Compound Exocytosis Research Articles

Compound Exocytosis in Pituitary Cells

Neurotransmitter and hormone release from vesicles involves fusion between the vesicle and the plasma membranes, a process termed exocytosis. Recently we reported that most of the spontaneous and stimulated exocytotic events in pituitary lactotrophs are transient and repetitive, appearing in bursts lasting more than 100 s. However, whether this is also the case in compound vesicle-to-vesicle exocytosis is unknown. Here we investigated compound exocytotic events in resting and stimulated lactotrophs by using optical and cell-attached patch-clamp capacitance measurements. Elementary compound exocytotic events were characterized by multiple-amplitude on-steps in synaptopHluorin fluorescence and in membrane capacitance signals. Multiple-amplitude on-steps appeared either as a relatively large upward step, indicating that vesicles were fused with each other prior to fusion of the vesicle membrane with the plasma membrane (multivesicular exocytosis), or as a time-dependent stepwise signal increase, indicating sequential fusion of two or more vesicles with the plasma membrane (sequential exocytosis). In the majority of membrane capacitance recordings (>90%), multiple-amplitude on-steps terminated as multiple-amplitude off-steps. These complex amplitude events were repetitive, indicating that transient fusion pore openings reflect repetitive interactions of a single vesicle or vesicles in a cluster with the plasma membrane. Out of many mechanisms, these interactions may enable the diffusion of fusion proteins from the plasma membrane to the membrane of the primary fused vesicles, consequently enabling vesicle-to-vesicle fusion. The incidence of compound exocytotic events increased by 33% after stimulation, which is consistent with the enhanced efficiency of hormone secretion after the stimulus.

Rescuing the Subprime Meltdown in Insulin Exocytosis in Diabetes

Neuroendocrine pancreatic islet beta-cells secrete the hormone insulin in response to glucose stimulation and adapt efficiently to increased demand by peripheral tissues to maintain glucose homeostasis. Insulin is packed within dense-core granules, which traffic and dock onto the plasma membrane whereby a Ca(2+) stimulus evokes exocytosis by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), complex-mediated, membrane fusion. Recent studies have unveiled postdocking steps mediated by "priming" factors that influence SNARE complex assembly to confer fusion readiness to the docked granules. This review will summarize recent insights into the priming role for Munc13 in the exocytosis of insulin granules. We present evidence for the interaction of Munc13-1 with exocytotic substrates involved in cAMP-mediated potentiation of insulin release, the latter we show to mediate enhanced granule-to-granule fusion events underlying compound exocytosis. We thus also further review the current understanding of granule-to-granule fusion. As agents acting on cAMP signaling are clinically used to augment insulin release in diabetes, this better understanding of priming steps may reveal additional novel therapeutic strategies to increase the capacity for insulin release to improve the treatment of diabetes.

Evidence That Vesicles Undergo Compound Fusion on the Synaptic Ribbon

The ribbon synapse can release a stream of transmitter quanta at very high rates. Although the ribbon tethers numerous vesicles near the presynaptic membrane, most of the tethered vesicles are held at a considerable distance from the plasma membrane. Therefore, it remains unclear how their contents are released. We evoked prolonged bouts of exocytosis from a retinal bipolar cell, fixed within seconds, and then studied the ribbons by electron microscopy. Vesicle density on ribbons was reduced by approximately 50% compared with cells where exocytosis was blocked with intracellular ATP-gammaS. Large, irregularly shaped vesicles appeared on the ribbon in cells fixed during repetitive stimulation of exocytosis, and in some cases the large vesicles could be traced in adjacent sections to cisternae open to the medium. The large cisternal structures were attached to the ribbon by filaments similar to those that tether synaptic vesicles to the ribbon, and they occupied the base of the ribbon near the plasma membrane, where normal synaptic vesicles are found in resting cells. We suggest that the cisternae attached to ribbons represent synaptic vesicles that fused by compound exocytosis during strong repetitive stimulation and, thus, that vesicles tethered to the ribbon can empty their contents by fusing to other vesicles docked at the presynaptic membrane. Such compound fusion could explain the extremely high release rates and the multivesicular release reported for auditory and visual ribbon synapses.

