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Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins

Nature Cell Biology volume 4, pages 286–293 (2002)Cite this article

Abstract

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins within cells. Here, we demonstrate that GFP can be used as a CALI reagent to locally inactivate proteins in living cells. We show that focused laser irradiation of EGFP–α-actinin expressed in Swiss 3T3 fibroblasts results in the detachment of stress fibres from focal adhesions (FAs), whereas the integrity of FAs, as determined by interference reflection microscopy (IRM), is preserved. Moreover, consistent with a function for focal adhesion kinase (FAK) in FA signalling and not FA structure, laser irradiation of EGFP–FAK did not cause either visible FA damage or stress fibre detachment, although in vitro CALI of isolated EGFP–FAK decreased its kinase activity, but not its binding to paxillin. These data indicate that CALI of specific FA components may be used to precisely dissect the functional significance of individual proteins required for the maintenance of this cytoskeletal structure. In vitro CALI experiments also demonstrated a reduction of EGFP–α-actinin binding to the cytoplasmic domain of the β1 integrin subunit, but not to actin. Thus, α-actinin is essential for the binding of microfilaments to integrins in the FA. CALI-induced changes in α-actinin result in the breakage of that link and the subsequent retraction of the stress fibre.

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Figure 1: FA-targeted CALI of EGFP–α-actinin.
Figure 2: FA and stress fibres are not damaged by CALI irradiation.
Figure 3: A typical example of 'slow' retraction of stress fibre after CALI irradiation at the FA.
Figure 4: The effect of CALI on the actin-binding activity of EGFP–α-actinin in a cosedimentation assay.
Figure 5: The effect of CALI on the integrin-binding activity of α-actinin.
Figure 6: CALI irradiation of EGFP–FAK does not cause detachment of irradiated stress fibres or any visible damage to the FA.
Figure 7: The effect of CALI on the catalytic activity of FAK and EGFP–FAK.
Figure 8: A highly simplified schematic representation of the FA.

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Acknowledgements

We thank M. Lotano, M. Edlund and J. Cohen for their helpful assistance. We are especially grateful to M. Schaller and J. Broome for their help and advice in setting up the in vitro kinase assay. This work was supported by National Institutes of Health grants GM 35325, the Cell Migration Consortium IK54GM64346 and by P60-DE13079 from the National Institute for Dental and Cranial Research.

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC, USA

    Zenon Rajfur, Partha Roy & Ken Jacobson

  2. Department of Cell and Molecular Physiology, University of North Carolina, Chapel Hill, NC, USA

    Carol Otey

  3. Departments of Anesthesiology, Cell Biology and Pediatrics, Johns Hopkins University, Baltimore, MD, USA

    Lewis Romer

  4. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA

    Ken Jacobson

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  1. Zenon Rajfur
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Correspondence to Ken Jacobson.

Supplementary information

Figure S1

Wild-type Swiss 3T3 fibroblasts maintain function after a typical CALI dose of laser light. (PDF 135 kb)

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Rajfur, Z., Roy, P., Otey, C. et al. Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins. Nat Cell Biol 4, 286–293 (2002). https://doi.org/10.1038/ncb772

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