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Regulated exocytosis, a universal process of eukaryotic cells, involves the merging between the vesicle membrane and the plasma membrane, plays a key role in cell-to-cell communication, particularly in the release of hormones and neurotransmitters. There are a number of barriers a vesicle needs to pass to discharge vesicle content to the extracellular space. At the pre-fusion site vesicles need to be transported to the sites on the plasma membrane where the merger may begin. Classically cytoskeleton was considered an important barrier for vesicle translocation and was thought to be disintegrated to allow vesicle access to the plasma membrane [1]. However, it was considered later that cytoskeletal elements may also play a role at the post-fusion stage, promoting the vesicle merger with the plasma membrane and fusion pore expansion [ 4 , 22 , 23 ]. In this Special Issue of Cell Calcium entitled "Regulated Exocytosis", the authors address outstanding issues related to vesicle chemical messenger release by regulated exocytosis, including that related to the question whether vesicle content discharge is complete or only partial upon the merging of the vesicle membrane with the plasma membrane triggered by Ca2+. Among processes that limit vesicle discharge at the post-fusion stage is the accumulation of cholesterol in some vesicles [19] , a process that has recently been associated with cell aging [20]. [ABSTRACT FROM AUTHOR]

Summary Conversion from fibroblasts to neurons has recently been successfully induced. However, the underlying mechanisms are poorly understood. Here, we find that depletion of p53 alone converts fibroblasts into all three major neural lineages. The induced neuronal cells express multiple neuron-specific proteins and generate action potentials and transmitter-receptor-mediated currents. Surprisingly, depletion does not affect the well-known tumorigenic p53 target, p21. Instead, knockdown of p53 upregulates neurogenic transcription factors, which in turn boosts fibroblast-neuron conversion. p53 binds the promoter of the neurogenic transcription factor Neurod2 and regulates its expression during fibroblast-neuron conversion. Furthermore, our method provides a high efficiency of conversion in late-passage fibroblasts. Genome-wide transcriptional analysis shows that the p53-deficiency-induced neurons exhibit an expression profile different from parental fibroblasts and similar to control-induced neurons. The results may help to understand and improve neural conversion mechanisms to develop robust neuron-replacement therapy strategies. [ABSTRACT FROM AUTHOR]

• Dynamin 1 controls vesicle size by regulating membrane pit size. • Dynamin 1 is more crucial for endocytosis than recognized at synapses. Following calcium-triggered vesicle exocytosis, endocytosis regenerates vesicles to maintain exocytosis and thus synaptic transmission, which underlies neuronal circuit activities. Although most molecules involved in endocytosis have been identified, it remains rather poorly understood how endocytic machinery regulates vesicle size. Vesicle size, together with the transmitter concentration inside the vesicle, determines the amount of transmitter the vesicle can release, the quantal size, that may control the strength of synaptic transmission. Here, we report that, surprisingly, knockout of the GTPase dynamin 1, the most abundant brain dynamin isoform known to catalyze fission of the membrane pit's neck (the last step of endocytosis), not only significantly slowed endocytosis but also increased the synaptic vesicle diameter by as much as ∼40–64% at cultured hippocampal synapses. Furthermore, dynamin 1 knockout increased the size of membrane pits, the precursor for endocytic vesicle formation. These results suggest an important function of dynamin other than its well-known fission function – control of vesicle size at the pit formation stage. [Display omitted] [ABSTRACT FROM AUTHOR]

Summary: Calcium influx triggers and accelerates endocytosis in nerve terminals and nonneuronal secretory cells. Whether calcium/calmodulin-activated calcineurin, which dephosphorylates endocytic proteins, mediates this process is highly controversial for different cell types, developmental stages, and endocytic forms. Using three preparations that previously produced discrepant results (i.e., large calyx-type synapses, conventional cerebellar synapses, and neuroendocrine chromaffin cells containing large dense-core vesicles), we found that calcineurin gene knockout consistently slowed down endocytosis, regardless of cell type, developmental stage, or endocytic form (rapid or slow). In contrast, calcineurin and calmodulin blockers slowed down endocytosis at a relatively small calcium influx, but did not inhibit endocytosis at a large calcium influx, resulting in false-negative results. These results suggest that calcineurin is universally involved in endocytosis. They may also help explain the discrepancies among previous pharmacological studies. We therefore suggest that calcineurin should be included as a key player in mediating calcium-triggered and -accelerated vesicle endocytosis. [Copyright &y& Elsevier]

Summary: Rapid endocytosis, which takes only a few seconds, is widely observed in secretory cells. Although it is more efficient in recycling vesicles than in slow clathrin-mediated endocytosis, its underlying mechanism, thought to be clathrin independent, is largely unclear. Here, we report that cleavage of three SNARE proteins essential for exocytosis, including synaptobrevin, SNAP-25, and syntaxin, inhibited rapid endocytosis at the calyx of Held nerve terminal, suggesting the involvement of the three SNARE proteins in rapid endocytosis. These SNARE proteins were also involved in slow endocytosis. In addition, SNAP-25 and syntaxin facilitated vesicle mobilization to the readily releasable pool, most likely via their roles in endocytosis and/or exocytosis. We conclude that both rapid and slow endocytosis share the involvement of SNARE proteins. The dual role of three SNARE proteins in exo- and endocytosis suggests that SNARE proteins may be molecular substrates contributing to the exocytosis-endocytosis coupling, which maintains exocytosis in secretory cells. [Copyright &y& Elsevier]

Summary: Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis. [ABSTRACT FROM AUTHOR]

