Cobl-mediated dendritogenesis is suppressed by CaM inhibition.

<p>(<b>A–H</b>) End-point analysis of CaM inhibitor effects in primary hippocampal neurons (DIV4+2). (<b>A–F</b>) Hippocampal neurons transfected as indicated at DIV4 and treated with 0.1% (final) DMSO (<b>A,D</b>), CaM inhibitor W7 (<b>B,E</b>) and CaM inhibitor CGS9343B (both in 0.1% DMSO final) (<b>C,F</b>), respectively, 30 h after transfection. Cells were processed for anti-MAP2 immunostaining 2 d after transfection. Bar, 20 μm. (<b>G,H</b>) Quantitative examinations of dendrite number (<b>G</b>) and dendritic branch points (<b>H</b>) normalized to the GFP+DMSO controls of each assay. Note that Cobl-mediated dendrite formation is suppressed by the CaM inhibitors W7 and CGS9343B. Data represent mean ± SEM. GFP+DMSO, <i>n</i> = 197; GFP+W7, <i>n</i> = 120; GFP+CGS9343B, <i>n</i> = 169; GFP-Cobl+DMSO, <i>n</i> = 101; GFP-Cobl+W7, <i>n</i> = 53; GFP-Cobl+CGS93943B, <i>n</i> = 80. Statistical significances (to control, marked above column; between other conditions, indicated by lines) were determined using one-way ANOVA with Tukey’s post-test. *<i>p</i> < 0.05; **<i>p</i> < 0.01; ***<i>p</i> < 0.001. (<b>I</b>) 3-D-time-lapse imaging of GFP-Cobl dynamics and neuronal morphogenesis visualized by PM-mCherry before and after incubation with the CaM inhibitor CGS9343B. Shown is a selection (times as indicated) of MIPs recorded by spinning disc microscopy. Green <b>*</b>, protrusion that was initiated or grew when compared to previous image (examples numbered to allow for tracking); red <b>°</b>, protrusion that shrunk compared to previous image; yellow I, static protrusion. Note that whereas most neuronal structures were dynamic, CGS9343B addition (red line; 11:20 min:s) impeded this morphological dynamics and neuronal structures largely were static until the end of recording. Bar, 5 μm. Please also see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s020" target="_blank">S4 Movie</a>. (<b>J</b>) Quantitation of the frequencies of protrusion initiation before and after addition of DMSO and DMSO+inhibitor (CGS9343B), respectively. DMSO, <i>n</i> = 12; DMSO+inhibitor, <i>n</i> = 10 dendrite sections. For data underlying <b>G, H,</b> and <b>J</b> see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002233#pbio.1002233.s001" target="_blank">S1 Data</a>. Statistical significances, one-way ANOVA with Tukey’s post-test. ***<i>p</i> < 0.001.