Journal: The Journal of Neuroscience
Article Title: α-Neurexins Together with α2δ-1 Auxiliary Subunits Regulate Ca2+ Influx through Cav2.1 Channels
Figure Lengend Snippet: αNrxn modulates surface mobility of α2δ-1 and α2δ-3 auxiliary subunits differentially. A , Representative immunofluorescent images of surface α2δ-1 enriched in synaptic boutons, visualized by an antibody against the HA moiety of α2δ-1::HA cotransfected with synGCaMP6f into WT neurons (top) or TKO neurons (bottom). Scale bar, 5 μm. B , Quantification of colocalization between synGCaMP6f and surface α2δ-1-positive puncta in WT and TKO. Data are mean ± SEM; n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.433) by unpaired t test. C , Labeling of the surface population of HA-tagged α2δ-1 ( C 1 ) transfected into WT neurons using an antibody specific to the HA moiety. EGFP was cotransfected to visualize neurites ( C 2 ), merged images ( C 3 ) and an overlay of all trajectories of QD-tracked single α2δ-1 molecules in a subfield as indicated ( C 4 ); sample trajectories of QD-tracked single α2δ-1 molecules ( C 5 ). Scale bars: C 1 – D 3 , 10 μm; C 4 , D 4 , 2 μm; C 5 , D 5 , 0.5 μm. D , Labeling of surface α2δ-1 as in C using TKO neurons. E , Logarithmic distribution of diffusion coefficients for α2δ-1 on axons of WT and TKO neurons, showing more trajectories of higher mobility in TKO (see §) and fewer low mobility trajectories (see #); n = trajectories/cells; error bars (SEM) shown only in outward direction. F , Median and IQR (25–75%) of diffusion coefficients of α2δ-1 shown in E . Numbers of cells from four independent experiments (in bars). * p = 0.0277, by Kruskal–Wallis test with Dunn's post-test. G , Immunofluorescent images of surface α2δ-3 in synaptic boutons as in A . Scale bar, 5 μm. H , Quantification of colocalization between synGCaMP6f and surface α2δ-3-positive puncta in WT and TKO. Data are mean ± SEM. n = synGCaMP6f-positive puncta/neurons from three to four independent experiments per condition; n.s. = not significant ( p = 0.4835), by unpaired t test. I , Logarithmic distribution of diffusion coefficients as in E but for α2δ-3. With α2δ-3, more trajectories of higher mobility occurred in WT (see §), indicating a reverse effect when compared with α2δ-1 ( E ). J , Median and IQR (25–75%) of diffusion coefficients of α2δ-3 shown in I . Numbers of cells from four independent experiments (in bars). * p = 0.0347, by Kruskal–Wallis test with Dunn's post-test.
Article Snippet: To remove DNA and RNA aggregates, samples were passed six times through 1 ml syringes (30Gx1/2 inch) and subsequently subjected to Western blot analysis as described before, using the following antibodies: anti-Cav2.1 P/Q type (Synaptic Systems, catalog #152203) 1:1000, anti-α2δ-1 (Alomone Labs, catalog #ACC-015) 1:500, anti-α2δ-3 (Alomone Lab, catalog #ACC-103) 1:500, anti-Actin (Sigma-Aldirch, catalog #A50-60) 1:2000, and anti-Nrxn123 (Synaptic Systems, catalog #175003) 1:1000.
Techniques: Labeling, Transfection, Diffusion-based Assay