An electron-microscope and freeze-fracture study of the egg cortex of Brachydanio rerio

Summary We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/m² and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form domeshaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).     

Intracellular localization of lactosylceramide, the major human neutrophil glycosphingolipid

The abundance of lactosylceramide (LacCer; Gal beta 1-4Glc beta 1-1Cer) in human polymorphonuclear neutrophils (PMN) (about 10(9) molecules/cell) seemed inconsistent with an exclusive plasma membrane LacCer localization in these cells. Therefore, the distribution of LacCer between plasma membrane and intracellular compartments was analyzed. Binding of 125I-labeled monoclonal anti-LacCer antibody (T5A7) to intact cells indicated that only 0.1-0.2% of total LacCer was accessible to antibody. Chemical and immunochemical comparisons of organic extracts prepared from PMN cytoplasts (i.e. PMN depleted of nucleus and granules) and intact PMN demonstrated that less than 25% of PMN LacCer was plasma membrane-derived. Simultaneous particle volume and surface staining analyses suggested that selective LacCer loss from cytoplasts could not explain this result. Intracellular LacCer was demonstrated by intense staining of PMN frozen thin sections with T5A7 in indirect immunofluorescence tests. Two-color fluorescence studies using frozen thin sections of neutrophils previously surface-stained with saturating concentrations of T5A7 indicated that this staining did not reflect section artifact. Organic extracts of density gradient-fractionated PMN cavitates were analyzed by radioimmunoassay to determine whether LacCer associates with known populations of PMN granules. Antigen predominantly cosedimented with enzyme markers for primary and secondary granules rather than with plasma membrane marker. Thin layer chromatography of glycolipids extracted from these density gradient fractions identified LacCer as the only antigenic species and demonstrated that chemically detectable LacCer was primarily in granule-enriched rather than plasma membrane fractions. These data indicate that human PMN LacCer is predominantly intracellular and that the glycolipid may be a constituent of PMN lysosomal granules.     

Microfilaments and microtubules in calcium ionophore-induced secretion of lysosomal enzymes from human polymorphonuclear leukocytes.

Human peripheral blood leukocytes (PMN) are induced to release lysosomal enzymes by the calcium ionophore A23187 in the presence but not the absence of extracellular Ca++. Whereas secretion induced by particulate or immune stimuli is accompanied by an increase in visible microtubules and is inhibitable by colchicine, secretion induced by A23187 and Ca++ was not accompanied by an increase in microtubule numbers and was not inhibited by colchicine. Ca++ did not appear to regulate microtubule assembly in these cells since resting PMN had a mean of 22.3 +/- 2.0 microtubules in the centriolar region as compared to 22.3 +/- 1.1 in ionophore-treated cells and 24.9 +/- 1.5 in cells exposed to ionophore and 1 mM Ca++. Bipolar filaments, 10 nm thick and 300--400 nm long, were numerous in the pericortical cytoplasm of cells exposed to both reagents. Microtubules in these cells were decorated with an electron-opaque fibrillar material. PMN exposed to A23187 and Ca++ were contracted in two directions at right angles to each other: (a) Contractions parallel to the plasma membrane resulted in extensive plication of the cell membrane. The cytoplasm subjacent to the plicae contained dense filamentous webs. Plication was prevented by cytochalasin B or reversed by subsequent exposure to an endocytic stimulus such as zymosan. (b) Contractions perpendicular to the plasma membrane, toward the cytocenter, resulted in the formation of vacuoles in normal PMN and of membrane invaginations in cytochalasin B-treated PMN. Whereas contractions parallel to the plasma membrane could occur in the absence of enzyme release (ionophore alone) and enzyme release could occur in the absence of such contractions (ionophore plus calcium plus cytochalasin B), contraction toward the cytocenter occurred in all experimental conditions in which significant enzyme release was obtained. Thus, lysosomal enzyme secretion in PMN involves contractile movements in the plasma membrane toward the lysosomes rather than the reverse. These calcium-mediated contractile events are mediated by cytochalasin B-insensitive microfilaments but not by microtubule assembly.     

