Stages Of Exocytosis Research Articles

Phospholipase D1 produces phosphatidic acid at sites of secretory vesicle docking and fusion.

Phospholipase D1 (PLD1) activity is essential for the stimulated exocytosis of secretory vesicles where it acts as a lipid-modifying enzyme to produces phosphatidic acid (PA). PLD1 localizes to the plasma membrane and secretory vesicles, and PLD1 inhibition or knockdowns reduce the rate of fusion. However, temporal data resolving when and where PLD1 and PA are required during exocytosis is lacking. In this work, PLD1 and production of PA are measured during the trafficking, docking, and fusion of secretory vesicles in PC12 cells. Using fluorescently tagged PLD1 and a PA-binding protein, cells were imaged using TIRF microscopy to monitor the presence of PLD1 and the formation of PA throughout the stages of exocytosis. Single docking and fusion events were imaged to measure the recruitment of PLD1 and the formation of PA. PLD1 is present on mobile, docking, and fusing vesicles and also colocalizes with Syx1a clusters. Treatment of cells with PLD inhibitors significantly reduces fusion, but not PLD1 localization to secretory vesicles. Inhibitors also alter the formation of PA; when PLD1 is active, PA slowly accumulates on docked vesicles. During fusion, PA is reduced in cells treated with PLD1 inhibitors, indicating that PLD1 produces PA during exocytosis.

Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion.

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor-Derived Peptides for Regulation of Mast Cell Degranulation.

Vesicle-associated V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and target membrane-associated T-SNAREs (syntaxin 4 and SNAP-23) assemble into a core trans-SNARE complex that mediates membrane fusion during mast cell degranulation. This complex plays pivotal roles at various stages of exocytosis from the initial priming step to fusion pore opening and expansion, finally resulting in the release of the vesicle contents. In this study, peptides with the sequences of various SNARE motifs were investigated for their potential inhibitory effects against SNARE complex formation and mast cell degranulation. The peptides with the sequences of the N-terminal regions of vesicle-associated membrane protein 2 (VAMP2) and VAMP8 were found to reduce mast cell degranulation by inhibiting SNARE complex formation. The fusion of protein transduction domains to the N-terminal of each peptide enabled the internalization of the fusion peptides into the cells equally as efficiently as cell permeabilization by streptolysin-O without any loss of their inhibitory activities. Distinct subsets of mast cell granules could be selectively regulated by the N-terminal-mimicking peptides derived from VAMP2 and VAMP8, and they effectively decreased the symptoms of atopic dermatitis in mouse models. These results suggest that the cell membrane fusion machinery may represent a therapeutic target for atopic dermatitis.

Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties.

SummaryThe fusion pore is the first crucial intermediate formed during exocytosis, yet little is known regarding the mechanisms that determine the size and kinetic properties of these transient structures1. Here, we reduced the number of available SNAREs in neurons and observed changes in transmitter release suggestive of alterations in fusion pores. To address this, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state. Optical measurements revealed that increasing the number of SNARE complexes enhanced the rate of release from single pores, and enabled the escape of larger cargos. To determine whether this was due to changes in nascent pore size versus stability, we developed a novel approach, based on nanodiscs and planar lipid bilayer electrophysiology, that affords μsec time resolution at the single event level. Remarkably, both parameters were affected by SNARE copy number. Increasing the number of v-SNAREs per nanodisc from three to five caused a two-fold increase in pore size and decreased the rate of pore closure by more than three orders of magnitude. Moreover, trans-SNARE pairing was highly dynamic: flickering nascent pores closed upon addition of a v-SNARE fragment, revealing that the fully assembled, stable, SNARE complex does not form at this stage of exocytosis. Finally, a deletion at the base of the SNARE complex, that mimics the action of botulinum neurotoxin A, dramatically reduced fusion pore stability. In summary, trans-SNARE complexes are dynamic, and the number of SNAREs recruited to drive fusion determine fundamental properties of individual pores.

