Establishing synthetic ribbon-type active zones in a heterologous expression system
- Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Germany
- Auditory Neuroscience and Synaptic Nanophysiology Group, Max Planck Institute for Multidisciplinary Sciences, Germany
- IMPRS Molecular Biology, Göttingen Graduate School for Neuroscience and Molecular Biosciences, University of Göttingen, Germany
- Cluster of Excellence 'Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells' (MBExC 2067), University of Göttingen, Germany
- Institute for Neuropathology, University Medical Centre Göttingen, Germany
- Cellular Biophysics & Translational Cardiology Section, Heart Research Center Göttingen, University Medical Center Göttingen, Germany
- Department of Cardiology and Pneumology, University Medical Center Göttingen, Germany
- Institute of Anatomy and Embryology, University Medical Center Göttingen, Germany
Figures
Figure 1 with 1 supplement
Membrane targeting of Bassoon using a palmitoylation consensus sequence.
(A) Representative confocal images of human embryonic kidney 293 (HEK293) cells transfected with (i) GFP only (CtBP2/RIBEYE in magenta labeling nuclei, GFP in cyan), (ii) RIBEYE-GFP (CtBP2/RIBEYE in magenta, GFP in cyan), (iii) Bassoon (green), and (iv) RIBEYE-GFP and Bassoon. Note that antibodies against RIBEYE B-domain and the nuclear transcription factor CtBP2 result in similar staining patterns as CtBP2 is transcribed from the same gene as RIBEYE and is identical to the RIBEYE-B domain except for the first 20 N-terminal amino acids. The upper panel shows maximum projections, and the lower panel shows exemplary single sections from confocal stacks. Scale bar = 5 µm. (B) Schematic of construct for membrane targeting of Bassoon. The first 95 amino acids from full-length Bassoon were replaced with a palmitoylation consensus sequence from GAP43. Constructs without and with a C-terminal GFP tag were used, as depicted. (C) Sample confocal image (single section) showing membrane-targeted palm-Bassoon (green) expressed in HEK293 cells appears as puncta distributed along the periphery of the cell, marked by Na, K-ATPase α1 (blue). The inset shows a maximal projection of the confocal section. Scale bar = 5 µm. (D) Zoom-in from (C) shows colocalization of palm-Bassoon with membrane marker Na, K-ATPase α1. Scale bar = 1 µm. Schematic of construct for membrane targeting of Bassoon. (E) Quantification of Bassoon signal intensity at periphery vs inside of cell. Cells expressing palm Bassoon (N = 10 cells) clearly show a higher peripheral distribution compared to cells expressing full-length Bassoon (N = 10 cells), ****p<0.0001, Mann-Whitney-Wilcoxon test. Overlaid data points represent individual cells, crosses represent the mean values, central band indicates the median, whiskers represent 90/10 percentiles, and boxes represent 75/25 percentiles.
Figure 1—figure supplement 1
Subcellular distribution of RIBEYE and palm-Bassoon clusters in human embryonic kidney 293 (HEK293) cells.
Maximal projections of confocal sections of HEK293 cells transfected with RIBEYE-GFP (A, B, C) or palm-Bassoon-GFP (D, E, F), shown in green. Representative co-staining for calnexin (endoplasmic reticulum marker), GM130 (cis-Golgi marker), and LysoTracker (live labeling of lysosomes) has been shown, respectively, in blue. Neither RIBEYE clusters nor palm-Bassoon appears to localize in either of the three compartments. Note that (C) and (F) represent live cells, while the remaining images are from fixed cells. Scale bar = 10 μm.
Figure 2 with 1 supplement
Co-expression of RIBEYE with palm-Bassoon results in ribbon-type active zone (AZ)-like structures.
