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. 1999 Jun 14;145(6):1325-40.
doi: 10.1083/jcb.145.6.1325.

alpha-Dystroglycan is a laminin receptor involved in extracellular matrix assembly on myotubes and muscle cell viability

F Montanaro  1 , M Lindenbaum, S Carbonetto
Affiliations

alpha-Dystroglycan is a laminin receptor involved in extracellular matrix assembly on myotubes and muscle cell viability

F Montanaro et al. J Cell Biol. .

Abstract

alpha-Dystroglycan (alpha-DG) is a laminin-binding protein and member of a glycoprotein complex associated with dystrophin that has been implicated in the etiology of several muscular dystrophies. To study the function of DG, C2 myoblasts were transfected stably with an antisense DG expression construct. Myotubes from two resulting clones (11F and 11E) had at least a 40-50% and 80-90% reduction, respectively, in alpha-DG but normal or near normal levels of alpha-sarcoglycan, integrin beta1 subunit, acetylcholine receptors (AChRs), and muscle-specific kinase (MuSK) when compared with parental C2 cells or three clones (11A, 9B, and 10C) which went through the same transfection and selection procedures but expressed normal levels of alpha-DG. Antisense DG-expressing myoblasts proliferate at the same rate as parental C2 cells and differentiate into myotubes, however, a gradual loss of cells was observed in these cultures. This loss correlates with increased apoptosis as indicated by greater numbers of nuclei with condensed chromatin and more nuclei labeled by the TUNEL method. Moreover, there was no sign of increased membrane permeability to Trypan blue as would be expected with necrosis. Unlike parental C2 myotubes, 11F and 11E myotubes had very little laminin (LN) on their surfaces; LN instead tended to accumulate on the substratum between myotubes. Exogenous LN bound to C2 myotubes and was redistributed into plaques along with alpha-DG on their surfaces but far fewer LN/alpha-DG plaques were seen after LN addition to 11F or 11E myotubes. These results suggest that alpha-DG is a functional LN receptor in situ which is required for deposition of LN on the cell and, further, implicate alpha-DG in the maintenance of myotube viability.

