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Figure 2


Fig. 2. Colocalisation of ectopically expressed GFP-AMPK {gamma}1 subunit fusion protein with endogenous AMPK {alpha}1 subunit in wild-type (ple +/+) myocytes (A-F) and dislocation of AMPK in plectin-deficient (ple -/-) myotubes (G-I). Wild-type or plectin-deficient mouse myoblasts transfected with expression plasmids encoding a GFP-{gamma}1 subunit fusion protein were subjected to immunofluorescence microscopy either in their undifferentiated state (A-C), or after differentiation for 4 days (D-I), using antibodies to the {alpha}1 subunit of AMPK (A,D,G); the GFP-{gamma}1 fusion protein was visualised directly (B,E,H). Note, colocalisation of the two AMPK subunits in wild-type cells (C,F), indicating that the overexpressed {gamma}1-subunit became integrated into the native AMPK complex. In wild-type myocytes differentiated for 4 days, AMPK showed a filamentous arrangement (D-F) with a striated appearance in subsarcolemmal regions (see boxed area magnified in insert). In plectin-/- myotubes, expressing GFP-AMPK {gamma}1 subunit fusion protein, the regular arrangement of AMPK (as seen in wild-type myotubes) is lost (G-I). Both, the overexpressed GFP-{gamma}1 subunit fusion protein (H) and the endogenous AMPK {alpha}1 subunit (G) showed a more diffuse staining pattern compared with wild-type cells. Bars, 10 µm.





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