Article Figures & Data
Figures
- Figure 1.
Impaired vesicle tethering at AZs of a bruchpilot mutant (brpnude) lacking the last 17 C-terminal amino acids. A, Chemical induction of premature stop codons resulted in truncated versions of the BRP protein (BRP; dark blue; boxes indicate coiled-coil domains) of ∼99% (brpnude; gray), of ∼70% (brp1.3; green), and of ∼50% length (brp5.45; blue). Below, Illustration of the BRP protein (blue) within the AZ (red: Ca2+ channels; gray: synaptic vesicle) and the corresponding positions of the truncation (arrows) (Fouquet et al., 2009). B, All three BRP mutants (brpnude, brp1.3; brp5.45) showed severely impaired survival rates and walking skills. C, In the brpnude allele, a single base mutation at the C-terminal position 1724 leads to a premature stop codon and a BRP protein (BRPnude) lacking the last 17 of 1740 aa. D, STED images of neuromuscular junctions stained with a C-terminal BRP antibody (green; BRPNc82) and simultaneous confocal images of an N-terminal BRP antibody (magenta; BRPN-term). The distribution of both antibody signals appears unaltered at brpnude synapses (right) compared to controls (left). Arrows and arrowheads indicate planar and vertical AZs, respectively. E, Examples of conventionally embedded AZs of control and brpnude. Note fewer tethered vesicles in brpnude. F, Height and length of the platform of the electron-dense cytomatrix (T-bar) for control and brpnude. G, The number of docked vesicles per AZ section for control and brpnude (n = 22 and 25 AZs, respectively) and the number of vesicles found within three shells (see inset) each of 50 nm thickness surrounding the AZ (n = 20 and 31 AZs, respectively). Scale bars: D, 1 μm; E, 200 nm.
- Figure 2.
Normal Ca2+ channel clustering and basal release at brpnude synapses. A, GluRIID labels (magenta, confocal) were used to quantify the Ca2+ channel (CacGFP, green, STED) clusters in control animals (top row) and brpnude mutants (below). The Cac signals at synapses that appeared ideally planar (n = 16 each) were averaged after alignment with the postsynaptic GluRIID signal (right panels) or themselves (center panels). While the example images were scaled up individually, all four averages were scaled up with the same factor. B, Intensity profiles of the average Cac images in black for controls and in gray for brpnude mutants with SE bars and the corresponding Gaussian fits (standard deviations given by σ) in gray. C, Examples of EPSCs elicited at 0.2 Hz in control (black), brpnude (gray), brp1.3 (green), and brp5.45 mutants (blue; average of 10 each) in 1.0 mm Ca2+. The average peak EPSC amplitudes and rise and decay kinetics were normal in brpnude mutants. In brp1.3 and brp5.45 mutants EPSC amplitudes were reduced and rise times increased (n = 22, 18, 4, and 12 for control, brpnude, brp1.3, and brp5.45, respectively). D, Example traces of mEPSCs of control (black), brpnude (gray), brp1.3 (green), and brp5.45 mutants (blue). The average mEPSC amplitudes were normal in brpnude, brp1.3, and brp5.45 mutants (n = 6, 6, 4, and 6 for control, brpnude, brp1.3, and brp5.45, respectively). Scale bar in A, 250 nm.
- Figure 3.
Tethering vesicles to the AZ dense body prevents synaptic depression. A, Examples of EPSCs elicited by a paired-pulse protocol with 30 ms interpulse interval in control (black) and brpnude (gray). B, Average paired-pulse ratios plotted versus interpulse interval in 1.0 mm Ca2+ for control (black, n = 12) and brpnude (gray, n = 9; asterisk indicates significant differences) superimposed with exponential fits (time constants are indicated). Inset, 0.6 mm Ca2+. C, Average peak EPSC amplitudes during the 60 Hz train for control (black, n = 21) and brpnude (gray, n = 20). Inset, Average amplitudes of the first 5 EPSCs of the train. The steady-state amplitude (average of EPSC amplitudes marked by the bracket) was significantly reduced in brpnude mutants compared to controls. D, Steady-state amplitudes normalized to the steady-state amplitude of the respective control, for brpnude (gray), brp1.3 (green), brp5.45 (blue), and Cac RNAi synapses (red) in extracellular Ca2+ concentrations that lead to normal (“rescued”) basal EPSC amplitudes ([Ca2+]rescue) (see supplemental Fig. S2, available at www.jneurosci.org as supplemental material). E, Average peak EPSC amplitudes during the first rapid component of recovery after the train (τ1) for control (black, n = 21) and brpnude (gray, n = 20) fitted exponentially (lines). Time constants with SEs are indicated. Bottom, The time constants of the first component of recovery normalized to the corresponding time constant of the control (color code as in D). F, Corresponding average peak EPSC amplitudes during the second slower component of recovery after the train (τ2) with corresponding average data.
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