doi: 10.1371/journal.pbio.0050198.
Open syntaxin docks synaptic vesicles
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- PMID:17645391
- PMCID: PMC1914072
- DOI: 10.1371/journal.pbio.0050198
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Open syntaxin docks synaptic vesicles
Marc Hammarlund et al. PLoS Biol.2007 Aug.
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Abstract
Synaptic vesicles dock to the plasma membrane at synapses to facilitate rapid exocytosis. Docking was originally proposed to require the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins; however, perturbation studies suggested that docking was independent of the SNARE proteins. We now find that the SNARE protein syntaxin is required for docking of all vesicles at synapses in the nematode Caenorhabditis elegans. The active zone protein UNC-13, which interacts with syntaxin, is also required for docking in the active zone. The docking defects in unc-13 mutants can be fully rescued by overexpressing a constitutively open form of syntaxin, but not by wild-type syntaxin. These experiments support a model for docking in which UNC-13 converts syntaxin from the closed to the open state, and open syntaxin acts directly in docking vesicles to the plasma membrane. These data provide a molecular basis for synaptic vesicle docking.
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Figures
A neuromuscular junction from a VB acetylcholine motor neuron in a wild-type adult is shown. The center of the synapse is marked by the dense projection. Three distinct morphological classes of vesicles are visible: docked, within 30 nm, and cytoplasmic. Image was acquired at 150 K magnification.
(A) Shown is a sample electron micrograph from a wild-type animal showing a single VA motor neuron profile in the ventral nerve cord. This profile contains a dense projection (DP), shown in red, which is divided in two in this profile. The membrane region within 231 nm of the dense projection (active zone) is shown in orange, the region from 232–330 nm (VFZ, vesicle-free zone) is light gray, and the region farther than 330 nm from the dense projection (perisynaptic zone) is amber. Yellow, docked synaptic vesicles. MT, mitochondria. The linear distance from the edge of each docked vesicle to the edge of the dense projection was measured (d1). (B) Shown is a reconstruction of a sample VA motor neuron synapse from 38 serial electron micrographs. The plasma membrane (light blue), dense projection (red), and docked vesicles (yellow) are shown. Two single sections are represented by blue and green bands. The green section contains the dense projection and is the image shown in (A). The blue section illustrates the calculation of the distance d2 for docked vesicles in sections without a dense projection. This distance was calculated from the number of 33-nm sections between the vesicle and the dense projection (a) and the radial distance from an imaginary extension of the dense projection to the vesicle (b). (C) Distribution of all docked vesicles in sections containing a dense projection for acetylcholine and GABA neurons. Distances were sorted into 33-nm bins, and the number of measurements in each bin divided by the total number of sections. Data in this analysis are from four wild-type animals comprising 30 synapses and 140 profiles (acetylcholine) and 21 synapses and 100 profiles (GABA). As in (A) the active zone is shown in orange, the region from 232–330 nm (vesicle-free zone) is light gray, and the region farther than 330 nm from the dense projection (perisynaptic zone) is amber. (D) Shown is the distribution of all vesicles in reconstructed synapses for acetylcholine and GABA neurons. Distances were calculated as described above and sorted into 33-nm bins with respect to the distance from the dense projection. For those vesicles in sections not containing dense projections sorting was done based on a tube model of the synapse. Specifically, vesicles were considered in the active zone pool if they were docked within an imaginary stripe extending the length of the tube of a width of 231 nm on either side of the dense projection; these vesicles are indicated in orange. All vesicles not in this stripe, that is those on the far side of the tube, were considered in the perisynaptic zone; these vesicles are indicated in amber. Data in this analysis are from two wild-type animals comprising 11 synapses and 136 profiles (acetylcholine) and nine synapses and 168 profiles (GABA).
