title: 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 and electron microscopy, and modelling 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.
authors.affiliation: Laboratory of Molecular Biophysics, National Heart Lung and Blood Institute, National Institutes of Health, 50 South Drive, Bethesda, Maryland 20892, USA.
authors.forename: Kem A
authors.initials: KA
authors.lastname: Sochacki
authors.forename: Ben T
authors.initials: BT
authors.lastname: Larson
authors.forename: Deepali C
authors.initials: DC
authors.lastname: Sengupta
authors.forename: Mathew P
authors.initials: MP
authors.lastname: Daniels
authors.forename: Gleb
authors.initials: G
authors.lastname: Shtengel
authors.forename: Harald F
authors.initials: HF
authors.lastname: Hess
authors.forename: Justin W
authors.initials: JW
authors.lastname: Taraska
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D002966:Clathrin / Q000502:physiology / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology*; D008565:Membrane Proteins / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008857:Microscopy, Interference / Q000379:methods; D016716:PC12 Cells / Q000502:physiology; D051381:Rats; D013572:Synaptic Vesicles / Q000502:physiology / Q000648:ultrastructure; D050494:Vesicular Acetylcholine Transport Proteins / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D002966:Clathrin; D008565:Membrane Proteins; D050494:Vesicular Acetylcholine Transport Proteins