Kiss-and-coat and compartment mixing: coupling exocytosis to signal generation and local actin assembly

Regulated exocytosis is thought to occur either by "full fusion," where the secretory vesicle fuses with the plasma membrane (PM) via a fusion pore that then dilates until the secretory vesicle collapses into the PM; or by "kiss-and-run," where the fusion pore does not dilate and instead rapidly reseals such that the secretory vesicle is retrieved almost fully intact. Here, we describe growing evidence for a third form of exocytosis, dubbed "kiss-and-coat," which is characteristic of a broad variety of cell types that undergo regulated exocytosis. Kiss-and-coat exocytosis entails prolonged maintenance of a dilated fusion pore and assembly of actin filament (F-actin) coats around the exocytosing secretory vesicles followed by direct retrieval of some fraction of the emptied vesicle membrane. We propose that assembly of the actin coats results from the union of the secretory vesicle membrane and PM and that this compartment mixing represents a general mechanism for generating local signals via directed membrane fusion.     

The number of secretory vesicles remains unchanged following exocytosis.

Earlier studies using electron microscopy demonstrate that there is no loss of secretory vesicles following exocytosis. Depletion however, of vesicular contents resulting in the formation of empty or partially empty vesicles is seen in electron micrographs, post exocytosis, in a variety of cells. Our studies using atomic force microscopy (AFM) reveal that following stimulation of secretion, live pancreatic acinar cells having 100-180 nm in diameter fusion pores located at the apical plasma membrane, dilate only 25-35% during exocytosis. Since secretory vesicles in pancreatic acinar cells range in size from 200 nm to 1200 nm in diameter, their total incorporation at the fusion pore, would distend the structure much more then what is observed. These earlier results prompted the current study to determine secretory vesicle dynamics in live pancreatic acinar cells following exocytosis. AFM studies on live acinar cells reveal no loss of secretory vesicle number following exocytosis. Parallel studies using electron microscopy, further confirmed our AFM results. These studies demonstrate that following stimulation of secretion, membrane-bound secretory vesicles transiently dock and fuse to release vesicular contents.     

Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.     

The release of secretory vesicle in encysting Giardia lamblia

Giardia is an intestinal parasite that undergoes adaptation for survival outside the host. It secretes an extracellular cyst wall using a poorly understood process. An encystation-specific secretory vesicle (ESV) was previously described containing cyst wall proteins. The process of release of these vesicles has been suggested to occur after fragmentation of large ESV in small secretory vesicles, followed by exocytosis, but it was not demonstrated. The release of the ESV was studied by transmission electron microscopy. It was observed: (1) the moment of vesicle release; (2) that a large vesicle is exocytosed and does not fragment into small vesicles; (3) membrane fusion is distinct from traditional exocytosis since it is incomplete; (4) the occurrence of membrane fragmentation and that those membranes reseal to form ghosts; (5) these membrane ghosts may be endocytosed, adhered to flagellar surface or/and form empty vesicles in the extracellular medium.     

Regulation of fusion pore closure and compound exocytosis in neuroendocrine PC12 cells by SCAMP1

During exocytosis, neuroendocrine cells can achieve partial release of stored secretory products from dense core vesicles (DCVs) by coupling endocytosis directly at fusion sites and without full discharge. The physiological role of partial secretion is of substantial interest. Much is known about SNARE-mediated initiation of exocytosis and dynamin-mediated completion of endocytosis, but little is known about coupling events. We have used real-time microscopy to examine the role of secretory carrier membrane protein SCAMP1 in exo-endocytic coupling in PC12 cells. While reduced SCAMP1 expression is known to impede dilation of newly opened fusion pores during onset of DCV exocytosis, we now show that SCAMP1 deficiency also inhibits closure of fusion pores after they have opened. Inhibition causes accumulation of fusion figures at the plasma membrane. Closure is recovered by restoring expression and accelerated slightly by overexpression. Interestingly, inhibited pore closure resulting from loss of SCAMP1 appears to increase secondary fusion of DCVs to already-fused DCVs (compound exocytosis). Unexpectedly, reinternalization of expanded DCV membranes following compound exocytosis appears to proceed normally in SCAMP1-deficient cells. SCAMP1's apparent dual role in facilitating dilation and closure of fusion pores implicates its function in exo-endocytic coupling and in the regulation of partial secretion. Secondarily, SCAMP1 may serve to limit the extent of compound exocytosis.     