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Figures

Figure 1
Figure 1
Molecular characterization of antisense DG-expressing C2 myotubes. (A) Map of antisense DG expression vector (Materials and Methods). (B) Equal amounts of proteins extracted from C2, 11F, and 11E myotube cultures were probed with mAb IIH6, directed against a carbohydrate epitope specific to α-DG. Densitometric analysis of similar blots revealed consistent 50–60% and 80–90% decreases in α-DG expression in 11F and 11E extracts, respectively, compared with C2 extracts. The same blot was also probed with an antiserum specific for α-SG. Densitometry of this protein revealed no change in its expression in the antisense-expressing clones. (C) Western blot with mAb IIH6 indicating that the expression of α-DG is not significantly affected in the 9B, 10C, and 11A antisense clones. (D) Western blot with an antiserum to the DG core protein, confirming the decreased expression of α-DG in 11F and 11E myotube cultures. (E) C2, 11F, and 11E myotubes were fractionated into soluble and KCL-washed, light microsome fractions (Materials and Methods) and equal protein loads of each fraction were probed for the presence of α-DG with mAb IIH6. The presence of α-DG in the microsomal rather than soluble fractions is consistent with proper targeting of the residual α-DG to membranes in 11F and 11E cells. (F) Western blots probed for β-DG (43 kD) showing its decreased expression in 11F and 11E clones, but not 9B, 10C, or 11A clones.
Figure 4
Figure 4
LN expression in C2, 11F, and 11E cultures. (A) Differentiated cultures of C2, 11A, 11F, and 11E myotubes were double labeled with an antiserum recognizing all three chains of mouse LN-1 (α1β1γ1) and rhodamine-conjugated α-bungarotoxin (not shown). LN immunoreactivity on C2 and 11A myotubes is a composite pattern of dots, fine linear deposits and large patches. 9B and 10C myotubes showed an identical LN distribution on their surface (not shown). LN immunoreactivity between C2 myotubes is associated with myoblasts. In contrast, large portions of 11F myotubes are devoid of LN and alternate with regions showing a punctate pattern of immunoreactivity. The few LN patches present always correspond to AChR clusters (arrow). Essentially no LN immunoreactivity was detected on 11E myotubes. Few myoblasts are present in cultures of 11F and 11E cells and large LN deposits are often present in-between myotubes. These are not shown here since their intense fluorescence obscured the labeling on the surface of myotubes at this high magnification. The dotted lines show the myotube outline. Bar, 10 μm. (B) Cultures from C2, 11F, 11E, 9B, 10C, and 11A myotubes were directly extracted in reducing sample buffer (C) and the culture medium (M) was concentrated ∼100-fold. Equal amounts of protein were probed with an antiserum that recognizes the α1, β1, and γ1 chains of LN. Decreased expression of α- and β-DG in 11F and 11E cells did not result in decreased expression of LN by the cells (C) or increased secretion in the culture medium (M).
Figure 4
Figure 4
LN expression in C2, 11F, and 11E cultures. (A) Differentiated cultures of C2, 11A, 11F, and 11E myotubes were double labeled with an antiserum recognizing all three chains of mouse LN-1 (α1β1γ1) and rhodamine-conjugated α-bungarotoxin (not shown). LN immunoreactivity on C2 and 11A myotubes is a composite pattern of dots, fine linear deposits and large patches. 9B and 10C myotubes showed an identical LN distribution on their surface (not shown). LN immunoreactivity between C2 myotubes is associated with myoblasts. In contrast, large portions of 11F myotubes are devoid of LN and alternate with regions showing a punctate pattern of immunoreactivity. The few LN patches present always correspond to AChR clusters (arrow). Essentially no LN immunoreactivity was detected on 11E myotubes. Few myoblasts are present in cultures of 11F and 11E cells and large LN deposits are often present in-between myotubes. These are not shown here since their intense fluorescence obscured the labeling on the surface of myotubes at this high magnification. The dotted lines show the myotube outline. Bar, 10 μm. (B) Cultures from C2, 11F, 11E, 9B, 10C, and 11A myotubes were directly extracted in reducing sample buffer (C) and the culture medium (M) was concentrated ∼100-fold. Equal amounts of protein were probed with an antiserum that recognizes the α1, β1, and γ1 chains of LN. Decreased expression of α- and β-DG in 11F and 11E cells did not result in decreased expression of LN by the cells (C) or increased secretion in the culture medium (M).
Figure 5
Figure 5
Expression of LN α2 chain and integrin β1 subunit in C2, 11F, and 11E cells. (A) Cultures of C2, 11F, and 11E myotubes were double labeled with rhodamine-conjugated α-bungarotoxin and with antisera to either the LN α2 chain found in merosin, or the integrin β1 subunit. LN α2 chain is present at very low level on the surface of C2 myotubes and is concentrated at AChR clusters (arrows) on C2, 11F, and 11E myotubes. The integrin β1 subunit is expressed in large oval patches on the surface of C2 and 11F myotubes, and is often found in long strips running the length of 11E myotubes. The dotted lines show the myotube outline. Bar, 10 μm. (B) Cultures from C2, 11F, 11E, 9B, 10C, and 11A cultures were detergent extracted. Proteins were separated by SDS-PAGE and blots were simultaneously probed with an antiserum to the integrin β1 subunit and a mAb to muscle actin, to compensate for differences in the degree of differentiation among cultures. No decrease in expression of the integrin β1 subunit could be detected in the antisense clones compared with C2 cultures; rather the expression of this integrin subunit appears slightly increased in 11E cultures.