Recent Progress in Membrane Dynamics Research by Two-photon Microscopy

Two-photon microscopy is a less-invasive cross-sectional imaging technique for long-term visualization of living cells within deeper layers of organs. This microscopy is based on the multi-photon excitation process and has been used widely in medical and biological sciences. An attractive property of two-photon microscopy, multicolor excitation capability has enabled quantification of spatiotemporal patterns of [Ca(2+)]i, ion transport and single episodes of fusion pore openings during exocytosis. In pancreatic acinar cells, we have successfully demonstrated the existence of "sequential compound exocyotosis" for the first time. Sequential compound exocytosis has subsequently been identified in a wide variety of secretory cells including exocrine, endocrine and blood cells. Further exploration has revealed dynamics and physiological roles of actin cytoskeleton, and soluble NSF attachment receptor (SNARE) proteins. In addition, our newly developed method (TEPIQ method) can be used to determine fusion pores and the diameters of vesicles smaller than the diffraction-limited resolution. Recently, we have successfully observed neurons deeper than 0.9 mm from the brain cortex surface in an anesthetized mouse. We have also improved the spatial resolution needed to visualize fine structures of basal dendrites in layer V in vivo. This microscopy also can be used to visualize dendritic spines, axon terminals and miroglia cells, suggesting that we can follow long-term changes of neural or glial cells in a living mouse. Two-photon microscopy will thus be important in advancing the study of the molecular basis of physiological and pathological events in the human body.

Foxa2 Controls Vesicle Docking and Insulin Secretion in Mature β Cells

The winged-helix transcription factor Foxa2 regulates Pdx1 gene expression and fetal endocrine pancreas development. We show here by inducible gene ablation that Foxa2 inactivation in mature beta cells induces hyperinsulinemic hypoglycemia in Foxa2(loxP/loxP),Pdx1-CreERT2 adult mice. Mutant beta cells exhibited a markedly increased pool of docked insulin granules, some of which were engaged in sequential or compound exocytosis, consistent with increased first-phase glucose-stimulated insulin secretion. Expression of multiple genes involved in vesicular trafficking, membrane targeting, and fuel-secretion pathways is dependent on Foxa2. In addition, impaired cytosolic Ca(2+) oscillations and elevated intracellular cyclic AMP production accompanied this secretory defect and were likely contributors to the sensitization of the exocytotic machinery. Thus, in the absence of Foxa2, alterations in intracellular second-messenger signaling redistribute the insulin granules into the readily releasable pool. We conclude that Foxa2 is required for both fetal pancreas development and the function of mature beta cells.

Presence of SNAP-23 and syntaxin 4 in mouse and hamster peritoneal mast cells

Mast cells (MCs) play a crucial role in inflammatory reactions. Their presence and number in the peritoneal cavity is important to overcome and enhance resistance to peritoneal infection. When MCs are activated they release a variety of biological mediators from their granules, such as histamine, that contribute to the appropriate and rapid local immune response. Granular content is released using a process of compound exocytosis, also termed degranulation. SNAP-23 and syntaxin 4 are plasma membrane proteins involved in degranulation of rat MCs. Their presence, however, has not been studied in MCs of other rodent species. The aim of the present study was to investigate using immunocytochemistry whether SNAP-23 and syntaxin 4 are present in peritoneal MCs of the mouse and hamster. In addition, the diameter, percentage and histamine content of these cells were also analyzed. Our results demonstrate that SNAP-23 and syntaxin 4 are present in the mouse and hamster peritoneal MCs, suggesting that proteins involved in the secretory process in MCs are conserved among species. Likewise, we conclude that peritoneal MCs of mouse and hamster are heterogeneous in size, percentage and histamine content.

