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Imaging the coordination of multiple signalling activities in living cells

Nature Reviews Molecular Cell Biology volume 12, pages 749–756 (2011)Cite this article

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Abstract

Cellular signal transduction occurs in complex and redundant interaction networks, which are best understood by simultaneously monitoring the activation dynamics of multiple components. Recent advances in biosensor technology have made it possible to visualize and quantify the activation of multiple network nodes in the same living cell. The precision and scope of this approach has been greatly extended by novel computational approaches (referred to as computational multiplexing) that can reveal relationships between network nodes imaged in separate cells.

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Figure 1: Examples of experimental multiplexing.
Figure 2: Workflow of computational multiplexing.
Figure 3: Assessing the activity of RHO GTPases using computational multiplexing.
Figure 4: Determining the requirements for spatiotemporal sampling to allow computational multiplexing based on constitutive fluctuations.

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Acknowledgements

This work was funded by the US National Institutes of Health grants R01 GM90317 (to G.D. and K.H) and GM057464 (to K.H.) and the Cell Migration Consortium (U54 GM64346).

Author information

Author notes

  1. Christopher M. Welch and Hunter Elliott: C.M.W and H.E contributed equally to this work.

Authors and Affiliations

  1. Christopher M. Welch and Klaus M. Hahn are at the Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.,

    Christopher M. Welch & Klaus M. Hahn

  2. Hunter Elliott and Gaudenz Danuser are at the Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.,

    Hunter Elliott & Gaudenz Danuser

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Correspondence to Gaudenz Danuser or Klaus M. Hahn.

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Supplementary information

41580_2011_BFnrm3212_MOESM1_ESM.pdf

Supplementary information S1 (Figure) | Spectra of fluorophores used in ratiometric FRET multiplex experiments. (PDF 566 kb)

Supplementary information S2 (Table) | FRET-based multiplexing studies (PDF 218 kb)

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FURTHER INFORMATION

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Glossary

Fluorescence polarization anisotropy

A technique that measures the rotational diffusion of fluorophores by measuring the difference in the polarization of excitation and emission light. Changes in fluorescence polarization anisotropy indicate changes in the rotational diffusion of molecules that are induced by their interactions with other molecules.

Orthogonal wavelengths

Biosensor emission or excitation wavelengths that are sufficiently different to allow two fluorescent probes to be imaged separately in the same cell.

Pairwise cross-correlation analysis

A technique that uses a mathematical framework to define whether the variation of one time-resolved image activity is coupled or independent of the variation of another time-resolved image activity.

Quantum dots

Small semiconductor crystals that emit light of a longer wavelength on excitation with a shorter wavelength, akin to fluorophores.

Ratiometric imaging

An imaging technique in which biosensors are designed so that the ratio of emission or excitation at two different wavelengths reflects the biological activity being measured. This ratio is independent of the biosensor's fluorescence intensity, so eliminates the effects of cell thickness, uneven biosensor distribution, uneven illumination and other factors.

Spectral decomposition

A mathematical technique to separate the contribution of multiple fluorophore species to the image signal at a certain wavelength. This allows the separation of the signals of fluorophores with overlapping emission spectra.

Stokes shift

The difference between the excitation and emission wavelengths of a fluorescent probe.

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Welch, C., Elliott, H., Danuser, G. et al. Imaging the coordination of multiple signalling activities in living cells. Nat Rev Mol Cell Biol 12, 749–756 (2011). https://doi.org/10.1038/nrm3212

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