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First published online 11 April 2006
doi: 10.1242/jcs.02891

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Plectin scaffolds recruit energy-controlling AMP-activated protein kinase (AMPK) in differentiated myofibres

Martin Gregor^1,*, Aniko Zeöld^1,*, Susanne Oehler¹, Kerstin Andrä Marobela¹, Peter Fuchs¹, Günter Weigel², D. Graham Hardie³ and Gerhard Wiche^1,

¹ Department of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, A-1030 Vienna, Austria
² Department of Cardiothoracic Surgery, Medical University of Vienna, A-1090 Vienna, Austria
³ Division of Molecular Physiology, Wellcome Trust Biocentre, University of Dundee, Dundee, DD1 5EH, UK

View larger version (29K): [in a new window]   Fig. 1. Interaction of AMPK 1 subunit with plectin. (A) Schematic representation showing the domain structure of plectin and the fragment (ple-R5-6; I4218-G4543; GenBank accession number AY480038) used as bait in the yeast two-hybrid screening (Osmanagic-Myers and Wiche, 2004). The actin- and IF-binding domains (ABD, IFBD), the rod and C-terminal repeat domains 1-6 of plectin are indicated. The isolated AMPK 1 subunit clone contained the entire coding sequence comprising four CBS domains. (B) Plectin was immunoprecipitated from lysates of wild-type (+/+) myotubes using anti-plectin antiserum. Lysates (Lys) and immunoprecipitates (IP) were analysed by immunoblotting using antibodies immunoreactive with AMPK 1 subunit and plectin. Note Co-IP of AMPK 1, and absence of signal in control sample (C) run without precipitating antibodies and in sample from plectin-deficient myocytes (-/-). (C) Immunoprecipitation of 1, 2, and 1 AMPK subunits from lysates of differentiated wild-type myocytes (myotubes) using indicated antibodies, and detection of cosedimenting 1 and 1 AMPK subunits by immunoblotting.

View larger version (106K): [in a new window]   Fig. 2. Colocalisation of ectopically expressed GFP-AMPK 1 subunit fusion protein with endogenous AMPK 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-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 1 subunit of AMPK (A,D,G); the GFP-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 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 1 subunit fusion protein, the regular arrangement of AMPK (as seen in wild-type myotubes) is lost (G-I). Both, the overexpressed GFP-1 subunit fusion protein (H) and the endogenous AMPK 1 subunit (G) showed a more diffuse staining pattern compared with wild-type cells. Bars, 10 µm.

View larger version (85K): [in a new window]   Fig. 3. Colocalisation of plectin and AMPK in differentiated myotubes in a cross-striated pattern. Double immunofluorescence microscopy, using anti-plectin antiserum #46 and anti-AMPK 1 subunit antibodies, revealed distinct staining patterns in myoblasts (A-C). The staining patterns of AMPK (A) and plectin (B) were punctuated and filamentous, respectively, showing no colocalisation in distant cytoplasmic regions of myoblasts (C). By contrast, in differentiated myotubes (D-F) and on longitudinal sections of mouse skeletal muscle fibres (see Fig. 5), plectin and AMPK 1 showed extensive colocalisation, both in overlapping cross-striated patterns (F). Bars, 10 µm.

View larger version (36K): [in a new window]   Fig. 4. Differentiation-dependent and subunit-dependent interaction of plectin with AMPK. (A,B) Lysates prepared from wild-type (+/+) and plectin deficient (-/-) undifferentiated myoblasts (A), or differentiated (6 days) myotubes (B) were incubated with antiserum #46 to plectin (IP), or without antibodies (C) for 3 hours. Immunocomplexes formed were isolated using protein G beads, separated by 10% SDS-PAGE and subjected to immunoblotting using antiserum to plectin, a 1:1 mixture of antibodies to the 1 and 2 subunits of AMPK (total AMPK), or antibodies recognising AMPK 1 or 2 subunits individually. AMPK 1 was co-precipitated with plectin only from lysates prepared from differentiated myotubes; AMPK 2 did not co-precipitate with plectin at any stage. (C) Lysates prepared from myotubes as in B were incubated with antibodies to AMPK 1 or 2 and immunoprecipitates were analysed by immunoblotting using the antibodies indicated. Lys, immunoblots of lysates.