Simultaneous evanescent wave imaging of insulin vesicle membrane and cargo during a single exocytotic event

The classical model of secretory vesicle recycling after exocytosis involves the retrieval of membrane (the omega figure) at a different site. An alternative model involves secretory vesicles transiently fusing with the plasma membrane (the 'kiss and run' mechanism) [1,2]. No continuous observation of the fate of a single secretory vesicle after exocytosis has been made to date. To study the dynamics of fusion immediately following exocytosis of insulin-containing vesicles, enhanced green fluorescent protein (EGFP) fused to the vesicle membrane protein phogrin [3] was delivered to the secretory vesicle membrane of INS-1 beta-cells using an adenoviral vector. The behaviour of the vesicle membrane during single exocytotic events was then examined using evanescent wave microscopy [4-6]. In unstimulated cells, secretory vesicles showed only slow Brownian movement. After a depolarizing pulse, most vesicles showed a small decrease in phogrin-EGFP fluorescence, and some moved laterally over the plasma membrane for approximately 1 microm. In contrast, secretory vesicles loaded with acridine orange all showed a transient (33-100 ms) increase in fluorescence intensity followed by rapid disappearance. Simultaneous observations of phogrin-EGFP and acridine orange indicated that the decrease in EGFP fluorescence occurred at the time of the acridine orange release, and that the lateral movement of EGFP-expressing vesicles occurred after this. Post-exocytotic retrieval of the vesicle membrane in INS-1 cells is thus slow, and can involve the movement of empty vesicles under the plasma membrane ('kiss and glide').     

High calcium concentrations shift the mode of exocytosis to the kiss-and-run mechanism

Exocytosis, the fusion of secretory vesicles with the plasma membrane to allow release of the contents of the vesicles into the extracellular environment, and endocytosis, the internalization of these vesicles to allow another round of secretion, are coupled. It is, however, uncertain whether exocytosis and endocytosis are tightly coupled, such that secretory vesicles fuse only transiently with the plasma membrane before being internalized (the 'kiss-and-run' mechanism), or whether endocytosis occurs by an independent process following complete incorporation of the secretory vesicle into the plasma membrane. Here we investigate the fate of single secretory vesicles after fusion with the plasma membrane by measuring capacitance changes and transmitter release in rat chromaffin cells using the cell-attached patch-amperometry technique. We show that raised concentrations of extracellular calcium ions shift the preferred mode of exocytosis to the kiss-and-run mechanism in a calcium-concentration-dependent manner. We propose that, during secretion of neurotransmitters at synapses, the mode of exocytosis is modulated by calcium to attain optimal conditions for coupled exocytosis and endocytosis according to synaptic activity.     

Mechanisms of dense core vesicle recapture following "kiss and run" ("cavicapture") exocytosis in insulin-secreting cells