(A) (i) Representative confocal image (single section) of a human embryonic kidney 293 (HEK293) cell transfected with RIBEYE-GFP (magenta) and palm-Bassoon (green). Co-expression of RIBEYE with palm-Bassoon targets RIBEYE to the cell membrane. Inset shows maximum projection. Scale bar = 5 µm. (ii) Exemplary 2D stimulated emission depletion (STED) images for RIBEYE – palm-Bassoon juxtapositions acquired from cells as shown in (i). Scale bar = 500 nm; individual channels have been depicted with an intensity-coded look-up table with warmer colors indicating higher intensity. (i) Representative maximum projection of confocal sections of apical organ of Corti from a Wistar rat (postnatal day 18); data as published in Michanski et al., 2023, stainings were for CtBP2/RIBEYE (magenta; labeling synaptic ribbons and inner hair cell [IHC] nuclei) and Bassoon (green; spots juxtaposing with ribbons represent IHC AZs, spots not juxtaposing with ribbons represent efferent synapses formed by lateral olivocochlear neurons onto SGN boutons). Scale bar = 5 µm. (B) (ii) Juxtaposing RIBEYE and Bassoon spots imaged in 2D STED and confocal mode, respectively. Scale bar = 500 nm. Note the striking resemblance to reconstituted RIBEYE+palm-Bassoon structures in HEK293 cells as shown in (Aii). STED images are from 3 sample transfections, representative of 7 total transfections; individual channels have been depicted with an intensity-coded look-up table with warmer colors indicating higher intensity. (C) Quantification of RIBEYE and Bassoon signal intensity at periphery vs inside of cell shows a higher peripheral distribution of RIBEYE and Bassoon in HEK293+RIBEYE+palm-Bassoon cells (N=9 cells) as compared to HEK293+RIBEYE+Bassoon cells (N=9 cells); ****p<0.0001, Mann-Whitney-Wilcoxon test. Overlaid data points represent individual cells, crosses represent mean values, central band indicates the median, whiskers represent 90/10 percentiles, and boxes represent 75/25 percentiles. Distribution of volumes of RIBEYE and palm-Bassoon puncta from HEK293 cells expressing RIBEYE and palm-Bassoon (n = 20 cells, quantifications from 5 sample transfections). Volumes of synaptic ribbons from rat IHCs (n = 29 cells, 3 rats) have been plotted for comparison. (E) Box plot depicting data from (D). Volumes of RIBEYE and palm-Bassoon puncta are comparable to each other (p>0.99, Kruskal-Wallis test with post hoc Dunn’s multiple comparison), but on average, they are much smaller and considerably more variable when compared to volumes of RIBEYE and Bassoon puncta from rat IHCs, respectively (****p<0.0001, Kruskal-Wallis test with post hoc Dunn’s multiple comparison). Overlaid plus signs represent individual spots, crosses represent mean values, central band indicates the median, whiskers represent 90/10 percentiles, and boxes represent 75/25 percentiles. Quantification of RIBEYE and Bassoon signal intensity at periphery vs inside of cell shows a higher peripheral distribution of RIBEYE and Bassoon in HEK293+RIBEYE+palm-Bassoon cells (N=9 cells) as compared to HEK293+RIBEYE+Bassoon cells (N=9 cells); ****p<0.0001, Mann-Whitney-Wilcoxon test. Overlaid data points represent individual cells, crosses represent mean values, central band indicates the median, whiskers represent 90/10 percentiles, and boxes represent 75/25 percentiles. (F) RIBEYE puncta in HEK cells expressing RIBEYE and palm-Bassoon (nspots = 864, N = 20 cells) appear more spherical than Bassoon puncta in the same cells (nspots = 961, N = 20 cells; ****p<0.0001, Kruskal-Wallis test with post hoc Dunn’s multiple-comparison test). Note the similar trend in IHC synaptic ribbons where RIBEYE puncta are more spherical than Bassoon puncta (nspots = 290 for RIBEYE and nspots = 314 for Bassoon, N = 29 cells, 3 rats; ****p<0.0001, Kruskal-Wallis test with post hoc Dunn’s multiple-comparison test). Overlaid plus signs represent individual spots, crosses represent mean values, central band indicates the median, whiskers represent 90/10 percentiles, and boxes represent 75/25 percentiles.
Figure 2—figure supplement 1
Membrane localization of RIBEYE depends on expression levels of palm-Bassoon.
