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  • The EMBO Journal (2006) 25, 3725 - 3737
  • doi:10.1038/sj.emboj.7601256

Published online: 10 August 2006

Dissecting docking and tethering of secretory vesicles at the target membrane

Ruud F Toonen1,a, Olexiy Kochubey2,ab, Heidi de Wit1,a, Attila Gulyas-Kovacs2,ac, Bas Konijnenburg1, Jakob B Sørensen2, Jurgen Klingauf2 and Matthijs Verhage1

  1. Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam (VUA) and VU Medical Center (VUmc), Amsterdam, The Netherlands
  2. Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

Correspondence to:

Jakob B Sørensen, Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. Tel.: +49 551 201 1297; Fax: +49 551 201 1688; E-mail: jsoeren@gwdg.de

Jurgen Klingauf, Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. Tel.: +49 551 201 1629; Fax: +49 551 201 1688; E-mail: J.Klingauf@mpi-bpc.mpg.de

Matthijs Verhage, Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit (VU) Amsterdam and VU Medical Center (VUmc), De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands. Tel: +31 20 598 6936; Fax: +31 20 598 6926; E-mail: matthijs@cncr.vu.nl

aThese authors contributed equally to this work

bPresent address: Laboratory of Synaptic Mechanisms, Brain Mind Institute, EPFL, Station 15, 1015, Lausanne, Switzerland

cPresent address: Laboratory of Cardiac/Membrane Physiology, Rockefeller University, 1230 York Avenue, NY 10021, USA

Received 26 April 2006; Accepted 4 July 2006

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.

  • Keywords:

    • chromaffin,
    • docking,
    • exocytosis,
    • Munc18-1,
    • SNARE