The molecular mechanisms underlying "kiss and run" or "cavicapture" exocytosis of dense core secretory vesicles are presently unclear. Although dynamin-1 has previously been implicated in the recapture process in neurons, the recruitment of this fission protein to a single exocytosing vesicle has not been examined in real time during peptide release from pancreatic beta-cells. Imaged simultaneously in clonal insulin-secreting cells by dual color total internal reflection fluorescence microscopy, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y and green fluorescent protein (GFP)-tagged synaptotagmin-1 or synaptobrevin-2 rapidly diffused from sites of exocytosis, whereas the vesicle membrane protein phogrin and tissue plasminogen activator (tPA) were retained, consistent with fusion pore closure. Vesicle recovery frequently involved the recruitment of enhanced GFP-tagged dynamin-1, and GTPase-defective dynamin-1(K44E) increased the dwell time of tPA-mRFP at the plasma membrane. By contrast, recruitment of GFP chimeras of clathrin, epsin, and amphiphysin was not observed. Expression of dynamin-1(K535A), mutated in the pleckstrin homology domain, caused the apparent full fusion of vesicles, as reported by the additional release of tPA-mRFP (15-nm diameter) and enhanced GFP-tagged phogrin. We conclude that re-uptake of vesicles after peptide release by cavicapture corresponds to a novel form of endocytosis in which dynamin-1 stabilizes and eventually closes the fusion pore, with no requirement for "classical" endocytosis for retreat from the plasma membrane.     

Rab27b regulates exocytosis of secretory vesicles in acinar epithelial cells from the lacrimal gland

Tear proteins are supplied by the regulated fusion of secretory vesicles at the apical surface of lacrimal gland acinar cells, utilizing trafficking mechanisms largely yet uncharacterized. We investigated the role of Rab27b in the terminal release of these secretory vesicles. Confocal fluorescence microscopy analysis of primary cultured rabbit lacrimal gland acinar cells revealed that Rab27b was enriched on the membrane of large subapical vesicles that were significantly colocalized with Rab3D and Myosin 5C. Stimulation of cultured acinar cells with the secretagogue carbachol resulted in apical fusion of these secretory vesicles with the plasma membrane. Evaluation of morphological changes by transmission electron microscopy of lacrimal glands from Rab27b(-/-) and Rab27(ash/ash)/Rab27b(-/-) mice, but not ashen mice deficient in Rab27a, showed changes in abundance and organization of secretory vesicles, further confirming a role for this protein in secretory vesicle exocytosis. Glands lacking Rab27b also showed increased lysosomes, damaged mitochondria, and autophagosome-like organelles. In vitro, expression of constitutively active Rab27b increased the average size but retained the subapical distribution of Rab27b-enriched secretory vesicles, whereas dominant-negative Rab27b redistributed this protein from membrane to the cytoplasm. Functional studies measuring release of a cotransduced secretory protein, syncollin-GFP, showed that constitutively active Rab27b enhanced, whereas dominant-negative Rab27b suppressed, stimulated release. Disruption of actin filaments inhibited vesicle fusion to the apical membrane but did not disrupt homotypic fusion. These data show that Rab27b participates in aspects of lacrimal gland acinar cell secretory vesicle formation and release.     

Fusion pore regulation in peptidergic vesicles

Regulated exocytosis, which involves fusion of secretory vesicles with the plasma membrane, is an important mode of communication between cells. In this process, signalling molecules that are stored in secretory vesicles are released into the extracellular space. During the initial stage of fusion, the interior of the vesicle is connected to the exterior of the cell with a narrow, channel-like structure: the fusion pore. It was long believed that the fusion pore is a short-lived intermediate state leading irreversibly to fusion pore dilation. However, recent results show that the diameter of the fusion pore can fluctuate, suggesting that the fusion pore is a subject of stabilization. A possible mechanism is addressed in this article, involving the local anisotropicity of membrane constituents that can stabilize the fusion pore. The molecular nature of such a stable fusion pore to predict how interacting molecules (proteins and/or lipids) mediate changes that affect the stability of the fusion pore and exocytosis is also considered. The fusion pore likely attains stability via multiple mechanisms, which include the shape of the lipid and protein membrane constituents and the interactions between them.     

