Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jun 19;12(12):1665.
doi: 10.3390/cells12121665.

Rabconnectin-3α/DMXL2 Is Locally Enriched at the Synaptic Ribbon of Rod Photoreceptor Synapses

Affiliations

Rabconnectin-3α/DMXL2 Is Locally Enriched at the Synaptic Ribbon of Rod Photoreceptor Synapses

Alina Dittrich et al. Cells. .

Abstract

Ribbon synapses reliably transmit synaptic signals over a broad signalling range. Rod photoreceptor ribbon synapses are capable of transmitting signals generated by the absorption of single photons. The high precision of ribbon synapses emphasizes the need for particularly efficient signalling mechanisms. Synaptic ribbons are presynaptic specializations of ribbon synapses and are anchored to the active zone. Synaptic ribbons bind many synaptic vesicles that are delivered to the active zone for continuous and faithful signalling. In the present study we demonstrate with independent antibodies at the light- and electron microscopic level that rabconnectin-3α (RC3α)-alternative name Dmx-like 2 (DMXL2)-is localized to the synaptic ribbons of rod photoreceptor synapses in the mouse retina. In the brain, RC3α-containing complexes are known to interact with important components of synaptic vesicles, including Rab3-activating/inactivating enzymes, priming proteins and the vesicular H+-ATPase that acidifies the synaptic vesicle lumen to promote full neurotransmitter loading. The association of RC3α/DMXL2 with rod synaptic ribbons of the mouse retina could enable these structures to deliver only fully signalling-competent synaptic vesicles to the active zone thus contributing to reliable synaptic communication.