(A) Exemplary overview image of human embryonic kidney 293 (HEK293) cells co-transfected with RIBEYE (magenta) and palm-Bassoon (green) showing how to identify cells with SyRibbons. On average, about 10% of cells appear co-transfected, of which some show peripheral distribution of RIBEYE (examples marked by arrows). Scale bar = 100 μm for overview and 20 μm for zoom-in. (B) Peripheral distribution of RIBEYE appears to be dependent on expression levels of palm-Bassoon. Note the cell in the top panel with little to no palm-Bassoon expression and predominantly cytosolic RIBEYE puncta. Scale bar = 10 μm.
Figure 3 with 1 supplement
Cryo-correlative microscopy captures membrane-localized SyRibbons.
(A) RIBEYE-GFP signal on a 150-nm-thick lamella, revealed by fluorescent light microscopy within the cryo-focused ion beam (cryo-FIB) chamber (cryo-LM). Dotted lines delineate cell membranes. The orange arrow indicates a plasma membrane-proximal GFP fluorescence, whereas purple arrowheads point to cytosolic RIBEYE aggregates. The tomogram shown in (C) was acquired at the boxed region. The image underwent background subtraction for better visualization. (B) Transmission electron microscopy (TEM) image of the lamella in (A). Orange arrow and purple arrowheads locate the respective regions in (A), showing that GFP-positive spots correlated to electron-dense bodies. Dotted lines delineate plasma membranes. The tomogram shown in (C) was taken at the boxed region. (C) Tomogram slice showing a SyRibbon acquired at the boxed region in (B). PM: plasma membrane, ECS: extracellular space, ER: endoplasmic reticulum.
Figure 3—figure supplement 1
Fluorescence-guided milling and tomogram acquisition of SyRibbons.
(A) Vitrified group of human embryonic kidney (HEK) cells on an EM grid. The box indicates where a lamella was created using a cryo-focused ion beam (cryo-FIB). (B) Cryo-fluorescent light microscopy-guided milling of HEK cells. Top row: Cartoons showing different stages of milling, where dotted lines represent regions that are ablated in the next stage. GFP fluorescence (green dots) decreases as the lamella gets thinner. Bottom row: Fluorescence acquired along the milling process. Cells expressing membrane-localized GFP are targeted for cryo-FIB milling. Here, the junction between cell 1 and cell 2 containing multiple GFP puncta was the region of interest. On the polished lamella (150 nm, rightmost image), a few GFP spots remained, which could often be traced back to earlier stages. In some regions, smaller puncta were occluded by bright fluorescent spots when the lamella was thick and were only revealed at the last stage. The orange arrow indicates a peripheral signal, and purple arrowheads show cytosolic GFP. (C) Tomograms of SyRibbons. Some exhibit hollow cores (i, iii) or concentric layers (i, ii). Mito: mitochondrion, ER: endoplasmic reticulum.
Figure 4 with 1 supplement
Human embryonic kidney 293 (HEK293) cells expressing CaV1.3.
(A) Representative confocal section of a HEK293 cell stably expressing an inducible CaV1.3α transgene (untagged) along with constitutive transgenes for CaVβ3 and CaVα2δ1. Immunostainings have been performed using antibodies against CaV1.3 (green) and Na, K-ATPase α1 (blue, labeling the plasma membrane). Scale bar = 10 µm. (B) Representative confocal section of a HEK293 cell stably expressing CaVβ3 and CaVα2δ1, and transiently transfected with an N-terminal EGFP-tagged CaV1.3 construct. Immunostainings have been performed using antibodies against GFP (green) and Na, K-ATPase α1 (blue). Scale bar = 10 µm. (C) Representative confocal section of a HEK293 cell stably expressing CaVβ3 and CaVα2δ1 and transiently transfected with an N-terminal Halo-tagged CaV1.3 construct. Immunostainings have been performed using antibodies against CaV1.3 (green) and Halo-tag (magenta). Scale bar = 10 µm. Representative confocal section of a HEK293 cell stably expressing CaVβ3 and CaVα2δ1 and transiently transfected with an N-terminal EGFP-tagged CaV1.3 construct. Immunostainings have been performed using antibodies against GFP (green) and Na, K-ATPase α1 (blue). Scale bar = 10 µm. (D) Expression of either of the Ca2+ channel complexes results in clusters of comparable sizes (p>0.05, Kruskal-Wallis test with post hoc Dunn’s multiple-comparison test). Overlaid plus signs represent individual spots, crosses represent mean values, central band indicates the median, whiskers represent 90/10 percentiles, and boxes represent 75/25 percentiles.
