Fig. 4: Arp2/3 complex activation and binding by JMY is essential for dendritic arbor formation of developing neurons. | Communications Biology

Fig. 4: Arp2/3 complex activation and binding by JMY is essential for dendritic arbor formation of developing neurons.

From: JMY powers dendritogenesis and is regulated by CaM revealing a general, critical principle in neuromorphogenesis

Fig. 4

A MIPs of anti-MAP2 immunostained and GFP-expressing (reporter) primary hippocampal neurons transfected with plasmids indicated (transfected cells marked by asterisks) at DIV4 and treated with the Arp2/3 complex inhibitor CK666 and the solvent DMSO, respectively, 24 h later and fixed 30 h after transfection. Bars, 30 µm. Quantitative analyses of dendritic morphologies revealing that the JMY*/GFP + DMSO-mediated increases in dendritic branch points (B), dendritic terminal points (C), and total dendritic length (D) compared to GFP+DMSO control neurons were completely abolished upon addition of the Arp2/3 complex inhibitor CK666. E Sholl analyses show that the complete loss of JMY-mediated dendritic arbor complexity increases upon CK666 application occurs throughout the dendritic arbor. F Schematic representation of JMY and of a mutant lacking the Arp2/3 complex-binding CA domain. G–I Quantitative analyses of dendritic morphology of neurons transfected with either scr. RNAi (control), RNAi#1 against JMY or RNAi#1/JMY*, RNAi#1/JMY* ΔCA or RNAi#1/JMY*W981A showing that, in contrast to the successful rescue with JMY*, reexpression of JMY* ΔCA or JMY*W981A being unable to bind and/or activate the Arp2/3 complex were incapable of rescuing the RNAi#1-induced decrease in dendritic branch points (G), dendritic terminal points (H) and dendritic length (I) in comparison to scr. RNAi. (J) MIPs of hippocampal neurons transfected and fixed under the same conditions as for the morphometric analyses (G–I) with additional anti-JMY immunostainings and DAPI stainings showing that the mutants not able to rescue were successfully expressed and available in soma and dendrites. Bars, 30 µm. Data, mean ± SEM shown as bar/dot plots (B–D, G–I) and bar plot (E), respectively. B–E GFP+DMSO, n = 40; JMY*/GFP+DMSO, n = 40; GFP+CK666, n = 30; JMY*/GFP+CK666, n = 30 cells from two independent assays. G–I Scr. RNAi., RNAi#1 and RNAi#1/JMY* (part of the data were already included in Fig. 2B-D), n = 75; RNAi#1/JMY* ΔCA, n = 45; RNAi#1/JMY*W981A, n = 30 neurons from 2 to 5 independent assays. Numerical data are provided in Supplementary Data 1. Statistical assessments were conducted by comparing the factor transfection (GFP vs. JMY*/GFP, *) or treatment (DMSO vs. CK666, #) using Two-way ANOVA, Sidak′s test (B–D) as well as Three-way ANOVA with Tukey’s multiple comparisons test (E) and by using One-way ANOVA/Tukey′s post-test (G–I), respectively. * and #p < 0.05, **p < 0.01, ***p < 0.001, **** and ####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.

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