It has long been proposed that following vesicle fusion, a small pore might open and close rapidly without full dilation. Such ‘kiss-and-run’ vesicle fusion can in principle result in rapid vesicle recycling and influence the size and the kinetics of the resulting synaptic current. However, the existence of kiss-and-run remains highly controversial, as revealed by recent imaging and electrophysiological studies at several synapses, including hippocampal synapses, neuromuscular junctions and retinal bipolar synapses. Only a minor fraction of fusion events has been shown to be kiss-and-run, as determined using cell-attached capacitance recordings in endocrine cells, pituitary nerve terminals and calyx-type synapses. Further work is needed to determine whether kiss-and-run is a major mode of fusion and has a major role in controlling synaptic strength at synapses. [Copyright &y& Elsevier]

Studies from a variety of synapses indicate that the time course of endocytosis ranges from less than a second to hundreds of seconds. This raises questions about how the time course of endocytosis is regulated and why different rates of endocytosis are needed. Recent progress sheds light on these issues. Neuronal firing frequency and duration determine the time course of endocytosis. The dynamic nature of this time course could be a result of multiple endocytic pathways and/or of regulation by a variety of modulators. Because endocytosis is crucial for maintaining transmitter release during repetitive stimulation, regulation of endocytosis could thus provide a mechanism by which synaptic plasticity is achieved. [Copyright &y& Elsevier]

Provides information on the activation of presynaptic receptors for variety of neurotransmitter and neuromodulators that affects transmitter release at many synapses. Relationship between neurotransmitter release and presynaptic calcium concentration; Multiple types of calcium channels in presynaptic terminals; Inhibition of presynaptic calcium influx. INSETS: Box I. Techniques for loading nerve terminals..;Box 2. Analysis of relative contribution to...

For decades, two fusion modes were thought to control hormone and transmitter release essential to life; one facilitates release via fusion pore dilation and flattening (full collapse), and the other limits release by closing a narrow fusion pore (kiss-and-run). Using super-resolution stimulated emission depletion (STED) microscopy to visualize fusion modes of dense-core vesicles in neuroendocrine cells, we find that facilitation of release is mediated not by full collapse but by shrink fusion, in which the Ω-profile generated by vesicle fusion shrinks but maintains a large non-dilating pore. We discover that the physiological osmotic pressure of a cell squeezes, but does not dilate, the Ω-profile, which explains why shrink fusion prevails over full collapse. Instead of kiss-and-run, enlarge fusion, in which Ω-profiles grow while maintaining a narrow pore, slows down release. Shrink and enlarge fusion may thus account for diverse hormone and transmitter release kinetics observed in secretory cells, previously interpreted within the full-collapse/kiss-and-run framework. • Visualizing fused vesicular membrane shrinking and enlargement with STED microscopy • Shrinking is mediated by physiological osmotic pressure that squeezes fused vesicles • Vesicle shrinking facilitates content release by employing a large fusion pore • Vesicle enlargement slows down content release by employing a small fusion pore Shin et al. discover two fusion modes; one involves fused vesicle shrinking that employs a large pore to facilitate content release, and the other involves vesicle enlargement with a small pore that slows down release. Shrinking is energetically preferred over the generally assumed full-collapse fusion, because osmotic pressure squeezes fused vesicles. [ABSTRACT FROM AUTHOR]

The calyx of Held synapse plays an important role in the auditory system, relaying information about sound localization via fast and precise synaptic transmission, which is achieved by its specialized structure and giant size. During development, the calyx of Held undergoes anatomical, morphological, and physiological changes necessary for performing its functions. The large dimensions of the calyx of Held nerve terminal are well suited for direct electrophysiological recording of many presynaptic events that are difficult, if not impossible to record at small conventional synapses. This unique accessibility has been used to investigate presynaptic ion channels, transmitter release, and short-term plasticity, providing invaluable information about basic presynaptic mechanisms of transmission at a central synapse. Here, we review anatomical and physiological specializations of the calyx of Held, summarize recent studies that provide new mechanisms important for calyx development and reliable synaptic transmission, and examine fundamental presynaptic mechanisms learned from studies using calyx as a model nerve terminal. This article is part of a Special Issue entitled . [ABSTRACT FROM AUTHOR]

Repetitive nerve activity induces various forms of short-term synaptic plasticity that have important computational roles in neuronal networks. Several forms of short-term plasticity are caused largely by changes in transmitter release, but the mechanisms that underlie these changes in the release process have been difficult to address. Recent studies of a giant synapse — the calyx of Held — have shed new light on this issue. Recordings of Ca2+ currents or Ca2+ concentrations at nerve terminals reveal that regulation of presynaptic Ca2+ channels has a significant role in three important forms of short-term plasticity: short-term depression, facilitation and post-tetanic potentiation. [Copyright &y& Elsevier]

We have recently applied Lindau–Neher’s capacitance measurement technique to study vesicle trafficking at the calyx-type synapse in the rat medial nucleus of the trapezoid body (MNTB) in slice conditions. This application made the MNTB synapse an excellent model for the study of exocytosis and endocytosis at conventional active zones. However, the application was only made at calyces that are presumably equivalent to a single-compartment circuit because their passive current transients decayed mono-exponentially. Here, we determined whether the application could be extended to majority of calyces whose passive current transients decayed bi-exponentially. By comparison of calyces with mono- or bi-exponential decay in their passive current transients, we found similar properties in respect to: (1) the capacitance jump induced by trains of action-potential equivalent stimuli, which reflects exocytosis; (2) the size of a releasable vesicle pool; (3) the time course of the decay after the capacitance jump, which reflects endocytosis; and (4) the transient capacitance artifact observed in the presence of Cd2+ that blocks exocytosis. These similar properties were also obtained from modeling calyces as a single- or two-compartment circuit. Thus, capacitance measurements may be extended to the majority of calyces, which may facilitate the study of rapid vesicle trafficking at conventional active zones. [Copyright &y& Elsevier]