RIM-Binding Protein, a Central Part of the Active Zone, Is Essential for Neurotransmitter Release

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Science  16 Dec 2011:
Vol. 334, Issue 6062, pp. 1565-1569
DOI: 10.1126/science.1212991


The molecular machinery mediating the fusion of synaptic vesicles (SVs) at presynaptic active zone (AZ) membranes has been studied in detail, and several essential components have been identified. AZ-associated protein scaffolds are viewed as only modulatory for transmission. We discovered that Drosophila Rab3-interacting molecule (RIM)–binding protein (DRBP) is essential not only for the integrity of the AZ scaffold but also for exocytotic neurotransmitter release. Two-color stimulated emission depletion microscopy showed that DRBP surrounds the central Ca2+ channel field. In drbp mutants, Ca2+ channel clustering and Ca2+ influx were impaired, and synaptic release probability was drastically reduced. Our data identify RBP family proteins as prime effectors of the AZ scaffold that are essential for the coupling of SVs, Ca2+ channels, and the SV fusion machinery.

In response to Ca2+ influx, synaptic vesicles (SVs) fuse with the active zone (AZ) membrane. An elaborate protein-based cytomatrix covering the AZ membrane is meant to facilitate the release process, but its components and operational principles are poorly understood (14). We previously found Bruchpilot to be an essential building block of the Drosophila AZ cytomatrix (the T bar) (5). Here, we used antibodies to screen for additional cytomatrix components.

Rab3-interacting molecule (RIM)–binding proteins (RBPs) are enriched at presynaptic terminals (68) and interact with voltage-gated Ca2+ channels (VGCCs). Mammals harbor three rbp family loci; Drosophila has a single rbp gene (drbp) (9). We generated antibodies against Drosophila RBP (DRBP) (epitopes in Fig. 1A), which stained presynapses at neuromuscular junctions (NMJs) (Fig. 1B, left). The size of AZ cytomatrices is below the diffraction limit of light microscopy. To overcome this, we used a two-color stimulated emission depletion (STED) (10) microscope providing 50-nm resolution in the focal plane (Fig. 1B, right) (11). Relative to the Bruchpilot C-terminal label, DRBP C-terminal immunoreactivity localized toward the AZ center (Fig. 1C, left; model: Fig. 1F). Vertically, the DRBP label localized ~100 nm closer to the AZ membrane than the Bruchpilot C-terminal label (Fig. 1C, right; quantification in Fig. 1F). Postembedding immunogold labeling against DRBP C terminus yielded similar results (fig. S1). DRBP C- and N-terminal labels are similarly distributed in planar views (compare Fig. 1C and 1D, left), and in vertical views the centers of the DRBP C- and N-terminal signals are on average ~30 nm apart (compare Fig. 1C and 1D, right). The Cacophony (Cac) VGCC (12, 13) localizes beneath the scaffold formed by Bruchpilot in the AZ center (14). DRBP C terminus encircled a small (50 to 100 nm) AZ-central field of CacGFP (with green fluorescent protein) (12, 15) (Fig. 1E, left).

Fig. 1

Two-color STED analysis of DRBP organization at Drosophila NMJ synapses. (A) DRBP entails 3 Src homology 3 (SH3) domains interrupted by fibronectin type 3 (FN3) domains. The third SH3 domain interacts with both RIM and Ca2+ channels in yeast two-hybrid experiments (fig. S6). Positions of STOP codons introduced by chemical mutagenesis and antibody (Ab)–binding epitopes (AB) are shown (based on Flybase CG43073-PB). (B) Synaptic boutons stained with the indicated Abs showing AZs in planar [arrow, magnification in (C), left] and vertical [arrowhead, magnification in (C), right] views with confocal (left) and STED (right) resolution. (C to E) STED images of individual planar (left) and vertical (right) AZs stained with the indicated Abs. (F) Model of an AZ in oblique view, with peak-to-peak vertical distance measurements performed on two-color STED images. PRE, presynaptic, POST, postsynaptic. Scale bars: (B) 1.5 μm; (C to E) 200 nm.