View larger version (132K): [in a new window]   Fig. 5. Plectin deficiency alters the staining pattern of AMPK catalytic subunits 1 in skeletal muscle fibres. Frozen tissue sections of m. soleus isolated from 3-month-old wild-type (A-E,K-O; ple +/+) and muscle-specific plectin knockout (F-J,P-T; ple -/-) mice were immunolabelled using antibodies to AMPK 1 (A,F) and 2 (K,P) subunits, and plectin (B,G,L,Q). Note that plectin-specific staining in knockout tissue is restricted to connective tissue and blood vessels; only AMPK 1, but not 2, displayed extensive colocalisation with plectin (compare magnified boxed areas E and O); and fibres without plectin showed reduced AMPK 1 staining, and the striated staining pattern of the 1, rather than the 2 AMPK subunit was considerably deteriorated compared with wild-type (magnified boxed areas D, I and N, S, respectively). Bar, 10 µm.

View larger version (27K): [in a new window]   Fig. 6. Plectin-deficient myocytes display differentiation-dependent changes in AMPK subunit expression. (A) Total cell lysates, prepared from differentiating wild-type (+/+) and plectin-deficient myocytes (-/-) at the days (d) indicated, were subjected to immunoblotting using AMPK 1-, 2- and 1 subunit-specific antisera, as well as antibodies immunoreactive with all AMPK subunit isoforms (total AMPK). Equal amounts of proteins were loaded in each lane and protein bands were visualised as described in the text. The extent of differentiation of plectin+/+ and -/- myoblasts was monitored in parallel using antibodies to caveolin 3, myogenin and MyoD. (B-D) Signal intensities of AMPK subunit bands, densitometrically determined in three independent experiments (including that shown in A), were normalised to total AMPK. Error bars represent the s.e.m. of three independent experiments. Differences between values in wild-type (ple +/+) and plectin-deficient (ple -/-) cells were determined using an unpaired Student's t-test; *P<0.05.

View larger version (105K): [in a new window]   Fig. 7. Plectin is predominantly expressed in oxidative skeletal muscle fibres. Immunofluorescence microscopy of frozen sections of rat m. tibialis anterior using mAb 10F6 to plectin and NADH staining, performed on consecutive sections, are shown in (A) and (B), respectively. Note darkly stained mitochondria-rich oxidative type I fibres (I) and lightly stained glycolytic type II fibres (II) in B, and stronger plectin-staining in oxidative type I fibres in A. Bar, 25 µm.

View larger version (31K): [in a new window]   Fig. 8. Differential activation (phosphorylation) pattern of AMPK in differentiating wild-type (ple +/+) and plectin-deficient (ple -/-) myocytes: specificity and phosphatase independence. (A,B) Total cell lysates of wild-type or plectin-deficient myocytes, differentiated (diff.) for the time periods indicated (0-6 days), were subjected to immunoblotting using antibodies to unphosphorylated/phosphorylated (total) and phosphorylated (P-) forms of AMPK and p38 MAP kinase, respectively. (C,D) Signal intensities of phosphorylated AMPK and p38 MAP kinase protein bands were densitometrically determined in three independent experiments (including those shown in A,B), and normalised to total AMPK and p38 MAP kinase, respectively. Error bars represent the s.e.m. (E-G) Enzymatic activities of phosphatases (PP) 1, 2A, and 2C were measured in cell lysates obtained from undifferentiated myoblasts (E), or myoblasts differentiated for 3 (F) or 6 days (G), using a fluorescent substrate. Error bars represent the s.e.m. of three independent experiments. (C-G) Differences between values in ple (+/+) and ple (-/-) were determined using an unpaired Student's t-test; *P<0.05.

View larger version (35K): [in a new window]   Fig. 9. Elevated proportion of Thr172-P AMPK in muscle of newborn plectin-/- mice. (A) Immunoblot of skeletal muscle (diaphragm) and heart tissue homogenates prepared from plectin+/+ and plectin-/- newborn mice using antibodies to unphosphorylated/phosphorylated (total) and phosphorylated (P-) forms of AMPK. (B) Signal intensities of phosphorylated AMPK protein bands were densitometrically determined in three independent experiments (including that shown in A), and normalised to total AMPK. Error bars represent the s.e.m. of these experiments. Differences between plectin+/+ and plectin-/- values were determined using an unpaired Student's t-test; **P<0.01; P<0.001.

View larger version (11K): [in a new window]   Fig. 10. Cellular energy state. The ATP, ADP and AMP nucleotide pools in lysates of wild-type (ple +/+) and plectin-deficient (ple -/-) myocytes were determined by HPLC at various stages of differentiation and the corresponding energy charge (ATP + 1/2ADP) / (ATP + ADP + AMP) values (A) and the AMP:ATP nucleotide ratios (B) were calculated. Error bars represent the s.e.m. of three independent experiments.