Fusion is thought to open a pore to release vesicular cargoes vital for many biological processes, including exocytosis, intracellular trafficking, fertilization, and viral entry. However, fusion pores have not been observed and thus proved in live cells. Its regulatory mechanisms and functions remain poorly understood. With super-resolution STED microscopy, we observed dynamic fusion pore behaviors in live (neuroendocrine) cells, including opening, expansion, constriction, and closure, where pore size may vary between 0 and 490 nm within 26 milliseconds to seconds (vesicle size: 180-720 nm). These pore dynamics crucially determine the efficiency of vesicular cargo release and vesicle retrieval. They are generated by competition between pore expansion and constriction. Pharmacology and mutation experiments suggest that expansion and constriction are mediated by F-actin-dependent membrane tension and calcium/dynamin, respectively. These findings provide the missing live-cell evidence, proving the fusion-pore hypothesis, and establish a live-cell dynamic-pore theory accounting for fusion, fission, and their regulation.

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The function and pharmacology of γ-aminobutyric acid type A receptors (GABAARs) are of great physiological and clinical importance and have long been thought to be determined by the channel pore–forming subunits. We discovered that Shisa7, a single-passing transmembrane protein, localizes at GABAergic inhibitory synapses and interacts with GABAARs. Shisa7 controls receptor abundance at synapses and speeds up the channel deactivation kinetics. Shisa7 also potently enhances the action of diazepam, a classic benzodiazepine, on GABAARs. Genetic deletion of Shisa7 selectively impairs GABAergic transmission and diminishes the effects of diazepam in mice. Our data indicate that Shisa7 regulates GABAAR trafficking, function, and pharmacology and reveal a previously unknown molecular interaction that modulates benzodiazepine action in the brain.

Graphical AbstractHighlights d Visualizing fused vesicular membrane shrinking and enlargement with STED microscopy d Shrinking is mediated by physiological osmotic pressure that squeezes fused vesicles d Vesicle shrinking facilitates content release by employing a large fusion pore d Vesicle enlargement slows down content release by employing a small fusion pore SUMMARY 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 U-profile generated by vesicle fusion shrinks but maintains a large nondilating pore. We discover that the physiological osmotic pressure of a cell squeezes, but does not dilate, the U-profile, which explains why shrink fusion prevails over full collapse. Instead of kiss-and-run, enlarge fusion, in which U-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 fullcollapse/kiss-and-run framework.

The morphology of presynaptic specializations can vary greatly ranging from classical single-release-site boutons in the central nervous system to boutons of various sizes harboring multiple vesicle release sites. Multi-release-site boutons can be found in several neural contexts, for example at the neuromuscular junction (NMJ) of body wall muscles of Drosophila larvae. These NMJs are built by two motor neurons forming two types of glutamatergic multi-release-site boutons with two typical diameters. However, it is unknown why these distinct nerve terminal configurations are used on the same postsynaptic muscle fiber. To systematically dissect the biophysical properties of these boutons we developed a full three-dimensional model of such boutons, their release sites and transmitter-harboring vesicles and analyzed the local vesicle dynamics of various configurations during stimulation. Here we show that the rate of transmission of a bouton is primarily limited by diffusion-based vesicle movements and that the probability of vesicle release and the size of a bouton affect bouton-performance in distinct temporal domains allowing for an optimal transmission of the neural signals at different time scales. A comparison of our in silico simulations with in vivo recordings of the natural motor pattern of both neurons revealed that the bouton properties resemble a well-tuned cooperation of the parameters release probability and bouton size, enabling a reliable transmission of the prevailing firing-pattern at diffusion-limited boutons. Our findings indicate that the prevailing firing-pattern of a neuron may determine the physiological and morphological parameters required for its synaptic terminals.

Salvianolic acid A (Sal A), a bioactive compound isolated from the Chinese medicinal herb Danshen, is used for the prevention and treatment of cardiovascular diseases. However, the protective function of Sal A on preserving the role of blood-spinal cord barrier (BSCB) after spinal cord injury (SCI) is unclear. The present study investigated the effects and mechanisms of Sal A (2.5, 5, 10mg/kg, i.p.) on BSCB permeability at different time-points after compressive SCI in rats. Compared to the SCI group, treatment with Sal A decreased the content of the Evans blue in the spinal cord tissue at 24h post-SCI. The expression levels of tight junction proteins and HO-1 were remarkably increased, and that of p-caveolin-1 protein was greatly decreased after SCI Sal A. The effect of Sal A on the expression level of ZO-1, occluding, and p-caveolin-1 after SCI was blocked by the HO-1 inhibitor, zinc protoporphyrin IX (ZnPP). Also, Sal A inhibited the level of apoptosis-related proteins and improved the motor function until 21days after SCI. In addition, Sal A significantly increased the expression of microRNA-101 (miR-101) in the RBMECs under hypoxia. AntagomiR-101 markedly increased the RBMECs permeability and the expression of the Cul3 protein by targeting with 3'-UTR of its mRNA. The expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1 was significantly increased after agomiR-101 treatment. Therefore, Sal A could improve the recovery of neurological function after SCI, which could be correlated with the repair of BSCB integrity by the miR-101/Cul3/Nrf2/HO-1 signaling pathway.

Vesicle fusion at preestablished plasma membrane release sites releases transmitters and hormones to mediate fundamental functions like neuronal network activities and fight-or-flight responses. This half-a-century-old concept—fusion at well-established release sites in excitable cells—needs to be modified to include the sequential compound fusion reported here—vesicle fusion at previously fused Ω-shaped vesicular membrane. With superresolution STED microscopy in excitable neuroendocrine chromaffin cells, we real-time visualized sequential compound fusion pore openings and content releases in generating multivesicular and asynchronous release from single release sites, which enhances exocytosis strength and dynamic ranges in excitable cells. We also visualized subsequent compound fusion pore closure, a new mode of endocytosis termed compound kiss-and-run that enhances vesicle recycling capacity. These results suggest modifying current exo-endocytosis concepts by including rapid release-site assembly at fused vesicle membrane, where sequential compound fusion and kiss-and-run take place to enhance exo-endocytosis capacity and dynamic ranges.

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