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First published online 8 August 2006
doi: 10.1242/jcs.03096

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Non-muscle myosins 2A and 2B drive changes in cell morphology that occur as myoblasts align and fuse

Nathan T. Swailes^*, Melanie Colegrave, Peter J. Knight and Michelle Peckham

Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK

View larger version (51K): [in a new window]   Fig. 1. RT-PCR analysis at 0, 24, 48 and 72 hours of NM2A and NM2B expression levels in knockdown experiments. Data were obtained for untreated, sense, scrambled and antisense cells. (A) Example PCR results are shown for NM2A oligonucleotides and the corresponding ß-actin controls, and NM2B oligonucleotides. The data in each row were taken from the same experiment, and the same gel, but the results have been re-organised for display. (B) Time course of RNA levels for NM2A and NM2B in response to oligonucleotide treatment. RNA levels are normalised to actin mRNA in each extract and to the level at the zero time point. n=2 for NM2B; n=3 for NM2A and the error bars (s.e.m.) are shown for NM2A.

View larger version (121K): [in a new window]   Fig. 2. Effect of NM2A knockdown on myoblast alignment and fusion. The images show intensity-inverted, fluorescent, low-power images of differentiating myoblast cultures left untreated, or treated for 72 hours with sense, scrambled or antisense oligonucleotides for NM2A. The cells were co-stained for NM2A, actin and skeletal myosin. All these images were taken using the same settings, and contrast adjusted equally.

View larger version (129K): [in a new window]   Fig. 3. High power images of differentiating myoblast cultures after 36 hours of treatment, co-stained for NM2A, actin and nuclei. In all the control cultures, cells are present that have a characteristic bipolar shape (arrows), stain strongly for actin compared with non-differentiated cells, and are beginning to align and fuse. By contrast, in NM2A-knockdown cultures, cells have an irregular shape (arrows), and do not form groups of aligned bipolar cells. Bar, 20 µm.

View larger version (54K): [in a new window]   Fig. 4. Blended projection images of NM2A- and NM2B-knockdown cells: effects on cell shape and adhesion. (A) Projection images of NM2A- and NM2B-knockdown and control cells (NM2A-sense treated). The cells have been stained for actin (green), NM2A (for antisense NM2A and sense NM2A) or NM2B (red) and DNA (blue). Sites of interaction between cells are indicated by arrows. (B) A projection image for NM2B-knockdown cells before (0 hours) and after 36 or 72 hours of treatment. The cells have been stained for actin (green), NM2B (red) and DNA (blue). This demonstrates the elongation of the cells following NM2B-knockdown and the change in shape of the lamellipodia. Bar, is 20 µm for all six images.

View larger version (59K): [in a new window]   Fig. 5. NM2B-knockdown affects cell shape and alignment. (A) Low power images show intensity-inverted, fluorescent, low-power images of differentiating myoblast cultures left untreated or treated for 72 hours with sense, scrambled or antisense oligonucleotides for NM2B. These images were taken from less dense regions to show the dramatic increase in cell length that results from NM2B treatment, compared with controls. The cells were co-stained for NM2B and actin filaments. Images were taken using the same settings, and contrast adjusted equally for all the images. (B) Intensity-inverted, fluorescent, high-power images of differentiating myoblast cultures stained for actin after 36 hours of treatment with sense or antisense oligonucleotides to compare cell shape and alignment in groups. The arrows show aligned bipolar cells but, in the antisense treatment, elongated connections between cells (arrowheads) are evident.

View larger version (76K): [in a new window]   Fig. 6. High power images of myoblasts show NM2B knockdown greatly increases cell length after 36 hours of treatment. Aligned groups containing bipolar cells are present in all of the control conditions but, in NM2B-knockdown cells, the bipolar cells are highly elongated (see arrow) and there is less overlap between them. These images were taken using a x40 objective lens to enable visualisation of the highly elongated NM2B-knockdown cells.

