Histochemistry and Cell Biology

, Volume 138, Issue 4, pp 617–626 | Cite as

Immunofluorescent visualisation of focal adhesion kinase in human skeletal muscle and its associated microvasculature

  • Oliver J. Wilson
  • Christopher S. Shaw
  • Mark Sherlock
  • Paul M. Stewart
  • Anton J. M. WagenmakersEmail author
Original Paper


Within animal skeletal muscle, focal adhesion kinase (FAK) has been associated with load-dependent molecular and metabolic adaptation including the regulation of insulin sensitivity. This study aimed to generate the first visual images of the localisation of FAK within human skeletal muscle fibres and its associated microvasculature using widefield and confocal immunofluorescence microscopy. Percutaneous muscle biopsies, taken from five lean, active males, were frozen and 5-μm cryosections were incubated with FAK antibodies for visualisation in muscle fibres and the microvasculature. Anti-myosin heavy chain type I was used for fibre-type differentiation. Muscle sections were also incubated with anti-dihydropyridine receptor (DHPR) to investigate co-localisation of FAK with the t-tubules. FITC-conjugated Ulex europaeus Agglutinin I stained the endothelium of the capillaries, whilst anti-smooth muscle actin stained the vascular smooth muscle of arterioles. Fibre-type differences in the intensity of FAK immunofluorescence were determined with image analysis software. In transversely and longitudinally orientated fibres, FAK was localised at the sarcolemmal regions. In longitudinally orientated fibres, FAK staining also showed uniform striations across the fibre and co-staining with DHPR suggests FAK associates with the t-tubules. There was no fibre-type difference in sarcoplasmic FAK content. Within the capillary endothelium and arteriolar smooth muscle, FAK was distributed heterogeneously as clusters. This is the first study to visualise FAK in human skeletal muscle microvasculature and within the (sub)sarcolemmal and t-tubule regions using immunofluorescence microscopy. This technique will be an important tool for investigating the role of FAK in the intracellular signalling of human skeletal muscle and the endothelium of its associated microvasculature.


Focal adhesion kinase Skeletal muscle Fluorescence Confocal imaging 



The antibodies against human slow myosin (A4.840-c) used in the study were developed by Dr. Blau and were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. OJW is funded by a BBSRC targeted priority studentship into ageing.


