Within a live organism cells need to transfer information
Within a live organism, cells need to transfer information to each other. Exocytosis is one of the critical mechanism which releases variety of ligands into the extracellular space. When cells export material via exocytosis, they first pack into vesicles, then cause the vesicles to tether at the plasma membrane and finally trigger them to fuse with the plasma membrane. Among numbers of exocytosis in live systems, synaptic vesicular exocytosis from neurons has fastest kinetics, which typically occurs within 1 ms from the arrival of an BAY 80-6946 and following calcium influx (Südhof, 2013). At presynaptic terminals of neurons, neurotransmitters are packed into the synaptic vesicles (∼50 nm in diameter). Within presynaptic terminal of neurons (usually few μm in diameter) these synaptic vesicles are densely packed with their numbers widely vary, from tens to hundreds of thousands, depending on the sizes of the terminals (Schikorski and Stevens, 2001; Sätzler et al., 2002; Rizzoli and Betz, 2005; Rollenhagen and Lübke, 2006; Rollenhagen et al., 2007). Within the terminal, synaptic vesicles are especially dense around the presynaptic release sites called active zones, and some of vesicles are located close vicinity of the plasma membrane. These localizations have been assumed to support rapid neurotransmission between neurons (Verhage and Sørensen, 2006; Hallermann and Silver, 2013). To support fast transmitter release following the calcium influx, it has been assumed that those synaptic vesicles located just beneath the plasma membrane are “tethered” (anchoring of vesicles to the plasma membrane by intermediate molecules) or “docked” (physical attachment of vesicles to the plasma membrane) to the plasma membrane via anchoring proteins, and protein complexes essential for membrane fusions are pre-assemble before the time of calcium influx (Südhof, 2013; Imig et al., 2014; Kaeser and Regehr, 2017). Such mechanisms preceding the fusion are crucial since they support the sustained synaptic transmission by recruiting new vesicles to the presynaptic release sites, active zones, but our understanding of these steps is surprisingly limited, because most of techniques to examine exocytosis are inaccessible to them. The current hypotheses describing these steps are proposed based on indirect measurements mainly by electron microscopy and electrophysiology (Rollenhagen and Lübke, 2006; Körber and Kuner, 2016; Neher and Sakaba, 2008; Neher, 2015). Electron microscopy provides incomparable spatial resolution, but the time course of the phenomenon can only be obtained as a “time lapse” images of different preparations since the cells have to be fixed before the observation (Watanabe et al., 2014). In contrast, electrophysiological techniques provide superior time resolution, and the kinetics of exocytosis has been well studied in wide variety of preparations (Neher and Sakaba, 2008), but little is known about the preceding steps before fusion, such as tethering, docking and priming (molecular arrangement of the fusion-related proteins that take place after initial tethering of a synaptic vesicle but before exocytosis, such that the influx of calcium ions is all that is needed to trigger final exocytosis) since it cannot measure the kinetics of the phenomenon which doesn’t provide electrical signals. Therefore, back-calculations based on the kinetics of exocytosis has been used to speculate those pre-fusion activities (Katz, 1969; Diamond and Jahr, 1995; Neher and Sakaba, 2001). Fluorescence live imaging is best suited for investigating the dynamics of particle such as synaptic vesicles (Betz and Bewick, 1992; Sankaranarayanan and Ryan, 2000; Zhang et al., 2009), but their small sizes and dense localization at the active zones have compromised the observation of pre-fusion activities. Because the sizes of the synaptic vesicles (∼50 nm) are far below the diffraction limit of the light microscopy (typically ∼200 nm), it is impossible to distinguish one from another if they are densely packed. According to Betz and Angleson (1997), it is like “looking into a bucket of tennis ball through fogged glasses”. Total internal reflection fluorescent microscopy (TIRFM, Stout and Axelrod, 1989; Steyer et al., 1997; Steyer and Almers, 2001), which restricts fluorescence excitation to a very thin layer of approximately 100 mn near the plasma membrane, is one of the exceptions which can overcome the issues and address pre-fusion vesicle dynamics by fluorescence live imaging. TIRFM have been used for a large number of interesting studies other than exocytosis, such as single molecule imaging (Yanagida and Ishii, 2017), but we focus on fusion and pre-fusion dynamics of secreting vesicles in this review.