Fig 1: SPR-binding studies. After covalent immobilization of IgG, IgM, NPTX1, and NPTX2) via amine coupling, the ligand channels were covalently blocked with bovine serum albumin (BSA) along with an empty channel that served as negative control. The sensorgrams (pale lines) are generated with the subtraction of the responses detected on the BSA channel from those detected on specific ligand channels. Dark lines represent the data fitting with a single exponential (sc-gC1q2 and sc-gC1q2l) or a double exponential (sc-gC1q). A–D, sc-gC1q analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. E–H, sc-gC1q2 analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. I–L, sc-gC1q2l analyte interactions with IgG, IgM, NPTX1, and NPTX2 ligands, respectively. gC1q, globular part of C1q; IgG, immunoglobulin G; IgM, immunoglobulin M; NPTX, neuronal pentraxin; sc-gC1q, single-chain gC1q; SPR, surface plasmon resonance.
Fig 2: Investigation of the binding properties of single-cell gC1q (sc-gC1q) variants by ELISA. Human and mouse IgG proteins (hIgG and mIgG, respectively) and human IgM were immobilized and titrated with each form of sc-gC1q variants (A, B, and E). In order to use the same primary antibody in assays examining neuronal pentraxins, we coated the wells with NPTX1 or NPTX2 and titrated with the different forms of sc-gC1q (C and D; marked with different colors). Hill equations (lines) were fitted to the data (circles) using Origin8 software. Values represent mean (from three replicates) ± SEM. gC1q, globular part of C1q; IgG, immunoglobulin G; IgM, immunoglobulin M; NPTX, neuronal pentraxin.
Fig 3: Representative image of neuronal pentraxins along the axons. Mouse primary cortical neurons were immunostained for MAP2, Syp, and either NP1 or NP2. On the representative images, synaptic NPs are clearly apparent, whereas synapses without NP-staining are also visible. Solid circles show synaptophysin signals colocalized with either NP1 or NP2, while dashed circles show synapses, which do not contain the neuronal pentraxins.
Fig 4: The presence of NP1 in microglial engulfed C1q-tagged synapses. (A) Microglia were reconstructed from confocal image stacks using Iba1-staining (green) and engulfed, C1q- and Syp-colocalized NP1 spots (yellow, magenta, and cyan, respectively) were identified on mouse brain sections (white cycles). Orthogonal views (XY, YZ) demonstrate that microglia completely surround one of the phagocytosed C1q-tagged synaptic material with NP1 content. (B) Image analyses revealed that 64.80 ± 1.97% of C1q-tagged microglial Syp proteins are NP1 positive as well. (Means ± SEM are shown; n = 16 images recorded from brain sections of three mice. Statistically significant difference between groups was identified using two-tailed Student t-test of paired samples.)
Fig 5: Co-occurrence of the C1q-tag with NP1/2 on synaptosomes examined by flow cytometry. According to the gating criteria, C1q-labeled synaptosomes were almost exclusively (~97%) positive for NP1 (A) and NP2 (B). In contrast to C1q labeling, only ~24% of NP-labeled synaptosomes were positive for C1q. Density plots show representative measurements where the percentages of synaptosomes that belong to each quadrant were also indicated. The secondary antibody controls went through the same procedure as the fully labeled samples. Statistically significant differences were determined with two-tailed Student’s t-test of paired samples (Means ± SEM; n = 4 mice). To test the viability of synaptosomes, calcein-AM labeling was applied separately ( Supplementary Datasheet 2 ).
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