Impaired pericentrin localization and microtubule nucleation at the nuclear envelope in MD-1 myotubes.

<p>(<b>A</b>) Beta-tubulin (red) and pericentrin (green) double immunofluorescence staining of control and MD-1 myotubes. Chromatin was stained with DAPI (blue). Scale bar, 10 µm. Arrows indicate apparent pericentrin accumulation at the nuclear poles. (<b>B</b>) Control and MD-1 myotubes were treated with nocodazole followed by 5 min recovery in culture medium to allow microtubule regrowth. Samples were then fixed and stained as in panel A. Panels a and c show myotubes, whilst panels b and d show committed myoblasts from control and MD-1 cultures, respectively. Arrows in control cells indicate sites of microtubule regrowth at the nuclear poles, co-inciding with pericentrin localization. Arrowheads in patient cells indicate microtubule regrowth from cytoplasmic pericentrin foci. (<b>C</b>) Control and MD-1 myoblasts were fixed in methanol and subjected to immunofluorescence analysis using γ-tubulin antibodies to stain the centrosome and DAPI to stain the DNA. Distances between centrosomes and the nuclear periphery (µm) are indicated. Scale bar, 10 µm. (<b>D</b>) The number of microtubules nucleating from individual myotube nuclei prepared in B was counted and is presented as the mean ±S.D (n = 50 nuclei per sample). * p<0.05 as calculated using the Student's <i>t</i>-test. (<b>E</b>) Nucleus-centrosome distance was measured in 100 control and MD-1 myoblasts prepared in C, in two independent experiments, using Leica LAS AF Lite software and analysed using SPSS software. The median values (thick black lines) were 0.96 and 2.43 µm for control and MD-1 cells, respectively. <i>P</i> = 0.00012. ○ and * correspond to mild and extreme outliers, respectively.