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Fibre-type specific concentration of focal adhesion kinase at the sarcolemma: influence of fibre innervation and regeneration

Martin Flück^1,2,*, Andrew Ziemiecki³, Rudolf Billeter^2,4 and Markus Müntener⁵

¹ M. E. Müller-Institute for Biomechanics, University of Bern, Bühlestrasse 26, 3000 Bern 9, Switzerland
² Institute of Anatomy, University of Bern, Bühlestrasse 26, 3000 Bern 9, Switzerland
³ Department of Clinical Research, University of Bern, Bühlestrasse 26, 3000 Bern 9, Switzerland
⁴ School of Biomedical Sciences, University of Leeds, Leeds LS2 9NQ, UK
⁵ Institute of Anatomy, University of Zürich-Irchel and Department of Applied Biosciences ETH, Zürich, Switzerland

View larger version (33K): [in a new window]   Fig. 1. Schematic representation of a costamere and the focal adhesion complex (FAC). (A) Two laminin receptors, a dystrophin/glycoprotein complex and an integrin receptor complex are among the sarcolemmal structures (Pardo et al., 1983) that link the contractile apparatus of muscle fibres with the surrounding basal lamina. Components of both receptors, i.e. both dystrophin and the integrin-associated cytoskeletal proteins (talin, vinculin, -actinin), co-localise in subsarcolemmal complexes (Pardo et al., 1983) which connect through -actin and the intermediate-filament proteins desmin and vimentin to the Z-disk of skeletal muscle fibres (adapted from Patel and Lieber, 1997; Rybakova et al., 2000). (B) Integrin-based FACs of cultured mesodermal cells bridge cortical -actin to the extracellular matrix (ECM). The inset indicates schematically the proposed involvement of FAK (in red) in the formation of FACs. Occupancy of integrins with ECM ligand (1) causes phosphorylation (orange circle) of integrin-associated FAK (2) which, in turn, promotes recruitment of both cytoskeletal (paxillin, vinculin, talin, -actinin and -actin; coloured in dark green) and signalling molecules (3) (e.g. MAPK and c-src kinase, coloured in light green) to integrins (Miyamoto et al., 1995).

View larger version (38K): [in a new window]   Fig. 2. Characterisation of antibodies. Triton X-100 extracts of rat normal soleus muscle were subjected to immunoprecipitation with normal (Ø), polyclonal rabbit antisera A-17 against the N terminus (N) or the serum (C) of focal adhesion kinase (FAK) against the C-terminal portion (C) of FAK protein. Equal amounts of the precipitates, together with a sample of cell extract (E), were resolved by SDS—PAGE on 7.5 % gels and immunoblotted with either anti-FAK antiserum C-FAK (A) or anti-phosphotyrosine antibody (pTyr) (B). The sizes of molecular mass markers are indicated.

View larger version (17K): [in a new window]   Fig. 3. Focal adhesion kinase (FAK) expression in normal slow- and fast-twitch muscle. (A) Soluble deoxycholate protein extract (10 µg) of slow-twitch normal soleus (N-SOL) and fast-twitch normal extensor digitorum longus (N-EDL) muscle was subjected to immunoblotting analysis using the C-FAK serum. The position of the FAK protein is indicated by an arrow. (B) A Ponceau-S-stained transfer membrane prior to detection showing that approximately equal amounts of protein were loaded. The sizes of molecular mass (MW) markers are indicated.

View larger version (142K): [in a new window]   Fig. 4. Focal adhesion kinase (FAK) localisation in normal slow-twitch muscle. Immunocytochemical analysis of cross (A—D) and longitudinal (E,F) sections from slow-twitch normal soleus (N-SOL) muscle with the FAK N-terminal antiserum A-17 (A,E). Positive staining appears orange and nuclei appear blue. Control reactions of consecutive cryosections with normal rabbit serum are also shown (B,F). Arrows point to FAK-immunoreactivity at the sarcolemma. Consecutive sections were also stained for slow (C) or fast myosin (D) isoforms to determine the fibre types. All fibres, with the exception of two denoted type IIA, are of type I. Scale bars, 50 µm.