Each histogram shows two distributions: with the wild-type syntaxin allele (light bars) or with syntaxin overexpression (open or wild type, dark bars). Each bar graph shows a comparison of the number of docked vesicles between genotypes in the active zone pool (< 232 nm, orange bars) and the perisynaptic pool (> 300 nm, amber bars). To generate these pools, the number of docked vesicles in each pool was divided by the number of profiles to give a mean value for the number of vesicles in each pool per profile; bars show mean and standard error of mean. The green lines in the bar graphs in (B) and (D) show the number of vesicles in each pool in a matched wild-type control. All experiments that overexpress wild type or open syntaxin are in a syntaxin null genetic background (see Materials and Methods for complete genotypes). Only vesicles in profiles containing a dense projection were included in these analyses. See Table S1 for numbers and statistical analysis. (A) Expression of open instead of wild-type syntaxin does not affect the distribution of docked vesicles. Distribution of docked vesicles in the wild type (N2) and in open syntaxin overexpression in acetylcholine (left) or GABA synapses (right) is shown. For wild-type acetylcholinen = 1 animal, seven synapses, and 35 profiles; foropen-syntaxin acetylcholinen = 2 animals, ten synapses, and 57 profiles; foropen-syntaxin GABAn = 1 animal, four synapses, and 28 profiles; and for wild-type GABAn = 2 animals, seven synapses, and 49 profiles. (B) Open syntaxin rescues the docking defect in the active zone inunc-13(s69). Green lines indicate docked vesicles in the matched wild-type fixation. Forunc-13 acetylcholinen = 2 animals, eight synapses, and 34 profiles; forunc-13 open syntaxin acetylcholinen = 2 animals, ten synapses, and 55 profiles; forunc-13 GABAn = 2 animals, seven synapses, and 33 profiles; forunc-13 open-syntaxin GABA,n = 2 animals, eight synapses, and 47 profiles. (C) Overexpression of wild-type syntaxin does not affect the distribution of docked vesicles. For wild-type acetylcholinen = 1 animal, five synapses, and 21 profiles; forsyntaxin-OE acetylcholinen = 1 animal, five synapses, and 19 profiles; for wild-type GABAn = 1 animal, four synapses, and 17 profiles; forsyntaxin-OE GABAn = 1 animal, four synapses, and 12 profiles. (D) Overexpression of wild-type syntaxin does not rescue the docking defect in the active zone pool inunc-13(s69). Green lines indicate docked vesicles in the matched wild-type fixation. Forunc-13(s69) acetylcholinen = 1 animal, five synapses, and 17 profiles; forunc-13(s69) syntaxin-OE acetylcholinen = 1 animal, five synapses, and 19 profiles; forunc-13(s69) GABAn = 1 animal, five synapses, and 19 profiles; forunc-13(s69) syntaxin-OE GABAn = 1 animal, four synapses, and 19 profiles.
(A) GABA neuron development is presented. Sample images of wild type and syntaxin mosaic (EG3278) animals expressing GFP in the GABA neurons are shown. Right, anterior; top, dorsal. In both genotypes, commissures extend at regular intervals from the ventral to the dorsal nerve cord. The bright spots along the ventral nerve cord in the wild type are cell bodies. Cell bodies are also visible in the syntaxin mosaic, as are the larger coelomocytes, which express GFP as a marker for the syntaxin mosaic array. The number of commissures between the dorsal and ventral nerve cords is normal in syntaxin mosaic animals.n = 10 adults for each genotype. (B) Pre- and postsynaptic development is presented. Sample images are shown of the dorsal nerve cord of wild-type and syntaxin mosaic (EG3278) animals coexpressing a presynaptic marker (SNB-CFP, synaptobrevin-CFP,) and a postsynaptic marker (GABAA receptor-YFP). SNB-CFP is expressed in GABA neurons, which lack syntaxin in the mosaic animals. Normal colocalization was observed in both genotypes (n = 10 adults for each genotype). (C) The postsynaptic receptor field is presented. The postsynaptic response to exogenous GABA is normal in the syntaxin mosaic animals (EG3817) that lack syntaxin in the GABA neurons. Sample traces are shown on the left, and mean and standard error of mean data are shown on the right (n = 4 for each for each genotype). (D) EM reconstruction of the nerve cord in syntaxin mosaic animals (EG3817) lacking syntaxin in the GABA motor neurons is presented. The number and distribution of presynaptic specializations and of synaptic vesicle number is normal in syntaxin(−) neurons. The line graphs show the number of vesicles in each serial profile for the wild type (top) and the syntaxin mosaic (EG3817, bottom). Three profiles are presented on each graph: VD, blue; VA, brown; and VB, orange. Profiles containing a dense projection are indicated by a shaded bar of the corresponding color. The distribution of the dense projections in GABA syntaxin(−) neurons is similar to the wild-type pattern, with inhibitory and excitatory synapses alternating along the length of the nerve cord. Reconstructions are from 201 serial sections for the wild type and 199 serial sections for the mosaic strain.
