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First published online January 14, 2005
doi: 10.1242/10.1242/jcs.01630

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Remodelling of the nuclear lamina and nucleoskeleton is required for skeletal muscle differentiation in vitro

Ewa Markiewicz, Maria Ledran^* and Christopher J. Hutchison

Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham, DH1 4EB, UK

View larger version (44K): [in a new window]   Fig. 1. Changes in expression of lamins and LAP2 during myogenesis. Exponentially growing C2C12 myoblasts were induced to differentiate with 2% horse serum. Whole cell extracts were prepared at 0, 12, 18, 24, 36, 48, 72, 96 and 120 hour time points, and quantities were standardized to equal amounts of DNA as follows. Cell extracts were prepared at various time intervals and subjected to agarose-gel electrophoresis and stained with ethidium bromide. The genomic DNA was visualized as a slowly migrating band, separated from the faster migrating RNA specimens. To assure that detection was within a linear range, the extracts were analysed in serial dilutions. The intensity of bands corresponding to genomic DNA was analysed by densitometry using UVI bandmap software and the volume of each sample adjusted so that equal amounts of DNA were added to each lane of protein gels. Levels of expression of myogenin (a), lamin A (b), lamin B1 (c), lamin B2 (d), lamin C (e) and LAP2 (f) were analysed by western blotting and the intensity of each band evaluated by densitometry. The graphs below each blot are representative of three independent experiments.

View larger version (57K): [in a new window]   Fig. 2. Myoblast differentiation is accompanied by redistribution of lamins. C2C12 myoblasts were grown on glass coverslips and induced to differentiate with 2% horse serum. 0, 48-72 and 96-120 h after transfer to differentiation medium, cells were fixed and co-stained for either lamin A/C (a) or lamin B1(b) and myogenin (N.B. myoblasts do not differentiate synchronously and therefore the intensity of nuclear staining with anti-myogenin antibodies varies between cells within a single time point). z-axis sections of 48-hour myoblast cultures stained for lamin C were collected at 0.35 µm intervals and projected (c) (short arrows indicate a postmitotic myoblast, asterisks indicate mitotic myoblasts). Alternatively, z-axis series were obtained using a BioRad Lasersharp 2000 confocal microscope with the laser power adjusted to eliminate saturation of the image. Central sections were converted to PICT images without adjustment and surface plotting was performed using LaserPix software. (d) Original midsections stained with anti-lamin-A/C antibodies. Anti-myogenin staining was used to identify differentiated cells (arrows) but is not shown here. Surface plots of the same field are shown in (e). Red coloration shows the areas of most intense fluorescence (maximum output of 215 on a scale of 0-255), whereas blue shows the areas of least intense fluorescence (minimum output 0 on a scale of 0-255). 48-hour cultures were co-stained with anti-lamin-C and anti-lamin-B2 antibodies. Arrowheads indicate cells showing diffuse anti-lamin-C staining and weak anti-lamin-B2 staining (f). Images were collected on a BioRad Lasersharp 2000 confocal microscope. Bars, 10 µm.

View larger version (49K): [in a new window]   Fig. 3. Solubility properties of lamins and lamina-associated proteins during myogenesis. C2C12 myoblasts were induced to differentiate with 2% horse serum. At 0, 72 and 120 hours after transfer to differentiation medium, cells were harvested and subjected to nuclear isolation. Nuclei were either solubilized in SDS (nuclei) or sequentially extracted with hypotonic (LS) or hypertonic (HS) buffer. Samples were resolved by SDS-PAGE along with material resistant to extraction (INS), transferred to nitrocellulose and blotted with antibodies against lamin B1 (a), lamin B2 (b), lamin A (c) and lamin C (d), and the intensity of each band evaluated by densitometry and expressed as a proportion of each protein in whole nuclear extracts.