Figure 4—figure supplement 1
CaV1.3 does not appear to colocalize directly with RIBEYE or with palm-Bassoon.
(A) Representative confocal section of a human embryonic kidney 293 (HEK293) cell transfected with Halo-CaV1.3 (blue) and palm-Bassoon (green). The two proteins do not appear to colocalize. Scale bar = 5 μm. (B) Representative confocal section of a HEK293 cell transfected with Halo-CaV1.3 (blue) and RIBEYE-GFP (red). RIBEYE and CaV1.3 also do not appear to colocalize, speaking against a possible direct interaction between the two proteins. Scale bar = 5 μm.
Figure 5 with 2 supplements
RBP2 bridges palm-Bassoon and CaV1.3 to form supramolecular assemblies at the plasma membrane.
(A) Representative confocal images (single sections) of human embryonic kidney 293 (HEK293) cells (fixed) transfected with palm-Bassoon (green), RBP2 (red), and N-terminal Halo-tagged CaV1.3 (blue). The three colocalizing proteins appear to form supramolecular assemblies at the cell membrane. Scale bar = 5 µm for left panel showing cell overviews, 2 µm for right panels showing zoom-ins. (B) Representative confocal images (single sections) of live HEK293 cells transfected with palm-Bassoon-GFP (green), mKATE2-p2A-RBP2 (red, representing only mKATE2 signal and not RBP2 localization), and Halo-CaV1.3 (blue, labeled with Janelia Fluor 646 [JF646] HaloTag Ligand before imaging). Scale bar = 5 µm. (C) Exemplary super-resolution stimulated emission depletion (STED) images from live-labeled samples in (B) showing juxtaposition of palm-Bassoon and Halo-CaV1.3 signal at the plasma membrane in RBP2-positive cells. Scale bar = 1 µm. Representative confocal images (single sections) of HEK293 cells (fixed) transfected with palm-Bassoon (green), RBP2 (red), and N-terminal Halo-tagged CaV1.3 (blue). The three colocalizing proteins appear to form supramolecular assemblies at the cell membrane. Scale bar = 5 µm for left panel showing cell overviews, 2 µm for right panels showing zoom-ins.
Figure 5—figure supplement 1
Whole-cell Ca2+ current amplitudes increase upon co-expression of RBP2 and palm-Bassoon.
(A) Impact of co-expression of different ribbon-type active zone (AZ) proteins on CaV1.3α1/β3/α2δ1 Ca2+ currents. Color code: non-transfected controls expressing only CaV1.3 (black), co-expression of RBP2+palm-Bassoon (data depicted in turquoise), only palm-Bassoon (green), only RBP2 (blue), or only RIBEYE (magenta). Numbers of recorded cells/group have been indicated in the figure (bottom right). (B) Co-expression of RBP2 and palm-Bassoon seemingly increases the whole-cell Ca2+ current density in human embryonic kidney 293 (HEK293) cells expressing CaV1.3, [Ca2+]e = 10 mM. Expression of palm-Bassoon or RBP2 individually also results in a mild increase, but this trend is not statistically significant as shown in (D) when compared to controls and to cells expressing both the proteins. Expression of RIBEYE by itself causes no change in Ca2+ current density. Current density-voltage (IV) relation curves are depicted as mean Ca2+ current density with shaded area represents ± SEM. Top panel shows representative Ca2+ current traces. (C) We fitted a Boltzmann function to traces in (B) to determine the fractional activation of CaV1.3 channels. The voltage dependence of Ca2+ current influx does not appear to be different for any of the four scenarios. (D) Box plot depicting peak Ca2+ current density values from (A). p-Values are as follows: **PCaV only/CaV+RBP2+PBsn = 0.0041, PCaV only/CaV+PBsn = 0.3737, PCaV only/CaV+RBP2=0.1643, PCaV only/CaV+RIBEYE > 0.999; Kruskal-Wallis test with post hoc Dunn’s multiple-comparison test. (E, F) Box plots depicting voltage of half-maximal activation (Vhalf) and slope (k) of the Boltzmann fits in (C) show no statistically significant differences.
