Vesicle cycle in the presynaptic nerve terminal

Presynaptic nerve terminals contain a great number ofsynaptic vesicles filled with neurotransmitter. The transmission of information in synapses is mediated by release of transmitter from vesicles: exocytosis, after their fusion with presynaptic membrane. At the functioning synapses, the continuous recycling of synaptic vesicles occurs (vesicle cycle), which provides multiple reuse of vesicular membrane material during synaptic activity. Vesicle cycle consists of large number of steps, including vesicle fusion--exocytosis, formation of new vesicles--endocytosis, vesicle sorting, filling of vesicles with transmitter, intraterminal vesicle transport driving the vesicles to different vesicle pools and preparing to next exocytic event. At this paper, I presented the latest literature and our data regarding the steps and mechanisms of vesicle cycle at synapses. Special attention was paid to neuromuscular synapse as the most thoroughly investigated and as my favorite preparation.     

High- and Low-Mobility Stages in the Synaptic Vesicle Cycle

Synaptic vesicles need to be mobile to reach their release sites during synaptic activity. We investigated vesicle mobility throughout the synaptic vesicle cycle using both conventional and subdiffraction-resolution stimulated emission depletion fluorescence microscopy. Vesicle tracking revealed that recently endocytosed synaptic vesicles are highly mobile for a substantial time period after endocytosis. They later undergo a maturation process and integrate into vesicle clusters where they exhibit little mobility. Despite the differences in mobility, both recently endocytosed and mature vesicles are exchanged between synapses. Electrical stimulation does not seem to affect the mobility of the two types of vesicles. After exocytosis, the vesicle material is mobile in the plasma membrane, although the movement appears to be somewhat limited. Increasing the proportion of fused vesicles (by stimulating exocytosis while simultaneously blocking endocytosis) leads to substantially higher mobility. We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement.     

Calcium Dependence of Synaptic Vesicle Recycling Before and After Synaptogenesis

Abstract: Using an immunocytochemical assay to monitor synaptic vesicle exocytosis/endocytosis independently of neurotransmitter release, we have investigated some aspects of vesicle recycling in hippocampal neurons at different developmental stages. A calcium- and depolarization-dependent exocytotic/endocytotic recycling of synaptic vesicles was found to take place in neurons already before the formation of synaptic contacts. The analysis of synaptic vesicle recycling at different calcium concentrations revealed the presence of two release components: the first one activated by low calcium concentrations and sustaining vesicle recycling before synaptogenesis, and a second one activated by high calcium concentrations, which is specifically turned on after the establishment of synaptic contacts. These data suggest that formation of synapses correlates with the activation of a putative low-affinity calcium sensor, which allows synaptic vesicle exocytosis to be triggered and turned off over extremely short time scales, in response to large increases in the level of intracellular calcium.     

Okadaic acid disrupts synaptic vesicle trafficking in a ribbon-type synapse

Protein phosphorylation plays an essential role in regulating synaptic transmission and plasticity. However, regulation of vesicle trafficking towards and away from the plasma membrane is poorly understood. Furthermore, the extent to which phosphorylation modulates ribbon-type synapses is unknown. Using the phosphatase inhibitor okadaic acid (OA), we investigated the influence of persistent phosphorylation on vesicle cycling in goldfish bipolar cells. We followed uptake of FM1-43 during vesicle recycling in control and OA-treated cells. FM1-43 fluorescence spread to the center of control synaptic terminals after depolarization elicited Ca2+ influx. However, OA (1-50 nm) impaired this spatial spread of FM1-43 in a dose-dependent manner. Capacitance measurements revealed that OA (50 nm) did not modify either the amount or kinetics of exocytosis and endocytosis evoked by depolarizing pulses. The extremely low concentrations of OA (1-5 nm) sufficient to observe the inhibition of vesicle mobility implicate phosphatase 2A (PP2A) as a major regulator of vesicle trafficking after endocytosis. These results contrast with those at the neuromuscular junction where OA enhances lateral movement of vesicles between distinct vesicle clusters. Thus, our results suggest that phosphatases regulate vesicle translocation at ribbon synapses in a different manner than conventional active zones.     

