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Paxillin and focal adhesion kinase colocalise in human skeletal muscle and its associated microvasculature

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Abstract

Focal adhesion kinase (FAK) and paxillin are functionally linked hormonal- and mechano-sensitive proteins. We aimed to describe paxillin’s subcellular distribution using widefield and confocal immunofluorescence microscopy and test the hypothesis that FAK and paxillin colocalise in human skeletal muscle and its associated microvasculature. Percutaneous muscle biopsies were collected from the m. vastus lateralis of seven healthy males, and 5-μm cryosections were stained with anti-paxillin co-incubated with anti-dystrophin to identify the sarcolemma, anti-myosin heavy chain type I for fibre-type differentiation, anti-dihydropyridine receptor to identify T-tubules, lectin UEA-I to identify the endothelium of microvessels and anti-α-smooth muscle actin to identify vascular smooth muscle cells (VSMC). Colocalisation of anti-paxillin with anti-dystrophin or anti-FAK was quantified using Pearson’s correlation coefficient on confocal microscopy images. Paxillin was primarily present in (sub)sarcolemmal regions of skeletal muscle fibres where it colocalised with dystrophin (r = 0.414 ± 0.026). The (sub)sarcolemmal paxillin immunofluorescence intensity was ~2.4-fold higher than in sarcoplasmic regions (P < 0.001) with sarcoplasmic paxillin immunofluorescence intensity ~10 % higher in type I than in type II fibres (P < 0.01). In some longitudinally orientated fibres, paxillin formed striations that corresponded to the I-band region. Paxillin immunostaining was highest in endothelial and VSMC and distributed heterogeneously in both cell types. FAK and paxillin colocalised at (sub)sarcolemmal regions and within the microvasculature (r = 0.367 ± 0.036). The first images of paxillin in human skeletal muscle suggest paxillin is present in (sub)sarcolemmal and I-band regions of muscle fibres and within the microvascular endothelium and VSMC. Colocalisation of FAK and paxillin supports their suggested role in hormonal and mechano-sensitive signalling.

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Acknowledgments

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.

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Authors and Affiliations

  1. Exercise Metabolism Research Group, School of Sport and Exercise Sciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, UK

    Oliver J. Wilson & Helen Bradley

  2. Institute of Sport, Exercise and Active Living, Victoria University, Melbourne, Australia

    Christopher S. Shaw

  3. Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Tom Reilly Building, Byrom Street, Liverpool, L3 3AF, UK

    Anton J. M. Wagenmakers

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  1. Oliver J. Wilson
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  2. Helen Bradley
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Correspondence to Anton J. M. Wagenmakers.

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Wilson, O.J., Bradley, H., Shaw, C.S. et al. Paxillin and focal adhesion kinase colocalise in human skeletal muscle and its associated microvasculature. Histochem Cell Biol 142, 245–256 (2014). https://doi.org/10.1007/s00418-014-1212-3

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