Dissecting docking and tethering of secretory vesicles at the target membrane.
Secretory vesicles dock at their target in preparation for fusion. Using single-vesicle total internal reflection fluorescence microscopy in chromaffin cells, we show that most approaching vesicles dock only transiently, but that some are captured by at least two different tethering modes, weak and strong. Both vesicle delivery and tethering depend on Munc18-1, a known docking factor. By decreasing the amount of cortical actin by Latrunculin A application, morphological docking can be restored artificially in docking-deficient munc18-1 null cells, but neither strong tethering nor fusion, demonstrating that morphological docking is not sufficient for secretion. Deletion of the t-SNARE and Munc18-1 binding partner syntaxin, but not the v-SNARE synaptobrevin/VAMP, also reduces strong tethering and fusion. We conclude that docking vesicles either undock immediately or are captured by minimal tethering machinery and converted in a munc18-1/syntaxin-dependent, strongly tethered, fusion-competent state.
Toonen
Ruud F
RF
Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam (VUA) and VU Medical Center (VUmc), Amsterdam, The Netherlands.
Kochubey
Olexiy
O
de Wit
Heidi
H
Gulyas-Kovacs
Attila
A
Konijnenburg
Bas
B
Sørensen
Jakob B
JB
Klingauf
Jurgen
J
Verhage
Matthijs
M
D000199:Actins / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000502:physiology* / Q000648:ultrastructure; D005089:Exocytosis / Q000502:physiology*; D018628:Gene Dosage / Q000502:physiology; D008561:Membrane Fusion / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D051938:Munc18 Proteins / Q000235:genetics / Q000378:metabolism*; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism; D050683:R-SNARE Proteins / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism; D022142:Secretory Vesicles / Q000502:physiology* / Q000648:ultrastructure
D000199:Actins; D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C497938:Stxbp1 protein, mouse