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Interaction of metastasis-inducing S100A4 protein in vivo by fluorescence lifetime imaging microscopy

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Abstract

Elevated levels of the calcium-binding regulatory protein, S100A4, have been shown to be causative of a metastatic phenotype in models of cancer metastasis and to be associated with reduced patient survival in breast cancer patients. Recombinant S100A4 protein interacts in vitro in a calcium-dependent manner with the heavy chain of non-muscle myosin isoform A at a protein kinase C phosphorylation site. At present, the mechanism of metastasis induction by S100A4 in vivo is almost completely unknown. The binding of S100A4 to a C-terminal recombinant fragment of non-muscle myosin heavy chain in living HeLa cells has now been shown using confocal microscopy, fluorescence lifetime imaging microscopy and time-correlated single-photon counting. The association between S100A4 and non-muscle myosin heavy chain was studied by determining fluorescence resonance energy transfer-derived changes in the fluorescence lifetime of enhanced cyan fluorescent protein fused to S100A4 in the presence of a recombinant fragment of the C-terminal region of non-muscle myosin heavy chain (rNMMHCIIA) fused to enhanced yellow fluorescent protein. There was no interaction between the non-muscle myosin heavy chain fragment and a calcium-binding-deficient mutant of S100A4 protein which has been shown to be defective in the induction of metastasis in model systems in vivo. The results demonstrate, for the first time, not only direct interaction between S100A4 and a target rNMMHCIIA in live mammalian cells, but also that the interaction between S100A4 and the non-muscle myosin heavy chain in vivo could contribute to the mechanism of metastasis induction by a high level of S100A4 protein.

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Fig. 1A–C
Fig. 2
Fig. 3A–D
Fig. 4A, B
Fig. 5A–D

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Abbreviations

ECFP:

enhanced cyan fluorescent protein

EYFP:

enhanced yellow fluorescent protein

FLIM:

fluorescence lifetime imaging

FRET:

fluorescence resonance energy transfer

Mutant C:

S100A4 protein bearing an inactivating mutant in the C-terminal EF-hand loop

PBS:

phosphate-buffered saline

PCR:

polymerase chain reaction

rNMMHCIIA:

recombinant C-terminal fragment of non-muscle myosin heavy chain II, isoform A

TCSPC:

time-correlated single-photon counting

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Acknowledgements

This work was supported by the North West Cancer Research Fund and the Cancer and Polio Research Fund.

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Authors and Affiliations

  1. Cancer and Polio Research Fund Laboratories, School of Biological Sciences, University of Liverpool, Liverpool , L69 7ZB, UK

    Shu Zhang, Guozheng Wang, David G. Fernig, Philip S. Rudland & Roger Barraclough

  2. CLRC, Daresbury Laboratory, Daresbury, Warrington , WA4 4AD, UK

    Stephen E. D. Webb & Marisa Martin-Fernandez

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  1. Shu Zhang
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  2. Guozheng Wang
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  3. David G. Fernig
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  4. Philip S. Rudland
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  5. Stephen E. D. Webb
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  7. Marisa Martin-Fernandez
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Correspondence to Roger Barraclough.

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Zhang, S., Wang, G., Fernig, D.G. et al. Interaction of metastasis-inducing S100A4 protein in vivo by fluorescence lifetime imaging microscopy. Eur Biophys J 34, 19–27 (2005). https://doi.org/10.1007/s00249-004-0428-x

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  • DOI: https://doi.org/10.1007/s00249-004-0428-x

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