STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis

Abstract

Synaptic transmission is mediated by neurotransmitters that are stored in synaptic vesicles and released by exocytosis upon activation. The vesicle membrane is then retrieved by endocytosis, and synaptic vesicles are regenerated and re-filled with neurotransmitter. Although many aspects of vesicle recycling are understood, the fate of the vesicles after fusion is still unclear. Do their components diffuse on the plasma membrane, or do they remain together? This question has been difficult to answer because synaptic vesicles are too small (approximately 40 nm in diameter) and too densely packed to be resolved by available fluorescence microscopes. Here we use stimulated emission depletion (STED) to reduce the focal spot area by about an order of magnitude below the diffraction limit, thereby resolving individual vesicles in the synapse. We show that synaptotagmin I, a protein resident in the vesicle membrane, remains clustered in isolated patches on the presynaptic membrane regardless of whether the nerve terminals are mildly active or intensely stimulated. This suggests that at least some vesicle constituents remain together during recycling. Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light.

Protein scaffolds in the coupling of synaptic exocytosis and endocytosis

“…These proteins are mobile and intermix with newly exocytosed synaptic vesicle proteins 44,45 . By contrast, super-resolution stimulated emission depletion (STED) microscopy (BOX 4) has suggested that newly exocytosed synaptic vesicle proteins are clustered at the plasma membrane 46 . This apparent contradiction could be explained by differences between pHluorin-tagged synaptic vesicle membrane proteins and their native endogenous counterparts 47 .…”

“…Conflicting light-microscopic data regarding the mobility and state of clustering of newly exocytosed proteins [44][45][46][47] (FIG. 4b) may be explained by a model assuming that synaptic vesicle proteins initially decluster, but then undergo rapid reclustering (FIG.…”

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

“…Sample preparation: Primary cultures of neonatal rat hippocampal neurons were prepared as previously described [32]. Synaptotagmin was selectively labeled on the cell surface via monoclonal mouse antibodies (604.2) and Atto 647N-labeled goat anti-mouse Fab fragments as described elsewhere [21].…”

“…Synaptotagmin [33,34], a key protein in exocytosis, is a major constituent of synaptic vesicles [35] and is known to form clusters after exocytosis of neurotransmitter vesicles [32]. We imaged fluorescently la- beled synaptotagmin within axons of living cultured neurons at video-rate (28 frames per second) and a spatial resolution of 65 nm.…”

3‐D stimulated emission depletion microscopy with programmable aberration correction

“…By contriving the STED beam to have a minimum intensity (preferably zero) at the centre, the point spread function (PSF) of the microscope is effectively narrowed below the diffraction limit. This is often realised by propagating the STED beam through a suitable phase retarding plate [23,24] to impart a helical phase profile and achieve a so called "doughnut" shaped PSF at the focus of the objective lens. This approach is fundamentally limited only by the available power in the depletion beam and has realised lateral resolutions down tõ 6 nm [25] imaging NV centers in polycrystalline CVD diamond.…”