Shown is endogenous activity in (A) the wild type, (B) syntaxin mosaic EG3278, and (C) syntaxin mosaic EG3817 before and after the addition ofd -tubocurare (dTBC). EG3278 lacks syntaxin in both the acetylcholine and GABA neurons, while EG3817 lacks syntaxin in the GABA neurons. Before the addition ofd -tubocurare, activity represents input from acetylcholine and GABA neurons. (D) Before the addition ofd -tubocurare, wild-type animals exhibited 42.8 ± 6.5 fusions per second, EG3278 animals 0.02 ± 0.01 fusions per second (p < 0.0001), and EG3817 animals 4.3 ± 1.1 (p = 0.0016). After the addition ofd -tubocurare, activity represents input from GABA neurons only. Under these conditions, wild-type animals exhibited 28.5 ± 4.9 fusions per second, EG3278 animals 0.00 ± 0.00 fusions per second (p < 0.0001), and EG3817 0.06 ± 0.03 (p < 0.0001).d -tubocurare blocks all acetylcholine minis, since the drug blocks all minis inunc-49(e407) mutants, which lack the GABAA receptor [69] (21.0 ± 5.8 fusions per second before treatment and 0.0 ± 0.0 fusions per second after treatment). Recordings were performed in 5 mM Ca2+;n = 8 for the wild type,n = 2 for EG3278, andn = 5 for EG3817. Scale bars in photographs, 200 μm.
Each row shows two comparisons: total docked vesicles and the distribution of docked vesicles. For total docked vesicles (left), the mean number of docked vesicles per profile was calculated for each synapse (see Table S1 for complete methods and results). Bars show mean and standard error of the mean; *,p-values < 0.001. For vesicle distributions (right), the distance from the dense projection to each docked vesicle was determined, and these measurements were sorted into 33-nm bins. The number of vesicles in each bin was divided by the number of profiles to yield an average number of vesicles per profile in each bin. For both comparisons, only vesicles in profiles containing a dense projection were included. (A) Shown is a comparison of acetylcholine neurons in wild-type animals and mosaic animals with reduced syntaxin in the GABA and acetylcholine neurons (EG3278). Wild typen = 1 animal, five synapses, and 20 profiles and mosaicn = 1 animal, five synapses, and 24 profiles. (B) Shown is a comparison of GABA neurons in wild-type animals and mosaic animals with reduced syntaxin in the GABA and acetylcholine neurons (EG3278). Wild typen = 1 animal, four synapses, and 16 profiles and mosaicn = 1 animal, four synapses, and 22 profiles. (C) Shown is a comparison of acetylcholine neurons in wild-type animals and mosaic animals lacking syntaxin in the GABA neurons (EG3817). Wild typen = 2 animals, ten synapses, and 53 profiles and mosaicn = 2 animals, 11 synapses, and 66 profiles. (D) Shown is a comparison of GABA neurons in wild-type animals and mosaic animals (EG3817). Wild typen = 2 animals, eight synapses, and 38 profiles and mosaicn = 2 animals, ten synapses, and 51 profiles.
The average number of synaptic vesicles in single profiles containing a dense projection for each genotype is shown. Both undocked and docked vesicles were included in this analysis. Left, total vesicles for acetylcholine synaptic profiles; right, total vesicles for GABA synaptic profiles (Table S1 for complete list ofp-values). Bars show mean and standard error of the mean; *,p-values < 0.001 compared to wild type. Note that there is an unusually large increase in vesicle number at the GABA synapses of one of the mosaic syntaxin strains (EG3817). This increase is not observed at synapses of other syntaxin(−) genotypes.