We subjected the drbp locus to genetic analysis (fig. S2). A strain with a transposon inserted between exons 6 and 7 (MB02027, drbpMinos) was positioned in trans over a deficiency including drbp, as well as a neighboring locus [Df(3R)S201, short Df, see fig. S2]. In these larvae, DRBP levels at NMJs were reduced to one-third of control levels (Fig. 2A) (drbpMinos; arbitrary fluorescence intensity 36% ± 2, n = 27; control 100% ± 2, n = 21; P > 0.0001, Mann-Whitney U test). These drbp hypomorphic flies hatched below expected Mendelian ratios, and mutant larvae showed reduced locomotion (fig. S3). Chemical mutagenesis screening provided alleles with premature stop codons (drbpSTOP1, drbpSTOP2, and drbpSTOP3) (Fig. 1A and fig. S2). Animals carrying these alleles over Df only rarely reached adulthood, and mutant larvae barely moved (fig. S3). DRBP immunoreactivity at mutant larval NMJs was completely absent when we used either N- (fig. S4) or C-terminal antibodies (Fig. 2A). Thus, we consider these alleles to be null. Using pacman technology (16), we produced a genomic transgene encompassing the entire drbp locus (Rescue) (fig. S2). One copy of this construct partially restored NMJ staining (Fig. 2A) and partially rescued drbpSTOP1-3 adult vitality. Mutant larval NMJ terminals reached normal morphological size (Fig. 2B) (control: 516 ± 51 μm2, n = 20; drbpSTOP1: 554 ± 41 μm2, n = 20; P = 0.4903, Mann-Whitney U test), and had normal synapse numbers (Fig. 2B) (control: 454 ± 39, n = 20; drbpSTOP1: 489 ± 34, n = 20; P = 0.36, Mann-Whitney U test), as also seen in transmission electron microscopy (TEM) (fig. S5A, arrows). Postsynaptic glutamate receptor (GluR) fields appeared enlarged (Fig. 2B).

Fig. 2

drbp mutant synapses show ultrastructural defects. (A) Synapses of control animals were labeled by an Ab directed against the C terminus of DRBP. Synapses of drbpMinos mutants had severely reduced staining intensity, and drbpSTOP1 mutants completely lacked the DRBPC-Term signal. Staining was partially restored by the presence of one copy of the drbp genomic transgene (Rescue) in the null mutant background. (B) NMJs of muscles 6 and 7 labeled with the indicated Abs. (Insets) Magnifications of the boutons marked with arrows. Neither NMJ size (determined by horseradish peroxidase staining) nor AZ number per NMJ (determined by Bruchpilot+ punctae opposite postsynaptic GluR fields) were changed in drbp mutants compared with controls. Means ± SEM. n.s., Not significant (P > 0.05). (C) STED images of synapses stained with the indicated Abs. Control synapses showed typical donut-shaped BruchpilotC-Term signals in planar views (arrows, left), whereas in drbp mutants BruchpilotC-Term signal appeared misshapen (arrows, right). In vertical views, BruchpilotC-Term and GluR subunit IID (GluRIID) labels were strictly opposed (arrowheads, left), whereas in drbp mutants, BruchpilotC-Term signal often extended unusually far into the cytoplasm (arrowheads, right; ectopic material marked by asterisks). (D) T-bar EM images from controls, drbpMinos and drbpSTOP1 NMJs. T-bar pedestals marked with arrowheads and T-bar platforms with arrows. In drbpMinos, T bars appeared thinner, whereas drbpSTOP1 synapses lacked normally shaped T bars. (E) Electron tomography of control and drbpSTOP1 T bars. (Left) Virtual single section from reconstructed tomogram. Rendered model is shown in the middle (vertical view) and on the right (planar view from the bottom on the pedestal). Red, T bar; yellow, membrane proximal SVs. Scale bars: (A) 2 μm; (B) 20 μm, overview, 6 μm, insets; (C) 1 μm; (D) 100 nm.

