Fig 2: Microtubule Nucleation from the Nucleus Is Required for Proper Nuclear Positioning(A) 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 were immunostained with Akap450 (red), GM130 (gray), microtubules (MTs, green), and nuclei (DAPI, blue) following nocodazole washout. The scale bar represents 10 µm. See also Figure S4A.(B) C2C12 cells were transfected with the indicated siRNAs, differentiated for 48 hr, and stained for nuclei (DAPI, blue) and myosin heavy chain (MHC, white). Myotube outlines are marked by dashed lines, and nuclei are encircled.(C) Spreading factor analysis of nuclei in C2C12 myotubes as shown in (B) and for cells transfected with Nesprin-1 siRNA no. 2. Results are depicted as mean (blue line) with interquartile range (black bars) from three independent experiments. ***p < 0.001; **p < 0.01; Mann-Whitney test.(D) Spreading factor analysis of nuclei in differentiated human immortalized myotubes from a healthy control (wild-type) or SYNE1 (23560 G>T) patient cells. Results are depicted as mean (blue line) with interquartile range (black bars) from two independent experiments. ***p < 0.001; Mann-Whitney test. See also Movie S1.(E) C2C12 cells were transfected with the indicated siRNAs, differentiated for 48 hr, and stained for nuclei (DAPI, blue) and myosin heavy chain (MHC, white). Myotube outlines are marked by dashed lines, and nuclei are encircled.(F) Spreading factor analysis of nuclei in C2C12 myotubes shown in (E) and for myotubes transfected with Akap450 siRNA no. 2 and Pcnt (Pericentrin) siRNA no. 2. Results are depicted as mean (blue line) with interquartile range (black bars) from four independent experiments. ***p < 0.001; n.s., not statistically significant, Mann-Whitney test.(G) Snapshots of myotubes simulated with Cytosim. Nuclei are blue, and MTs in white. Three conditions are shown from top to bottom, with (+) and without (-) MTs nucleated from the NE and ±Kif5b anchored at the NE, as indicated. ***p < 0.001; Mann-Whitney test. See also Movie S2 and Figures S4B–S4D.(H) Spreading factor analysis of nuclei in simulated myotubes as shown in (G). Results are depicted as mean (blue line) with interquartile range (black bars). ***p < 0.001; **p < 0.01; Mann-Whitney test. See also Figure S4E.

Fig 3: Partial co-localization of nesprin-1-alpha2 with the centrosomal protein AKAP9 in human myotube cultures. The centrosomal scaffolding protein AKAP9 was partially localized at the nuclear envelope (a) and also between nuclei (d). Emerin (e) had a uniform nuclear envelope distribution whereas nesprin-1-alpha2 (N1a2: b) showed better, though partial, co-localization with AKAP9.

Fig 4: The Muscle-Specific Nesprin-1a 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-1a (GFP-N1a). 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-1a. 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-1a 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-1a (N1a), mycBirA*-Nesprin-1a with the LEWD motif mutated to LEAA (N1a [WD/AA]), or mycBirA*-Nesprin-2ß (N2ß) 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.

Fig 5: Myogenin expression is sufficient to induce nuclear envelope microtubule-organizing center (NE-MTOC) formation in non-muscle cells.(A) NIH3T3 fibroblasts were transfected with constructs encoding GFP, MyoD-GFP or myogenin-GFP (Myog-GFP). After three days, PCM-1 localization was assessed by immunostaining. Arrows indicate nuclei of transfected cells which have recruited PCM-1. Scale bars: 10 µm. (B) Quantification of (A) demonstrating that myogenin induces nuclear envelope localization of PCM-1 more efficiently than MyoD. Data are represented as individual biological replicates (n = 3), together with mean ± SD. ***: p < 0.001; 95% CI of difference Myog-GFP vs. MyoD-GFP = 30.99% to 49.22%. n = 3. (C-H) NIH3T3 Tet-ON mScarlet or MYOG-2A-mScarlet (MYOG-mScarlet) cells were treated with doxycycline (Dox) for three days. After immunostaining, nuclear envelope localization of PCM-1 (C-D), PCNT (E-F), and AKAP9 (G-H) was analyzed and quantified. Data are depicted as violin plots. Red line indicates the median, dotted lines indicate the 25% and 75% percentile. ***: p < 0.001. Scale bars: 20 µm (I) Immunostaining of MYOG-mScarlet cells treated with Dox for three days showing the presence of nesprin-1a+ nuclei. Scale bars: 20 µm (J) RT-PCR analysis of MYOG-mScarlet cells in the absence of Dox (-Dox) or treated with Dox for the indicated time points demonstrating that nesprin-1a is upregulated upon myogenin expression. Gapdh was used as equal input control. (K) ChIP-PCR analysis of Dox-treated MYOG-mScarlet cells using an anti-myogenin antibody or an IgG1 control showing that myogenin binds an E-box in the nesprin-1a promoter region. (L-M) Immunostaining of a-tubulin and subsequent quantification of nuclear envelope coverage after 30s of microtubule regrowth following cold-induced microtubule depolymerization in mScarlet or MYOG-mScarlet cells treated with Dox for three days. Data are depicted as violin plots. Red line indicates the median, dotted lines indicate the 25% and 75% percentile. ***: p < 0.001. Scale bars: 20 µm. N numbers indicate total number of analyzed nuclei pooled from three biological replicates. Figure 2—source data 1.Underlying data for graphs in Figure 2B, D, F and H. Figure 2—source data 2.Raw files and uncropped gels for Figure 2J. Figure 2—source data 3.Raw files and uncropped gels for Figure 2K.