Figure 5—figure supplement 2
Kinetics of CaV1.3 channels are not altered upon co-expression of various active zone (AZ) proteins.
(A) Average peak amplitude-normalized Ca2+ current traces in response to depolarizations of 500 ms from –91.2 to –21.2 mV (shaded area represents ± SEM). Expression of palm-Bassoon+RBP2 (turquoise), only palm-Bassoon (green), RBP2 (blue), or RIBEYE (magenta) does not seem to impact inactivation of CaV1.3 channels. (B) A power exponential function was fitted to the first 5 ms of current traces (as shown in Figure 5B) to obtain the activation time constant (mean ± SEM) at different voltages, which does not appear to be notably impacted.
Figure 6 with 1 supplement
Synthetic ribbon-type active zones recruit CaV1.3 Ca2+ channels.
(A) Experimental scheme for expression of Halo-tagged CaV1.3, palm-Bassoon, RIBEYE-GFP, and RBP2 in human embryonic kidney 293 (HEK293) cells. (B) Representative confocal images (single sections) of HEK293 cells transfected with RBP2 (not shown, expression confirmed using epifluorescence), RIBEYE-GFP (red), palm-Bassoon (green), Halo-CaV1.3 (blue) shows colocalization of the three latter proteins at the plasma membrane. Scale bar = 5 µm. (C) Confocal zoom-ins from (B). Line scans depict an increased CaV1.3 signal intensity (blue) at sites where RIBEYE (red) and palm-Bassoon (green) clusters localize. Pearson’s correlation coefficients (Pr) were calculated along the line profiles for RIBEYE and CaV1.3 signal intensity and indicate a high degree of correlation (~0.8) between the spatial localization of the two. Scale bar = 2 µm. (D) Super-resolution stimulated emission depletion (STED) images showing CaV1.3 clusters localizing at the base of the SyRibbons. Scale bar = 500 nm. (E) Distribution of volumes from confocal images of CaV1.3 clusters colocalizing with SyRibbons (orange, nspots = 634) and not colocalizing with SyRibbons (yellow, nspots = 1354) from HEK293 cells expressing CaV1.3, RBP2, and SyRibbons (N = 13 cells). Pooled volumes of all CaV1.3 clusters (with and without SyRibbons) from these cells have been shown in red (nspots = 1988). Volumes of CaV1.3 clusters from HEK293 cells expressing only Halo-CaV1.3 (black, nspots = 1281, N = 16 cells) and from cells expressing Halo-CaV1.3, RBP2, and palm-Bassoon (blue, nspots = 427, N = 6 cells) have been plotted for comparison. (F) Box plot depicting data from (E). CaV1.3 clusters appear larger on average in cells expressing CaV1.3, RBP2, and SyRibbons vs in cells expressing only CaV1.3 (**p=0.0044). Within cells expressing all four proteins, CaV1.3 clusters are much larger when colocalizing with SyRibbons vs when they do not (****p<0.0001). CaV1.3 clusters colocalizing with SyRibbons were in fact also larger than clusters in cells expressing only CaV1.3 and cells expressing only RBP2 and palm-Bassoon (****p<0.0001). Statistical test: Kruskal-Wallis test with post hoc Dunn’s multiple-comparison correction. Overlaid plus signs represent individual spots, crosses represent mean values, central band indicates the median, whiskers represent 90/10 percentiles, and boxes represent 75/25 percentiles.
Figure 6—figure supplement 1
Colocalization quantifications.
(A) Box-whisker plot depicting Pearson’s colocalization coefficient and Mander’s overlap coefficient between palm-Bassoon and RIBEYE, palm-Bassoon and CaV1.3, and RIBEYE and CaV1.3 in human embryonic kidney 293 (HEK293) cells transfected with the combinations of proteins described on the x-axis. (B) Costes method was implemented (100 repetitions) to test for statistical significance of the determined colocalizations. The bar plot depicts percentages of cells with p=1, indicating significant colocalization. A p-value less than 1 indicates that the measured colocalization is no better than random chance.
Figure 7
Ca2+ imaging reveals higher Ca2+ signal intensity underneath SyRibbons.