Activation of guinea pig polymorphonuclear leukocytes with soluble stimulators leads to nonrandom distribution of NADPH oxidase in the plasma membrane

Guinea pig polymorphonuclear leukocytes (PMN) were briefly activated with soluble stimulators such as sodium myristate (SM) or phorbol myristate acetate (PMA) and then disrupted by the nitrogen cavitation method to study the subcellular distribution of NADPH oxidase, which is responsible for O2 - generation. Fc-receptor and 5'-nucleotidase activities were measured as plasma membrane markers. 1) The homogenate was first fractionated by differential centrifugation. The O2- -generating activity of PMN activated either by SM or PMA was recovered in a 2 X 10(4) g pellet which contained a large amount of granules and about 50% of the plasma membrane markers, but not in a 1 X 10(5) g pellet which consisted of plasma membranes and few granules. 2) Further separation of the 2 X 10(4) g pellet from PMA-activated PMN was attempted by an iso-osmotic Percoll density gradient centrifugation. The O2- -generating activity was recovered in light fractions in which plasma membrane markers were found, but neither in specific nor in azurophil granules. The 1 X 10(5) g pellet showed a similar distribution of the plasma membrane markers to that of the 2 X 10(4) g pellet, except that the peak of the O2- -generating activity was much smaller on an identical density gradient. The results showed that NADPH oxidase is located in the plasma membranes precipitated by centrifugation at 2 X 10(4) X g but not in the ones precipitated at 1 X 10(5) X g. The results suggest that the plasma membrane of activated PMN has a mosaic distribution of NADPH oxidase.     

Observations on freeze-fractured membranes of a Trypanosome

Pure preparations of Trypanosoma brucei, free from plasma and cellular components were isolated from rat blood, and concentrated into loose pellets by low-speed centrifugation. Pellets were either processed for thin sectioning as a control for general morphology, or glycerol-treated after glutaraldehyde fixation for preparation of freeze-fracture replicas. Concentration of cells of 50,000–100,000/mm² of sectioned or fractured surface facilitated identification of fracture faces of the cell body, invaginated flagellar pocket and flagellum. Particle distribution and A and B faces of these regions of the cell are described. A collar of B face particles occurs around the neck of the flagellar pocket, possibly associated with a junction controlling ingress of ingested materials to coated vesicles formed along the membrane defining the pocket. A and B faces of the flagellum and adjoining surface of the cell body have shown that the only intra-membrane specialization corresponding to the miniature ‘maculae adherentes’ described previously in thin sections is probably an uninterrupted series of small clusters (3–6) of 80 Å particles on the A face of the flagellar membrane. It is proposed that these arrays represent attachment points for strands linking the axoneme and paraxial rod to the flagellar surface, and are not directly concerned with the physical adhesion of the flagellum to the cell body surface—a linkage that appears to be established within the extracellular gap between these apposed surfaces of the cell. The potential use of freeze-etching in further study of the external antigens of the infective cell is discussed.     

Anchorage of secretion-competent dense granules on the plasma membrane of bovine platelets in the absence of secretory stimulation

The ultrastructural changes in electropermeabilized bovine platelets that accompany the Ca2(+)-induced secretion of serotonin were investigated in ultra-thin sections of chemically fixed cells. Such preparations permitted us to study both the localization of and the structures associated with serotonin-containing dense granules. Localization of dense granules within cells was examined by measuring the shortest distances between the granular membranes and the plasma membrane. About 40% of total granules were located close to the plasma membrane at an average distance of 10.8 +/- 1.6 nm. 71% of the total number of granules were localized at a similar average distance of 12.5 +/- 2.7 nm in intact platelets. The percentage of granules apposed to the plasma membrane corresponded closely to the percentage of total serotonin that was maximally secreted after stimulation of the permeabilized (38 +/- 4.9%) and the intact platelets (72 +/- 3.6%). Furthermore, the percentage of granules anchored to the membrane, but not of those in other regions of permeabilized cells, decreased markedly when cells were stimulated for 30 s by extracellularly added Ca2+. The decrease in the numbers of granules in the vicinity of the plasma membrane corresponded to approximately 22% of the total number of dense granules that were used for measurements of the distances between the two membranes and corresponded roughly to the overall decrease (15%) in the average number of the granules per cell. Most dense granules were found to be associated with meshwork structures of microfilaments. Upon secretory stimulation, nonfilamentous, amorphous structures found between the plasma membrane and the apposed granules formed a bridge-like structure that connected both membranes without any obvious accompanying changes in the microfilament structures. These results suggest that the dense granules that are susceptible to secretory stimulation are anchored to the plasma membrane before stimulation, and that the formation of the bridge-like structure may participate in the Ca2(+)-regulated exocytosis.     

Control of secondary granule release in neutrophils by Ral GTPase

Neutrophil (polymorphonuclear leukocyte; PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocytosis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, we found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules but not primary or tertiary granules and caused dysregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, whereas chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Overexpression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, we found that Ral, which was associated with the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral translocation intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattractant stimulation.     

Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

ELECTRON MICROSCOPIC DEMONSTRATION OF A DITHIOTHREITOL-LABILE VITELLINE COAT SURROUNDING THE UNFERTILIZED EGG OF COMANTHUS JAPONICA (ECHINODERMATA: CRINOIDEA)*

In Comanthus, the unfertilized egg is surrounded by a vitelline coat, which is separated from the underlying plasma membrane by a space several hundred Ångstroms wide. By electron microscopy, the vitelline coat is a distinct layer 100 to 150 Å thick, which consists of finely granular material of moderate electron density. Treatment for 3 min in 0.01 M dithiothreitol in sea water buffered to pH 9.2 almost completely removes the vitelline coat and causes the irregularly shaped egg to become spherical. After such DTT-treated eggs have been washed for 2 min in sea water, they cannot be fertilized, but they can undergo a cortical reaction when treated with ionophore A23187. This cortical reaction consists of the exocytosis of cortical granule material directly into the surrounding sea water. By several hours after DTT treatment, most of the eggs, whether exposed to ionophore or not, fragment into spheres of diverse sizes.     

Alkaline phosphatase activity in human polymorphonuclear leukocytes

Synopsis Alkaline phosphatase has been localized ultracytochemically in PMN of man with normal and elevated levels of this enzyme. Contrary to guinea-pig PMN, no activity appears to be present in the specific granules. Instead, the plasma membrane and the membrane of the endocytic vacuoles show a strong staining. However, the demonstration of this activity depends on the preparatory procedure employed for PMN isolation. the use of dextran and Ficoll-Hypaque in the isolation procedure induces a marked increase in alkaline phosphatase staining of the PMN plasma membrane. Strongly increased activity at this site has been found in PMN from cancer patients. In most of them, additional staining has been observed in atypical vesicles and sometimes in the Golgi apparatus. These findings are discussed in the light of some previously reported controversial biochemical and cytochemical data on the distribution of alkaline phosphatase in human PMN.     

Functional activity of enucleated human polymorphonuclear leukocytes

Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.     

Fine Structure of Bacillus megaterium during Microcycle Sporogenesis

Ultrathin sections were prepared from cultures of Bacillus megaterium QM B1551 undergoing microcycle sporogenesis (initial spore to primary cell to second-stage spore without intervening cell division) on a chemically defined medium. The cytoplasmic core of the dormant spore was surrounded by plasma membrane, cell-wall primordium, cortex, outer cortical layer, and spore coats. Early in the cycle, the coat opened at the germinal groove, the cortex swelled, ribosomes and a chromatinic area associated with large mesosomes (which may later be incorporated into the expanding plasma membrane) appeared in the core, and the cell wall became defined at the site of the cell wall primordium. Poly-β-hydroxybutyrate granules began to appear in the primary cell at about 3 hr. By 7 hr, the forespore of the second-stage spore was delineated by typical double membranes. Between 7 and 12 hr, second-stage cell-wall primordium and cortex developed between the separating forespore membranes. The inner membrane became the plasma membrane of the second-stage spore, and the outer membrane eventually disintegrated within the second-stage spore cortex. A densely staining double layer (spore-coat primordium) developed external to the outer forespore membrane. The inner spore coat and the outer cortical layer of the second-stage spore developed from this primordium. The outer part of the spore coat, probably of sporangial origin, was laid down on the external surface of the inner spore coat. By 12 hr, second-stage spores were almost mature. By 20 hr, the mature endospores, with a thickened outer coat, were often still enclosed by degenerate primary cell wall and by the outer cortical layer and spore coat of the initial spore.     

The respiratory burst of bovine neutrophils. Role of a b type cytochrome and coenzyme specificity