View larger version (18K): [in a new window]   Fig. 9. Quantification of sarcolemmal focal adhesion kinase (FAK) expression in different fibre types of normal and foreign-reinnervated rat skeletal muscles: Fibres were classified into different types and subdivided on the criteria of FAK immunoreactivity into a sarcolemmal FAK-positive and sarcolemmal FAK-negative fibre population. The percentage of sarcolemmal FAK-positive fibres for each fibre type was then counted as described in Materials and methods. The histograms display the mean + S.E.M. (N=4-6) of the calculated percentage of sarcolemmal FAK-immunoreactive fibres in (A) normal soleus muscle (N-SOL, white columns), cross-reinnervated (X-SOL) soleus muscle (black columns) and transplanted and foreign-reinnervated (T-SOL) soleus muscle (grey columns), and in (B) normal extensor digitorum longus (N-EDL, white columns) and transplanted and reinnervated (T-EDL) extensor digitorum longus muscle (black columns). Individual values were compared using a bilateral 2-test for statistical significance. An asterisk denotes a significant difference (P<0.001) in the percentage of sarcolemmal FAK-immunoreactive fibres in a muscle fibre type between the experimental muscle types marked with a bracket.

View larger version (82K): [in a new window]   Fig. 5. Staining specificity of different focal adhesion kinase (FAK) antibodies. Immmunocytochemical analysis of FAK in parallel cryosections of fast-twitch normal extensor digitorum longus (N-EDL) muscle with polyclonal A-17 (A) and monoclonal FAK antiserum 2A7 (B). Positive staining is orange and nuclei appear blue. A control reaction with normal rabbit serum is also shown (C). Arrows indicate FAK immunoreactivity at the sarcolemma. Scale bar, 100 µm.

View larger version (127K): [in a new window]   Fig. 6. Focal adhesion kinase (FAK) localisation in fast-twitch muscle. (A) Immunocytochemical analysis of fast-twitch normal extensor digitorum longus (N-EDL) muscle for FAK protein with serum A-17. Fibre typing was carried out by detecting the expression of slow (C) and fast (D) myosin isoforms and histochemical analysis of myofibrillar ATPase activity after preincubation at pH 10.5 (E). Arrows point to FAK-immunoreactivity at the sarcolemma. A negative control reaction with normal rabbit serum is also shown (B). The fibre types are indicated. Scale bar, 100 µm.

View larger version (75K): [in a new window]   Fig. 7. Sarcolemmal focal adhesion kinase (FAK) immunoreactivity in muscle fibres during the slow- to fast-twitch transformation. Sarcolemmal FAK immunoreactivity (A, FAK antiserum A-17; B, control reaction) and fibre type (C, fast myosin) were determined on consecutive cryosections of soleus muscle 10 months after cross-reinnervation (X-SOL) with the nerve supply of the fast-twitch extensor digitorum longus muscle. The fibre types are indicated. Scale bar, 100 µm.

View larger version (119K): [in a new window]   Fig. 8. Sarcolemmal focal adhesion kinase (FAK) immunoreactivity in muscle fibres during the fast- to slow-twitch transformation. Sarcolemmal FAK immunoreactivity (A, serum A-17; B, control reaction) and fibre type (C, slow myosin; D, fast myosin) were determined on consecutive cryosections from an extensor digitorum longus muscle 8 months after autografting and reinnervation (T-EDL) with the nerve of the slow-twitch soleus muscle. The fibre types are indicated. Scale bar, 100 µm.

View larger version (22K): [in a new window]   Fig. 10. Summary of the findings on the regulation of sarcolemmal focal adhesion kinase (FAK) immunoreactivity by fibre type (innervation pattern) and regeneration. A model is proposed whereby an increased association between FAK and the sarcolemma is explained by frequent fibre recruitment and basement membrane remodelling and is correlated with increased turnover and density of costameres.