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.     

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.     

Coupling exo- and endocytosis: An essential role for PIP2 at the synapse

Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~ 200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Coupling exo- and endocytosis: an essential role for PIP₂ at the synapse

Chemical synapses are specialist points of contact between two neurons, where information transfer takes place. Communication occurs through the release of neurotransmitter substances from small synaptic vesicles in the presynaptic terminal, which fuse with the presynaptic plasma membrane in response to neuronal stimulation. However, as neurons in the central nervous system typically only possess ~200 vesicles, high levels of release would quickly lead to a depletion in the number of vesicles, as well as leading to an increase in the area of the presynaptic plasma membrane (and possible misalignment with postsynaptic structures). Hence, synaptic vesicle fusion is tightly coupled to a local recycling of synaptic vesicles. For a long time, however, the exact molecular mechanisms coupling fusion and subsequent recycling remained unclear. Recent work now indicates a unique role for the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP(2)), acting together with the vesicular protein synaptotagmin, in coupling these two processes. In this work, we review the evidence for such a mechanism and discuss both the possible advantages and disadvantages for vesicle recycling (and hence signal transduction) in the nervous system. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Exocytotic fusion pore stability and topological defects in the membrane with orientational degree of ordering

Regulated exocytosis is a process that strongly depends on the formation and stability of the fusion pore. It was indicated experimentally and theoretically that narrow and highly curved fusion pore may be stabilized by accumulation of anisotropic membrane components possessing orientational ordering. On the other hand, narrow fusion pore may also undergo repetitive opening and closing, disruption in the so called kiss and run process or become completely opened in the process of full fusion of the vesicle with the membrane. In this paper we attempt to elucidate the subtle interplay between the stabilizing and destabilizing processes in the fusion neck. A possible physical mechanism which may lead to disruption of the stable fusion pore or complete fusion of the vesicle with the membrane is discussed. It is indicated that topologically driven defects of the in-plane orientational membrane ordering in the region of the fusion pore may disrupt the fusion. Alternatively, it may facilitate repetitive opening and closing of the fusion pore or induce full fusion of the vesicle with the target membrane.     

Excess cholesterol inhibits glucose‐stimulated fusion pore dynamics in insulin exocytosis

Type 2 diabetes is caused by defects in both insulin sensitivity and insulin secretion. Glucose triggers insulin secretion by causing exocytosis of insulin granules from pancreatic β‐cells. High circulating cholesterol levels and a diminished capacity of serum to remove cholesterol from β‐cells are observed in diabetic individuals. Both of these effects can lead to cholesterol accumulation in β‐cells and contribute to β‐cell dysfunction. However, the molecular mechanisms by which cholesterol accumulation impairs β‐cell function remain largely unknown. Here, we used total internal reflection fluorescence microscopy to address, at the single‐granule level, the role of cholesterol in regulating fusion pore dynamics during insulin exocytosis. We focused particularly on the effects of cholesterol overload, which is relevant to type 2 diabetes. We show that excess cholesterol reduced the number of glucose‐stimulated fusion events, and modulated the proportion of full fusion and kiss‐and‐run fusion events. Analysis of single exocytic events revealed distinct fusion kinetics, with more clustered and compound exocytosis observed in cholesterol‐overloaded β‐cells. We provide evidence for the involvement of the GTPase dynamin, which is regulated in part by cholesterol‐induced phosphatidylinositol 4,5‐bisphosphate enrichment in the plasma membrane, in the switch between full fusion and kiss‐and‐run fusion. Characterization of insulin exocytosis offers insights into the role that elevated cholesterol may play in the development of type 2 diabetes.     

Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling

Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing ～19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH‐sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin‐tagged proteins. While the native proteins had a clustered distribution, the GFP‐tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin‐tagged vesicle proteins are less well integrated in clusters.     

Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.     

Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion

Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.     

Redistribution of synaptophysin and synapsin I during alpha-latrotoxin- induced release of neurotransmitter at the neuromuscular junction

The distribution of two synaptic vesicle-specific phosphoproteins, synaptophysin and synapsin I, during intense quantal secretion was studied by applying an immunogold labeling technique to ultrathin frozen sections. In nerve-muscle preparations treated for 1 h with a low dose of alpha-latrotoxin in the absence of extracellular Ca2+ (a condition under which nerve terminals are depleted of both quanta of neurotransmitter and synaptic vesicles), the immunolabeling for both proteins was distributed along the axolemma. These findings indicate that, in the presence of a block of endocytosis, exocytosis leads to the permanent incorporation of the synaptic vesicle membrane into the axolemma and suggest that, under this condition, at least some of the synapsin I molecules remain associated with the vesicle membrane after fusion. When the same dose of alpha-latrotoxin was applied in the presence of extracellular Ca2+, the immunoreactivity patterns resembled those obtained in resting preparations: immunogold particles were selectively associated with the membrane of synaptic vesicles, whereas the axolemma was virtually unlabeled. Under this condition an active recycling of both quanta of neurotransmitter and vesicles operates. These findings indicate that the retrieval of components of the synaptic vesicle membrane is an efficient process that does not involve extensive intermixing between molecular components of the vesicle and plasma membrane, and show that synaptic vesicles that are rapidly recycling still have the bulk of synapsin I associated with their membrane.     