(A) Experimental scheme for expression of SyRibbons, RBP2, and CaV1.3 in human embryonic kidney 293 (HEK293) cells for patch clamp in combination with Ca2+ imaging using the low-affinity Ca2+ indicator Calbryte590. (B) Exemplary cell used for Ca2+ imaging. Cells were identified by peripheral RIBEYE-GFP puncta as shown (indicative of RIBEYE and palm-Bassoon co-expression) and mKATE2 signal (indicative of RBP2 expression). (C) Current density-voltage (IV) relations from whole-cell patch clamp recordings of HEK293 cells expressing CaV1.3+RBP2+SyRibbons (n=20, in orange) and only CaV1.3 (n=10, in black). Lines represent mean current traces, and shaded area represents ± SEM. Whole-cell Ca2+ current density does not appear to change upon co-expression of RBP2 and SyRibbons (p=0.293, t-test). (D) The voltage dependence of Ca2+ current influx does not seem to be altered upon co-expression of RBP2 and SyRibbons at the whole-cell level. (E) A depolarizing pulse to +2 mV was applied to the cells for 500 ms, and the increase in Calbryte590 fluorescence was measured by acquiring images at a frame rate of 20 Hz using a spinning disk confocal microscope. (F) Representative image of a HEK293 cell expressing SyRibbons, RBP2, and CaV1.3 stimulated as described in (E). RIBEYE-GFP signal has been shown in magenta; the change in Calbryte590 fluorescence intensity (ΔF) upon Ca2+ binding (the frame at the onset of depolarization to +2mV) has been shown in green. Note the distinct Ca2+ signal ‘hotspots’ that colocalize with SyRibbons. Scale bar = 10 µm. (G) Zoom-ins from (F). Frames before, at the onset of, and after stimulation have been shown. Note the localized Ca2+ influx at the base of the SyRibbons. Scale bar = 1 µm. (H) Line profiles drawn tangentially to the membrane in composite ∆F image of Calbryte590 signal and RIBEYE-GFP show a high correlation between the localization of SyRibbons and peak intensity of Calbryte590 fluorescence increase (Pr = ~0.7). Plots show intensity profiles along line scans for RIBEYE-GFP (magenta) and Calbryte590 (light gray for frames before stimulus, bold green for peak intensity at onset of stimulus, light green lines for decaying intensity during ongoing stimulus, and dark gray for frames post-stimulus). (I) Regions of interest (ROIs) of 2 µm diameter were drawn at sites with (magenta circles) and without (green circles) SyRibbons and the corresponding Calbryte590 ΔF/F0 values were calculated for each ROI. (J) Plot of average (shaded area represents ± SEM) ΔF/F0 values from ROIs with and without SyRibbons (218 and 139 ROIs, respectively, from N = 24 cells, data from 7 transfections). The voltage dependence of Ca2+ current influx does not seem to be altered upon co-expression of RBP2 and SyRibbons at the whole-cell level. (K) On average, Calbryte590 ΔFmax/F0 was higher for ROIs with SyRibbons than ROIs without them in a given cell (*p=0.018, paired t-test). Dots represent mean of ΔFmax/F0 from ROIs with (magenta) and without (green) SyRibbons averaged per cell, error bars represent ± SEM.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell Line (Homo sapiens) | ChanTest Stably Transfected Human Cav1.3/β3/α2δ-1-HEK293 cells | Charles River Laboratories, Cleveland, OH, USA | Cat. No. CT6232 | Tetracycline-inducible human CaV1.3 pore-forming α1-D subunit transgene (CACNA1D, NM_000720.2), constitutive expression of CaVβ3 (CACNB3, NM_000725.2) and CaVα2δ–1 (CACNA2D1, NM_000722.2). Sex: HEK cells originate from a female fetus. |