The Molecular Architecture of Ribbon Presynaptic Terminals

The primary receptor neurons of the auditory, vestibular, and visual systems encode a broad range of sensory information by modulating the tonic release of the neurotransmitter glutamate in response to graded changes in membrane potential. The output synapses of these neurons are marked by structures called synaptic ribbons, which tether a pool of releasable synaptic vesicles at the active zone where glutamate release occurs in response to calcium influx through L-type channels. Ribbons are composed primarily of the protein, RIBEYE, which is unique to ribbon synapses, but cytomatrix proteins that regulate the vesicle cycle in conventional terminals, such as Piccolo and Bassoon, also are found at ribbons. Conventional and ribbon terminals differ, however, in the size, molecular composition, and mobilization of their synaptic vesicle pools. Calcium-binding proteins and plasma membrane calcium pumps, together with endomembrane pumps and channels, play important roles in calcium handling at ribbon synapses. Taken together, emerging evidence suggests that several molecular and cellular specializations work in concert to support the sustained exocytosis of glutamate that is a hallmark of ribbon synapses. Consistent with its functional importance, abnormalities in a variety of functional aspects of the ribbon presynaptic terminal underlie several forms of auditory neuropathy and retinopathy.     

SNARE protein recycling by αSNAP and βSNAP supports synaptic vesicle priming

Neurotransmitter release proceeds by Ca(2+)-triggered, SNARE-complex-dependent synaptic vesicle fusion. After fusion, the ATPase NSF and its cofactors α- and βSNAP disassemble SNARE complexes, thereby recycling individual SNAREs for subsequent fusion reactions. We examined the effects of genetic perturbation of α- and βSNAP expression on synaptic vesicle exocytosis, employing a new Ca(2+) uncaging protocol to study synaptic vesicle trafficking, priming, and fusion in small glutamatergic synapses of hippocampal neurons. By characterizing this protocol, we show that synchronous and asynchronous transmitter release involve different Ca(2+) sensors and are not caused by distinct releasable vesicle pools, and that tonic transmitter release is due to ongoing priming and fusion of new synaptic vesicles during high synaptic activity. Our analysis of α- and βSNAP deletion mutant neurons shows that the two NSF cofactors support synaptic vesicle priming by determining the availability of free SNARE components, particularly during phases of high synaptic activity.     

Role of Drosophila Rab5 during endosomal trafficking at the synapse and evoked neurotransmitter release

During constitutive endocytosis, internalized membrane traffics through endosomal compartments. At synapses, endocytosis of vesicular membrane is temporally coupled to action potential-induced exocytosis of synaptic vesicles. Endocytosed membrane may immediately be reused for a new round of neurotransmitter release without trafficking through an endosomal compartment. Using GFP-tagged endosomal markers, we monitored an endosomal compartment in Drosophila neuromuscular synapses. We showed that in conditions in which the synaptic vesicles pool is depleted, the endosome is also drastically reduced and only recovers from membrane derived by dynamin-mediated endocytosis. This suggests that membrane exchange takes place between the vesicle pool and the synaptic endosome. We demonstrate that the small GTPase Rab5 is required for endosome integrity in the presynaptic terminal. Impaired Rab5 function affects endo- and exocytosis rates and decreases the evoked neurotransmitter release probability. Conversely, Rab5 overexpression increases the release efficacy. Therefore, the Rab5-dependent trafficking pathway plays an important role for synaptic performance.     

Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes

The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.     

Visualizing postendocytic traffic of synaptic vesicles at hippocampal synapses.

We have investigated mechanisms in postendocytic processing of synaptic vesicles at hippocampal synapses, using synaptobrevin/vesicle-associated membrane protein (VAMP) tagged with variants of the green fluorescent protein. Following exocytosis, VAMP is retrieved at synaptic and adjoining axonal regions. Retrieved VAMP-containing vesicles return to synaptic vesicle clusters at a rate slower than endocytosis. Vesicles containing a different protein, synaptophysin, recluster at a similar rate, suggesting common vesicular intermediates for the two proteins. Activity prolongs the time taken by endocytosed vesicles to return to synapses. Exogenous calcium buffers slow endocytosis but have no additional effect on the time course of reclustering. In contrast, the protein kinase inhibitor staurosporine does not affect endocytosis but slows reclustering. Finally, since VAMP can move freely on surface membranes, sustained synaptic activity leads to mixing of this vesicular component between adjacent synapses.     

