Preferred Sites of Exocytosis and Endocytosis Colocalize during High- But Not Lower-Frequency Stimulation in Mouse Motor Nerve Terminals

Abstract

The spatial relationship of exocytosis and endocytosis in motor nerve terminals has been explored, with varied results, mostly in fixed preparations and without direct information on the utilization of each exocytic site. We sought to determine these spatial properties in real time using synaptopHluorin (spH) and FM4-64. Earlier we showed that nerve stimulation elicits the appearance of spH fluorescence hot spots, which mark preferred sites of exocytosis. Here we show that nerve stimulation in the presence of the styryl dye FM4-64 evokes hot spots of FM4-64 fluorescence. Their size, density, and rate of appearance are similar to the spH hot spots, but their rate of disappearance after stimulation was much slower (t 1/2 ϳ9 min vs ϳ10 s for spH hot spots), consistent with FM4-64 spots identifying bulk endocytosis and subsequent slow intracellular dispersion of nascent vesicles. Simultaneous imaging of both fluorophores revealed a strong colocalization of spH and FM4-64 spots, but only during high (100 Hz) stimulation. At 40 Hz stimulation, exocytic and endocytic spots did not colocalize. Our results are consistent with the hypothesis that hot spots of endocytosis, possibly in the form of bulk uptake, occur at or very near highly active exocytic sites during high-frequency stimulation.

Live imaging of bulk endocytosis in frog motor nerve terminals using FM dyes

“…If compound exocytosis occurs, FM dye and spH signals should appear simultaneously, at the same site. Instead, FM spots and spH spots did not perfectly colocalize (Gaffield et al 2009a), suggesting that at least some, and possibly all, of the FM spots do not reflect compound exocytosis.…”

“…Recently, we used low concentrations of FM dye to identify the locations of prominent endocytosis in mouse motor nerve terminals during high-frequency stimulation (Gaffield et al 2009a). Here, we extended this technique to frog motor nerve terminals, where we identified and characterized fluorescent structures, similar to tubule-like cisternae in appearance, that formed near sites of synaptic vesicle clusters during nerve stimulation.…”

Why does endocytosis in single cells care which side up?

“…It has been observed previously that clathrindependent endocytosis is polarized at the cell leading edge in migrating cells. 10 Interestingly, accumulations of endocytic sites or "hot spots" of endocytosis are found in a variety of specialized cells such as neurons and hepatocytes 11,12 We studied the distribution of Tfn and EGF, two ligands of receptor-mediated endocytosis, one-minute after their addition to cells using "density estimation" that allows quantification of spatial organization. We made the unexpected observation that ligands were not distributed equally all around the cell but concentrated at different sites of the cell during uptake.…”

Fluorescent AM1-43 and FM1-43 probes for dental sensory nerves and cells: Their labeling mechanisms and applications

“…Since the pioneering work by Betz et al [1] there have been a number of reports of FMI-43 labeling of cells via endocytosis of labeled membranes including labeling of endocytotic recycling synaptic vesicles of motor nerve fibers and endocytotic labeling of nerve fibers and terminals, lateral line hair cells, retinal photoreceptor cells, and even plant cells by FM1-43 and related dyes [2][3][4]6,8,9,55]. A review has also been published on FM1-43 labeling of Drosophila neuromuscular junctions via an endocytotic pathway [5].…”

“…A variety of cells and tissues are labeled with FM1-43 and AM1-43 FM1-43 and related fluorescent dyes have been reported to label a motor nerve terminal [1,4], Drosophila neuromuscular junctions [5], pituitary lactotroph granules [6], intestinal brush borders [7], cultured hippocampal neurons [8], photoreceptor cells of the retina [9], and plant cells [10] as results of endocytosis. FM1-43 and related fluorescent dyes have also Summary This mini-review first introduces the fluorescent styryl dyes, AM1-43 and FM1-43, which stain a variety of sensory cells and tissues including sensory nerve fibers.…”