The function of the nervous system relies on synaptic transmission. Synaptic transmission is mediated by calcium-triggered vesicle fusion, followed by vesicle endocytosis that recycles vesicles. Although significant progress has been made in understanding these processes, much remains unknown. My goal is to advance our understanding of these synaptic signaling processes. The progress of the last year is described below. 1. Membrane fusion is vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused -shaped structure in live neuroendocrine chromaffin cells and pancreatic -cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening at the plasma membrane. Unexpectedly, the transition to full fusion is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key evidence missed in the last three decades of study in support of the hemi-fusion hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion. 2. Vesicle fusion is executed via formation of an -shaped structure (-profile), followed by closure (kiss-and-run) or merging of the -profile into the plasma membrane (full fusion). Although -profile closure limits release but recycles vesicles economically, -profile merging facilitates release but couples to classical endocytosis for recycling. Despite its crucial role in determining exocytosis/endocytosis modes, how -profile merging is mediated is poorly understood in endocrine cells and neurons containing small 30-300nm vesicles. Here, using confocal and super-resolution STED imaging, force measurements, pharmacology and gene knockout, we show that dynamic assembly of filamentous actin, involving ATP hydrolysis, N-WASP and formin, mediates -profile merging by providing sufficient plasma membrane tension to shrink the -profile in neuroendocrine chromaffin cells containing 300nm vesicles. Actin-directed compounds also induce -profile accumulation at lamprey synaptic active zones, suggesting that actin may mediate -profile merging at synapses. These results uncover molecular and biophysical mechanisms underlying -profile merging. 3. 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.
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