Rapid Endocytosis and Vesicle Recycling in Neuroendocrine Cells

Endocytosis is a crucial process for neuroendocrine cells that ensures membrane homeostasis, vesicle recycling, and hormone release reliability. Different endocytic mechanisms have been described in chromaffin cells, such as clathrin-dependent slow endocytosis and clathrin-independent rapid endocytosis. Rapid endocytosis, classically measured in terms of a fast decrease in membrane capacitance, exhibits two different forms, “rapid compensatory endocytosis” and “excess retrieval.” While excess retrieval seems to be associated with formation of long-lasting endosomes, rapid compensatory endocytosis is well correlated with exocytotic activity, and it is regarded as a mechanism associated to rapid vesicle recycling during normal secretory activity. It has been suggested that rapid compensatory endocytosis may be related to the prevalence of a transient fusion mode of exo-endocytosis. In the latter mode, the fusion pore, a nanometric-sized channel formed at the onset of exocytosis, remains open for a few hundred milliseconds and later abruptly closes, releasing a small amount of transmitters. By this mechanism, endocrine cell selectively releases low molecular weight transmitters, and rapidly recycles the secretory vesicles. In this article, we discuss the cellular and molecular mechanisms that define the different forms of exocytosis and endocytosis and their impact on vesicle recycling pathways.     

Ca(2+) influx and neurotransmitter release at ribbon synapses

Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis.     

Systematic heterogeneity of fractional vesicle pool sizes and release rates of hippocampal synapses

Hippocampal neurons in tissue culture develop functional synapses that exhibit considerable variation in synaptic vesicle content (20–350 vesicles). We examined absolute and fractional parameters of synaptic vesicle exocytosis of individual synapses. Their correlation to vesicle content was determined by activity-dependent discharge of FM-styryl dyes. At high frequency stimulation (30 Hz), synapses with large recycling pools released higher amounts of dye, but showed a lower fractional release compared to synapses that contained fewer vesicles. This effect gradually vanished at lower frequencies when stimulation was triggered at 20 Hz and 10 Hz, respectively. Live-cell antibody staining with anti-synaptotagmin-1-cypHer 5, and overexpression of synaptopHluorin as well as photoconversion of FM 1-43 followed by electron microscopy, consolidated the findings obtained with FM-styryl dyes. We found that the readily releasable pool grew with a power function with a coefficient of 2/3, possibly indicating a synaptic volume/surface dependency. This observation could be explained by assigning the rate-limiting factor for vesicle exocytosis at high frequency stimulation to the available active zone surface that is proportionally smaller in synapses with larger volumes.     

Synaptobrevin is essential for fast synaptic-vesicle endocytosis

Synaptobrevin-2 (VAMP-2), the major SNARE protein of synaptic vesicles, is required for fast calcium-triggered synaptic-vesicle exocytosis. Here we show that synaptobrevin-2 is also essential for fast synaptic-vesicle endocytosis. We demonstrate that after depletion of the readily releasable vesicle pool, replenishment of the pool is delayed by knockout of synaptobrevin. This delay was not from a loss of vesicles, because the total number of pre-synaptic vesicles, docked vesicles and actively recycling vesicles was unaffected. However, altered shape and size of the vesicles in synaptobrevin-deficient synapses suggests a defect in endocytosis. Consistent with such a defect, the stimulus-dependent endocytosis of horseradish peroxidase and fluorescent FM1-43 were delayed, indicating that fast vesicle endocytosis may normally be nucleated by a SNARE-dependent coat. Thus, synaptobrevin is essential for two fast synapse-specific membrane trafficking reactions: fast exocytosis for neurotransmitter release and fast endocytosis that mediates rapid reuse of synaptic vesicles.     