Annexin A2, an essential partner of the exocytotic process in chromaffin cells

Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, such as neuroendocrine cells. In chromaffin cells, cytosolic annexin A2 is recruited to the plasma membrane upon cell stimulation. Here, we review the latest evidence detailing the functional importance of annexin A2 in different stages of exocytosis. These include the recruitment of annexin A2 to the plasma membrane near soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, the role of annexin A2 in the formation of lipid domains at exocytotic sites, and finally the annexin A2 bundling of actin microfilaments associated with chromaffin granules. These structures induce first the coalescence of lipid domains required for the formation of the exocytotic site, and in the second time, exert mechanical force on the granule to favor fusion pore expansion and squeeze the granule to facilitate catecholamine release. Annexin A2 is a calcium-, actin-, and lipid-binding protein implicated in exocytosis in different cell types, including neuroendocrine cells. Upon cell stimulation, annexin A2 translocates from the cytosol to the plasma membrane of chromaffin cells and bundles actin filaments associated with chromaffin granules. This promotes the formation of lipid domains required for granule docking, and facilitates catecholamine release by compressing the granule. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

Src kinases regulate de novo actin polymerization during exocytosis in neuroendocrine chromaffin cells.

The cortical actin network is dynamically rearranged during secretory processes. Nevertheless, it is unclear how de novo actin polymerization and the disruption of the preexisting actin network control transmitter release. Here we show that in bovine adrenal chromaffin cells, both formation of new actin filaments and disruption of the preexisting cortical actin network are induced by Ca2+ concentrations that trigger exocytosis. These two processes appear to regulate different stages of exocytosis; whereas the inhibition of actin polymerization with the N-WASP inhibitor wiskostatin restricts fusion pore expansion, thus limiting the release of transmitters, the disruption of the cortical actin network with cytochalasin D increases the amount of transmitter released per event. Further, the Src kinase inhibitor PP2, and cSrc SH2 and SH3 domains also suppress Ca2+-dependent actin polymerization, and slow down fusion pore expansion without disturbing the cortical F-actin organization. Finally, the isolated SH3 domain of c-Src prevents both the disruption of the actin network and the increase in the quantal release induced by cytochalasin D. These findings support a model where a rise in the cytosolic Ca2+ triggers actin polymerization through a mechanism that involves Src kinases. The newly formed actin filaments would speed up the expansion of the initial fusion pore, whereas the preexisting actin network might control a different step of the exocytosis process.

Critical Role of Cortical Vesicles in Dissecting Regulated Exocytosis: Overview of Insights Into Fundamental Molecular Mechanisms

Regulated exocytosis is one of the defining features of eukaryotic cells, underlying many conserved and essential functions. Definitively assigning specific roles to proteins and lipids in this fundamental mechanism is most effectively accomplished using a model system in which distinct stages of exocytosis can be effectively separated. Here we discuss the establishment of sea urchin cortical vesicle fusion as a model to study regulated exocytosis-a system in which the docked, release-ready, and late Ca(2+)-triggered steps of exocytosis are isolated and can be quantitatively assessed using the rigorous coupling of functional and molecular assays. We provide an overview of the insights this has provided into conserved molecular mechanisms and how these have led to and integrate with findings from other regulated exocytotic cells.

Evolution of the Land Plant Exocyst Complexes

Exocyst is an evolutionarily conserved vesicle tethering complex functioning especially in the last stage of exocytosis. Homologs of its eight canonical subunits – Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 – were found also in higher plants and confirmed to form complexes in vivo, and to participate in cell growth including polarized expansion of pollen tubes and root hairs. Here we present results of a phylogenetic study of land plant exocyst subunits encoded by a selection of completely sequenced genomes representing a variety of plant, mostly angiosperm, lineages. According to their evolution histories, plant exocyst subunits can be divided into several groups. The core subunits Sec6, Sec8, and Sec10, together with Sec3 and Sec5, underwent few, if any fixed duplications in the tracheophytes (though they did amplify in the moss Physcomitrella patens), while others form larger families, with the number of paralogs ranging typically from two to eight per genome (Sec15, Exo84) to several dozens per genome (Exo70). Most of the diversity, which can be in some cases traced down to the origins of land plants, can be attributed to the peripheral subunits Exo84 and, in particular, Exo70. As predicted previously, early land plants (including possibly also the Rhyniophytes) encoded three ancestral Exo70 paralogs which further diversified in the course of land plant evolution. Our results imply that plants do not have a single “Exocyst complex” – instead, they appear to possess a diversity of exocyst variants unparalleled among other organisms studied so far. This feature might perhaps be directly related to the demands of building and maintenance of the complicated and spatially diverse structures of the endomembranes and cell surfaces in multicellular land plants.

