Figures and data
Membrane targeting of Bassoon using a palmitoylation consensus sequence.
A Representative confocal images of 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. Upper panel shows maximum projections and 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). Inset shows maximal projection of confocal section. Scale bar = 5µm.
D Zoom-in from (C) shows colocalisation of palm-Bassoon with membrane marker Na, K-ATPase α1. Scale bar = 1µm.
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.
Subcellular distribution of RIBEYE clusters in HEK293 cells.
Maximal projections of confocal sections of HEK293 cells transfected with RIBEYE-GFP (green). Representative co-staining for (A) calnexin (endoplasmic reticulum marker), (B) GM130 (cis-Golgi marker) and (C) LAMP1 (lysosomal marker) have been shown respectively (blue). RIBEYE clusters do not appear to localize in either of the three compartments. Scale bar = 10 µm.
Co-expression of RIBEYE with palm-Bassoon results in ribbon-type AZ-like structures
A (i) Representative confocal image (single section) of a 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 = 5µm. (ii) Exemplary 2-D STED images for RIBEYE – palm-Bassoon juxtapositions acquired from cells as shown in (i); Scale bar = 500nm; individual channels have been depicted with an intensity-coded look-up table with warmer colors indicating higher intensity.
B (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 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 = 5µm. (ii) Juxtaposing RIBEYE and Bassoon spots imaged in 2-D STED and confocal mode respectively. Scale bar = 500nm. 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.
D 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 multi-comparison), but on an average 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 multi-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.
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.
Membrane localization of RIBEYE depends on expression levels of palm-Bassoon
A Exemplary overview image of HEK293 cells co-transfected with RIBEYE (magenta) and palm-Bassoon (green) showing how to identify cells with SyRibbons. On an 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.
SyRibbons employ RBP2 to tether CaV1.3 channels.
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.
D Expression of either of the Ca2+ channel complexes result 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.
CaV1.3 does not appear to colocalize directly with RIBEYE or with palm-Bassoon
A Representative confocal section of a 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.
RBP2 bridges palm-Bassoon and CaV1.3 to form supra-molecular assemblies at the plasma membrane.
A 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 supra-molecular 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), RBP2-p2A-mKATE2 (red, representing only mKATE2 signal and not RBP2 localization) and Halo-CaV1.3 (blue, labeled by with JF-646 HaloTag Ligand before imaging). Scale bar = 5 µm.
C Exemplary super-resolution 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.
Whole-cell Ca2+ current amplitudes increase upon co-expression of RBP2 and palm-Bassoon.
A Impact of co-expression of different ribbon-type 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 HEK293 cells expressing CaV1.3, [Ca]e = 10mM. Expression of palm-Bassoon or RBP2 individually also results in a mild increase, but this trend is not statistically significant as shown in (D). Expression of RIBEYE by itself causes no change in Ca2+ current density. Plots show lines representing mean Ca2+ current density traces and shaded area represents ± SEM.
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.
Kinetics of CaV1.3 channels are not altered upon co-expression of various 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) do 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 do not appear to be notably impacted.
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 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 STED images showing CaV1.3 clusters localizing at the base of the SyRibbons. Scale bar = 500nm.
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) have been plotted for comparison.
F Box plot depicting data from (E). CaV1.3 clusters appear larger on an average in cells expressing CaV1.3, RBP2 and SyRibbons versus 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 versus when they do not (****P < 0.0001,). Finally, when comparing these clusters with and without SyRibbons in cells expressing all four proteins versus clusters in cells expressing only CaV1.3, we observe a significant difference for the former (****P < 0.0001,), but not the latter (P = 0.099,). 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.
Ca2+ imaging reveals higher Ca2+ signal intensity underneath SyRibbons.
A Experimental scheme for expression of SyRibbons, RBP2 and CaV1.3 in 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, 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 +2mV 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 co-localize 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 ΔF 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 grey for frames before stimulus, bold green for peak intensity at onset of stimulus, light green lines for decaying intensity during ongoing stimulus and dark grey 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).
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.
List of plasmids