Exocytic Process Analyzed with Two-photon Excitation Imaging in Endocrine Pancreas

To elucidate the spatiotemporal profiles of final secretory stage, we have established two-photon extracellular polar tracer (TEP) imaging, with which we can quantify all exocytic events in the plane of focus within the intact tissues. With such technique, we can estimate the precise diameters of vesicles independently of the spatial resolution of optical microscope, and measure the fusion pore dynamics at nanometer resolution. At insulin exocytosis in the pancreatic islets, it took two seconds for the fusion pore to dilate from 1.4 nm in diameter to 6 nm in diameter, and such unusual stability of the pore may be due to the crystallization of the intragranular contents. Opening of the pore was preceded by unrestricted lateral diffusion of lipids along the inner wall of the pores, supporting the idea that this structure was mainly composed of membrane lipids. TEP imaging has been also applied to other representative secretory glands, and has revealed hitherto unexpected diversity in spatial organizations of exocytosis and endocytosis, which are relevant for physiology and pathology of secretory tissues. In the pancreatic islet, compound exocytosis was characteristically inhibited (<5%), partly due to the rarity of SNAP25 redistribution into the exocytosed vesicle membrane. Such mechanisms necessitate transport of insulin granules to the cell surface for fusion, and possibly rendering exocytosis more sensitive to metabolic state. Two-photon imaging will be powerful tools to elucidate molecular and cellular mechanisms of exocytosis and related disease, and to develop new therapeutic agencies as well as diagnostic tools.

Assays for Regulated Exocytosis of Mast Cell Granules

Mast cells are important effectors in innate and adaptive immune responses. They contain numerous secretory granules filled with inflammatory mediators in their cytoplasm. Exocytosis of granular content does not take place until the cell receives an appropriate stimulus such as the aggregation of IgE antibody bound to high-affinity IgE receptors by specific antigen. This process is therefore referred to as regulated exocytosis. A characteristic of mast cell exocytosis is that it does not involve release of a few individual granules, but rather, a large fraction of the granular content is released due to compound exocytosis, implicating the occurrence of granule-to-granule and granule-to-plasma membrane fusion. This unit describes assays that measure the release of granular content from mast cells. They include in vitro colorimetric-, radiolabel-, or antibody detection-based assays for substances stored in the granules. Given that animal models for basophil and mast cell activation are being used with increasing frequency, the unit also includes protocols to measure exocytosis of mast cell granule content in vivo.

Elimination of plasma membrane phosphatidylinositol (4,5)-bisphosphate is required for exocytosis from mast cells

The inositol lipid phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2] is involved in a myriad of cellular processes, including the regulation of exocytosis and endocytosis. In this paper, we address the role of PtdIns(4,5)P2 in compound exocytosis from rat peritoneal mast cells. This process involves granule-plasma membrane fusion as well as homotypic granule membrane fusion and occurs without any immediate compensatory endocytosis. Using a novel quantitative immunofluorescence technique, we report that plasma membrane PtdIns(4,5)P2 becomes transiently depleted upon activation of exocytosis, and is not detected on the membranes of fusing granules. Depletion is caused by phospholipase C activity, and is mandatory for exocytosis. Although phospholipase C is required for Ca2+ release from internal stores, the majority of the requirement for PtdIns(4,5)P2 hydrolysis occurs downstream of Ca2+ signalling - as shown in permeabilised cells, where the inositol (1,4,5)-trisphosphate-Ca2+ pathway is bypassed. Neither generation of the PtdIns(4,5)P2 metabolite, diacylglycerol (DAG) or simple removal and/or sequestration of PtdIns(4,5)P2 are sufficient for exocytosis to occur. However, treatment of permeabilised cells with DAG induces a small potentiation of exocytosis, indicating that it may be required. We propose that a cycle of PtdIns(4,5)P2 synthesis and breakdown is crucial for exocytosis to occur in mast cells, and may have a more general role in all professional secretory cells.