(A and B) Docked vesicles in the wild type andunc-13(s69) are presented. Only vesicles in axon profiles containing a dense projection were included in these analyses. (A) Docked vesicles from acetylcholine and GABA synapses combined are shown. Docking in the active zone pool (orange) is greatly reduced while docking in the perisynaptic pool (amber) is increased inunc-13(s69) mutants. (B) Distribution of docked vesicles in the wild type andunc-13(s69) in acetylcholine (left) or GABA synapses (right) is shown. For wild-type acetylcholinen = 2 animals, 13 synapses, and 52 profiles; forunc-13 acetylcholinen = 2 animals, eight synapses, and 34 profiles; for wild-type GABAn = 2 animals, nine synapses, and 42 profiles; and forunc-13 GABAn = 2 animals, seven synapses, and 33 profiles. (C) Distribution of docked vesicles in the wild type andunc-13(e1091) in acetylcholine (left) or GABA synapses (right) is shown. For wild-type acetylcholinen = 1 animal, seven synapses, and 35 profiles; forunc-13 acetylcholinen = 1 animal, seven synapses, and 33 profiles; for wild-type GABAn = 1 animal, four synapses, and 20 profiles; and forunc-13 GABAn = 1 animal, four synapses, and 28 profiles.
All experiments that overexpress wild type or open syntaxin are in a syntaxin null genetic background (see Materials and Methods). Error bars represent standard error of mean in all cases. (A–D) Open syntaxin bypasses evoked response defects ofunc-13 mutants. All traces for wild type,unc-13 open-syntaxin, andunc-13 evoked responses (left) are shown (A) at 5 mM calcium and (B) and at 1 mM calcium. (C) The mean peak amplitude for evoked responses is: at 5 mM calcium (wild type 2.0 ± 0.2 nA,n = 7;unc-13 open-syntaxin 0.8 ± 0.1 nA,n = 7;unc-13 0.015 ± 0.004 nA,n = 9) and at 1 mM calcium (wild type 1.7 ± 0.2 nA,n = 6;unc-13 open-syntaxin 0.25 ± 0.04 nA,n = 6;unc-13 0.005 ± 0.003 nA,n = 8). (D) Mean charge transfer evoked is: at 5 mM calcium (wild type 21.0 ± 3.4 pC,n = 7;unc-13 open-syntaxin 7.4 ± 1.3 pC,n = 7;unc-13 0.22 ± 0.04 pC,n = 9) and at 1 mM calcium (wild type 14.6 ± 2.0 pC,n = 6;unc-13 open-syntaxin 2.4 ± 0.4 pC,n = 6;unc-13 0.10 ± 0.03 pC,n = 8). (E) Evoked responses are delayed and asynchronous inunc-13 open-syntaxin animals. The cumulative plot for the fraction of total charge transfer as a function of time after the beginning of the stimulus artifact is shown. Individual points represent average cumulative current transfer at 2-ms intervals for each genotype.unc-13 open-syntaxin animals (in red) show a delay in release during evoked response at 1 mM calcium. (A and B) (F and G) Open syntaxin restores endogenous release (minis) inunc-13 mutants. (A) Right, representative traces of endogenous activity are shown in the wild type,unc-13 open-syntaxin, andunc-13 at 5 mM calcium. (F) Left, mean mini frequency at 5 mM calcium is shown (wild type 54.5 ± 6.0 fusions per second,n = 6;unc-13 open-syntaxin 14.4 ± 3.3 fusions per second,n = 6;unc-13 0.6 ± 0.1 fusions per second,n = 9). Right, mean mini amplitude is not altered in mutants (wild type 37.1 ± 6.3 pA,n = 6;unc-13 open-syntaxin 29.7 ± 3.1 pA,n = 6;unc-13 34.0 ± 2.9 pA,n = 9). (B) Right, representative traces of endogenous activity in the wild type,unc-13 open-syntaxin, andunc-13 at 1 mM calcium. (G) Left, mean mini frequency at 1 mM calcium (wild type 41.1 ± 4.8 fusions per second,n = 12);unc-13 open-syntaxin 7.9 ± 1.4 fusions per second,n = 13;unc-13 0.3 ± 0.1 fusions per secondn = 8). Right, mean mini amplitude is not altered in mutants (wild type 37.5 ± 3.3 pA,n = 6;unc-13 open-syntaxin 29.5 ± 2.7 pA,n = 6;unc-13 24.1 ± 3.8 pA,n = 9).
References
- Südhof TC. The synaptic vesicle cycle: A cascade of protein-protein interactions. Nature. 1995;375:645–653. - PubMed
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