMs) or receptors associated with the external surface of the plasma membrane. A major feature of cell surfaces is the presence of carbohydrate structures (N- or O-glycosylation) on many of these protein and lipid molecules. The term "glycocalyx" was introduced by
Ultrastructural spatial distribution of cell surface antigens on leukocytes has been accomplished by immunogold localization using field-emission scanning electron microscopy (FESEM) (2 nm in "infinitely thin" biological samples (
A number of different CAM families are present in the glycocalyx of the human platelet, including the following: (a) a group of integrins with affinities for collagen (a2B1), laminin (a6,B1), vitronectin (a3B1), and the abundant promiscuous receptor GPIIbIIIa (CD41/CD61), which interacts with von Willibrand factor, thrombospondin, fibronectin, fibrinogen, and vitronectin; (b) leucine-rich glycoproteins, such as GPI-IX (CD42a/CD42b,bß); (c) two immunoglobulin-type glycoproteins, Pecam-1 and HLA class 1 ß2 microglobulin; and (d) the C-type lectin family member P-selectin (CD62P) (
The aim of this study was to demonstrate that use of cryomethods for preservation of cell structure together with high-resolution cryoFESEM could detect individual CAMs in the glycocalyx using a model system based on spread human platelets. Our strategy consisted of selecting distinct molecules that could be recognized by immunogold labeling. These molecules also display key structural differences resulting in unique topographical shapes. As shown in Fig 1, three major protein molecules in the platelet glycocalyx were selected for detection, i.e., the rod-shaped P-selectin molecule [CD62P; 13,000 copies per activated platelet (,bß; 25,000 copies per platelet (
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Materials and Methods |
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Human Blood Platelets
Human blood for this study was drawn after informed consent from normal donors know to be free of any medications. As previously described (
Antibodies
Mouse monoclonal antibodies against P-selectin were obtained from Dr. Rodger McEver (University of Oklahoma Health Sciences Center, Oklahoma City, OK: G1, directed against the NH2-terminal lectin domain and S12 against the short consensus repeat region) and Drs. Eugene Butcher and Aaron Warnick (Department of Pathology, Stanford University, Palo Alto, CA: WAPS12.2). Mouse MAb W5 against GPIb and T10, a blocking antibody directed against a combined region of GPIIbIIIa, were also provided by Dr. McEver. All primary antibodies were mouse monoclonal IgG and were diluted to concentrations of 1020 µg/ml in 10% normal sheep serum in phosphate-buffered saline, pH 7.4 (NSSPBS). Goat anti-mouse IgG conjugated to 10-nm colloidal gold was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA) and used at dilutions of 1:101:20 in 10% NSSPBS.
Cell Adhesion
Platelets were allowed to spread or adhere on carbon-coated sapphire discs (2.9-mm diameter, 0.05 mm thick; Rudolf Bruegger AG, Minusio, Switzerland). To spread platelets, a drop of the platelet suspension was placed on the sapphire discs and the cells were allowed to settle for 20 min at RT. Non-attached cells were washed off in Hank's buffer. The spread cell monolayer was pre-fixed for 10 min in Hank's buffer with 0.01% glutaraldehyde, then washed in the same buffer before immunolabeling.
Antibody Labeling
The prefixed adherent platelets on the discs were incubated on the top of drops of PBS with 10% normal sheep serum (NShSPBS) for 10 min. The discs were then blotted and transferred onto the first or primary antibody to a specific CAM. After a 30-min incubation on the first antibody, the samples were washed multiple times on drops of NShSPBS and then transferred to a drop of immunogold marker (goat anti-mouse IgG conjugated to 10-nm colloidal gold diluted 1:20 in NShSPBS). After a 30-min incubation period the samples were washed in PBS. All incubations were done at RT.
Post-immunolabeling Chemical Fixation
Immunolabeled spread platelet monolayers were fixed in 3% glutaraldehyde in 0.1 M Na-cacodylate buffer with 7.5% sucrose, pH 7.4, for at least 30 min. The samples were washed in the same buffer. Some samples were postfixed in 2% OsO4 in 0.1 M Na-cacodylate with 7.5% sucrose, pH 7.4, for 20 min, and then washed in the same buffer.
Sample Preparation for CryoFESEM
Immunolabeled fixed platelets were rinsed with distilled water and most of the overlying water was blotted away from the side to reduce sample thickness for better freezing results. The sapphire discs were frozen by plunging into liquid propane chilled by liquid N2 and then were cryotransferred to a modified holder for the Balzers BA-360 unit (TechnoTrade; Manchester, NH) for double-layer coating by the method of 710-nm carbon from above (90° angle). The partially freeze-dried, double-layer coated samples were cryotransferred under liquid nitrogen into a Gatan cryostage and then inserted into the Hitachi S-900 in-lens FESEM (Niessi Sangyo America; Mountain View, CA) for cryo-observation at -95C and at 10 keV accelerating voltage. High-resolution backscattered electron images were collected using a modified YAG scintillator (
Measuring Height of CAMs
The height of CAMs in cryosamples was measured in high-magnification stereo pairs using the Sterecon program, a 3D, interactive, contour-based segmentation and measurement program (
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Results |
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The activated spread human platelet was chosen as a model system for demonstrating individual CAMs because it possesses three unique CAMs that differ in topographic appearance (Fig 1) and because MAbs were available for all three. Unlike their normal discoid shape, human platelets fixed after spreading were seen by cryoFESEM as thin, rounded but flattened monolayers of cytoplasm (Fig 2A) with occasional pseudopods extending from the margin of the cell. High-resolution backscattered electron imaging of immunogold labeling was accomplished using atomic number contrast in which gain of the YAG detector was set so that 10-nm colloidal gold particles appeared as round white spheres. Spread fixed platelets labeled for P-selectin revealed that P-selectin molecules were distributed from edge to edge of the exposed surface of the fully spread cells (Fig 2 and Fig 3). Immunogold-labeled P-selectin was seen as dimers or multimers of colloidal gold on rod-like extensions protruding from the surface of the platelet membrane. The vertical projection of P-selectin dimers above the membrane could clearly be seen by stereo imaging (Fig 3A) and by the length of the shadow resulting from the unidirectional shadowing of the cell at 45° with Ta/W (Fig 3B). In fortuitous backscattered-electron images at high magnification, individual molecules of P-selectin were recognizable (inset, Fig 3B). Small unlabeled knob-like protrusions were seen in the background between the labeled P-selectin CAM. In regions of the cell surface where an obstruction interfered with the angle of the metal coating, it was difficult to assess the height of immunogold-labeled P-selectin owing to lack of sufficient shadow contrast (Fig 2B).
