Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells.

Abstract

The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membranes is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin . The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.Recently, we showed that cytochemical and immunochemical techniques can be combined with freeze-fracture to gain generalized access for direct labeling of the fracture faces of biological membranes as well as of exposed groups in crossfractured cytoplasm (14-16) . The results of these "fracturelabel" techniques can be assessed either by observation of thin sections of freeze-fractured specimens ("thin-section fracturelabel") (15, 16) or of platinum/carbon replicas of critical point dried, freeze-fractured preparations ("critical-point-drying fracture-label") (14).Initial application of thin-section fracture-label showed that numerous concanavalin A binding sites could be labeled on the membrane faces of freeze-fractured plasma membranes, endoplasmic reticulum, and nuclear envelope membranes of leukocytes, HeLa cells, and hepatocytes (16). We report here the results of thin-section and critical-point-drying fracturelabel of isolated rat exocrine and endocrine pancreatic cells. We used colloidal gold and ferritin conjugates to determine and compare the patterns of distribution of wheat-germ agglutinin (WGA) and concanavalin A (Con A) binding sites on the fracture face of their plasma and intracellular membranes. Our results show that whereas Con A binding sites are present on nuclear envelope, endoplasmic reticulum, secretory vesicle, and plasma membranes, wheat-germ agglutinin binding sites are absent from the nuclear envelope and the endoplasmic reticu-THE JOURNAL OF CELL BIOLOGY " VOLUME 91 NOVEMBER 1981 361-372 lum. These results are consistent with current views on the pathways of glycosylation of membrane proteins (5,12, 18,19,22,23,24) and do not support the reflux of fully glycosylated products to the endoplasmic reticulum (3,7,25,29). They demonstrate the capacity of fracture-label to investigate the topochemistry of plasma and intracellular membranes in situ . MATERIALS AND METHODS CellsPancreas tissue was excised from 3-to 8-d-old rats (Sprague-Dawley) and digested with collagenase type IV (5 g/ml in Hanks' solution, from Worthington Biochemical Corp ., Freehold, N . J .) for 6 min at 37°C, and isolated cells or small clusters of acinar and islet cells were obtained. The cells and cell clusters were harvested by centrifugation (800 rpm, 6 min), washed twice in Hanks' solution and fixed in 1% glutaraldehyde for 2 h at 4°C . A...

High density of transmembrane glycoproteins on the flagellar surface of boar sperm cells.

“…They contrast with the images seen in mens, reorganization of freeze-fracture membrane halves re-suits in a rugose structure (Fig. 9) where membrane-intercalated particles are not identifiable (27,28,(41)(42)(43) . Observation of replicas of boar sperm flagella illustrated clearly the high density of both Con A and WGA receptors along protoplasmic membrane halves of the different segments of the sperm tail (Figs .…”

“…Thin sections were observed by transmission electron microscopy after counterstaining with lead citrate only . 41). At the midpiece, the axoneme is surrounded by nine dense fibers that are, on the outside, circumferentially covered by mitochondria (illustrated here in Figs.…”

“…10, view of midpiece segment of sperm flagellum ; Figs . shadowing, according to their different texture as observed with the dissecting microscope (27,41). In frozen "sandwiched" preparations, only fracture faces can be revealed (28).…”

Freeze-fracture cytochemistry: partition of glycophorin in freeze-fractured human erythrocyte membranes.

“…We must note, however, that lectin labeling experiments cannot be used to decide whether the molecules labeled remained structurally intact or whether covalent bonds in polypeptide chains were ruptured during fracture as suggested by previous biochemical analysis of the exoplasmic half of erythrocyte membranes (9). Yet, while labeling of outer surface sites on exoplasmic faces appears intriguing and obscures the molecular rearrangements that make it possible, it corresponds to a reliable experimental fact observed in a variety of plasma and intracellular membranes (2,3,14) and advantageous in the study of partition of membrane components upon fracture .…”

“…They allow the observation of numerous fractured membranes and permit a better and easier assessment of the pattern of distribution over entire fracture faces . Thin-section fracture-label is, however, of particular value when it is necessary to identify the type of cell or cellular organelle being labeled (14).…”