Effects of Carbachol and Adenosine on Neurotransmitter Secretion Induced by Potassium Chloride, Ionomycin,and Sucrose

We compared the effects of adenosine and cholinergic agonist carbachol on spontaneous secretion during local application of K+, ionomycin, and sucrose increasing Ca2+ concentration in the nerve terminal. Adenosine and carbachol had no effect on Ca2+ entry, but modulated later stages of exocytosis.

Fusion of isolated synaptic vesicles as a model of the terminal stage of regulated exocytosis

In the study of membrane fusion, which is the terminal stage of exocytosis, we used a simplified model consisting of homotypic membranes of isolated synaptic vesicles (SV) obtained from the synaptosomal fraction of rat brain tissue. It was shown that fusion of SV develops in the presence of cytoplasmic proteins and 10−7 to 10−5 M Ca2+ ions. This conclusion was made based on changes in the intensity of fluorescence of a probe, R18. Calcium ions were found to be the most effective activators of the membrane fusion when the effects of bivalent cations, Ca2+, Sr2+, and Ba2+, were compared. ATP induced membrane fusion both in the presence and in the absence of Ca2+, and the effects of ATP and Ca2+ were additive. These findings allow us to believe that there are factors in the system containing SV and soluble proteins of synaptosomes, which initiate fusion of the membranes under the influence of not only Ca2+ but also ATP. The intensity of Ca2+-dependent fusion of SV dropped after trypsin treatment, i.e., proteolysis resulted in modulation of the sensitivity of vesicular proteins and/or a change in their capability of evoking membrane fusion. Monoclonal antibodies against synaptotagmin and synaptobrevin inhibited fusion of SV, but only partly. Our results support the concept that Ca2+-regulated membrane fusion is possible without the involvement of the entire SNARE complex.

Regulation of the exocytotic machinery by cAMP-dependent protein kinase: implications for presynaptic plasticity.

For over a decade, the enhancement of regulated exocytosis by cAMP-dependent protein kinase (PKA) has remained unexplained at the molecular level. The fact that this phenomenon has been observed in such a wide variety of secretory cell types, from pancreatic beta-cells to neurons, suggests that it is an important and fundamental mechanism. Extensive analysis of the phosphorylation of exocytotic proteins has yielded few substrates of PKA in vitro, and fewer still have had physiological effects attributed to their phosphorylation. Here we review two proteins that do fulfil these criteria: the synaptic vesicle proteins cysteine string protein (CSP) and Snapin. Phosphorylation of these proteins by PKA produces changes in their respective protein-protein interactions, and has been attributed to modulation of the vesicle priming (Snapin) and vesicle fusion (CSP) stages of exocytosis. We also discuss how the function of CSP and Snapin phosphorylation might fit into an interesting aspect of the PKA-dependent enhancement of exocytosis: presynaptic plasticity in the brain.

Potential Shifts and Intracellular Signaling in Smooth Muscle and Exocrine Cells: Role of the Secretory Potential (a Working Hypothesis)

