ABSTRACT Sec1-related proteins function in most, if not all, membrane trafficking pathways in eukaryotic cells. The Sec1-related protein required in neurons for synaptic vesicle exocytosis

is UNC-18. Several models for UNC-18 function during vesicle exocytosis are under consideration. We have tested these models by characterizing _unc-18_ mutants of the nematode

_Caenorhabditis elegans_. In the absence of UNC-18, the size of the readily releasable pool is severely reduced. Our results show that the near absence of fusion-competent vesicles is not

caused by a reduction in syntaxin levels, by a mislocalization of syntaxin, by a defect in fusion or by a failure to open syntaxin during priming. Rather, we found a reduction of docked

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ACKNOWLEDGEMENTS We thank J. Rand for providing _unc-18(md299)_ and antisera to UNC-18, M. Bastiani for providing the confocal microscope, the _Caenorhabditis_ Genetics Center for providing

_unc-18(e234)_ and _unc-18(e81)_, E. Maryon for providing the _Pt09a9.10:gfp_ reporter construct, and J.-L. Bessereau, P. Rostaing and A. Triller for assistance with developing

immuno-electron microscopy techniques. This work was supported by National Institutes of Health grants to J.E.R. and E.M.J., a US Public Health Service grant to M.L.N., and an INSERM _Poste

Vert_ grant to R.M.W. AUTHOR INFORMATION Author notes * Robby M Weimer Present address: École Normale Supérieure, INSERM U497, 75005, Paris, France * Robby M Weimer and Janet E Richmond:

These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, 84112-0840, Utah, USA Robby M

Weimer, Warren S Davis & Erik M Jorgensen * Department of Biological Sciences, University of Illinois at Chicago, Chicago, 606067, Illinois, USA Janet E Richmond * Department of Anatomy

and Neurobiology, Washington University School of Medicine, Saint Louis, 63110, Missouri, USA Gayla Hadwiger & Michael L Nonet Authors * Robby M Weimer View author publications You can

also search for this author inPubMed Google Scholar * Janet E Richmond View author publications You can also search for this author inPubMed Google Scholar * Warren S Davis View author

publications You can also search for this author inPubMed Google Scholar * Gayla Hadwiger View author publications You can also search for this author inPubMed Google Scholar * Michael L

Nonet View author publications You can also search for this author inPubMed Google Scholar * Erik M Jorgensen View author publications You can also search for this author inPubMed Google

Scholar CORRESPONDING AUTHOR Correspondence to Erik M Jorgensen. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION

SUPPLEMENTARY FIG. 1. The size of the readily releasable pool is reduced in _unc-18(md299)_ mutants as assayed by application of hyperosmotic saline in 0 mM external calcium. The total

number of miniature postsynaptic currents were measured during a 1.5 second application of hyperosmotic saline in 0 mM Ca2+ in the presence of 5 mM EGTA and the total quanta released plotted

as mean ± SEM (_n_ = 4 and 3 for wild-type and _unc-18(md299),_ respectively). The baseline deflection observed after application of hyperosmotic saline is due to current leak in the

muscle. (PDF 82 kb) SUPPLEMENTARY FIG. 2. Syntaxin is not mislocalized from axonal plasma membrane in the absence of UNC-18. (A and B) Electron micrographs of neuromuscular junctions from

wild-type and _unc-18(e234)_ animals stained with anti-syntaxin antibody a939 (ref. 1 below) then 15nm colloidal gold conjugated secondary antibody. In each panel, a column of micrographs

represent sections in series; the closed arrowheads indicate a synapse, the open arrowheads label gold particles and the bar equals 500nm. For immunoEM analysis, Bristol N2 and

EN666[_unc-18(e234)_] hermaphrodites were prepared for post-embedded immunolabeling using a protocol adapted from studies of other invertebrates (ref. 2 below). Specifically, young adults

were immobilized in _Escherichia coli_ by high pressure freeze, fixed in anhydrous methanol with 4% paraformaldehyde and 1.5% uranyl acetate at -90°C, infiltrated at -45°C with lowicryl HM20

(Polyscience Inc.) which was then polymerized by UV exposure. Ultrathin sections (~40nm) were collected, incubated with the a939 antibody, labeled with gold colloidal suspensions of 15nm

diameter coupled to GAR-IgG (BBInternational) following the manufactures recommendations (Aurion) and then viewed with a Philips CM20 electron microscope. (C) The anti-syntaxin antibody

localized gold particles to axonal processes of wild-type and _unc-18(e234)_ animals. For both strains, the bead density was calculated in each section by dividing the number of beads inside

or outside the nerve cord by the area of the nerve cord or that of the surrounding tissue, and then plotted as the average density (10-6 beads/nm2) ± SEM (wild-type, inside 2.1 ± 0.2,

outside 0.4 ± 0.04 n = 48 sections; _unc-18(e234)_, inside 1.4 ± 0.1, outside 0.3 ± 0.03 _n_ = 28 sections). (D) More specifically, the anti-syntaxin antibody localized gold particles to the

plasma membrane of wild-type and unc-18(e234) neurons. The distance of gold particles from the plasma membrane within neurons was measured then plotted as the average distance ± SEM

(wild-type, 15.7 ± 1.7nm _n_ = 277 beads; _unc-18(e234)_, 19.8 ± 2.7nm _n_ = 195 beads). The theoretical distance between the gold particle coupled to a secondary antibody and the antigen

recognized by a primary antibody is roughly 30nm. Thus, the accumulation of gold particles within 30nm of the plasma membrane in _unc-18(e234)_ demonstrates that syntaxin is localized to the

plasma membrane in the absence of UNC-18. References: 1. Saifee, O., Wei, L. & Nonet, M. L. The _Caenorhabditis elegans unc-64_ locus encodes a syntaxin that interacts genetically with

synaptobrevin. _Mol. Biol. Cell_ 9, 1235-1252 (1998). 2. McDonald, K. High-pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabeling. _Methods

Mol. Biol._ 117, 77-97 (1999). (PDF 236 kb) SUPPLEMENTARY METHODS (PDF 12 KB) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Weimer, R., Richmond, J.,

Davis, W. _et al._ Defects in synaptic vesicle docking in _unc-18_ mutants. _Nat Neurosci_ 6, 1023–1030 (2003). https://doi.org/10.1038/nn1118 Download citation * Received: 04 June 2003 *

Accepted: 15 July 2003 * Published: 14 September 2003 * Issue Date: 01 October 2003 * DOI: https://doi.org/10.1038/nn1118 SHARE THIS ARTICLE Anyone you share the following link with will be

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