A new method of preparation of bovine polymorphonuclear leukocytes (PMN) is described. The subcellular distribution of cytochrome b in resting and activated bovine PMN was compared to that of the O2-.-generating oxidase (assessed as NADPH cytochrome c reductase inhibited by superoxide dismutase). In resting PMN and in PMN activated by phorbol myristate acetate (PMA), cytochrome b was located into two membrane fractions, one of which was enriched in plasma membrane and cosedimented with alkaline phosphatase, while the other consisted of a denser material cosedimenting with markers of the specific and azurophil granules, i.e. the vitamin-B12-binding protein and myeloperoxidase respectively. During activation of PMN by PMA, 15-20% cytochrome b migrated from dense granules to the plasma membrane. The distribution of the O2-. generating oxidase and cytochrome b in subcellular particles was studied during the course of phagocytosis of PMA-coated latex beads by bovine PMN. At the onset of the respiratory burst, the phagocytic vacuoles arising from internalization of the plasma membrane were enriched in oxidase and alkaline phosphatase, but their specific content of cytochrome b was limited; in contrast, cytochrome b was predominant in denser membrane fractions cosedimenting with myeloperoxidase and the vitamin-B12-binding protein. After a few minutes of phagocytosis, a fraction of light vacuoles, slightly denser than the phagocytic vacuoles, became enriched in O2-.-generating oxidase, cytochrome b, the vitamin-B12-binding protein and myeloperoxidase. These vacuoles probably arose from the fusion of the phagocytic vacuoles with dense granules. In bovine PMN supplemented with glucose and maintained in anaerobiosis, activation by PMA induced slow reduction of cytochrome b (60-70% in 15 min at 37 degrees C). Similar results were obtained with cytoplasts after activation by PMA (30% reduction in 3 min at 37 degrees C). Cytochrome b in a particulate fraction obtained by centrifugation at 100 000 X g of an homogenate of PMA-activated PMN, was slowly reduced upon addition of NADPH under anaerobiosis (less 20% in 20 min at 37 degrees C). No reduction occurred in the 100 000 X g fraction prepared from non-activated PMN. The Soret band of cytochrome b reduced by dithionite was displaced by CO only by 1-2 nm. At subsaturating concentrations, CO had no effect on the rate of O2 uptake by activated bovine PMN.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ordered membrane particles in rhabdomeric microvilli of the housefly (Musca domestica L.)

Rhabdomeric microvilli of the housefly were freeze-fractured (FF) and thin sectioned (TS) for ultrastructural examination. Ordered files of closely packed membrane particles (82 Å wide, 250 Å long) were seen (FF) on the microvillar membrane (usually E face). The long axis of each particle was canted about 45° to that of the microvillus. Occasionally particles in this array appeared on the P face. It is hypothesized that ordered particles may represent either a photopigment precursor stock, a second photolabile pigment, or the newly discovered sensitizing, UV-absorbing, photostable visual pigment. In the underlying membrane leaflet (P face) were found spherical (85 Å diameter) unoriented particles in a concentration of about 6,000/μm². The size, shape and density of these structures are compatible with those of rhodopsin particles. These particles also covered the basal area of each microvillus. The findings from TS material were difficult to correlate with those from FF replicas. At high magnification the former showed that the plasma membrane of the transected microvillus is composed of spherical, hollow subunits (averaging 43 Å diameter), sometimes fused to form double, 86 Å units. These substructures were closely packed and continuous around the microvillus. This beaded plasma membrane, in rare cases, was doubled around the microvillus. In other instances the plasma membranes were continuous between neighboring microvilli. The physiological implications of these ultrastructural features are discussed.     

Ultrastructural cytochemistry of membrane-bound phosphatases in preimplantation mouse embryos.

The time of appearance and the ultrastructural localization of the enzyme activity of alkaline phosphatase (AlkPase), 5′-nucleotidase (5′Nuc), Mg²⁺-ATPase, transport ATPase, cyclic AMP phosphodiesterase (cAMP-PDase), and adenylate cyclase (AC) were investigated in unfertilized eggs and in mouse preimplantation embryos. Enzyme activity was associated only with the plasma membrane. AlkPase activity appeared only in limited areas of the plasma membrane of one-cell embryos and increased in the eight-cell and morula stages. In blastocysts, the enzyme activity was concentrated mainly in the trophoblast cells. 5′Nuc activity appeared first in four- or eight-cell embryos and the highest activity was observed in trophoblast cells in the blastocyst and in plasma membrane between cells forming inner cell mass. Mg²⁺-activated ATPase activity was present in all embryos and in unfertilized egg plasma membrane. Transport (Na⁺K⁺)-ATPase appeared only in the closely apposed membranes of adjacent cells in morulae and blastocysts. A very low cAMP-PDase activity appeared between adjacent cells in two-cell embryos, and the highest activity was observed on the outer surface of the plasma membrane of trophoblasts. AC was the only enzyme whose activity was located on the inner (cytoplasmic) side of the plasma membrane and appeared as early as the one-cell stage embryo. The relation between the time of the appearance of enzyme activity and the preparation of embryos for implantation and upon embryonic proliferative activity is discussed.     

Membrane interactions between secretion granules and plasmalemma in three exocrine glands

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.     