Compound Exocytosis: Mechanisms and Functional Significance

Compound exocytosis occurs in many cell types. It represents a specialized form of secretion in which vesicles undergo fusion with each other as well as with the plasma membrane. In most cases, compound exocytosis occurs sequentially, with deeper-lying vesicles fusing, after a delay, with vesicles that have already fused with the plasma membrane. However, in some cells, vesicles can also apparently fuse with each other intracellularly before any interaction with the plasma membrane. In this review, we discuss the general features of compound exocytosis, and the features that are specific to particular cells. We consider mechanisms that might impose the requirement for vesicles to fuse with the plasma membrane before they become able to fuse with each other, the possibility that there are biochemical differences between vesicle-plasma membrane fusion events and subsequent secondary homotypic vesicle fusion events, and the role that cytoskeletal elements might play in the stabilization of fused vesicles, in order to permit secondary fusion events. Finally, we discuss the likely physiological significance of compound exocytosis in the various cell types in which it exists.

Sequential compound exocytosis of large dense‐core vesicles in PC12 cells studied with TEPIQ (two‐photon extracellular polar‐tracer imaging‐based quantification) analysis

We investigated exocytosis of PC12 cells using two-photon excitation imaging and extracellular polar tracers (TEP imaging) at the basal region of PC12 cells adjacent to the glass cover slip. TEPIQ (two-photon extracellular polar-tracer imaging-based quantification) analysis revealed that most exocytosis was mediated by large dense-core vesicles (LVs) with a mean diameter of 220 nm, and that exocytosis of LVs occurred slowly with a mean latency of approximately 7 s even though exocytosis was induced with large increases in cytosolic Ca2+ concentration by uncaging of a caged-Ca2+ compound. We also found that 97% of exocytic LVs remained poised at the plasma membrane, 72% maintained their fusion pores in an open conformation for more than 30 s, and 76% triggered sequential compound exocytosis of vesicles that were located deeper in the cytosol. Sequential compound exocytosis by PC12 cells was confirmed by electron microscopic investigation with photoconversion of diaminobenzidine by FM1-43 (a polar membrane tracer). Our data suggest that pre-stimulus docking of LVs to the plasma membrane does not necessarily hasten the fusion reaction, while docking and resulting stability of exocytic LVs facilitates sequential compound exocytosis, and thereby allowing mobilization of deep vesicles.

Glucagon-Like Peptide 1 Regulates Sequential and Compound Exocytosis in Pancreatic Islet β-Cells

Glucagon-like peptide 1 (GLP-1) has been postulated to potentiate insulin secretion by cAMP-mediated enhancement of mobilization and priming of secretory granules, but the precise exocytic events are unknown. We used epi-fluorescent microscopy of the fluorescent dye FM1-43, which incorporates into the plasma membrane and the exocytosing secretory granules (appearing as plasma membrane hotspots). KCl evoked exocytosis of 1.8 +/- 0.5 hotspots/rat beta-cell at the cell periphery, 82% of which are single transient increases of low amplitudes (151 +/- 7%), suggesting single secretory granule exocytosis; and the remaining 18% are stepwise increases in plasma membrane hotspots with higher amplitudes (170 +/- 9%), suggesting sequential secretory granule to secretory granule exocytic fusions. Addition of GLP-1 increased the hotspots to 6.0 +/- 0.7/beta-cell and exhibited a larger number of stepwise (41%) than transient (10%) increases with higher amplitudes of 259 +/- 19 and 278 +/- 23%, respectively. More interestingly, GLP-1 also evoked a robust and sustained pattern (49%) with even higher amplitudes of 354 +/- 18%, which are likely accelerated sequential secretory granule-secretory granule fusions. Electron microscopy studies collaborated with these imaging results, showing that GLP-1 increased the number of docked secretory granules at the plasma membrane and also increased the number of events showing direct contact of oncoming secretory granules with secretory granules undergoing exocytosis. We conclude that the potentiation of insulin secretion by GLP-1 is contributed by the mobilization of more insulin secretory granules to dock at the plasma membrane and the acceleration of sequential secretory granule-secretory granule fusions.

