Imaging the post-fusion release and capture of a vesicle membrane protein

Abstract

The molecular mechanism responsible for capturing, sorting, and retrieving vesicle membrane proteins following triggered exocytosis is not understood. Here we image the post-fusion release and then capture of a vesicle membrane protein, the vesicular acetylcholine transporter, from single vesicles in living neuroendocrine cells. We combine these measurements with super-resolution interferometric photo-activation localization microscopy (iPALM), electron microscopy, and modeling to map the nanometer-scale topography and architecture of the structures responsible for the transporter’s capture following exocytosis. We show that after exocytosis, the transporter rapidly diffuses into the plasma membrane, but most travels only a short distance before it is locally captured over a dense network of membrane-resident clathrin-coated structures. We propose that the extreme density of these structures acts as a short-range diffusion trap. They quickly sequester diffusing vesicle material and limit its spread across the membrane. This system could provide a means for clathrin-mediated endocytosis to quickly recycle vesicle proteins in highly excitable cells.

Exocytosis and Endocytosis: Modes, Functions, and Coupling Mechanisms

“…These results suggest that the endocytosis trigger takes place at or near the release site, challenging a seemingly established principle that endocytosis takes place at the periactive zone (6, 69). Consistent with this suggestion, FM dye uptake at snake neuromuscular junctions is near the active zone (86), a retrievable vesicle pool at the plasma membrane of hippocampal boutons was implied to be within ~100–300 nm of the active zone (111), clathrin may be located at Drosophila active zones (112), and endocytic sites are within 500 nm of exocytic sites in PC12 cells (137). …”

Correlative super-resolution fluorescence and metal-replica transmission electron microscopy

“…Cells were imaged by total internal reflection fluorescence (TIRF) in imaging buffer as described previously 22 . Each image shown is an average of five subsequent 100 ms frames each 500 ms apart.…”

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

“…With the advent of superresolution microscopy (SRM), including techniques based on single molecule localization microscopy (SMLM), visualization of biology at spatial resolutions on the order of 10–20 nm has revealed exciting, new biological phenomena previously invisible to diffraction-limited light microscopy10111213. SMLM has been implemented in two (2D) and three dimensions (3D)141516 and has also been extended to tissue samples, where frozen brain sections were examined using stochastic optical reconstruction microscopy (STORM)17, a common form of SMLM.…”