| Recombinant DNA reagent | pAAV-CMV-HBA-RIBEYE-eGFP (plasmid) | This study | Addgene plasmid # 241988, RRID:Addgene_241988 | pAAV-GFP backbone, CMV-HBA promoter, human RIBEYE-GFP, Ampicillin resistance, WPRE regulatory element |
| Recombinant DNA reagent | pCS2+-CMV-Bassoon (plasmid) | This study | Addgene plasmid # 250160, RRID:Addgene 250160 | pCS2+ backbone, CMV promoter, rat Bassoon, Kanamycin resistance, SV40 poly(A) |
| Recombinant DNA reagent | C1-CMV-palm-Bassoon(95–3938)-EGFP (plasmid) | This study | Addgene plasmid # 250161, RRID:Addgene_ 250161 | pEGFP-C1 backbone, CMV enhancer, palm-rat Bassoon (95–3938)-EGFP, Kanamycin resistance, SV40 poly(A) |
| Recombinant DNA reagent | C1-CMV-palm-Bassoon(95–3938)-STOP (plasmid) | This study | Addgene plasmid # 250162, RRID:Addgene_ 250162 | pEGFP-C1 backbone, CMV enhancer, palm-rat Bassoon (95–3938)-STOP, Kanamycin resistance, SV40 poly(A) |
| Recombinant DNA reagent | pHTN-CMV-EGFP-CaV1.3 (plasmid) | Schmitz et al., 2000 | – | pHTN backbone, CMV enhancer-promoter, EGFP-human CACNA1D, Ampicillin resistance, SV40 poly(A) |
| Recombinant DNA reagent | pHTN-CMV-HaloTag-CaV1.3 (plasmid) | Schmitz et al., 2000 | – | pHTN backbone, CMV enhancer-promoter, HaloTag-human CACNA1D, Ampicillin resistance, SV40 poly(A) |
| Recombinant DNA reagent | f(syn)w-CMV-mKATE2-p2A-RBP2 (plasmid) | This study | Addgene plasmid # 241989, RRID:Addgene_241989 | F(syn)w backbone, CMV enhancer, mKATE2-p2A-RBP2, Ampicillin resistance, WPRE, SV40 poly(A) |
| Recombinant DNA reagent | pAAV-CMV-RBP2 (plasmid) | This study | Addgene plasmid # 241990, RRID:Addgene_241990 | pAAV backbone, CMV enhancer-HBA, RBP2, Ampicillin resistance, WPRE, SV40 poly(A) |
| Antibody | Anti-GFP (Chicken polyclonal) | Abcam | Ab13970, RRID:AB_300798 | IF(1:200) |
| Antibody | Anti-CtBP2 (Mouse monoclonal IgG1) | BD Biosciences | 612044, RRID:AB_399431 | IF(1:200) |
| Antibody | Anti-RIBEYE-A (Guinea pig polyclonal) | Synaptic Systems | 192104, RRID:AB_2800537 | IF(1:200) |
| Antibody | Anti-RIBEYE-A (Rabbit polyclonal) | Synaptic Systems | 192103, RRID:AB_2086775 | IF(1:200) |
| Antibody | Anti-RIBEYE-B (Rabbit polyclonal) | Synaptic Systems | 192003, RRID:AB_2261205 | IF(1:200) |
| Antibody | Anti-Bassoon (Chicken polyclonal) | Synaptic Systems | 141016 | IF(1:200) |
| Antibody | Anti-Bassoon (Mouse monoclonal IgG2a) | Abcam | Ab82958, RRID:AB_1860018 | IF(1:200) |
| Antibody | Anti-Na, K-ATPase α1 (Mouse monoclonal IgG1) | Abcam | Ab7671, RRID:AB_306023 | IF(1:200) |
| Antibody | Anti-Calnexin (Rabbit polyclonal) | Abcam | Ab22595, RRID:AB_2069006 | IF(1:200) |
| Antibody | Anti-GM130 (Mouse monoclonal IgG1) | BD Biosciences | 610822, RRID:AB_398142 | IF(1:200) |
| Antibody | Anti-LAMP1 (CD107A, Mouse monoclonal IgG1) | eBioscience | 14-1079-80, RRID:AB_467426 | IF(1:200) |
| Antibody | Anti-Halo (Mouse monoclonal) | Promega | G9211, RRID:AB_2688011 | IF(1:200) |