Two phases of zymogen granule lifetime in mouse pancreas: ghost granules linger after exocytosis of contents

Different cell types show widely divergent mechanisms and kinetics of exocytosis. We investigated these processes in pancreatic acinar cells by using video-rate 2-photon microscopy to image entry of extracellular dye into individual zymogen granules undergoing exocytosis. Fluorescence signals display two distinct phases; an initial peak that then decays over several seconds to a prolonged plateau. Several observations suggest that the first component reflects the binding of dye to the granule contents and their subsequent release into the acinar duct. These observations include: the peak/plateau fluorescence ratio differs between different dyes; the initial fluorescence decay mirrors the loss of granule contents as monitored by differential interference contrast microscopy; and the fall in vesicular fluorescence is accompanied by a rise in fluorescence in the adjacent duct lumen. We thus propose the use of extracellular fluorescent probes as a convenient means to monitor the kinetics of loss of proteinaceous content from secretory granules. In pancreatic acinar cells the fusion pore remains open much longer than required to ensure secretion of the granule contents, and instead the persistent empty 'ghost-granule' may act as a conduit to which secondary granules can fuse and release their contents by compound exocytosis.

Two-photon microscopic analysis of acetylcholine-induced mucus secretion in guinea pig nasal glands

The spatiotemporal changes in intracellular free Ca 2+ concentration ([Ca 2+] i) as well as fluid secretion and exocytosis induced by acetylcholine (ACh) in intact acini of guinea pig nasal glands were investigated by two-photon excitation imaging. Cross-sectional images of acini loaded with the fluorescent Ca 2+ indicator fura-2 revealed that the ACh-evoked increase in [Ca 2+] i was immediate and spread from the apical region (the secretory pole) of acinar cells to the basal region. Immersion of acini in a solution containing a fluorescent polar tracer, sulforhodamine B (SRB), revealed that fluid secretion, detected as a rapid disappearance of SRB fluorescence from the extracellular space, occurred exclusively in the luminal region and was accompanied by a reduction in acinar cell volume. Individual exocytic events were also visualized with SRB as the formation of Ω-shaped profiles at the apical membrane. In contrast to the rapidity of fluid secretion, exocytosis of secretory granules occurred with a delay of ∼70 s relative to the increase in [Ca 2+] i. Exocytic events also occurred deep within the cytoplasm in a sequential manner with the latency of secondary exocytosis being greatly reduced compared with that of primary exocytosis. The delay in sequential compound exocytosis relative to fluid secretion may be important for release of the viscous contents of secretory granules into the nasal cavity.

Syncollin is required for efficient zymogen granule exocytosis.

Syncollin is a 13 kDa protein that is present in the exocrine pancreas, where the majority of the protein is tightly attached to the luminal surface of the zymogen granule membrane. We have addressed the physiological role of syncollin by studying the phenotype of syncollin KO (knockout) mice. These mice show pancreatic hypertrophy and elevated pancreatic amylase levels. Further, secretagogue-stimulated amylase release from pancreatic lobules of syncollin KO mice was found to be reduced by about 45% compared with wild-type lobules, and the delivery of newly synthesized protein to zymogen granules was delayed, indicating that the mice have a pancreatic secretory defect. As determined by two-photon imaging, the number of secretagogue-stimulated exocytotic events in acini from syncollin KO mice was reduced by 50%. This reduction was accounted for predominantly by a loss of later, 'secondary' fusion events between zymogen granules and other granules that had already fused with the plasma membrane. We conclude that syncollin is required for efficient exocytosis in the pancreatic acinar cell, and that it plays a particularly important role in compound exocytosis.