1 Franko National University, Lviv, Ukraine. Correspondence should be addressed to V. V. Manko (e-mail: vvmanko@franko.lviv.ua). Due to certain reasons, results of electrophysiological studies of secretory cells of the exocrine glands have until now been interpreted to a great extent within the framework of concepts proposed for excitatory cells and, especially, for smooth muscle cells. The statement that “... changes in the functioning of the cell are based on changes of its membrane potential” was transferred from physiology of the excitatory cells to physiology of the secretory cells. Indeed, there are some clear analogies between electrophysiological processes and their structural basis in secretory cells of the exocrine glands and smooth muscle cells. These are the relatively low values of the membrane potential, the existence of close cell-to-cell contacts, the presence of a certain set of Ca transport systems, the absence of an endoplasmic reticulum characterized by a sufficiently perfect structure (as in the case of skeletal muscle cells), and, as a consequence, the significant role of extracellular Ca in the coupling of the membrane and intracellular processes. Probably, these features are related not to a common origin of the above cell types but to slow temporal characteristics of the functions performed by these cells. These similarities allowed investigators to transfer interpretations of the experimental data obtained on smooth muscles onto secretory cells, which obviously promoted studies of the latter units. Nonetheless, direct applications of such an approach began to hinder to a certain extent electrophysiological studies of exocrine cells. Obvious discrepancies became clear when it was necessary to correlate directions of changes of the membrane potential of these cells with their secretory activity. It is well known that exocrine secretory cells differ from smooth muscle cells in their inability to generate action potentials, and only the so-called secretory potential is recorded from these cells after the action of a primary messenger. The use of modern microcytofluorometry techniques showed that the natural functional activities of smooth muscle and exocrine cells are initiated according to fundamentally different schemes. In the former case, this is such a chain: action of the primary messenger → shift of the membrane potential → activation of Ca channels of the plasma membrane and/or intracellular stores → an increase in the level of cytosol Ca → changes in the functional activity (contraction). In other words, changes in the membrane potential in this case are the reason for changes in the latter activity. At the same time, in the secretory cells changes in their membrane potential are not a reason for but a result of the processes realized within the cell; the chain of events is as follows: action of the primary messenger → activation of intracellular Ca channels → increase in the cytosol Ca concentration → activation of Ca-sensing channels → change in the membrane potential. In this scheme, the importance of changes in the membrane potential remains unclear: Why should it be changed when the secretion process has already been activated? We hypothesize that the secretory potential can: (i) modify the Ca signal indirectly, via Ca transport systems of the plasma membrane and, in such a way, influence the secretion, and/or (ii) directly limit or promote certain stages of exocytosis. Both these assumptions can be considered only working hypotheses. It is probable that they are both wrong. Nonetheless, the secretory potential performs some function, and if we take this into consideration, we shall be able to better interpret the variability of secretory potentials recorded from different exocrine cells.

Desensitization of insulin secretory response to imidazolines, tolbutamide, and quinine: II. Electrophysiological and fluorimetric studies

Prolonged in vitro exposure (18 h) of pancreatic islets to insulin secretagogues that block ATP-dependent K + channels (K ATP channels), such as sulfonylureas, imidazolines, and quinine, induced a desensitization of insulin secretion (Rustenbeck et al., pages 1685–1694, this issue). To elucidate the underlying mechanisms, K ATP channel activity, plasma membrane potential and the cytosolic Ca 2+ concentration ([Ca 2+] i) were measured in mouse single B-cells. In B-cells desensitized by phentolamine or quinine (100 μM each) K ATP channel activity was virtually absent and could not be elicited by diazoxide. Desensitization by alinidine (100 μM) induced a marked reduction of K ATP channel activity, which could be reversed by diazoxide, whereas exposure to idazoxan (100 μM) or tolbutamide (500 μM) had no lasting effect on K ATP channel activity. Correspondingly, phentolamine-, alinidine-, and quinine-desensitized B-cells were markedly depolarized, whereas B-cells that had been exposed to tolbutamide or idazoxan had an unchanged resting membrane potential. The increase in [Ca 2+] i normally elicited by phentolamine and alinidine was suppressed after desensitization by these compounds, whereas the [Ca 2+] i increase by re-exposure to quinine was markedly reduced and that by tolbutamide only minimally affected as compared with control-cultured B-cells. The increase in [Ca 2+] i elicited by a K + depolarization was diminished in secretagogue-pretreated B-cells, the extent depending on the secretagogue. This effect was closely correlated with the degree of depolarization after pretreatment with the respective secretagogue. In conclusion, the apparently uniform desensitization of secretion by K ATP channel blockers is due to different effects at two stages located distally in the stimulus-secretion coupling: either at the stage of [Ca 2+] i regulation, where the increase is depressed as a consequence of a persistent depolarization (e.g. in the case of phentolamine or alinidine) and/or at the stage of exocytosis, which responds only weakly to substantial increases in [Ca 2+] i (in the case of tolbutamide).

