Fig. 1: JMY is a CaM binding protein, and JMY overexpression in primary hippocampal neurons promotes dendritic branching.

A Immunoblots of coprecipitates of GFP, GFP-JMY, and fragments thereof expressed in L929 cells using a matrix with immobilized CaM and conditions without and with Ca²⁺ (2 µM). B Immunoblot analyses of coprecipitation experiments of endogenous JMY from HeLa cell lysates with immobilized CaM and quantitative examinations thereof. Endogenous JMY interacts with CaM in a Ca^2+-dependent manner (consistently shown under two different Ca²⁺ concentrations). White lines indicate lanes omitted from the blot (due to change of order). C Coimmunoprecipitation analyses of endogenous JMY with CaM from HeLa cells. Endogenous CaM immunoprecipitated by anti-CaM antibodies specifically coimmuniprecipitated endogenous JMY, as detected by immunobloting analyses. D Representative maximum intensity projections (MIPs) of HEK293 cells showing a reconstitution of JMY/CaM complexes. Note the recruitment of endogenous JMY (green in merges) to mitochondrially anchored mCherry-CaM (Mito-mCherry-CaM) but not to Mito-mCherry (red in merges). Mitochondria were marked with MitoTracker (blue in merges). Complex formation thus appears in white in the merged images (arrows mark examples). Bars, 10 μm. E MIPs of anti-JMY (green in merge) and anti-MAP2 (red in merge) coimmunolabeling in rat hippocampal neurons (DIV6). Growth cones displaying JMY enrichments are marked by arrows. Boxed areas represent areas magnified below. Bar, 10 μm. F Anti–JMY immunoblotting of lysates (30 μg protein per lane) from cultures of cortical and hippocampal rat neurons. Anti-β-tubulin immunoblotting is shown for comparison. G Representative MIPs of GFP and JMY*/GFP overexpressing primary hippocampal neurons immunostained with anti-MAP2 antibodies. Transfected neurons are marked by red asterisks. Bars, 30 μm. H–L Quantitative morphometric analyses of GFP and JMY*/GFP overexpressing primary hippocampal neurons transfected at DIV4 and fixed 30 h later. Untagged JMY* and GFP (reporter) were co-expressed as separate proteins from the same mRNA using an internal ribosome entry site (IRES). Anti-MAP2-based morphology tracing and reconstruction revealed a gain-of-function phenotype for JMY*/GFP expressing cells with significantly increased dendritic branch points (H), dendritic terminal points (I), total dendritic length (J), Sholl intersections (K) and dendritic branch depth levels 2-4 (L) when compared to only GFP expressing control neurons. Data, mean ± SEM shown as bar/dot plots (B, H–J) and bar plots (K, L), respectively. B n = 2 assays. H–L n = 30 cells of each condition from 2 independent assays. Numerical data and images of uncropped and unedited blots are provided in Supplementary Data 1 and 2, respectively. Statistical significances, One-way ANOVA/Tukey’s post-test (B), Mann–Witney U-test (H, I), Welch′s t-test (J), and Two-way ANOVA/Bonferroni′s test (K, L), respectively. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. For exact p values see figure panels. Note that ****p < 0.0001 are too small values to be reported by the software used.