br Application of TIRFM to visualizing exocytosis TIRFM
Application of TIRFM to visualizing exocytosis TIRFM was first used to visualize exocytosis from large secretory granules from chromaffin hippo signaling (Steyer et al., 1997). Before that, visualization of secretory organelle was strictly limited to a few exceptions, such as mast cell of mutant mice, or sea urchin eggs, where granules have exceptionally large sizes (Breckenridge and Almers, 1987; Whalley et al., 1995). Fluorescence labeling of synaptic vesicles has been introduced by Betz and Bewick (1992) using FM 1-43. It is an excellent marker for synaptic vesicles, and its application revealed how synaptic vesicles are recycled for a next round of exocysosis (Betz and Bewick, 1992), as well as kinetics of net amount of exocytosis and endocytosis (Ryan et al., 1993; Lagnado et al., 1996; Smith and Betz, 1996). Steyer et al. (1997) loaded granules with fluorescent dye acridine orange, which diffuses across plasma membranes and is trapped in acidic compartment such as secretory granules, and saw them spread out and vanish upon stimulations. Granules of chromaffin cells show a typical size for the cells which release hormones (∼300 nm), and this work opened up the ways to investigate secretory mechanisms of a variety of important molecules from endocrine cells, such as insulin from pancreatic beta cells (Tsuboi et al., 2000; Ohara-Imaizumi et al., 2002; Obermüller et al., 2005). Another advantage of using endocrine cells is its affinity with molecular manipulations. There, one can label the target protein with a fluorescence protein using molecular techniques such as electroporation or virus infection, and dynamics or distributions of many exocytosis-related proteins have been revealed (Merrifield et al., 2002; Sieber et al., 2007; An et al., 2010; Gandasi and Barg, 2014). Shortly after the first chromaffin cell study (Steyer et al., 1997), TIRFM was applied to visualize fusion and pre-fusion dynamics of much smaller (∼30 nm) synaptic vesicles at the goldfish retinal bipolar cells by the same group (Zenisek et al., 2000). Because the synaptic vesicles are packed together tightly at the active zones, TIRFM alone is not enough to resolve individual vesicles. They labeled only a very small fraction of vesicles with non-membrane permeable fluorescent dye, FM 1-43 (Betz and Bewick, 1992), which is inserted into the outer leaflet of the cell membrane. After exocytosis, the membrane of synaptic vesicles is retrieved by endocytosis, and there is some delay between exocytosis and endocytosis (Saheki and De Camilli, 2012). Thus, if the terminal in FM-dye containing solution is stimulated weakly to trigger only a small fraction of synaptic vesicles for exocytosis, they are loaded with FM-dye during their brief stay at the cell surface, and retrieved into the terminal via endocytosis. After washout of the surface FM-dye, the only remaining fluorescent objects are a small fraction of FM-loaded synaptic vesicles which go through a exo-endocytosis cycle during the loading period. At the bipolar cell, only one percent or less of the vesicles were labeled to resolve single synaptic vesicles by TIRFM. Zenisek et al. (2000) visualize that when a FM-labeled vesicle enters into evanescent field, its brightness increases as it moves toward the plasma membrane, and when exocytosis occurs, the fluorescence is incorporated into the plasma membrane. Visualization of individual FM-labeled vesicles at the retinal photoreceptor or bipolar cell by TIRFM was followed by several other groups (Midorikawa et al., 2007; Chen et al., 2013), and these studies have revealed that at least half of the released vesicles were “newcomers” that were continuously recruited to active zones during stimulation and fused with the plasma membrane shortly after tethering. This mechanism may be an efficient way of supplying vesicles for a synapse that operates by graded potentials (Werblin and Dowling, 1969). However, it has been still remained to be elucidated how synaptic vesicles behave before exocytosis at conventional spiking neurons where the timing of exocytosis is synchronized to the incoming action potential within a ms range (Schneggenburger and Rosenmund, 2015).