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Reversible cryo-arrest for imaging molecules in living cells at high spatial resolution

Nature Methods volume 13, pages 665672 (2016) | Download Citation

Abstract

The dynamics of molecules in living cells hampers precise imaging of molecular patterns by functional and super-resolution microscopy. We developed a method that circumvents lethal chemical fixation and allows on-stage cryo-arrest for consecutive imaging of molecular patterns within the same living, but arrested, cells. The reversibility of consecutive cryo-arrests was demonstrated by the high survival rate of different cell lines and by intact growth factor signaling that was not perturbed by stress response. Reversible cryo-arrest was applied to study the evolution of ligand-induced receptor tyrosine kinase activation at different scales. The nanoscale clustering of epidermal growth factor receptor (EGFR) in the plasma membrane was assessed by single-molecule localization microscopy, and endosomal microscale activity patterns of ephrin receptor A2 (EphA2) were assessed by fluorescence lifetime imaging microscopy. Reversible cryo-arrest allows the precise determination of molecular patterns while conserving the dynamic capabilities of living cells.

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Acknowledgements

The authors would like to thank M. Reichl, J. Luig and P. Glitz for excellent technical assistance and A. Krämer for help in writing the manuscript. This study was funded by the Fraunhofer Society and the Max Planck Society for the Promotion of Science (CryoSystems grant to G.R.F. and P.I.H.B.) and the European Research Council (ERC AdG 322637 to P.I.H.B.).

Author information

Author notes

    • Martin E Masip
    • , Jan Huebinger
    •  & Jens Christmann

    These authors contributed equally to this work.

Affiliations

  1. Department of Systemic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany.

    • Martin E Masip
    • , Jan Huebinger
    • , Jens Christmann
    • , Ola Sabet
    • , Frank Wehner
    • , Antonios Konitsiotis
    •  & Philippe I H Bastiaens
  2. Faculty of Chemistry and Chemical Biology, Technical University of Dortmund, Dortmund, Germany.

    • Jens Christmann
    •  & Philippe I H Bastiaens
  3. Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany.

    • Günther R Fuhr

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Contributions

P.I.H.B., F.W. and G.R.F. conceived the project; M.E.M., J.H. and J.C. developed and tested the cryo-stage, performed and analyzed cell survival assay of HeLa cells, anisotropy and FLIM of EGFR–PTB; O.S., J.H. and M.E.M. performed and analyzed LIFEA2 FLIM measurements; J.H. developed the cryo-arrest protocol, performed and analyzed FRAP, confocal imaging of lipids, cell survival assay of additional cell lines and activity measurements of MAPKs; A.K., J.H. and M.E.M. performed and analyzed RNA extraction and qRT-PCR experiments; J.C., J.H. and M.E.M. performed and analyzed single-particle tracking and PALM; P.I.H.B., J.H., J.C. and M.E.M. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Philippe I H Bastiaens.

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    Supplementary Text and Figures

    Supplementary Figures 1–10

Videos

  1. 1.

    Time-lapse single molecule imaging of labeled EGFR in the basal membrane of HeLa cells at 37 °C.

    Representative region of the basal membrane of a HeLa cell expressing SNAP-EGFR labeled with Cy3. The video shows the diffusion of the receptor at 37 °C. Exposure time of each frame is 31.6 ms.

  2. 2.

    Time-lapse single molecule imaging of labeled EGFR in the basal membrane of HeLa cells at -45 °C.

    Representative region of the basal membrane of a HeLa cell expressing SNAP-EGFR labeled with Cy3. The video shows the diffusion of the receptor at -45 °C. Exposure time of each frame is 31.6 ms.

  3. 3.

    3D projection of LIFEA2 activity.

    The video shows a 3D projection of a confocal FLIM z-scan through a representative HeLa cell cryo-arrested 5' after stimulation with pre-clustered ephrinA1-Fc (2 μg ml-1).

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DOI

https://doi.org/10.1038/nmeth.3921

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