Figure 6. Effect of Nesprin-2 LEAA mutation on interaction with BicD2.

(A, B) GST-BicD2 CT pull-downs from HeLa cell lysates expressing GFP-tagged N2G SR, Mini N2G or N2G SR LEAA were evaluated for GFP co-immunoprecipitation. (B) Quantification of the GFP signal intensity relative to N2G SR. The LEAA mutation severely disrupted the Nesprin-2-BicD2 interaction. (C, D) E16 rat brain was electroporated with N2G SR or N2G SR LEAA, and subsequently imaged in fixed brain slices at E20. (C) Representative images of the neocortex with electroporated cells in green (GFP) and stained with DAPI. Bracket shows the margins of the CP. (D) Quantification of the proportion of cells reaching the CP. Electroporation of the N2G SR LEAA construct causes a decrease in the number of cells reaching this region. (E, F) E16 rat brain was electroporated with N2G SR or N2G SR LEAA together with PACT-DsRed, and imaged in fixed brain slices at E20. (E) Representative images of electroporated neurons in the CP showing centrosome (Cent.) position (arrow) relative to that of the soma. (F) Quantification of the N-C distance. Electroporation of the N2G SR LEAA causes an increase in N-C spacing. Data presented as scatter dot plot with bar representing mean±s.d. in B and D, and as scatter dot plot with bar representing median with range in F. One-way ANOVA was used in B, and Mann Whitney test for non-parametric distributions was used in D and F (**P<0.01; ****P<0.0001). Data in B arises from n=3 independent experiments. The GFP signal for each condition was normalized to GST BicD2 CT signal. For each experiment, GFP signal of the different conditions was normalized to N2G SR. Data in D include at least 1666 electroporated cells from at least 6 embryos, per condition. Data in F include at least 239 electroporated cells from at least 4 embryos, per condition. C scale bar, 100μm. E scale bar, 10μm.