Bruchpilot spots in drbp mutants appeared largely unaltered at confocal resolution (Fig. 2B). However, with STED, Bruchpilot signals emerged as atypically organized, no longer forming regular donuts but adopting irregular shapes (compare Fig. 2C, arrows left to right). Bruchpilot signals extended into the cytoplasm unusually far (Fig. 2C, asterisks), not matching postsynaptic GluR fields in either planar (Fig. 2C, arrows) or vertical views (Fig. 2C, arrowheads). Organization of the AZ cytomatrix in drbp mutants was clearly affected in electron microscopy (EM) of both conventionally embedded samples (fig. S5B) and high-pressure frozen- and freeze-substituted (HPF) (14, 17, 18) samples (Fig. 2D). The drbp hypomorph (drbpMinos/Df) still formed structures resembling T-bar platforms (Fig. 2D, arrows), but they resided on thinner pedestals (Fig. 2D, arrowheads) (T-bar width: controls = 85.5 ± 10, drbpMinos = 56.3 ± 5.5, P < 0.05; T-bar height: controls = 55.8 ± 1.4, drbpMinos = 48.5 ± 2.2, P < 0.01, Student’s t test). At AZ membranes of drbp nulls, abnormally shaped electron-dense material was found, but no regular T bars (Fig. 2D). In three-dimensional electron tomography reconstructions, it became obvious that the entire cytomatrix was severely misshapen (Fig. 2E). We occasionally observed free-floating electron-dense material detached from the AZ plasma membrane in drbp nulls (fig. S5, C and D). These atypical electron densities still tethered SVs (fig. S5B, arrows; insets in fig. S5, C and D), a function mediated by the C-terminal end of Bruchpilot (19). With EM, overall SV density in NMJ boutons appeared unaltered (fig. S5A). The number of membrane-proximal SVs (up to a 5-nm distance) counted over the whole AZ, however, were reduced in drpbSTOP1 animals [control: 3.2 ± 0.2, n of AZs = 26; drbpMinos: 3.1 ± 0.3, n of AZs = 17, P = 0.79; drbpSTOP1: 2.1 ± 0.3, n of AZs = 16; P < 0.005, Student’s t test; scored with HPF, as standard aldehyde fixation is imprecise for this purpose (20)]. For functional analysis, two-electrode voltage-clamp recordings were performed at larval NMJs. In drbp hypomorphs, evoked excitatory junctional current (eEJCs) amplitudes were reduced by about half (Fig. 3A). In drbp nulls, synaptic transmission was practically abolished (Fig. 3A) [control: –52.4 ± 4.9 nA, n = 10; drbpSTOP1: –2.6 ± 0.2 nA, n = 4; drbpMinos: –23.0 ± 5.3 nA, n = 8; P < 0.001; one-way analysis of variance (ANOVA) Tukey’s post test]. We facilitated synaptic release by recording in elevated extracellular Ca2+. In 2 mM Ca2+, eEJC amplitudes were reduced to ~10% of control levels in all three null alleles. One copy of the genomic drbp transgene (fig. S2) completely rescued this phenotype (Fig. 3B) (control: –113.7 ± 7.2 nA, n = 12; drbpSTOP1: –11.7 ± 2.8 nA, n = 9; drbpSTOP2: –9.8 ± 3.1 nA, n = 6; drbpSTOP3: –9.6 ± 1.6 nA, n = 8; Rescue: –119.1 ± 12.7 nA, n = 7; P < 0.001; one-way ANOVA, Tukey’s post test). Frequency and amplitude of miniature excitatory junctional currents (mEJCs) were unchanged in drbpSTOP1 (Fig. 3C), despite enlarged GluR fields. Thus, the number of quanta (i.e., SVs) released per individual action potential (AP) (quantal content, Fig. 3D) was dramatically reduced in the absence of DRBP.

Fig. 3

drbp mutants suffer from defective evoked neurotransmitter release. (A) eEJC sample traces and quantification for control, drbpMinos, and drbpSTOP1 (1 mM extracellular Ca2+, 0.2 Hz). (B) eEJC sample traces and quantification for control, drbp null mutants, and genomic rescue recorded at 2 mM extracellular Ca2+. (C) (Left) mEJC sample traces. (Middle) Mean cumulative histogram of mEJC amplitudes (mean ± standard deviation; two-tailed Kolmogorov-Smirnov test: P = 0.89). (Right) mEJC frequency (control: 0.92 ± 0.14, n = 10; drbpSTOP1: 1.01 ± 0.19, n = 11; Student’s t test: P = 0.72). (D) Quantal content of drbpSTOP1 eEJCs was significantly reduced (control: 120.8 ± 6.9, n = 9; drbpSTOP1: 9.5 ± 1.8, n = 11; P < 0.001; Student’s t test). Means ± SEM (unless otherwise noted).

