Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site

Abstract

Docking of secretory vesicles at the plasma membrane is a poorly understood prerequisite for exocytosis. Current models propose raft-like clusters containing syntaxin as docking receptor, but direct evidence for this is lacking. Here we provide quantitative measurements of several exocytosis proteins (syntaxin, SNAP25, munc18, munc13 and rab3) at the insulin granule release site and show that docking coincides with rapid de novo formation of syntaxin1/munc18 clusters at the nascent docking site. Formation of such clusters prevents undocking and is not observed during failed docking attempts. Overexpression of syntaxins’ N-terminal Habc-domain competitively interferes with both cluster formation and successful docking. SNAP25 and munc13 are recruited to the docking site more than a minute later, consistent with munc13’s reported role in granule priming rather than docking. We conclude that secretory vesicles dock by inducing syntaxin1/munc18 clustering in the target membrane, and find no evidence for preformed docking receptors.

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Figure 1: Stable docking requires clustered syntaxin.
Figure 2: Exocytosis occurs only from docked granules.
Figure 3: Time course of protein binding during granule events.
Figure 4: Munc18 and rab3a colocalize with granules deeper in the cell.
Figure 5: Syntaxin Habc fragment prevents granule-associated clusters and docking.
Figure 6: Syntaxin clusters arise from short temporary binding of single molecules to the docking site.
Figure 7: Model of secretory granule docking.

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Acknowledgements

We thank W. Almers and E. Gylfe for equipment; E. Gylfe, P. Rorsman, J. Taraska and R. Chandler for comments on earlier versions of the manuscript; H. Mulder for cells; J. Rettig, W. Almers, R. Burgoyne and Q. Low for plasmids; and J. Saras for technical assistance. The work was supported by the Swedish Science Council, Swedish Diabetes Society, European Foundation for the Study of Diabetes, Diabetes Wellness Network Sweden, Barndiabetesfonden, and the foundations of NovoNordisk-, Göran Gustafsson-, Family Ernfors-, Zetterlings- and OE&E Johanssons.

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N.R.G. and S.B. performed experiments and analysis. S.B. wrote the paper.

Correspondence to Sebastian Barg.

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