The Plasma Membrane Q-SNARE Syntaxin 2 Enters the Zymogen Granule Membrane during Exocytosis in the Pancreatic Acinar Cell

During exocytosis in the pancreatic acinar cell, zymogen granules fuse directly with the apical plasma membrane and also with granules that have themselves fused with the plasma membrane. Together, these primary and secondary fusion events constitute the process of compound exocytosis. It has been suggested that the sequential nature of primary and secondary fusion is a consequence of the requirement for plasma membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors, such as syntaxin 2, to enter the membrane of the primary fused granule. We have tested this possibility by determining the location of syntaxin 2 in unstimulated and stimulated pancreatic acini. Syntaxin 2 was imaged by confocal immunofluorescence microscopy. Fused granules were detected both through their filling with the aqueous dye lysine-fixable Texas Red-dextran and through the decoration of their cytoplasmic surfaces with filamentous actin. In unstimulated cells, syntaxin 2 was exclusively present on the apical plasma membrane. In contrast, after stimulation, syntaxin 2 had moved into the membranes of fused granules, as judged by its location around dye-filled structures of 1-mum diameter that were coated with filamentous actin. At long times of stimulation (5 min), the majority (85%) of dye-filled granules were also positive for syntaxin 2. In contrast, at shorter times (1 min), more dye-filled granules (29%) were syntaxin 2-negative. We conclude that syntaxin 2 enters the membrane of a fused zymogen granule after the opening of the fusion pore, and we suggest that this movement might permit the onset of secondary fusion.

Stabilization of Exocytosis by Dynamic F-actin Coating of Zymogen Granules in Pancreatic Acini

Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini. Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound. Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho. Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis. Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation. Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating. Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.

Presence of SNAP-25 in rat mast cells

Mast cells participate in inflammation and allergies by releasing active mediators stored in numerous cytoplasmic granules. Degranulation implies compound exocytosis which involves a combination of granule–granule and granule–plasma membrane fusions. One of the most important proteins in the exocytotic process in neural and endocrine cells is the synaptosomal associated protein of 25 kDa (SNAP-25). In the present study, using a highly specific monoclonal antibody against SNAP-25, we have demonstrated by immunocytochemistry, western blot and immunoelectron microscopy the presence of SNAP-25 in rat peritoneal mast cells. Likewise we localized the protein mainly on the membrane of the secretory granules. Thus while the precise function of SNAP-25 in mast cells remains to be elucidated, it may be envolved in granule–granule fusion needed in degranulation.

Zymogen granule exocytosis is characterized by long fusion pore openings and preservation of vesicle lipid identity.

The dynamics of the fusion pore that forms between a secretory vesicle and the plasma membrane are important in the regulation of both exocytosis and endocytosis. Here, we describe characteristics of fusion during zymogen granule exocytosis in exocrine pancreatic acinar cells. By using fluorescence recovery after photobleaching techniques, we show that the fusion pore remains open to allow free aqueous exchange with the vesicle lumen. There is no lipid interchange between the plasma and granule membranes during this time, and at the end of its life, the intact granule shrinks in situ, probably by a gradual pinching off of membrane patches. We propose that the protracted fusion pore lifetime is adapted to permit compound exocytosis, whereby the lingering primary granule acts as a conduit through which the contents of a secondary granule can be released. The lack of lipid intermixing may then facilitate selective recycling of granule membrane and preservation of apical membrane integrity.

Compound Exocytosis and Cumulative Fusion in Eosinophils

Focal release of cytotoxic proteins by eosinophils onto the target surface plays an important role in parasite killing. Degranulation was stimulated by intracellular application of calcium and guanosine 5'-3-O-(thio)triphosphate via the recording patch pipette or via streptolysin-O permeabilization. Exocytotic fusion was monitored by capacitance measurements, whereas release of fluorescent weak bases, which accumulate selectively within eosinophil granules, was followed by fluorescence imaging. Several distinct types of granule fusion events were directly observed by simultaneous capacitance and fluorescence measurements. These are fusion of a single granule with the plasma membrane, intracellular granule-granule fusion, fusion of large compounds of pre-fused granules with the plasma membrane (compound exocytosis), and sequential fusion of granules to granules previously fused to the plasma membrane. Extensive granule-granule fusion was also observed by electron microscopy of permeabilized cells. All these fusion mechanisms contribute to focal release. The coexistence of distinct modes of exocytosis suggests that their regulation may modulate effector functions of eosinophils during helminth infection and allergic response.