At paired-pulse stimulation, drbpSTOP1 synapses exhibited an unusually strong facilitation (Fig. 4A). During a 10-Hz stimulation train, a protocol that leads to marked depression of eEJCs in controls, drbpSTOP1 mutants showed sustained facilitation and reached a steady-state level more than double the initial amplitude (Fig. 4B). When stimulated with five consecutive pulses at 100 Hz, drbpSTOP1 eEJCs exhibited substantial recovery and almost reached absolute control levels on the fifth pulse (Fig. 4C). Altogether, these results suggest that drbp null mutants suffer from a severely reduced release probability. However, as SV release can be substantially elevated under high-frequency stimulation, we conclude that the core fusion machinery is still operational in drbp mutants, and we hypothesize that the reduced SV release evoked by single APs is mainly due to defects upstream of the SV fusion process.

Fig. 4

drbp mutants show altered short-term synaptic transmission and deficits in Ca2+ channel abundance. (A) (Top) Sample traces of paired pulse stimulation for control (black) and drbpSTOP1 (gray). (Bottom) Paired pulse ratio was significantly increased in drbpSTOP1 at 30 ms, as well as 10 ms interstimulus interval (ISI) (P < 0.001 for 30 ms ISI; P < 0.001 for 10 ms ISI; Student’s t test). (B) (Top) Sample traces of 10-Hz stimulation for control (black) and drbpSTOP1 (gray). (Bottom) Mean eEJC amplitudes are plotted in bins of two (controls: closed symbols, n = 10; drbpSTOP1: open symbols, n = 8). (C) Sample traces of five-pulse stimulation at 100 Hz for control (black) and drbpSTOP1 (gray) (top) and quantification (bottom) illustrate strong facilitation at drbpSTOP1 NMJs. (D) drbpSTOP1 eEJCs (left), but not mEJCs (right), had a significantly higher rise time than controls. (E) (Left) Confocal image showing 1b boutons filled with Oregon-Green 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid–1 (BAPTA-1) and sample line scans across single boutons (dashed line, top) of single action-potential evoked spatially averaged Ca2+ transients (average of 10 scans). (Right) Changes in fluorescence quantified as ΔF/F revealed ~30% reduced peak amplitudes of evoked Ca2+ signals in drbpSTOP1 mutants. Decay time constant τ remained unchanged (control: 150 ± 10 ms, n = 20; drbpSTOP1 153 ± 10 ms, n = 44; P = 0.86, Student’s t test). Baseline fluorescence did not differ between genotypes. (F) Synapses of control animals, drbpSTOP1 and drbpMinos mutants were stained for CacGFP (green) and GluRIID (magenta). Ca2+ channel density was consistently reduced by about 25% measured over the whole NMJ in all mutant alleles. Measuring CacGFP intensity per PSD yielded a slightly stronger decrease in drbpSTOP1 animals. Scale bar: 2 μm. Means ± SEM.

The eEJC rise times were slightly but significantly increased (Fig. 4D) (control 1.00 ± 0.05 ms, n = 12; drbpSTOP1 1.34 ± 0.09 ms, n = 9; P < 0.01; Student’s t test), whereas mEJC rise times were unchanged (control 1.20 ± 0.06 ms; n = 10; drbpSTOP1 1.21 ± 0.07 ms, n = 10; P = 0.9; Student’s t test). Thus, evoked SV fusion events in drbp mutants appeared desynchronized with the invasion of the presynaptic terminal by an AP. Because this synchronization depends on the spatiotemporal pattern of AP-triggered Ca2+ influx into the nerve terminal (21), changes in the abundance of Ca2+ channels might contribute to the observed defect. Indeed, Ca2+ channel signals, as evaluated by expression of CacGFP (5, 22), were slightly but consistently reduced by ~25% measured over the whole NMJ in all three drbp null alleles (Fig. 4F) (control 100% ± 2, n = 136; drbpSTOP1 74% ± 3, n = 52; drbpSTOP2 80% ± 3, n = 19; drbpSTOP3 70% ± 3, n = 45). For the drbp hypomorph, Ca2+ channel signal was reduced by about 25% as well (Fig. 4F) (drbpMinos 71% ± 1, n = 27; P < 0.001, Mann-Whitney U test), whereas the evoked response was only reduced to 44% (Fig. 3A). Measuring CacGFP intensity per postsynaptic density (PSD) yielded a slightly stronger 36% decrease in drbpSTOP1 animals compared with controls (control 100% ± 4, n = 48 NMJs; drbpSTOP1 64% ± 3, n = 52; P < 0.001, Mann-Whitney U test). This decrease in Ca2+ channel signal intensity was reflected by a ~30% decrease in calcium influx in response to single APs as determined by the ΔF/F amplitude of spatially averaged Ca2+ signals recorded from drbpSTOP1 mutant boutons (Fig. 4E) (control: 0.85 ± 0.06, n = 20 boutons; drbpSTOP1 0.57 ± 0.03, n = 44 boutons; P < 0.001, Student’s t test).