Figure 2
Figure 2
Morphology of C2, 11F, and 11E cells. Cultures of C2, 11F, and 11E cells were stained with Coomassie blue at day 0, or 2 or 4 d in fusion medium. Cell morphology as well as differentiation of the antisense-expressing clones resembles that of C2 cells, apart from a decreased density of myotubes most obvious in 11E cultures at 4 d. Bar, 250 μm.
Figure 3
Figure 3
α-DG expression on the surface of C2, 11F, and 11E myotubes. Cultures of C2, 11F, and 11E myotubes were immunostained with mAb IIH6 to α-DG. For the same exposure time, α-DG staining in C2 myotubes (C2, top) was extremely intense compared with 11F and especially 11E cells. Since the specific signal was amplified using the biotin-streptavidin system (see Materials and Methods), differences in fluorescence intensity are qualitative, not quantitative. A shorter exposure time for C2 cells (C2, bottom) revealed a fine punctate immunoreactive pattern on the myotube surface. In 11F and 11E cells the density but not the pattern of expression of α-DG was affected. The dotted lines show the myotube outline. Bar, 10 μm.
Figure 6
Figure 6
Addition of exogenous LN results in aggregates of α-DG in C2 and 11F but not 11E myotubes. After treatment with exogenous LN (12 nM), the distribution of surface α-DG and LN on C2, 11F, and 11E was visualized with mAb IIH6 and the anti-LN antiserum. LN was found to elicit the formation of plaques (arrows) of surface α-DG and LN in C2 myotubes. A few such plaques were seen on 11F myotubes (arrows) but their area and number are greatly reduced relative to C2 myotubes. No α-DG or LN plaques were seen on 11E myotubes, which did not seem able to bind the exogenous LN. Photomicrographs for α-DG and LN are from separate fields. Dotted lines show the myotube outline. Bar, 10 μm.
Figure 7
Figure 7
Decreased levels of α-DG expression correlate with increased cell death. DAPI staining and TUNEL labeling of day 4 cultures of C2, 11F, 11E, 9B, 10C, and 11A cells reveal differences in cell density and in the level of apoptotic cell death between the clones. 11E cultures show a marked decrease in the number of adherent cells compared with all other clones. Bar, 100 μm.
Figure 8
Figure 8
Quantification of cell loss and apoptotic cell death. (A) Spectrofluorometric quantification (Materials and Methods) of cell number after transfer to fusion medium beginning with day 0 at which time the culture is a monolayer of confluent myoblasts. A significant decrease in the number of cells in α-DG–deficient cells is detectable from day 2 (11E) in fusion medium and is maintained until day 4. In contrast, 9B and 10C clones that express normal levels of α-DG do not show a marked decrease in cell number. Values represent mean ± SEM from two experiments. Values for day 0 were used as a reference for each cell line. (B) Proportion of proliferating cells as determined by BrdU incorporation (Materials and Methods). Decreased expression of α-DG in 11F and 11E cells is not associated with a decreased ability of these cells to proliferate. Values represent mean ± SEM from three experiments. (C) Percentage of TUNEL-labeled nuclei relative to the total number of nuclei as determined by DAPI staining. Data are from one representative experiment. Values represent means ± SEM derived from three separate dishes; 20 fields were quantified per dish using a 63× objective. * and ** represent statistically significant differences (P < 0.01 and P < 0.001, respectively).
Figure 9
Figure 9
Decreased levels of α-DG expression do not correlate with loss of membrane integrity. Live C2 and 11E cultures were stained with the vital dye Trypan blue that is normally excluded from cells with an intact plasma membrane. (A) Quantification of the percentage of myotubes with one or more nuclei stained with Trypan blue. No statistically significant difference was found between C2 and 11E myotubes (ANOVA, Fisher's t-test). 11F cells were not assayed. Data represent the mean ± SEM from three separate experiments. (B) Quantification of the percentage of Trypan blue stained nuclei belonging to myoblasts versus myotubes. No significant differences were found for either myoblasts or myotubes between C2 (open bars) and 11E (shaded bars) cultures (ANOVA, Fisher's t-test). Data represent the mean ± SEM from three separate experiments.
Figure 9
Figure 9
Decreased levels of α-DG expression do not correlate with loss of membrane integrity. Live C2 and 11E cultures were stained with the vital dye Trypan blue that is normally excluded from cells with an intact plasma membrane. (A) Quantification of the percentage of myotubes with one or more nuclei stained with Trypan blue. No statistically significant difference was found between C2 and 11E myotubes (ANOVA, Fisher's t-test). 11F cells were not assayed. Data represent the mean ± SEM from three separate experiments. (B) Quantification of the percentage of Trypan blue stained nuclei belonging to myoblasts versus myotubes. No significant differences were found for either myoblasts or myotubes between C2 (open bars) and 11E (shaded bars) cultures (ANOVA, Fisher's t-test). Data represent the mean ± SEM from three separate experiments.
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