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Determination of supramolecular structure and spatial distribution of protein complexes in living cells

Nature Photonics volume 3, pages 107113 (2009) | Download Citation

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Abstract

Resonant energy transfer from an optically excited donor molecule to a non-excited acceptor molecule residing nearby is widely used to detect molecular interactions in living cells. To date, resonant energy transfer has been used to obtain stoichiometric information, such as the number of proteins forming a complex, for a handful of proteins, but only after performing sequential scans of the emission wavelengths, excitation wavelengths, or sometimes both. During this lengthy process of measurement, the molecular makeup of a cellular region may change, limiting the applicability of resonant energy transfer to the determination of cellular averages. Here, we demonstrate a method for the determination of protein complex size, configuration, and spatial distribution in single living cells. It relies on a spectrally resolved two-photon microscope, a simple but competent theory, and a judicious selection of fluorescent tags. This approach eventually may lead to tracking the dynamics of individual molecular complexes inside living cells.

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Acknowledgements

This work was supported by a grant from the Wisconsin Institute for Biomedical and Health Technology (grant no. W620 to V.R.), seed funds from the UWM Research Growth Initiative (grant no. X014 to V.R.), and a grant from Canadian Institutes of Health Research (grant no. MOP43990 to J.W.W.). We thank D. Gillman for useful suggestions regarding the computer routines for instrument control, M. J. Woodside for his assistance with the lifetime imaging, and S. Angers for very useful discussions on BRET. We thank K. J. Blumer for providing YFP.

Author information

Author notes

    • Mike Melnichuk

    Present address: Department of Physics, University of Michigan, Ann Arbor, Michigan 48109-1040, USA

Affiliations

  1. Department of Physics, University of Wisconsin-Milwaukee, 1900 E Kenwood Boulevard, Milwaukee, Wisconsin 53211, USA

    • Valerica Raicu
    • , Michael R. Stoneman
    • , Russell Fung
    • , Mike Melnichuk
    • , David B. Jansma
    • , Sasmita Rath
    • , Michael Fox
    •  & Dilano K. Saldin
  2. Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, USA

    • Valerica Raicu
  3. Leslie L. Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario M5S 3M2, Canada

    • Luca F. Pisterzi
    •  & James W. Wells

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Contributions

V.R. coordinated the project, designed the TPM, built the optical set-up, developed the theory and the algorithms for image reconstruction and data analysis, analysed data, and wrote the paper. M.S., S.R. and M.F. carried out experiments using the TPM and analysed data. R.F. wrote computer codes for hardware control and for image reconstruction. M.M. interfaced the scanners with the computer and built an earlier version of the optical set-up. D.B.J. made the genetic constructs. L.P. and J.W.W. determined the RET efficiency using FLIM. D.K.S. participated in theoretical modelling. All authors contributed to manuscript editing.

Corresponding authors

Correspondence to Valerica Raicu or David B. Jansma.

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DOI

https://doi.org/10.1038/nphoton.2008.291

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