  1. Avraham HK, Lee TH, Koh Y, Kim TA, Jiang S, Sussman M, Samarel AM, Avraham S (2003) Vascular endothelial growth factor regulates focal adhesion assembly in human brain microvascular endothelial cells through activation of the focal adhesion kinase and related adhesion focal tyrosine kinase. J Biol Chem 278:36661–36668PubMedCrossRefGoogle Scholar
  2. Baar K, Esser K (1999) Phosphorylation of p70(S6k) correlates with increased skeletal muscle mass following resistance exercise. Am J Physiol 276:C120–C127PubMedGoogle Scholar
  3. Baron V, Calleja V, Ferrari P, Alengrin F, Van Obberghen E (1998) p125Fak focal adhesion kinase is a substrate for the insulin and insulin-like growth factor-I tyrosine kinase receptors. J Biol Chem 273:7162–7168PubMedCrossRefGoogle Scholar
  4. Bergstrom J (1975) Percutaneous needle biopsy of skeletal muscle in physiological and clinical research. Scand J Clin Lab Invest 35:609–616PubMedCrossRefGoogle Scholar
  5. Bisht B, Dey CS (2008) Focal Adhesion Kinase contributes to insulin-induced actin reorganization into a mesh harboring Glucose transporter-4 in insulin resistant skeletal muscle cells. BMC Cell Biol 9:48PubMedCrossRefGoogle Scholar
  6. Bisht B, Goel HL, Dey CS (2007) Focal adhesion kinase regulates insulin resistance in skeletal muscle. Diabetologia 50:1058–1069PubMedCrossRefGoogle Scholar
  7. Bisht B, Srinivasan K, Dey CS (2008) In vivo inhibition of focal adhesion kinase causes insulin resistance. J Physiol 586:3825–3837PubMedCrossRefGoogle Scholar
  8. de Boer D, Ring C, Wood M, Ford C, Jessney N, McIntyre D, Carroll D (2007) Time course and mechanisms of mental stress-induced changes and their recovery: hematocrit, colloid osmotic pressure, whole blood viscosity, coagulation times, and hemodynamic activity. Psychophysiology 44:639–649PubMedCrossRefGoogle Scholar
  9. Durieux AC, D’ Antona G, Desplanches D, Freyssenet D, Klossner S, Bottinelli R, Fluck M (2009) Focal adhesion kinase is a load-dependent governor of the slow contractile and oxidative muscle phenotype. J Physiol 587:3703–3717PubMedCrossRefGoogle Scholar
  10. Ervasti JM (2003) Costameres: the Achilles’ heel of Herculean muscle. J Biol Chem 278:13591–13594PubMedCrossRefGoogle Scholar
  11. Fluck M, Carson JA, Gordon SE, Ziemiecki A, Booth FW (1999) Focal adhesion proteins FAK and paxillin increase in hypertrophied skeletal muscle. Am J Physiol 277:C152–C162PubMedGoogle Scholar
  12. Fluck M, Ziemiecki A, Billeter R, Muntener M (2002) Fibre-type specific concentration of focal adhesion kinase at the sarcolemma: influence of fibre innervation and regeneration. J Exp Biol 205:2337–2348PubMedGoogle Scholar
  13. Glover EI, Phillips SM, Oates BR, Tang JE, Tarnopolsky MA, Selby A, Smith K, Rennie MJ (2008) Immobilization induces anabolic resistance in human myofibrillar protein synthesis with low and high dose amino acid infusion. J Physiol 586:6049–6061PubMedCrossRefGoogle Scholar
  14. Goel HL, Dey CS (2002a) Focal adhesion kinase tyrosine phosphorylation is associated with myogenesis and modulated by insulin. Cell Prolif 35:131–142PubMedCrossRefGoogle Scholar
  15. Goel HL, Dey CS (2002b) Insulin stimulates spreading of skeletal muscle cells involving the activation of focal adhesion kinase, phosphatidylinositol 3-kinase and extracellular signal regulated kinases. J Cell Physiol 193:187–198PubMedCrossRefGoogle Scholar
  16. Gordon SE, Fluck M, Booth FW (2001) Selected contribution: skeletal muscle focal adhesion kinase, paxillin, and serum response factor are loading dependent. J Appl Physiol 90:1174–1183 (discussion 1165)PubMedGoogle Scholar
  17. Huang D, Khoe M, Ilic D, Bryer-Ash M (2006) Reduced expression of focal adhesion kinase disrupts insulin action in skeletal muscle cells. Endocrinology 147:3333–3343PubMedCrossRefGoogle Scholar
  18. Ilic D, Kovacic B, McDonagh S, Jin F, Baumbusch C, Gardner DG, Damsky CH (2003) Focal adhesion kinase is required for blood vessel morphogenesis. Circ Res 92:300–307PubMedCrossRefGoogle Scholar
  19. Ishida T, Peterson TE, Kovach NL, Berk BC (1996) MAP kinase activation by flow in endothelial cells. Role of beta 1 integrins and tyrosine kinases. Circ Res 79:310–316PubMedCrossRefGoogle Scholar
  20. Karlsson HK, Zierath JR (2007) Insulin signaling and glucose transport in insulin resistant human skeletal muscle. Cell Biochem Biophys 48:103–113PubMedCrossRefGoogle Scholar
  21. Klossner S, Durieux AC, Freyssenet D, Flueck M (2009) Mechano-transduction to muscle protein synthesis is modulated by FAK. Eur J Appl Physiol 106:389–398PubMedCrossRefGoogle Scholar
  22. Knight JB, Yamauchi K, Pessin JE (1995) Divergent insulin and platelet-derived growth factor regulation of focal adhesion kinase (pp 125FAK) tyrosine phosphorylation, and rearrangement of actin stress fibers. J Biol Chem 270:10199–10203PubMedCrossRefGoogle Scholar
  23. Lauritzen HP, Ploug T, Prats C, Tavare JM, Galbo H (2006) Imaging of insulin signaling in skeletal muscle of living mice shows major role of t-tubules. Diabetes 55:1300–1306PubMedCrossRefGoogle Scholar
  24. Lauritzen HP, Galbo H, Brandauer J, Goodyear LJ, Ploug T (2008a) Large GLUT4 vesicles are stationary while locally and reversibly depleted during transient insulin stimulation of skeletal muscle of living mice: imaging analysis of GLUT4-enhanced green fluorescent protein vesicle dynamics. Diabetes 57:315–324PubMedCrossRefGoogle Scholar