Toxines bactériennes : outils pour l’étude des protéines G impliquées dans les mécanismes de l’exocytose dans les cellules neuroendocrines

In neuroendocrine cells, regulated exocytosis is a multistep process that comprises the recruitment and priming of secretory granules, their docking to the exocytotic sites, and the subsequent fusion of granules with the plasma membrane leading to the release of secretory products into the extracellular space. Using bacterial toxins which specially inactivate subsets of G proteins, we were able to demonstrate that both trimeric and monomeric G proteins directly control the late stages of exocytosis in chromaffin cells. Indeed, in secretagogue-stimulated chromaffin cells, the subplasmalemmal actin cytoskeleton undergoes a specific reorganization that is a prerequisite for exocytosis. Our results suggest that a granule-bound trimeric Go protein controls the actin network surrounding secretory granules through a pathway involving the GTPase RhoA and a downstream phosphatidylinositol 4-kinase. Furthermore, the GTPase Cdc42 plays a active role in exocytosis, most likely by providing specific actin structures to the late docking and/or fusion steps. We propose that G proteins tightly control secretion in neuroendocrine cells by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis. Our data highlight the use of bacterial toxins, which proved to be powerful tools to dissect the exocytotic machinery at the molecular level.

Immunohistochemical localization of cellubrevin on secretory granules in pancreatic B-cells.

Cellubrevin is one of the proteins involved in the docking and fusion of secretory granules to the plasma membrane. It has been reported that cellubrevin is widely distributed in both neural and non-neural cells, including insulin-secreting B-cells. This study aims to demonstrate by immunohistochemical techniques that cellubrevin is localized in insulin-secreting cells and further to examine whether it might occur in glucagon- and somatostatin-secreting cells in the pancreatic islet in the rat and mouse. We used the polyclonal antibody against the N-terminal peptide whose specificity was confirmed by Western blot analysis. Double immuno-staining demonstrated that cellubrevin was localized in insulin-containing cells, but both glucagon-containing and somatostatin-containing cells lacked the immuno-reactivity. Immuno-electron microscopic analysis revealed the localization of cellubrevin on the margin of secretory granules near the plasma membrane but not in the granules closer to the nucleus. These observations support the view that cellubrevin in the pancreatic islet is expressed on the membrane of the secretory granules in B-cells at the stage of exocytosis.

Cyclosporin A inhibits degranulation of rat basophilic leukemia cells and human basophils. Inhibition of mediator release without affecting PI hydrolysis or Ca2+ fluxes.

Cyclosporin A (CSA) inhibits IgE receptor-mediated exocytosis from rat basophilic leukemia (RBL) cells and human peripheral blood basophils in a dose-dependent manner over the therapeutic range of CSA concentrations achieved in vivo. Half-maximal inhibition was observed at 0.2 micrograms/ml CSA. The effect of CSA on several biochemical parameters involved in receptor-mediated activation of RBL cells was examined. Maximum inhibition of secretion occurred when CSA was added 5 min before activation, and inhibition was nearly maximum when the drug was added 2 min before the cells were triggered. The same results were observed when RBL cells were stimulated with A23187, a calcium ionophore. These results suggest a mechanism other than inhibition of protein synthesis is involved. Inhibition by CSA of release by either secretagogue persisted, even if CSA was removed from the buffer before the cells were triggered. No inhibition was observed of either receptor-mediated phosphatidylinositol hydrolysis, 45Ca2+ uptake, or the rise in the intracellular concentration of free Ca2+ under the same conditions that produced greater than 80% inhibition of serotonin release. These results demonstrate that the early events in signal transduction are not affected, and suggest that the intracellular target for CSA participates in a later stage of exocytosis. Furthermore, the data suggest that CSA suppresses cells other than T lymphocytes and predict that patients on CSA therapy may have altered response to allergens.

Polyphosphoinositide breakdown and subsequent exocytosis in the Ca2+/ionophore-induced acrosome reaction of mammalian spermatozoa

