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. 2017 Oct 9;27(19):2999–3009.e9. doi: 10.1016/j.cub.2017.08.031

Figure 2.

Figure 2

The Muscle-Specific Nesprin-1α Isoform Is Required for Recruiting Centrosomal Proteins to the Nucleus

(A and B) Representative epi-fluorescence images of differentiated human immortalized myotubes from a healthy control (wild-type) or from a patient carrying a nonsense mutation within the SYNE1 (23560 G>T) gene immunostained for Pericentrin (Pcnt, red), Akap450 (red), or PCM1 (red) and (A) Myogenin (MYOG, gray) as differentiation marker or (B) the cis-Golgi marker GM130 (green) and nuclei (DAPI, blue). The scale bar represents 10 μm. See also Figure S2G.

(C) Representative epi-fluorescence images of C2C12 myoblasts transfected with dsRed-PACT and GFP or GFP-Nesprin-1α (GFP-N1α). Cells were stained for nuclei (DAPI, blue) and Myogenin (not shown). The scale bar represents 10 μm.

(D) Quantification of dsRed-PACT recruitment to the NE in non-differentiated, Myogenin-negative C2C12 cells expressing GFP or GFP-Nesprin-1α. Error bars ± SD; n represents total number of nuclei from three independent experiments.

(E) C2C12 wild-type or Nesprin-1 CRISPR mutant cells transduced with mycBirA∗-Nesprin-1α without and with 1 μg/mL doxycycline (−/+DOX) were differentiated for 48 hr, fixed, and stained for Nesprin-1 (green, clone 9F10), Pericentrin (Pcnt, red), and Myogenin (MYOG, gray). The scale bar represents 10 μm. See also Figures S2H–S2J.

(F) Quantification of Pericentrin recruitment to the NE in Myogenin-(MYOG)-positive nuclei as described in (E). Error bars ± SEM; n represents total number of nuclei from three independent experiments. ∗∗∗p < 0.001; n.s., not statistically significant, Tukey’s multiple comparisons test following one-way ANOVA.

(G) Schematic representation of the different myc-BirA∗-Nesprin constructs used for the experiments shown in (H).

(H) C2C12 wild-type, untransduced Nesprin-1 CRISPR mutant cells or CRISPR mutant cells transduced with mycBirA∗-Nesprin-1α (N1α), mycBirA∗-Nesprin-1α with the LEWD motif mutated to LEAA (N1α [WD/AA]), or mycBirA∗-Nesprin-2β (N2β) were incubated with doxycycline and differentiated for 48 hr, fixed, and stained for myosin heavy chain (MHC, green), Akap450 (red), and nuclei (DAPI, blue). The scale bar represents 10 μm. See also Figure S2K.