Consistent with previous findings in mammals (6, 7, 23) we found a biochemical interaction between DRBP and both Drosophila Ca2+ channel α1 subunit Cac (yeast two-hybrid, human embryonic kidney (HEK) cell culture membrane recruitment assay) (fig. S6) and the Drosophila homolog of AZ protein RIM (yeast two-hybrid) (fig. S6, A and B). Interactions were specifically mediated by highly homologous PXXP motifs that bind to the third DRBP SH3 domain (fig. S6, A and B). Recently, RIM has been suggested to tether Ca2+ channels to the AZ membrane by means of (i) a direct interaction with the Ca2+ channel α1 subunit and (ii) an interaction with RBP (23). Mouse rim-1 and rim-2 double knockouts (DKOs) (23, 24), however, showed a rather moderate reduction in SV release in comparison with the dramatic drbp null phenotype. Thus, DRBP might bundle multiple interactions that are not necessarily downstream of RIM. Mechanistically, loss of DRBP leads to defects in Ca2+ channel abundance, Ca2+ influx, SV docking, and cytomatrix organization that probably all contribute to the severity of the drbp-release deficit.

Of note, in Bruchpilot mutants, cytomatrix and Ca2+ channel clustering defects are more pronounced than in drbp nulls (5, 14). Functionally, drbp and bruchpilot phenotypes appear similar: Both demonstrate decreased and desynchronized evoked SV release with atypical short-term facilitation. However, the deficits in evoked SV release are much more severe in drbp nulls than in bruchpilot nulls [i.e., release occurs at 5% versus 30% (5) of the respective wild-type level]. DRBP levels were clearly reduced in bruchpilot mutants (fig. S7), whereas gross Bruchpilot levels were not altered in drbp mutants (Fig. 2B). Given that even a partial loss of DRBP causes marked reduction in SV release (Fig. 3A), deficits in bruchpilot mutants might be explained, at least in part, by a concomitant loss of DRBP, and DRBP probably serves functions beyond the structural and Ca2+ channel–clustering roles of Bruchpilot.

Taken together, we identified DRBP as a central part of the AZ cytomatrix. How, in detail, DRBP functionally integrates into this protein network is subject to future analyses. Notably, the short-term plasticity phenotype of drbp mutants is reminiscent of mammalian munc13-1 KO and caps-1 and caps-2 DKO mutants (25, 26), which implicates functional links between priming factors and DRBP. Consistent with the functional importance of the DRBP protein family suggested by our study, human genetics recently identified two rbp loci associated with autism with high confidence (27, 28).

Supporting Online Material

Materials and Methods

Figs. S1 to S7

References (2939)

References and Notes

  1. Acknowledgments: This work was supported by Deutsche Forschungsgemeinschaft (DFG) grants (SFB 665, SFB 958, and EXC 257) to S.J.S and D.S., as well as Bundesministerium für Bildung und Forschung (The German Federal Agency of Education and Research) funding for Deutsche Zentrum für Neurodegenerative Erkrankungen (DZNE) to D.S. Also, M.S. was supported by a Ph.D. fellowship from the Max Delbrück Center for Molecular Medicine and a Boehringer Ingelheim Fonds Ph.D. fellowship. E.K. and S.W. were supported by Ph.D. fellowships from the graduate school GRK 1123 funded by the DFG. M.M. was supported by a fellowship of the Swiss National Science Foundation (PBSKP3-123456/1).
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