Synaptic Vesicle Endocytosis

National Institute Of Neurological Disorders And Stroke

Investigators

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Abstract

Neurons contact each other mostly by synaptic transmission at synapses. The maintenance of synaptic transmission relies on vesicle endocytosis, which recycles fused vesicles for the second round of exocytosis. My goal is to improve our understanding of the cellular and molecular mechanisms underlying synaptic vesicle endocytosis, which are the building block for the maintenance of synaptic transmission and thus the signaling process of the nervous system. Our progress in the last year is described below. The progress of the last year is described below I should mention that a large part of our efforts has been devoted to overcoming the severe flooding in December of 2022 in the building. These efforts include room and equipment repair as well as restoring the order of the experiments to the pre-flood level. 1. Recent advances in stimulated emission depletion (STED) microscopy offer an unparalleled avenue to study membrane dynamics of endocytosis, i.e. the membrane transformation from flat-shaped to round-shaped vesicles in real-time. Here we describe a method of using state-of-the-art STED microscopy to image these membrane dynamics in bovine chromaffin cells. This method can potentially be applied to study other membrane budding dynamics in other sub-cellular systems and other cell model systems. 2. Membrane budding entails forces to transform flat membranes into vesicles essential for cell survival. Accumulated studies have identified coat-proteins (e.g., clathrin) as potential budding factors. However, forces mediating many non-coated membrane buddings remain unclear. We developed an in-vitro system in which dynamin is the only protein acting on DOPS-liposomes. With this system, we found that dynamin can generate forces to pull the membrane, a new type of force previously unrecognized. This force may contribute to the generation of endocytic forces in vivo to produce a vesicle during endocytosis. 3. Endocytosis involves the flat-to-round membrane transformation. In the past, we captured these transitions by scanning the microscopic XZ plane at a single Y-axis location. Such a single XZ-plane imaging has a high speed to capture rapid changes but is of low chance (because only a single plane is imaged). We developed a 3-D volume scanning that increases the chances of capturing the endocytic membrane transformation significantly. 4. Synaptic exocytosis is mediated by the soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex, composed of synaptobrevin, SNAP-25, and syntaxin. After exocytosis, the SNARE complex is disassembled by NSF, and endocytosis follows to recycle vesicles. NSF and three SNARE proteins are generally not considered to be involved in endocytosis. Here we report that these four proteins were involved in two widely observed forms of endocytosis, rapid and slow endocytosis, at both large calyx-type synapses and conventional hippocampal synapses. This finding calls for modification of the current endocytosis model to include four core exocytosis proteins. Together with published biochemical data, we suggest that after exocytosis, SNARE proteins disassembled from the SNARE complex by NSF bind and recruit endocytosis proteins to mediate endocytosis. This model may help develop a mechanistic explanation for why endocytosis follows exocytosis with a similar amount, an enigma since the discovery of endocytosis in the 1970s. 5. Neurotransmitter in vesicles is released through a fusion pore when vesicles fuse with the plasma membrane. Subsequent retrieval of the fused vesicle membrane is the key step in recycling exocytosed vesicles. Application of advanced electrophysiological techniques to a large nerve terminal, the calyx of Held, has led to recordings of endocytosis, individual vesicle fusion and retrieval, and the kinetics of the fusion pore opening process and the fission pore closure process. These studies have revealed three kinetically different forms of endocytosis -- rapid, slow, and bulk endocytosis; and two forms of fusion -- full collapse and kiss-and-run. Calcium influx triggers all kinetically distinguishable forms of endocytosis at calyces by activation of calmodulin/calcineurin signaling pathway and protein kinase C, which may dephosphorylate and phosphorylate endocytic proteins. Polymerized actin may provide mechanical forces to bend the membrane, forming membrane pits, the precursor for generating vesicles. We provide a review of these research advancements.