View larger version (54K): [in a new window]   Fig. 7. Immunofluorescent images of NM2B-knockdown cells after 72 hours of treatment in dense cultures shows that cells can still fuse. Cells were co-stained for skeletal myosin (red), actin (green) and DNA (blue). To the left are low-power images that show the presence of myotubes positive for skeletal myosin. To the right are high-power images each showing a myotube formed by NM2B-knockdown cells, compared with a myotube formed in control conditions (sense NM2B-treated). Five nuclei seen in the NM2B-knockdown cells are all very close together, whereas the two nuclei in a similar length of myotube formed by control cells are widely spaced.

View larger version (83K): [in a new window]   Fig. 8. Still images from a time-lapse experiment for NM2A- or NM2B-knockdown. Three sets of montages are shown, one each for untreated, NM2A- or NM2B-knockdown cells, from a single time-lapse DIC imaging experiment. The cells were left untreated or treated with antisense oligonucleotides for 24 hours before the start of filming, and filmed for the next 18 hours immediately after adding additional oligonucleotides. The numbers shown in the corner of each frame are the time elapsed since the first frame (time=0) in minutes (frame interval was 5 minutes). The arrows for the untreated cells show the progress of a single cell as it forms part of an aligned group of bipolar cells. The arrows for the NM2A-knockdown experiment show a single cell that tends to have a longer tail than controls but is able to retract. The arrows for the NM2B-knockdown experiment show a cell that becomes highly extended as it interacts with, and fails to detach from, other cells in the culture, and eventually its tail breaks (at t=185 minutes). See also supplementary material Movies 1-3.

View larger version (81K): [in a new window]   Fig. 9. Western analysis of NM2A and NM2C expression in myoblasts (MB) and 3 day myotubes (MT). Western analysis shows a positive band of the correct size for NM2A in myoblasts and myotubes. By contrast, despite using a longer exposure time (and hence higher background), a positive band of the correct size for NM2C was only observed in myotubes and not in myoblasts. Protein loading in each of the lanes was the same.

View larger version (68K): [in a new window]   Fig. 10. Distributions of NM2A and NM2B by immuno-electron microscopy. (A) A single confocal slice from a group of aligned bipolar cells co-immunostained for NM2B, actin and DNA (DAPI). The majority of actin and non-muscle myosin (NM2B) is found at the plasma membrane (arrow). The arrowhead denotes a cell that is in the final stages of cell-division, that remains associated with the group of aligned cells. The staining pattern for NM2A is similar, and the staining shown here is similar to that reported earlier (Wells et al., 1997). (B) Representative electron micrographs of sections near the ventral surface of aligned myoblasts labelled with a primary antibody raised against NM2A, or a primary antibody raised against NM2B. The gold particles can be seen clearly, and only on the cytoplasmic side of the plasma membrane. For comparison, a control section is shown in which the primary antibody was omitted. No gold particles are observed in the control cells. (C) Distribution of gold particles for NM2A in three aligned cells, with respect to the plasma membrane. The mean±s.d. for each cell is shown, where n=10, and represents the number of different sampling positions, at 5 µm intervals from one end of the cell to the other along each of the cells. Nine sections (three dorsal, three middle and three ventral) were averaged together for each sampling position after the proportion of gold particles within each 100 nm bin was calculated. In total, the distance of 10,966 particles from the plasma membrane was measured and used in this analysis. Labels for the x-axis are as shown for E. (D) Distribution of gold particles for NM2B with respect to the plasma membrane in three aligned cells. The mean±s.d. is shown as for NM2A in C. In total, the distance of 9180 particles from the plasma membrane was measured and used in this analysis. Labels for the x-axis are as shown for E. (E) A direct comparison of the distributions of NM2A (mean for three cells shown in C) and NM2B (mean for three cells shown in D) gold labelled particles in the six aligned cells. As no significant differences (by chi-square test) were observed in the distribution of particles at each of 5 µm sampling positions along the cells, or between cells, the data were pooled into a single figure for the three cells for each myosin isoform. For NM2A and NM2B, the mean±s.d. is shown, where n=3 (number of cells).

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