An investigation was made of the modifications in phospholipids that occur during the exocytotic event known as the 'sperm acrosome reaction'. Phospholipids were prelabelled with 32P, and exocytosis was induced with Ca2+ and the ionophore A23187. When incubated with [32P]Pi in various media suitable for supporting sperm survival or fertilization in vitro, spermatozoa from all five species examined (ram, boar, guinea pig, mouse and human) incorporated 32P rapidly into the components of the phosphoinositide cycle. There were differences both between species and between media with respect to the actual rate of incorporation of label, and also between species with respect to other phospholipids labelled. Treatment of spermatozoa with Ca2+ and A23187 to induce the acrosome reaction resulted in a rapid breakdown of phosphatidylinositol 4, 5-bisphosphate and phosphatidylinositol 4-phosphate, which was complete within 3 min; there was also a great increase in labelling of phosphatidate. Occurrence of acrosome reactions in the sperm population was only observed after 5-10 min and reached a maximum response of greater than 90% after more than 30 min. The phosphoinositide breakdown was related to subsequent exocytosis: after EGTA/ionophore treatment, neither inositide breakdown nor exocytosis took place; however, later addition of Ca2+ resulted in immediate inositide breakdown, and exocytosis followed, with a delay relative to Ca2+ addition exactly similar to that following standard Ca2+/ionophore treatment. Neomycin inhibited both inositide breakdown and subsequent exocytosis provided it was added together with Ca2+ and ionophore; however, if the drug was added 3 min after Ca2+ and ionophore (by which time inositide breakdown was already complete), exocytosis was not inhibited. Ca2+ seemed to have several consecutive roles in the acrosome reaction. Low (micromolar) levels of free Ca2+ were needed both for phosphoinositide breakdown and for an event downstream of this breakdown; no other bivalent cation could substitute for Ca2+ in either event, and inositide breakdown was actually inhibited by Mg2+. In addition, millimolar levels of Ca2+ were needed for later stages of exocytosis, although this requirement could be satisfied by Sr2+. We conclude that breakdown of polyphosphoinositides is an essential early process after Ca2+ entry in the chain of events that lead to exocytosis in the mammalian sperm acrosome reaction.

Exocytotic release of neurotransmitter substances from nerve endings in the taste buds of rat circumvallate papillae.

Exocytotic release of neurotransmitters from nerve endings was demonstrated ultrastructurally in the taste buds of rat circumvallate papillae by stimulation of high K+ and Ca2+ Ringer perfusion and application of tannic acid-Ringer incubation (TARI) method. Omega-shaped images of large cored vesicles and small clear vesicles, indicating exocytotic release of their contents, were found only in the non-synaptic sites. Occasionally exocytosis occurred at sites facing other nerve fibers. Many coated pits were also seen, which presumably represent membrane retrieval at a later stage of exocytosis. It is likely that the taste buds receive more than one type of innervation.

Membrane specializations of the cells of the Harderian gland of the rabbit with particular reference to the mechanism of exocytosis.

Membrane specializations of the cells of the red and white lobe of the Harderian gland, especially membrane differentiations involved in exocytosis, as well as granule interactions were investigated by means of the freeze-fracture and thin-section techniques. Gap junctions are numerous between both types of glandular cells. Tight junctions consist of about five strands, loosely arranged and often fragmented. Large secretion droplets of both cell types containing different kinds of lipids are formed by fusion. The adjacent granule bilayers are devoid of intramembrane particles (IMP) at the fusion site. During exocytosis the large secretion droplets of the red-lobe cells often cause the plasma membrane to bulge out extensively into the lumen. The overlying plasmalemma as well as the granule membrane show IMP-free patches. Secretion granules of the white-lobe cells, smaller in volume, deform the plasma membrane only slightly when lying in close apposition. The distribution of IMP on the plasmalemma of the white-lobe cells remains random at the side of impending fusion. Thin sections sometimes reveal, in this stage of exocytosis, a single unit membrane but never a pentalaminar structure. Both cellular components of the Harderian gland, when pretreated identically, show different membrane reactions with respect to IMP-clearing during exocytosis. This leads to the conclusion that IMP-clearing may not be responsible for membrane fusion but for other complex events in the regulation of membrane-to-membrane interactions.

Freeze-fracture study of the chromaffin cell during exocytosis: Evidence for connections between the plasma membrane and secretory granules and for movements of plasma membrane-associated particles

Exocytosis was studied in acetylcholine-stimulated bovine adrenal medulla. During a pre-exocytotic stage, chromaffin granules are found in juxtaposition to the plasma membrane and separated from it by an electron dense space 25--27 A in width. Freeze-fracture studies show this stage to be characterized by connections between the granules and the plasma membrane. These connections are apparently cytoplasmic but bridge both membranes; they are presumably proteinaceous, but their exact nature remains to be elucidated. Later stages of exocytosis were also studied by the freeze-fracture technique; a typical feature is the lack of intramembrane particles around the fusion site. Both connections and membrane particle movement are discussed in terms of recent biochemical findings.