title: Munc18-1 protein molecules move between membrane molecular depots distinct from vesicle docking sites.
abstract: Four evolutionarily conserved proteins are required for mammalian regulated exocytosis: three SNARE proteins, syntaxin, SNAP-25, and synaptobrevin, and the SM protein, Munc18-1. Here, using single-molecule imaging, we measured the spatial distribution of large cohorts of single Munc18-1 molecules correlated with the positions of single secretory vesicles in a functionally rescued Munc18-1-null cellular model. Munc18-1 molecules were nonrandomly distributed across the plasma membrane in a manner not directed by mode of interaction with syntaxin1, with a small mean number of molecules observed to reside under membrane resident vesicles. Surprisingly, we found that the majority of vesicles in fully secretion-competent cells had no Munc18-1 associated within distances relevant to plasma membrane-vesicle SNARE interactions. Live cell imaging of Munc18-1 molecule dynamics revealed that the density of Munc18-1 molecules at the plasma membrane anticorrelated with molecular speed, with single Munc18-1 molecules displaying directed motion between membrane hotspots enriched in syntaxin1a. Our findings demonstrate that Munc18-1 molecules move between membrane depots distinct from vesicle morphological docking sites.
authors.affiliation: Life-Physical Sciences Interface Laboratory, Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot Watt University, Edinburgh EH14 4AS, Scotland, United Kingdom.
authors.forename: Annya M
authors.initials: AM
authors.lastname: Smyth
authors.forename: Lei
authors.initials: L
authors.lastname: Yang
authors.forename: Kirsty J
authors.initials: KJ
authors.lastname: Martin
authors.forename: Charlotte
authors.initials: C
authors.lastname: Hamilton
authors.forename: Weiping
authors.initials: W
authors.lastname: Lu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cousin
authors.forename: Colin
authors.initials: C
authors.lastname: Rickman
authors.forename: Rory R
authors.initials: RR
authors.lastname: Duncan
mesh_terms: D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001703:Biophysics / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D051938:Munc18 Proteins / Q000378:metabolism*; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D050600:SNARE Proteins / Q000378:metabolism
chemical_list: D051938:Munc18 Proteins; D050600:SNARE Proteins; D049452:Green Fluorescent Proteins