| Antibody | Anti-RBP2 (Rabbit polyclonal) | Synaptic Systems | 316103, RRID:AB_2619739 | IF(1:200) |
| Antibody | Anti-CaV1.3 (Rabbit polyclonal) | Alomone | ACC005, RRID:AB_2039775 | IF(1:100) |
| Antibody | Alexa Fluor 488 conjugated anti-chicken (Goat polyclonal) | Invitrogen | A11039, RRID:AB_2534096 | IF(1:200) |
| Antibody | Alexa Fluor 488 conjugated anti-guinea pig (Goat polyclonal) | Invitrogen | A11073, RRID:AB_2534117 | IF(1:200) |
| Antibody | Alexa Fluor 488 conjugated anti-rabbit (Goat polyclonal) | Invitrogen | A11008, RRID:AB_143165 | IF(1:200) |
| Antibody | Alexa Fluor 488 conjugated anti-mouse (Goat polyclonal) | Invitrogen | A11001, RRID:AB_2534069 | IF(1:200) |
| Antibody | Alexa Fluor 405 conjugated anti-rabbit (Goat polyclonal) | Invitrogen | A31556, RRID:AB_221605 | IF(1:200) |
| Antibody | Alexa Fluor 546 conjugated anti-mouse (Goat polyclonal) | MoBiTec | A11003, RRID:AB_2534071 | IF(1:200) |
| Antibody | Alexa Fluor 568 conjugated anti-chicken (Goat polyclonal) | Abcam | Ab175711, RRID:AB_2827757 | IF(1:200) |
| Antibody | Alexa Fluor 568 conjugated anti-guinea pig (Goat polyclonal) | Invitrogen | A11075, RRID:AB_2534119 | IF(1:200) |
| Antibody | Alexa Fluor 568 conjugated anti-rabbit (Goat polyclonal) | Thermo Fisher | A11011, RRID:AB_143157 | IF(1:200) |
| Antibody | Alexa Fluor 647 conjugated anti-mouse (Goat polyclonal) | Invitrogen | A21236, RRID:AB_2535805 | IF(1:200) |
| Antibody | Alexa Fluor 647 conjugated anti-rabbit (Goat polyclonal) | Invitrogen | A21244, RRID:AB_2535812 | IF(1:200) |
| Antibody | STAR Red conjugated anti-chicken (Goat monoclonal IgY) | Abberior | 2-0102-011-2 | IF(1:200) |
| Antibody | STAR 635p conjugated anti-mouse (Goat polyclonal) | Abberior | ST635P-1001-500UG, RRID:AB_2893232 | IF(1:200) |
| Antibody | STAR 635p conjugated anti-rabbit (Goat polyclonal) | Abberior | 2-0012-007-2 | IF(1:200) |
| Antibody | STAR 580 conjugated anti-mouse (Goat polyclonal) | Abberior | ST580-1001-500UG, RRID:AB_2923543 | IF(1:200) |
| Software, algorithm | ImageJ, Fiji | NIH | RRID:SCR_003070 | https://imagej.nih.gov/ij/, http://fiji.sc |
| Software, algorithm | Illustrator | Adobe | RRID:SCR_010279 | |
| Software, algorithm | Imaris 9.6 | Oxford Instruments | RRID:SCR_007370 | http://www.bitplane.com/imaris/imaris |
| Software, algorithm | Igor Pro 6 and 7 | WaveMetrics | RRID:SCR_000325 | http://www.wavemetrics.com/products/igorpro/igorpro.htm |
| Software, algorithm | Prism | GraphPad | RRID:SCR_015807 | https://www.graphpad.com/scientific-software/prism/ |
| Software, algorithm | PatchMaster | HEKA Electronics, Germany | RRID:SCR_000034 | http://www.heka.com/products/products_main.html#soft_pm |
| Software, algorithm | VisiView 5.0 | Visitron Systems GmbH | RRID:SCR_022546 | |
| Other | Janelia Fluor 646 HaloTag Ligand | Promega | Cat. No. GA1120 | Final concentration: 200 nM |
| Other | Calbryte590 | AAT Bioquest | Cat. No. 20706 | Effective concentration: 100 µM, kd = 1.4 µM |
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Establishing synthetic ribbon-type active zones in a heterologous expression system
eLife 13:RP98254.
https://doi.org/10.7554/eLife.98254.3