  25. Lauritzen HP, Ploug T, Ai H, Donsmark M, Prats C, Galbo H (2008b) Denervation and high-fat diet reduce insulin signaling in t-tubules in skeletal muscle of living mice. Diabetes 57:13–23PubMedCrossRefGoogle Scholar
  26. Lebrun P, Mothe-Satney I, Delahaye L, Van Obberghen E, Baron V (1998) Insulin receptor substrate-1 as a signaling molecule for focal adhesion kinase pp125(FAK) and pp60(src). J Biol Chem 273:32244–32253Google Scholar
  27. Li S, Kim M, Hu YL, Jalali S, Schlaepfer DD, Hunter T, Chien S, Shyy JY (1997) Fluid shear stress activation of focal adhesion kinase. Linking to mitogen-activated protein kinases. J Biol Chem 272:30455–30462PubMedCrossRefGoogle Scholar
  28. Mikines KJ, Sonne B, Farrell PA, Tronier B, Galbo H (1988) Effect of physical exercise on sensitivity and responsiveness to insulin in humans. Am J Physiol 254:E248–E259PubMedGoogle Scholar
  29. Narici MV, Flueck M, Koesters A, Gimpl M, Reifberger A, Seynnes OR, Niebauer J, Rittweger J, Mueller E (2011) Skeletal muscle remodeling in response to alpine skiing training in older individuals. Scand J Med Sci Sports 21(Suppl 1):23–28PubMedCrossRefGoogle Scholar
  30. Pardo JV, Siliciano JD, Craig SW (1983) A vinculin-containing cortical lattice in skeletal muscle: transverse lattice elements (“costameres”) mark sites of attachment between myofibrils and sarcolemma. Proc Natl Acad Sci USA 80:1008–1012PubMedCrossRefGoogle Scholar
  31. Peng X, Ueda H, Zhou H, Stokol T, Shen TL, Alcaraz A, Nagy T, Vassalli JD, Guan JL (2004) Overexpression of focal adhesion kinase in vascular endothelial cells promotes angiogenesis in transgenic mice. Cardiovasc Res 64:421–430PubMedCrossRefGoogle Scholar
  32. Perseghin G, Price TB, Petersen KF, Roden M, Cline GW, Gerow K, Rothman DL, Shulman GI (1996) Increased glucose transport-phosphorylation and muscle glycogen synthesis after exercise training in insulin-resistant subjects. N Engl J Med 335:1357–1362PubMedCrossRefGoogle Scholar
  33. Ploug T, van Deurs B, Ai H, Cushman SW, Ralston E (1998) Analysis of GLUT4 distribution in whole skeletal muscle fibers: identification of distinct storage compartments that are recruited by insulin and muscle contractions. J Cell Biol 142:1429–1446PubMedCrossRefGoogle Scholar
  34. Quach NL, Rando TA (2006) Focal adhesion kinase is essential for costamerogenesis in cultured skeletal muscle cells. Dev Biol 293:38–52PubMedCrossRefGoogle Scholar
  35. Schlaepfer DD, Hauck CR, Sieg DJ (1999) Signaling through focal adhesion kinase. Prog Biophys Mol Biol 71:435–478PubMedCrossRefGoogle Scholar
  36. Shen TL, Park AY, Alcaraz A, Peng X, Jang I, Koni P, Flavell RA, Gu H, Guan JL (2005) Conditional knockout of focal adhesion kinase in endothelial cells reveals its role in angiogenesis and vascular development in late embryogenesis. J Cell Biol 169:941–952PubMedCrossRefGoogle Scholar
  37. Shikata Y, Birukov KG, Garcia JG (2003) S1P induces FA remodeling in human pulmonary endothelial cells: role of Rac, GIT1, FAK, and paxillin. J Appl Physiol 94:1193–1203PubMedGoogle Scholar
  38. Tong P, Khayat ZA, Huang C, Patel N, Ueyama A, Klip A (2001) Insulin-induced cortical actin remodeling promotes GLUT4 insertion at muscle cell membrane ruffles. J Clin Invest 108:371–381PubMedGoogle Scholar
  39. Wang W, Hansen PA, Marshall BA, Holloszy JO, Mueckler M (1996) Insulin unmasks a COOH-terminal Glut4 epitope and increases glucose transport across t-tubules in skeletal muscle. J Cell Biol 135:415–430PubMedCrossRefGoogle Scholar
  40. Wang Y, Flores L, Lu S, Miao H, Li YS, Chien S (2009) Shear stress regulates the Flk-1/Cbl/PI3K/NF-kappaB pathway via actin and tyrosine kinases. Cell Mol Bioeng 2:341–350PubMedCrossRefGoogle Scholar
  41. Wilkinson SB, Phillips SM, Atherton PJ, Patel R, Yarasheski KE, Tarnopolsky MA, Rennie MJ (2008) Differential effects of resistance and endurance exercise in the fed state on signalling molecule phosphorylation and protein synthesis in human muscle. J Physiol 586:3701–3717PubMedCrossRefGoogle Scholar
  42. Wu X, Davis GE, Meininger GA, Wilson E, Davis MJ (2001) Regulation of the L-type calcium channel by alpha 5beta 1 integrin requires signaling between focal adhesion proteins. J Biol Chem 276:30285–30292PubMedCrossRefGoogle Scholar
  43. Wu MH, Guo M, Yuan SY, Granger HJ (2003) Focal adhesion kinase mediates porcine venular hyperpermeability elicited by vascular endothelial growth factor. J Physiol 552:691–699PubMedCrossRefGoogle Scholar
  44. Zeng G, Nystrom FH, Ravichandran LV, Cong LN, Kirby M, Mostowski H, Quon MJ (2000) Roles for insulin receptor, PI3-kinase, and Akt in insulin-signaling pathways related to production of nitric oxide in human vascular endothelial cells. Circulation 101:1539–1545PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Oliver J. Wilson
    • 1
  • Christopher S. Shaw
    • 1
  • Mark Sherlock
    • 2
  • Paul M. Stewart
    • 2
  • Anton J. M. Wagenmakers
    • 1
    Email author
  1. 1.Exercise Metabolism Research Group, School of Sport and Exercise SciencesThe University of BirminghamBirminghamUK
  2. 2.Centre for Endocrinology, Diabetes and Metabolism, School of Clinical and Experimental MedicineThe University of BirminghamBirminghamUK

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