Membrane fusion during secretion: cortical granule exocytosis in sex urchin eggs as studied by quick-freezing and freeze-fracture

Exocytosis of cortical granules was observed in sea urchin eggs, either quick-frozen or chemically fixed after exposure to sperm. Fertilization produced a wave of exocytosis that began within 20 s and swept across the egg surface in the following 30 s. The front of this wave was marked by fusion of single granules at well-separated sites. Toward the rear of the wave, granule fusion became so abundant that the egg surface left with confluent patches of granule membrane. The resulting redundancy of the egg surface was accommodated by elaboration of characteristic branching microvilli, and by an intense burst of coated vesicle formation at approximately 2 min after insemination. Freeze-fracture replicas of eggs fixed with glutaraldehyde and soaked in glycerol before freezing displayed forms of granule membrane interaction with the plasma membrane which looked like what other investigators have considered to be intermediates in exocytosis. These were small disks of membrane contact or membrane fusion, which often occurred in multiple sites on one granule and also between adjacent granules. However, such membrane interactions were never found in eggs that were quick-frozen fixation, or in eggs fixed and frozen without exposure to glycerol. Glycerination of fixed material appeared to be the important variable; more concentrated glycerol produced a greater abundance of such "intermediates." Thus, these structures may be artifacts produced by dehydrating chemically fixed membranes, and may not be directly relevant to the mechanism by which membranes naturally fuse.     

Myelin P0-Glycoprotein: Predicted Structure and Interactions of Extracellular Domain

Protein zero (P₀), a transmembrane glycoprotein, accounts for over 50% of the total protein in PNS myelin. The extracellular domain of P₀ (P₀-ED) is similar to the immunoglobulin variable domain, carrying one acceptor sequence for N-linked glycosylation. The x-ray diffraction analysis of PNS myelin has demonstrated reversible transitions that depend on pH and ionic strength, resulting in three distinct structures characterized by widths of about 36 Å, 50 Å (native), and 90 Å between the extracellular surfaces of the membranes. In the current work, we considered the constraints imposed by these x-ray diffraction data on the orientation of P₀-ED, and we propose how this immunoglobulin-like domain could be accommodated in the variable widths of the extracellular space between myelin membranes. The modeling made use of the finding that β-strand predictions for P₀-ED are virtually superimposable with those of the V_H domain of the phosphocholine-binding immunoglobulin M603 of mouse, which has a similar number of residues as P₀-ED and a structure that has been solved crystallographically. The dimensions of P₀-ED from the space-filling model, developed using PC- based molecular modeling software, were found to be 44 Å× 25 Å× 23 Å. On the assumption that neither the shape nor the orientation of P₀-ED changes appreciably, then the different widths at the extracellular apposition would easily accommodate P₀-ED from apposed membranes if the molecules were oriented so that the β- strands were approximately perpendicular to the membrane surface. The apposed P₀-EDs would fully overlap at the closest apposition of the membranes, partially overlap in the native state, and align end to end in the incompletely swollen state. The P₀-ED regions analogous to the complementarity-determining regions of immunoglobulins can account for the recognition of P₀-ED from apposed membranes in the incompletely swollen state. Two of the faces of P₀-ED that show charge complementarity could account for the homophilic interactions of P₀-ED from apposed membranes in the native state. This association can be stabilized further by hydrophobic interactions. The N- linked nonasaccharide after energy minimization fit into a cavity, which was at the base of P₀-ED and which was lined with three positively charged residues. Thus, the carbohydrate may help to maintain the orientation of P₀ at the membrane surface. Our model shows how the single immunoglobulin-like domain of P₀ can account for distinct structural states of myelin membrane packing by homophilic interactions.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Docking is not a prerequisite but a temporal constraint for fusion of secretory granules

We examined secretory granule dynamics using total internal reflection fluorescence microscopy in normal pancreatic β cells and their mutants devoid of Rab27a and/or its effector, granuphilin, which play critical roles in the docking and recruitment of insulin granules to the plasma membrane. In the early phase of glucose stimulation in wild-type cells, we observed marked fusion of granules recruited from a relatively distant area, in parallel with that from granules located underneath the plasma membrane. Furthermore, despite a lack of granules directly attached to the plasma membrane, both spontaneous and evoked fusion was increased in granuphilin-null cells. In addition to these granuphilin-null phenotypes, Rab27a/granuphilin doubly deficient cells showed the decreases in granules located next to the docked area and in fusion from granules near the plasma membrane in the early phase of glucose-stimulated secretion, similar to Rab27a-mutated cells. Thus, the two proteins play nonoverlapping roles in insulin exocytosis: granuphilin acts on the granules underneath the plasma membrane, whereas Rab27a acts on those in a more distal area. These findings demonstrate that, in contrast to our conventional understanding, stable attachment of secretory granules to the plasma membrane is not prerequisite but temporally inhibitory for both spontaneous and evoked fusion.