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. 2018 Sep 11;12:304. doi: 10.3389/fncel.2018.00304

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Copyright © 2018 Shaib, Staudt, Harb, Klose, Shaaban, Schirra, Mohrmann, Rettig and Becherer.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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CAPS1 is localized to synapses. Co-cultured adult Synaptobrevin2-mRFP (Syb2-mRFP) knock-in DRG neurons with E18 CAPS DKO spinal cord (SC) neurons were fixed with 4% paraformaldehyde at day in vitro (DIV) 7 and stained with antibodies directed against CAPS1 or CAPS2 and synapsin to mark synapses. This culture condition ensured that anti-CAPS labeling was localized to DRG and not SC interneurons (SC neurons) and that DRG neurites could be readily identified by Syb2-mRFP red fluorescence. (Ai) Left: bright field image of one SC neuron and a network of neurites. Right: overlay of a maximal intensity projection (MIP) image of Syb2-mRFP (magenta), synapsin (yellow) and CAPS1 (cyan) labeling. (Aii) Enlarged portion of a neurite outlined in (Ai). The individual channels (Syb2-mRFP, synapsin and CAPS1) and their overlay are displayed from top to bottom. (Aiii) Line profile along the neurite presented in (Aii). Note that CAPS1 is enriched at synapses (puncta in which Syb2-mRFP and synapsin co-localize). (Bi) Left: overlay of a bright field image and anti-neurofilament 200 (NF200; blue) labeling of a peptidergic DRG neuron. Overlay of a MIP image of Syb2-mRFP (magenta), synapsin (yellow) and CAPS1 (cyan) labeling. (Bii) Enlarged portion of a neurite outlined in (Bi). The individual channels (Syb2-mRFP, synapsin and CAPS1), their overlay, and the NF200 labeling are presented from top to bottom (Biii) Line profile along the neurite shown in (Bii). (C) Average line profile diagram for CAPS1 over synapses that were selected according to co-localized high fluorescence intensities of the Syb2-mRFP and synapsin signals (n = 130). (D) Average line profile diagram over synapses for CAPS2, Syb2-mRFP and synapsin (n = 31). (E) Co-localization was tested with Manders’ coefficient measured on isolated neurites of DRG neurons. Localizations of Syb2-mRFP to synapsin and its inverse, CAPS1 to Syb2-mRFP and CAPS1 to synapsin, CAPS2 to Syb2-mRFP and CAPS2 to synapsin are presented. Averages are indicated as a black line with SEM (n = 21 and 10 for CAPS1 and CAPS2, respectively). (F) Schematic representation of the experimental design. Yellow dots indicate SC-SC neuron homotypic synapses in which, by design, only synapsin can be observed. Magenta dots likely correspond to SVs or LDCVs at extra-synaptic locations. CAPS1 was found at low levels throughout the DRG-neuron (light blue, see (B,C)) and enriched at heterotypic synapses (white dots) but can also co-localize with Syb2-mRFP (violet dots).