Fig. 7: 497 P Mutation abolishes localization of SEMA4D and Sema7A to the cell surface and growth cones and reduces neuronal migration and axon projection in vivo.
From: Semaphorin heterodimerization in cis regulates membrane targeting and neocortical wiring
a Surface biotinylation followed by avidin pull down of human sp-myc-SEMA4D (abbr. hS4D) or human sp-myc-SEMA4D-497P (abbr. hS4D-497P) in the presence or absence of mouse HA-Sema7A (abbr. mS7A). Cell Surface to INtracellular (CS/IN) ratio of Sema4D was calculated by first normalizing each fraction to all Sema4D detected in whole cell lysate (WCL) where the complete calculation is CS/IN = (CS/WCL)/(IN/WCL). Plotted is mean+SD, using n = 3 separate experiments. Repeated measures ANOVA with Tukey’s multiple comparison’s. SEMA4D vs SEMA4D + Sema7A padjusted = 0.0061, SEMA4D+Sema7A vs SEMA4D-497P padjusted = 0.0008, SEMA4D + Sema7A vs SEMA4D-497P + Sema7A padjusted = 0.0007. b Proximity Ligation Assay (PLA) detecting interaction between mouse HA-Sema7A and human sp-myc-SEMA4D or human sp-myc-Sema4D-497P. Scalebar = 10μm. c Midline panoramas and quantifications of human SEMA4D + GFP and SEMA4D-497P + GFP in utero electroporations. Two-tailed t-test with Welch’s correction, SEMA4D vs SEMA4D-497P p = 0.0345. nbrains SEMA4D = 4, SEMA4D-497P = 4. Box and whisker plot whiskers as min-max with box bounds at lower and upper quartiles and center line at the median. Scale bar in panoramas is 500 μm, while scale bar in midline magnification is 100 μm. d Neuronal migration profiles following in utero electroporation with SEMA4D or SEMA4D-497P, presented in the same format described in Fig. 1. Scalebar at bottom of SEMA4D IUE is 100μm. Representative electroporations are shown adjacent to raw data points and half violin plots of the total distribution of neurons across all brains with mean of means±SD cortical position on the right. Two-tailed t test, SEMA4D vs SEMA4D-497P p < 0.0001. nbrains SEMA4D n = 6, SEMA4D-497P n = 7. e Graphical model depicting general findings that Sema7A expression is regulated by Satb2, which promotes surface localization of Sema4D:Sema7A heterodimers (left). Upon deletion of Satb2 (middle), decreased Sema7A expression means heterodimerization with Sema4D is less able to occur, which coincides with deficits in axon extension and neuronal migration. Similarly, the Sema4D 497P mutation (right), which affects normal glycosylation and trafficking of Sema4D to the plasma membrane, results in deficits in neuronal migration and axon extension. For simplicity, panels denote p < 0.05 as *; p < 0.01 as **; and p < 0.001 as ***. All source data are provided in the Source Data file.