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Immunogold labeling for GPIb in the GPI-IX complex revealed a unique pattern for immunostaining over the entire surface of the platelet unlike that seen for the rod-shaped P-selectin or the integrin GPIIbIIIa (Fig 4A4C). Stereo analysis of the immunogold labeling for GPIb
revealed linear arrays that protruded above the platelet membrane (Fig 4C). Careful examination of the immunolabeled arrays often revealed the presence of one or two colloidal gold markers in these linear protrusions. The presence of two colloidal gold markers may represent the postulated stochiometric ratio of two GPI-IX complexes with one GPV molecule in a linear array on the surface of the membrane (Fig 1, Fig 4A, and Fig 4B).
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The topographic appearance of the integrin GPIIbIIIa as small knob-like protrusions from the membrane surface was distinct from that of the rod-shaped P-selectin or the linear arrays seen for the GPI-IX complex. Immunogold labeling for GPIIbIIIa was detected over small knob-like projections in the dorsal surface of the fully spread platelet membrane (Fig 5). Occasionally, immunogold labeling for GPIIbIIIa was seen as dimers or multimers of immunogold particles. Careful examination of the immunolabeled membrane in Fig 5 clearly showed that not all knob-like projections were labeled with immunogold, which is consistent with the presence of other integrin molecules on the platelet membrane.
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Controls for the immunogold labeling of CAM included omission of the primary antibody and the omission of the secondary antibody labeled with colloidal gold. In both cases, the end result was the absence of immunogold label on the surface molecules. In addition, the use of MAbs to CAMs having uniquely different topography showed the lack of crossreactivity among these different molecules, thus providing additional evidence for their specificity.
The projected height of the rod-shaped P-selectin molecules above the dorsal membrane of the flattened spread platelet was 48.2 ± 3.6 nm (n=4), a value similar to that reported by above the membrane revealed an average height of 37.2 ± 3.3 nm (n=8), which favorably compares with the range of 3553 nm reported by a number of investigators (
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Discussion |
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The present results demonstrate that cryoFESEM possesses sufficient resolution to detect individual CAMs in the glycocalyx in a model system based on the activated and fully spread human platelet. Using immunogold labeling, three CAMs in the platelet glycocalyx were identified and confirmed as having unique topographic appearances. P-selectin (CD62P) was seen as rod-shaped protrusions above the platelet membrane, GPIb was seen in the GPI-IX complex (CD42a/CD42b
,bß) as part of a linear array projecting above the membrane, and GPIIbIIIa (CD41/CD61) was seen as small knob-like protrusions above the platelet membrane.
Our results with immunolabeling for P-selectin support earlier work of
2,ß2,
2, and
, where
represents GPIb
, ß represents GPIbß,
represents GPIX, and
is GPV (Fig 1). Because GPV is associated with two GPI-IX complexes and the entire complex binds to the actin cytoskeleton, it was suggested that the physical association of GPV-(GPI-IX)2 might exist in a linear array on the cell surface (Fig 1). This interpretation was supported by our high-resolution stereo imaging of this complex (Fig 4A4C) which demonstrated a linear array of surface projections with two gold markers associated with the complex. Linear arrays of immunogold staining for GPIb
have also been reported by
and actin-binding protein in the platelet cytoskeleton using high-resolution transmission EM and immunogold labeling.
The greatest variation in the measurement of individual CAMs was seen with the values obtained for labeled (22.3 ± 2.7 nm) and unlabeled (16.9 ± 4.2 nm) integrins. Although these are close to the reported values of 20-22 nm (
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The detection and measurement of individual CAMs in the glycocalyx of human platelets have been accomplished using cryoFESEM and cryopreparative methods (Fig 6). Many factors can influence the resolution obtained in cryoFESM, including the type of specimen ("infinitely thin" vs bulky), the accelerating voltage, contamination/radiation damage, temperature, and the nature of the metal coating used to generate the secondary electron or backscattered-electron signal (2 nm thick), the signal comes chiefly from the thickness of the metal layer and the practical limitation on resolution is the lateral diffusion of electrons which sets this limit for SEM. According to
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Acknowledgments |
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Sterecon analysis work was supported by NIH NCRR grant RR01219 to the Resource for Visualization of Biological Complexity at the Wadsworth Center.
We wish to thank Drs Rodger McEver and Eugene Butcher for their generous supply of antibodies. We also thank Marcie Krumweide, Chris Frethem, and Cecile Ottenwaelter for their excellent technical assistance, and the University of Minnesota Graduate School and the Minnesota Medical Foundation for financial support.
Received for publication March 15, 2001; accepted March 30, 2001.
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