Recycling endosomes of polarized epithelial cells actively sort apical and basolateral cargos into separate subdomains

The plasma membranes of epithelial cells plasma membranes contain distinct apical and basolateral domains that are critical for their polarized functions. However, both domains are continuously internalized, with proteins and lipids from each intermixing in supranuclear recycling endosomes (REs). To maintain polarity, REs must faithfully recycle membrane proteins back to the correct plasma membrane domains. We examined sorting within REs and found that apical and basolateral proteins were laterally segregated into subdomains of individual REs. Subdomains were absent in unpolarized cells and developed along with polarization. Subdomains were formed by an active sorting process within REs, which precedes the formation of AP-1B-dependent basolateral transport vesicles. Both the formation of subdomains and the fidelity of basolateral trafficking were dependent on PI3 kinase activity. This suggests that subdomain and transport vesicle formation occur as separate sorting steps and that both processes may contribute to sorting fidelity.     

Exocytosis of catecholamine (CA)-containing and CA-free granules in chromaffin cells

Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.     

A model for exocytosis based on the opening of calcium-activated potassium channels in vesicles

It is proposed that the role of calcium in calcium-induced exocytosis is to open Ca-activated K channels present in vesicle membranes. The opening of these channels coupled with anion transport across the vesicle membranes would result in an influx of K and anions, increasing the osmotic pressure of the vesicles. For those vesicles situated very close to the cell plasma membrane, this would lead to fusion with the membrane and exocytosis of the vesicle contents. This model can account for facilitation and other key properties of transmitter release. In addition, the model predicts that vesicles with a higher transmitter content, and hence higher initial osmotic pressure, would be preferentially discharged. The model also predicts that a faster response can be obtained for small vesicles than for large vesicles, providing a rationale as to why neurotransmitters, which must be released quickly, are packaged in small vesicles.     

Plasma membrane resident 'fusion complexes' mediate reconstituted exocytosis

Calcium-triggered exocytosis is thought to be mediated by membrane-associated protein complexes. In sea urchin eggs, high concentrations of calcium activate multiple 'fusion complexes' per cortical vesicle-plasma membrane docking site. Some of these fusion complexes are known to reside in the vesicle membrane. It is not known if fusion complexes also reside in the plasma membrane, or if plasma membrane-resident fusion complexes require cognate partners in the vesicle membrane. Using reconstitution, we show that N-ethylmaleimide treatment of either vesicles or plasma membrane fragments prior to reconstitution does not completely inhibit exocytosis. Treatment of both components did result in complete inhibition. Upon reconstitution, cortical vesicles and the early endosomes formed by compensatory endocytosis both contributed, on average, two fusion complexes per reconstituted docking site. The plasma membrane contributed, on average, two fusion complexes per docking site when assembled with cortical vesicles, but only one complex when reconstituted with endosomes. We conclude that there are at least two types of plasma membrane-resident fusion complexes that participate in reconstituted cortical vesicle-plasma membrane fusion. The activity of one of these fusion complexes is target-specific for cortical vesicles, while the second type also supports fusion with endosomes.     

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').     

Synaptic vesicle exocytosis. Does a lingering kiss lead to fusion?

Direct optical measurements of single synaptic vesicles undergoing exocytosis at a synapse reveal rapid and complete transfer of membrane marker from the vesicle to the plasma membrane (; this issue of Neuron). Contact between the two membranes is consistent with free lipid exchange, such as might result from full fusion of the vesicle and plasma membranes.     

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.     

Kinetic diversity in the fusion of exocytotic vesicles.

The speed at which secretory vesicles fuse with the plasma membrane is a key parameter for neuronal and endocrine functions. We determined the precise time courses for fusion of small clear and large dense-core vesicles in PC12 and chromaffin cells by simultaneously measuring both plasma membrane areas and release of vesicular contents. We found that instantaneous increases in cytosolic Ca2+ concentration evoked vesicle fusion, but with time constants that varied over four orders of magnitude among different types of vesicles and cells. This indicates that the molecular machinery for the final Ca2+-dependent fusion steps of exocytosis is highly variable and is as critical as Ca2+ signalling processes in determining the speed and amount of secretion of neurotransmitters and hormones. Our results suggest a new possibility that the molecules responsible for the final fusion reaction that leads to vesicle fusion are key determinants for neuronal plasticity and hormonal disorders.     

Quantitative Analysis of Membrane Trafficking in Regulation of Cdc42 Polarity

Vesicle delivery of Cdc42 has been proposed as an important mechanism for generating and maintaining Cdc42 polarity at the plasma membrane. This mechanism requires the density of Cdc42 on secretory vesicles to be equal to or higher than the plasma membrane polarity cap. Using a novel method to estimate Cdc42 levels on post‐Golgi secretory vesicles in intact yeast cells, we: (1) determined that endocytosis plays an important role in Cdc42's association with secretory vesicles (2) found that a GFP‐tag placed on the N‐terminus of Cdc42 negatively impacts this vesicle association and (3) quantified the surface densities of Cdc42 on post‐Golgi vesicles which revealed that the vesicle density of Cdc42 is three times more dilute than that at the polarity cap. This work suggests that the immediate consequence of secretory vesicle fusion with the plasma membrane polarity cap is to dilute the local Cdc42 surface density. This provides strong support for the model in which vesicle trafficking acts to negatively regulate Cdc42 polarity on the cell surface while also providing a means to recycle Cdc42 between the cell surface and internal membrane locations.      

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium‐triggered fusion

Synaptic vesicles fuse with the plasma membrane in response to Ca²⁺ influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large‐scale, automated cryo‐electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca²⁺‐triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high‐energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.