Neurotransmitter release at ribbon synapses in the retina

The synapses of photoreceptors and bipolar cells in the retina are easily identified ultrastructurally by the presence of synaptic ribbons, electron-dense bars perpendicular to the plasma membrane at the active zones, extending about 0.5 microm into the cytoplasm. The neurotransmitter, glutamate, is released continuously (tonically) from these 'ribbon synapses' and the rate of release is modulated in response to graded changes in the membrane potential. This contrasts with action potential-driven bursts of release at conventional synapses. Similar to other synapses, neurotransmitter is released at ribbon synapses by the calcium-dependent exocytosis of synaptic vesicles. Most components of the molecular machinery governing transmitter release are conserved between ribbon and conventional synapses, but a few differences have been identified that may be important determinants of tonic transmitter release. For example, the presynaptic calcium channels of bipolar cells and photoreceptors are different from those elsewhere in the brain. Differences have also been found in the proteins involved in synaptic vesicle recruitment to the active zone and in synaptic vesicle fusion. These differences and others are discussed in terms of their implications for neurotransmitter release from photoreceptors and bipolar cells in the retina.     

Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.     

Streamlined synaptic vesicle cycle in cone photoreceptor terminals

Cone photoreceptors tonically release neurotransmitter in the dark through a continuous cycle of exocytosis and endocytosis. Here, using the synaptic vesicle marker FM1-43, we elucidate specialized features of the vesicle cycle. Unlike retinal bipolar cell terminals, where stimulation triggers bulk membrane retrieval, cone terminals appear to exclusively endocytose small vesicles. These retain their integrity until exocytosis, without pooling their membranes in endosomes. Endocytosed vesicles rapidly disperse through the terminal and are reused with no apparent delay. Unlike other synapses where most vesicles are immobilized and held in reserve, only a small fraction (<15%) becomes immobilized in cones. Photobleaching experiments suggest that vesicles move by diffusion and not by molecular motors on the cytoskeleton and that vesicle movement is not rate limiting for release. The huge reservoir of vesicles that move rapidly throughout cone terminals and the lack of a reserve pool are unique features, providing cones with a steady supply for continuous release.     

Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

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.     

Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin

The synaptic vesicle cycle encompasses the pre-synaptic events that drive neurotransmission. Influx of calcium leads to the fusion of synaptic vesicles with the plasma membrane and the release of neurotransmitter, closely followed by endocytosis. Vacated release sites are repopulated with vesicles which are then primed for release. When activity is intense, reserve vesicles may be mobilized to counteract an eventual decline in transmission. Recently, interplay between endocytosis and repopulation of the readily releasable pool of vesicles has been identified. In this study, we show that exo-endocytosis is necessary to enable detachment of synapsin from reserve pool vesicles during synaptic activity. We report that blockage of exocytosis in cultured mouse hippocampal neurons, either by tetanus toxin or by the deletion of munc13, inhibits the activity-dependent redistribution of synapsin from the pre-synaptic terminal into the axon. Likewise, perturbation of endocytosis with dynasore or by a dynamin dominant-negative mutant fully prevents synapsin redistribution. Such inhibition of synapsin redistribution occurred despite the efficient phosphorylation of synapsin at its protein kinase A/CaMKI site, indicating that disengagement of synapsin from the vesicles requires exocytosis and endocytosis in addition to phosphorylation. Our results therefore reveal hitherto unidentified feedback within the synaptic vesicle cycle involving the synapsin-managed reserve pool.     

植物胞吞和胞吐的耦合调控

真核细胞通过胞吞和胞吐作用将大分子和颗粒性物质运出或运送至质膜,其中包括一些具有重要生物学功能的蛋白质。胞吞和胞吐途径之间的耦合对维持质膜的完整性以及调控质膜蛋白的丰度和活性至关重要。动物中,突触小泡的胞吞和胞吐在时空上紧密耦合已被证明是持续神经传递的必要条件。近年来,随着对植物囊泡运输的深入研究,越来越多的证据表明,植物细胞的胞吞和胞吐间同样存在耦合调控,且在植物生长发育和对外界环境的响应中扮演重要角色。该文综述了植物协同调控胞吞和胞吐的生理学意义,并结合网格蛋白介导囊泡运输的最新研究进展探讨了其可能的耦合机制。