Keywords: DMXL2; Rab3; rabconnectin-3α; retina; ribbon synapse; vesicular H+-ATPase.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
RC3α/DMXL2 expression in the mouse retina. (A) Retinal lysate from wild-type mice was probed by Western blot (WB) with affinity-purified rabbit polyclonal anti-RC3α/DMXL2. A high molecular weight band at ≈340 kDa was detected by the antibody in retinal lysates. (B) Peptide arrays that correspond to the RC3α/DMXL2 region against which this polyclonal antibody was generated, were incubated with affinity-purified rabbit polyclonal anti-RC3α/DMXL2 antibody. (B1) The location of all peptide spots was visualized by UV illumination. (B2) shows the result of the immunolabelling of the peptide array with affinity-purified rabbit polyclonal anti-RC3α/DMXL2 (ECL detection). Peptide spots #4, #5 and #6 strongly reacted with the anti-RC3α/DMXL2 polyclonal antibody. (B3) The amino acid sequence of spots #4, #5 and #6, that strongly reacted with the polyclonal RC3α/DMXL2 antibody, are highlighted in red. (C) The peptide sequence of mouse RC3α/DMXL2 (NP766359) that reacted with the affinity-purified rabbit polyclonal anti-RC3α/DMXL2 (highlighted in red) was aligned with the corresponding sequence from human RC3α/DMXL2 (AAL93215). The corresponding sequences are highly conserved between mouse and human RC3α/DMXL2 (73% amino acid identities).
Figure 2
Figure 2
RC3α/DMXL2 is highly expressed in photoreceptor synapses in the OPL in close vicinity to synaptic ribbons. (A) 0.5 μm-thin retina sections incubated with affinity-purified rabbit polyclonal antibody against RC3α/DMXL2. RC3α/DMXL2 immunosignals are strongly enriched in the OPL in which photoreceptor ribbon synapses are found. (B1B3,C1C3) 0.5 μm-thin sections of the retina double-immunolabelled with rabbit anti-RC3α/DMXL2 (green channel) and with mouse anti-RIBEYE (2D9) (red channel). Signals from green and red channels (B1,C1/B2,C2) were overlaid in (B3/C3). Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 5 μm.
Figure 3
Figure 3
Control exposures. (A1A3) 0.5 μm-thin retina sections were immunolabelled with rabbit anti-RC3α/DMXL2 (green channel) and with mouse anti-RIBEYE (2D9) (red channel), as in Figure 2. (B1B3) The same immunolabelled retina section as shown in (A1A3), but with the 488 nm laser turned off (with all PMT detection settings remaining unchanged). (C1C3) Same double-immunolabelled retina section as shown in (A1A3), but with the 568 nm laser turned off (with all PMT detection settings remaining unchanged). Signals from green and red channels (A1,B1,C1/A2,B2,C2) were overlaid in (A3,B3,C3). Abbreviations: OPL, outer plexiform layer. Scale bars: 5 μm.
Figure 4
Figure 4
(A) Schematic domain structure of RC3α/DMXL2. Amino- and carboxyterminal WD40 repeats (blue boxes) and the central Rav1p_C domain (orange box) are schematically depicted. Monoclonal antibodies (2G2 and 12D8) were generated against a peptide stretch downstream of the Rav1p_C-domain (aa1919–aa2029, highlighted in pink). (B) SDS-PAGE of the purified GST-tagged fusion proteins (Coomassie Blue stained gel). Lane 1: RC3α/DMXL2 (aa1919–aa2029)-GST, lane 2: GST alone. (C1,D1) WB analyses of purified GST fusion proteins probed with RC3α/DMXL2 monoclonal antibodies. RC3α/DMXL2 (aa1919–aa2029)-GST was applied in lanes 3 and 5; GST in lanes 4 and 6 of (C1,C2,D1,D2). In (C1), RC3α/DMXL2-GST and GST were probed with anti- RC3α/DMXL2 (2G2). In (C2) the same blot was re-probed with anti-GST to verify equal loading. In (D1), RC3α/DMXL2 (aa1919–2029)-GST and GST were probed with anti-RC3α/DMXL2 (12D8). In (D2) the same blot was re-probed with anti-GST to verify equal loading. (E1,E2,F1,F2) show peptide arrays that cover the protein region of RC3α/DMXL2 against which the antibodies were generated (aa1919–aa2029). These peptide arrays were probed with the indicated monoclonal anti-RC3α/DMXL2 antibodies to determine the precise binding epitope of the antibodies. (E1,F1) UV light was used to visualize the location of all peptide spots. (E2) shows the peptide array that was incubated with monoclonal anti-RC3α/DMXL2 2G2; (F2) shows the peptide array incubated with monoclonal anti-RC3α/DMXL2 12D8. Peptide spot #1 (KKDQLDSVSGRMENGPSESK) strongly reacted with the 2G2 monoclonal antibody and peptide spot #4 (ADWSAVTSSQFDWSQPMVTV) with the 12D8 monoclonal antibody. (G) Amino acid sequences of all peptide spots. The peptide sequences of peptide spots #1 and #4, that strongly reacted with anti-RC3α/DMXL2 monoclonal antibodies 2G2 and 12D8, are highlighted in red.
Figure 5
Figure 5
Single immunolabelling of mouse retina sections with the monoclonal RC3α/DMXL2 antibodies 2G2 and 12D8. In (A,C), 10 μm—thick cryostat sections of the mouse retina were immunolabelled with the indicated antibodies; in (E,F) 0.5 μm thin resin sections of the mouse retina. (B,D) represent control incubations in which the primary antibody was omitted. All other steps of the immunolabelling procedure were identical. Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer. Scale bars: 5 μm.
Figure 6
Figure 6
RC3α/DMXL2 is strongly expressed in photoreceptor synapses of the OPL in close vicinity to the synaptic ribbons. (A1A3,B1B3,C1C3,D1D3,E1E3) Retina sections double-immunolabelled with mouse anti-RC3α/DMXL2 2G2 (A1,B1,C1,D1,E1) and with rabbit antibodies against CSP (A2), PSD-95 (B2), Cav1.4 (C2) and RIBEYE (D2,E2). Signals from green channels (A1,B1,C1,D1,E1) and red channels (A2,B2,C2,D2,E2) were overlaid in (A3,B3,C3,D3,E3). In (A1A3,D1D3,E1E3), 10 μm-thick cryostat sections were used for immunolabelling; in (B1B3,C1C3) 0.5 μm—thin resin sections. Abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer. Scale bars: 5 μm.
Figure 7
Figure 7
Post-embedding immunogold labelling of ultrathin LR Gold sections from the mouse retina (AF). Rod photoreceptor synapses were immunolabelled with monoclonal anti-RC3α/DMXL2 2G2 antibody (AE). (F) shows a representative negative control incubation in which the primary antibody was omitted. All other steps of the immunolabelling procedure remained the same. Please note that membrane contrast of membranes is limited because a post-embedding approach (without usage of OsO4) was applied. Secondary goat anti-mouse antibodies were conjugated to ultrasmall (1.4 nm diameter) gold particles that were subsequently silver-intensified. Abbreviations: sr, synaptic ribbon; pre, presynaptic; ho, postsynaptic dendrites of horizontal cells. Scale bars: 300 nm (AF).

Similar articles

Cited by

References

    1. Sjöstrand F.S. Ultrastructure of retinal rod synapses of the guinea pig eye as revealed by three-dimensional reconstructions from serial sections. J. Ultrastruct. Res. 1958;2:122–170. doi: 10.1016/S0022-5320(58)90050-9. - DOI - PubMed
    1. Vollrath L., Huss H. The synaptic ribbons of the guinea-pig pineal gland under normal and experimental conditions. Z. Zellforsch. Mikrosk. Anat. 1973;139:417–429. doi: 10.1007/BF00306595. - DOI - PubMed
    1. Nakajima Y., Wang D.W. Morphology of afferent and efferent synapses in hearing organ of goldfish. J. Comp. Neurol. 1974;156:403–416. doi: 10.1002/cne.901560403. - DOI - PubMed
    1. Krstic R. Ultracytochemistry of the synaptic ribbons in the rat pineal organ. Cell Tissue Res. 1976;166:135–143. doi: 10.1007/BF00215133. - DOI - PubMed
    1. McNulty J.A. Ultrastructural observations on synaptic ribbons in the pineal organ of the goldfish. Cell Tissue Res. 1980;210:249–256. doi: 10.1007/BF00237613. - DOI - PubMed

Publication types

Substances

Grants and funding

Work of the authors was supported by a research grant from the Rolf M. Schwiete foundation (grant 2021-022), SFB894 (TP A7 and TP P3, Deutsche Forschungsgemeinschaft) and DFG Schm797/8-1.

LinkOut - more resources