View larger version (61K): [in a new window]   Fig. 4. Changes in solubility properties of LAP2 during myogenesis. (a) C2C12 myoblast cultures were induced to differentiate and, after 0, 72 and 120 hours, cells were harvested and subjected to nuclear isolation. Nuclei were either solubilized in SDS (nuclei) or sequentially extracted with hypotonic (LS) or hypertonic (HS) buffer. Samples were resolved by SDS-PAGE along with material resistant to extraction (INS), transferred to nitrocellulose and blotted with antibodies against LAP2 and the intensity of each band evaluated by densitometry. (b) C2C12 myoblasts were grown on glass coverslips and induced to differentiate with 2% horse serum. After 0, 48, 72, 96 and 120 hours of differentiation, cells were fixed and immunostained with anti-lamin, anti-LAP2 and anti-myogenin antibodies, and observed using a confocal microscope. Arrows show postmitotic myoblasts and arrowheads show mitotic myoblasts. (c) 48-hour cultures of differentiating myoblasts were stained with anti-lamin C and LAP15 (anti-LAP2) antibodies. Bars, 10 µm.

View larger version (77K): [in a new window]   Fig. 5. Transfection of C2C12 myoblasts with mutants of lamin A prevents nuclear envelope organization and inhibits myoblast differentiation. C2C12 cells grown on glass coverslips were transiently transfected with wild-type lamin A or lamin A with point mutation W520S. 24 hours after transfection, cells were either incubated in growth medium for next 24 hours (undifferentiated, a) or induced to differentiate with 2% horse serum (differentiated, b). The cultures were fixed and co-stained for the presence of the exogenous lamin A (with anti-FLAG antibodies) and endogenous myogenin (a,b), endogenous lamin B1 (c,d) or endogenous lamin C (e,f). (a,b) Examples of nuclei scored as having nucleoplasmic staining (see Fig. 6) are indicated with * or +; examples of cells scored as having nuclear envelope staining are indicated with @. Following transfection with any FLAG vector, differentiation events are delayed by approximately 48 hours. However, cultures transfected with FLAG-W520S did not differentiate at all. Images were obtained using a BioRad laser sharp 2000 confocal microscope. Bars, 10 µm.

View larger version (27K): [in a new window]   Fig. 6. Transfection of C2C12 myoblasts with dominant mutants of lamin A prevents nuclear-envelope organization and inhibits myoblast differentiation. C2C12 myoblasts were transfected in triplicate experiments with wild-type (wt) lamin A or lamin A W520S. 24 hours after transfection, cells were induced to differentiate with 2% horse serum. At 24-hour intervals, cells were fixed and stained with anti-myogenin antibody and anti-FLAG antibody. Two hundred transfected cells were counted for strong staining of exogenous lamins in nucleoplasm (a) or at the nuclear envelope (b), or for expression of myogenin (c). Results show the mean ± the standard deviation from triplicate experiments.

View larger version (80K): [in a new window]   Fig. 7. Association of Rb with LAP2 and disruption of Rb-LAP2 complexes by mutant lamin A. The expression and localization of Rb and LAP2 were investigated by double immunofluorescence and confocal microscopy by co-staining undifferentiated (a) and differentiated myoblasts (b) with anti-LAP2 antibodies (left) and anti-RbS780 (centre). Micrographs are displayed as individual black and white or two-colour merged images (right). The formation of multiprotein complexes containing Rb, LAP2 and lamin C was investigated by immunoprecipitation (c-e) from cultures containing undifferentiated myoblasts (left), differentiated myoblasts (centre) and myotubes (right). Cell extracts were immunoprecipitated with anti-LAP2 antibodies (c,e) or anti-lamin C antibodies (d) and blotted with anti-RbS780 (c,d) or anti-LAP2 antibodies (e). sup, supernatant; Ppt, precipitate; *, hyperphosphorylated Rb; -, hypophosphorylated Rb (c,d); arrow in e, LAP2. Expression of mutant (mut) lamin A disrupts LAP2 and leads to loss of expression of Rb (f-i). C2C12 myoblasts were transfected with wild-type (f,h) or mutant (g,i) lamin A constructs and fixed and stained with antibodies against LAP2 (f,g) or RbS780 (h,i) before (top) or after (bottom) cultures were induced to differentiate. Each montage shows the exogenous lamin (left), the endogenous LAP2 or Rb (middle) and two-colour merged images (right). Arrowheads (g, bottom) show transfected cells in which LAP2 is no longer detected. Arrows (i, bottom) show transfected cells in which RbS780 is no longer detected. Bars, 10 µm.

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