Synaptic Vesicle Exocytosis

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

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. Our goal is to advance the understanding of these synaptic signaling processes. The progress of the last year is described below. 1. 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 Omega-shaped vesicular membrane. With superresolution STED microscopy in excitable neuroendocrine chromaffin cells, we visualized in real-time sequential compound fusion pore openings and content release, generating multivesicular and asynchronous release from single release sites, which enhances exocytosis strength and dynamic range in excitable cells. These results suggest a need for modifying the current exocytosis concept by including rapid release-site assembly at fused vesicle membrane, where sequential compound fusion takes place to enhance exocytosis capacity and dynamic range. 2. 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 approximately 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. 3. Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated Omega-shape profile until it merges completely at the plasma membrane. To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C delta (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at 20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells. 4. Real-time confocal and super-resolution imaging reveals membrane dynamics of exo- and endocytosis, including hemi-fusion, fusion pore opening, expansion, constriction, closure (kiss-and-run), fused-vesicle shrinking (shrink fusion), and flat membrane transition to vesicles via intermediate Lambda- and Omega-shape structures. Here, we describe a protocol for imaging these membrane dynamics, including primary culture of bovine adrenal chromaffin cells, fluorescent probe application, patch-clamp to deliver depolarization and evoke exo- and endocytosis, electron microscopy (EM), and real-time confocal and stimulated emission depletion (STED) microscopy. 5. Vesicle exo- and endocytosis mediate important biological functions, including synaptic transmission. We wrote an article commenting on a newly developed technique: the fluorescently tagged C2 domain of phospholipase A2 binds to membrane phosphatidylcholine and thus labels vesicle membrane, allowing for super-resolution and electron microscopic visualization of vesicle trafficking. 6. Cytoskeletal filamentous actin (F-actin) has long been considered a molecule that may regulate exo- and endocytosis. However, its exact roles remained elusive. Recent studies shed new light on many crucial roles of F-actin in regulating exo- and endocytosis. Here, we review this progress in the study of secretory cells, particularly neurons and endocrine cells. These studies reveal that F-actin promotes vesicle replenishment to the readily releasable pool most likely via active zone clearance, which may sustain synaptic transmission and overcome short-term depression of synaptic transmission during repetitive firing. By enhancing plasma membrane tension, F-actin promotes fusion pore expansion, vesicular content release, and a fusion mode called shrink fusion involving fusing vesicle shrinking. Not only F-actin, but also the F-actin assembly pathway, including ATP hydrolysis, N-WASH, and formin, are involved in mediating these roles of exo- and endocytosis. Neurological disorders, including spinocerebellar ataxia 13 caused by Kv3.3 channel mutation, may involve impairment of F-actin and its assembly pathway, leading in turn to impairment of exo- and endocytosis at synapses that may contribute to neurological disorders.