Research ArticleCELLULAR NEUROSCIENCE

Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones

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Science Advances  15 Apr 2020:
Vol. 6, no. 16, eaay7193
DOI: 10.1126/sciadv.aay7193
  • Fig. 1 Super-resolution (dSTORM) imaging reveals the nanoscale organization of mGluR4 at parallel fiber AZs.

    (A) Schematic view of the organization of the mouse cerebellum, showing the ordered arrangement of the parallel fibers, which originate from granule cells and form dense synapses with the dendritic spines of Purkinje cells. mGluR4s located on the presynaptic membrane of parallel fiber synapses regulate synaptic transmission by inhibiting the release of glutamate (yellow box). Two different planes were used for cutting the cerebellum: coronal (blue; parallel to parallel fibers) and parasagittal (red; perpendicular to parallel fibers). (B) Two-color dSTORM imaging of mGluR4 (magenta) and bassoon (green). An image of a coronal section acquired in a region corresponding to the molecular layer of the cerebellum is shown. The corresponding wide-field fluorescence image is given for comparison. (C and D) Enlarged views of representative AZs imaged by dSTORM in either coronal (C) or parasagittal (D) sections. Note the different orientations of the AZs relative to the imaging plane. AZs captured en face as in the top example in (C) were used in subsequent analyses. Images in (C) and (D) are representative of two and four independent experiments, respectively.

  • Fig. 2 Organization of mGluR4 at parallel fiber AZs.

    (A) Principle of the analysis. En face AZs were identified on the basis of the bassoon localizations (green dots) using the DBSCAN algorithm (53). Gray color indicates the AZ area identified by the analysis. mGluR4 nanoclusters (magenta circles) were subsequently identified on the basis of the mGluR4 localizations (magenta dots) using DBSCAN (see fig. S1 for details). (B) Histogram reporting the surface areas of the analyzed en face AZs. (C) Comparison of mGluR4 localization densities inside and outside AZs. n = 799 AZs. Data are means ± SEM. ****P < 0.0001 by two-sided paired t test. (D) Ripley’s H function analysis investigating the clustering of mGluR4 and bassoon on different length scales. Data were compared with a Neyman-Scott distribution (n = 20 and σ = 20 nm) to simulate randomly distributed localization clusters and with random uniformly distributed localizations. H maxima were observed at approximately 240 nm (bassoon), 100 nm (mGluR4), and 60 nm (Neyman-Scott).

  • Fig. 3 Analysis of mGluR4 stoichiometry by single-molecule microscopy.

    (A) Schematic view of the mGluR4 construct carrying an N-terminal SNAP-tag (SNAP-mGluR4), which was used for the analysis. The construct was transiently expressed in CHO cells at low densities, corresponding to 0.45 ± 0.08 (SD) fluorescently labeled mGluR4s/μm2, and labeled at 1:1 stoichiometry with a saturating concentration of an Alexa Fluor 647 benzylguanine derivative, which binds covalently and irreversibly to the SNAP-tag. Cells were sequentially fixed and imaged by TIRF microscopy. (B) Representative TIRF image of a fixed CHO cell expressing the SNAP-mGluR4 construct. Dots represent individual receptor particles, which were identified with an automated single-particle detection algorithm. (C) Representative distribution of the intensity of mGluR4 particles in a cell expressing the SNAP-mGluR4 construct. Data were fitted with a mixed Gaussian model. The result of a mixed Gaussian fitting after partial photobleaching (dotted black line) was used to precisely estimate the intensity of single fluorophores in each image sequence. a.u., arbitrary units. (D) Relative abundance of monomers, dimers, and higher-order oligomers or nanoclusters detected by the analysis. Data are means ± SEM of 11 cells from three independent experiments (12,012 particles). (E) Estimation of the number of primary antibodies binding to one mGluR4. CHO cells transiently transfected to express wild-type mGluR4 at low densities—0.55 ± 0.07 (SD) fluorescently labeled mGluR4s/μm2—were incubated with either a limiting dilution (1:106) or a saturating concentration (1:100) of the primary antibody against mGluR4 and labeled with an Alexa Fluor 647–conjugated secondary antibody. Cells were then imaged and analyzed as in (B) and (C). Representative images and results of 20 (17,257) and 22 (13,553) cells from three independent experiments, respectively (number of particles in brackets), are shown.