Aging differentially affects multiple aspects of vesicle fusion kinetics

How fusion pore formation during exocytosis affects the subsequent release of vesicle contents remains incompletely understood. It is unclear if the amount released per vesicle is dependent upon the nature of the developing fusion pore and whether full fusion and transient kiss and run exocytosis are regulated by similar mechanisms. We hypothesise that if consistent relationships exist between these aspects of exocytosis then they will remain constant across any age. Using amperometry in mouse chromaffin cells we measured catecholamine efflux during single exocytotic events at P0, 1 month and 6 months. At all ages we observed full fusion (amperometric spike only), full fusion preceded by fusion pore flickering (pre-spike foot (PSF) signal followed by a spike) and pure "kiss and run" exocytosis (represented by stand alone foot (SAF) signals). We observe age-associated increases in the size of all 3 modes of fusion but these increases occur at different ages. The release probability of PSF signals or full spikes alone doesn't alter across any age in comparison with an age-dependent increase in the incidence of "kiss and run" type events. However, the most striking changes we observe are age-associated changes in the relationship between vesicle size and the membrane bending energy required for exocytosis. Our data illustrates that vesicle size does not regulate release probability, as has been suggested, that membrane elasticity or flexural rigidity change with age and that the mechanisms controlling full fusion may differ from those controlling "kiss and run" fusion.     

Membrane Bending Energy and Fusion Pore Kinetics in Ca-Triggered Exocytosis

A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca²⁺-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca²⁺-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca²⁺-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape.     

Secretory Vesicles Are Preferentially Targeted to Areas of Low Molecular SNARE Density

Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of vesicles with the cell surface. SNARE (soluble N-ethymaleimide sensitive factor attachment protein receptor) proteins are the catalytic core of the secretory machinery, driving vesicle and plasma membrane merger. Plasma membrane SNAREs (tSNAREs) are proposed to reside in dense clusters containing many molecules, thus providing a concentrated reservoir to promote membrane fusion. However, biophysical experiments suggest that a small number of SNAREs are sufficient to drive a single fusion event. Here we show, using molecular imaging, that the majority of tSNARE molecules are spatially separated from secretory vesicles. Furthermore, the motilities of the individual tSNAREs are constrained in membrane micro-domains, maintaining a non-random molecular distribution and limiting the maximum number of molecules encountered by secretory vesicles. Together our results provide a new model for the molecular mechanism of regulated exocytosis and demonstrate the exquisite organization of the plasma membrane at the level of individual molecular machines.     

Characterization of sequential exocytosis in a human neuroendocrine cell line using evanescent wave microscopy and “virtual trajectory” analysis

Secretion of hormones and other bioactive substances is a fundamental process for virtually all multicellular organisms. Using total internal reflection fluorescence microscopy (TIRFM), we have studied the calcium-triggered exocytosis of single, fluorescently labeled large, dense core vesicles in the human neuroendocrine BON cell line. Three types of exocytotic events were observed: (1) simple fusions (disappearance of a fluorescent spot by rapid diffusion of the dye released to the extracellular space), (2) "orphan" fusions for which only rapid dye diffusion, but not the parent vesicle, could be detected, and (3) events with incomplete or multi-step disappearance of a fluorescent spot. Although all three types were reported previously, only the first case is clearly understood. Here, thanks to a combination of two-color imaging, variable angle TIRFM, and novel statistical analyses, we show that the latter two types of events are generated by the same basic mechanism, namely shape retention of fused vesicle ghosts which become targets for sequential fusions with deeper lying vesicles. Overall, approximately 25% of all exocytotic events occur via sequential fusion. Secondary vesicles, located 200-300 nm away from the cell membrane are as fusion ready as primary vesicles located very near the cell membrane. These findings call for a fundamental shift in current models of regulated secretion in endocrine cells. Previously, sequential fusion had been studied mainly using two-photon imaging. To the best of our knowledge, this work constitutes the first quantitative report on sequential fusion using TIRFM, despite its long running and widespread use in studies of secretory mechanisms.     

Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells.

Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.     

Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle

During exocytosis, secretory vesicles of mast cells generate a current transient that marks the opening of the fusion pore, the first aqueous connection that forms between the vesicle lumen and the cell exterior. By recording and analyzing such current transients, we have tracked the conductance of the fusion pore over the first millisecond of its existence. The first opening of the pore occurs rapidly, generally within 100 microseconds at 23 degrees C. The electric conductance of the pore is a few hundred picosiemens at first, but gradually increases over the subsequent milliseconds. Evidently the pore opens abruptly and then dilates. The initial conductance of the pore suggests a diameter comparable to that of a large ion channel. From an analysis of "capacitance flicker" we infer that a pore can increase its diameter severalfold and still close again completely. This suggests that several early events in membrane fusion are reversible.     

Spatiotemporal Detection and Analysis of Exocytosis Reveal Fusion “Hotspots” Organized by the Cytoskeleton in Endocrine Cells

Total internal reflection fluorescence microscope has often been used to study the molecular mechanisms underlying vesicle exocytosis. However, the spatial occurrence of the fusion events within a single cell is not frequently explored due to the lack of sensitive and accurate computer-assisted programs to analyze large image data sets. Here, we have developed an image analysis platform for the nonbiased identification of different types of vesicle fusion events with high accuracy in different cell types. By performing spatiotemporal analysis of stimulus-evoked exocytosis in insulin-secreting INS-1 cells, we statistically prove that individual vesicle fusion events are clustered at hotspots. This spatial pattern disappears upon the disruption of either the actin or the microtubule network; this disruption also severely inhibits evoked exocytosis. By demonstrating that newcomer vesicles are delivered from the cell interior to the surface membrane for exocytosis, we highlight a previously unappreciated mechanism in which the cytoskeleton-dependent transportation of secretory vesicles organizes exocytosis hotspots in endocrine cells.     

Trafficking of astrocytic vesicles in hippocampal slices

The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.     

Multiple stimulation-dependent processes regulate the size of the releasable pool of vesicles

In neuroendocrine cells and neurones, changes in the size of a limited pool of readily releasable vesicles contribute to the plasticity of secretion. We have studied the dynamic alterations in the size of a near-membrane pool of vesicles in living neuroendocrine cells. Using evanescent wave microscopy we monitored the behaviour of individual secretory vesicles at the plasma membrane. Vesicles undergo sequential transitions between several states of differing fluorescence intensity and mobility. The transitions are reversible, except for the fusion step, and even in nonstimulated conditions the vesicles change states in a dynamic equilibrium. Stimulation selectively speeds up the three forward transitions leading towards exocytosis. Vesicles lose mobility in all three dimensions upon approach of the plasma membrane. Their movement is directed and targeted to the docking fusion sites. Sites of vesicle docking and exocytosis are distributed non-uniformly over the studied “footprint” region of the cell. While some areas are the sites of repeated vesicle docking and fusion, others are completely devoid of spots. Vesicular mobility at the membrane is confined, as if the vesicle were imprisoned in a cage or tethered to a binding site. Received: 10 August 1998 / Revised version: 24 September 1998 / Accepted: 24 September 1998     

Correlation between vesicle quantal size and fusion pore release in chromaffin cell exocytosis

A significant number of exocytosis events recorded with amperometry demonstrate a prespike feature termed a "foot" and this foot has been correlated with messengers released via a transitory fusion pore before full exocytosis. We have compared amperometric spikes with a foot with spikes without a foot at chromaffin cells and found that the probability of detecting a distinct foot event is correlated to the amount of catecholamine released. The mean charge of the spikes with a foot was found to be twice that of the spikes without a foot, and the frequency of spikes displaying a foot was zero for small spikes increasing to approximately 50% for large spikes. It is hypothesized that in chromaffin cells, where the dense core is believed to nearly fill the vesicle, the expanding core is a controlling factor in opening the fusion pore, that prefusion of two smaller vesicles leads to excess membrane, and that this slows pore expansion leading to an increased observation of events with a foot. Clearly, the physicochemical properties of vesicles are key factors in the control of the dynamics of release through the fusion pore and the high and variable frequency of this release makes it highly significant.