  • Fig. 4 Arrangement of mGluR4 relative to bassoon and CaV2.1 channels by distance-based colocalization analysis.

    (A) Two-color dSTORM imaging of mGluR4 (magenta) and CaV2.1 channels (green). Left: Representative super-resolved dSTORM image revealing the organization of mGluR4 relative to CaV2.1. Middle: Enlarged views corresponding to the regions delimited by the white boxes. Right: Images of same regions, where mGluR4 localizations are shown in red over CaV2.1 localizations in gray (areas with no or low mGluR4 localization densities, corresponding to sites outside AZs, are not shown). White arrows, areas of colocalization between mGluR4 and CaV2.1. (B) Distance-based colocalization analysis. The colocalization index values calculated over increasing distance, corresponding to the SD of the Gaussian filter applied to the bassoon or CaV2.1 channel, are reported. Results were compared to those obtained with an equal number of random uniformly distributed mGluR4 localizations (dashed lines). Data are means ± SEM of 7 or 10 dSTORM images from two independent preparations each, coimmunostained for mGluR4 and bassoon or mGluR4 and CaV2.1, respectively. Differences are statistically significant by two-way ANOVA followed by Holm-Sidak’s test. (C) Principle of the analysis. A Gaussian filter with increasing SD is applied to the bassoon channel, allowing the estimation of colocalization between mGluR4 and bassoon over increasing distances (see Materials and Methods for details). **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus random localizations. ##P < 0.01 and ###P < 0.001 versus mGluR4-bassoon.

  • Fig. 5 Arrangement of mGluR4 relative to bassoon and Munc-18-1 by distance-based colocalization analysis.

    (A) Representative two-color dSTORM imaging of mGluR4 (magenta) and Munc-18-1 (green). Left: Super-resolved dSTORM image revealing the organization of mGluR4 relative to Munc-18-1. Middle: Enlarged views corresponding to the regions delimited by the white boxes. Right: Images of same regions, where mGluR4 localizations are shown in red over Munc-18-1 localizations in gray (areas with no or low mGluR4 localization densities, corresponding to sites outside AZs, are not shown). White arrows, areas of colocalization between mGluR4 and Munc-18-1. (B) Distance-based colocalization analysis. Data shown are means ± SEM of seven or three dSTORM images from two independent preparations coimmunostained for mGluR4 and bassoon and one preparation coimmunostained for mGluR4 and Munc-18-1, respectively. Results were compared to those obtained with an equal number of random uniformly distributed mGluR4 localizations (dashed lines). Differences are statistically significant by two-way ANOVA followed by Holm-Sidak’s test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 versus random localizations. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 versus mGluR4-bassoon.

  • Fig. 6 Schematic representation of mGluR4 nanoscale organization within the AZ.

    Our data reveal a high level of spatial organization at parallel fiber AZs, where we find mGluR4 in close proximity to CaV2.1 channels and Munc-18-1. This places mGluR4 right next to both the channels implicated in calcium influx (CaV2.1) and a key regulator of the SNARE complex (Munc-18-1), which, upon calcium entry, is responsible for the fusion of synaptic vesicles and the resulting release of neurotransmitters. These findings provide an ultrastructural basis to understand the mechanisms implicated in the regulation of synaptic transmission by mGluR4 and possibly other presynaptic GPCRs.

Supplementary Materials

  • Supplementary Materials

    Super-resolution imaging reveals the nanoscale organization of metabotropic glutamate receptors at presynaptic active zones

    Sana Siddig, Sarah Aufmkolk, Sören Doose, Marie-Lise Jobin, Christian Werner, Markus Sauer, Davide Calebiro

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