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Tandem fluorescent protein timers for in vivo analysis of protein dynamics

Nature Biotechnology volume 30, pages 708–714 (2012)Cite this article

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Abstract

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

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Figure 1: Tandem fluorescent protein fusions as fluorescent timers.
Figure 2: Analysis of differential inheritance of cellular structures with the mCherry-sfGFP timer.
Figure 3: Inheritance of nuclear pore complexes during yeast mitosis.
Figure 4: Analysis of protein turnover with the mCherry-sfGFP tFT.
Figure 5: Identification of components of the N-end rule pathway.

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Acknowledgements

We are grateful to Y. Belyaev, S. Terjung and the Advanced Light Microscopy Facility of EMBL for support with microscopy; A. Riddell, A.P. Gonzalez and the FACS Facility of EMBL for flow cytometry analyses; the CellNetworks cluster for funding to M.K., the German Research Foundation for funding to E.S. (SFB638), the Howard Hughes Medical Institute for funding to P.J.K., the European Molecular Biology Organization for funding to A.Kh. (EMBO ALTF 1124-2010), the EU-FP7 Network of Excellence in Systems Microscopy for funding to J.D.B. and W.H., and the Novartis Stiftung for funding to A.Ka. We thank P.I. Bastiaens, D. Gilmour and A. Kinkhabwala for discussions.

Author information

Author notes

  1. Andreas Kaufmann

    Present address: Present address: ETH Zurich, Light Microscopy and Screening Center, Zurich, Switzerland.,

  2. Anton Khmelinskii and Philipp J Keller: These authors contributed equally to this work.

  3. Anna Bartosik, Matthias Meurer and Joseph D Barry: These authors provided equivalent support for this work.

Authors and Affiliations

  1. Center for Molecular Biology of the University of Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany

    Anton Khmelinskii, Anna Bartosik, Matthias Meurer, Balca R Mardin, Elmar Schiebel & Michael Knop

  2. European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany

    Anton Khmelinskii, Philipp J Keller, Anna Bartosik, Matthias Meurer, Andreas Kaufmann, Susanne Trautmann, Malte Wachsmuth & Michael Knop

  3. Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Philipp J Keller

  4. European Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany

    Joseph D Barry & Wolfgang Huber

  5. German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany

    Susanne Trautmann, Gislene Pereira & Michael Knop

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  1. Anton Khmelinskii
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Contributions

M.K. conceived and, together with E.S., A.Kh. and P.J.K., designed the project. A.Kh., M.M., A.B., A.Ka., S.T. and P.J.K. did all yeast experiments. B.R.M. did mammalian cell work. G.P. contributed reagents. P.J.K., J.D.B., A.Kh., W.H., M.W. and M.K. developed theory. P.J.K., J.D.B. and A.Kh. developed analytical tools. M.K., A.Kh. and P.J.K. wrote the manuscript with input from E.S. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Elmar Schiebel or Michael Knop.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods, Supplementary Notes 1–6, Supplementary Theory, Supplementary Tables 1,2 and Supplementary Figures 1–16 (PDF 2971 kb)

Supplementary Table 3

Results of the screens for components of the N-end rule pathway (XLS 866 kb)

Supplementary Movie 1

Movie corresponding to Figure 3d (i, ii) (MOV 556 kb)

Supplementary Movie 2

Movie corresponding to Figure 3d (iii) (MOV 1374 kb)

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Khmelinskii, A., Keller, P., Bartosik, A. et al. Tandem fluorescent protein timers for in vivo analysis of protein dynamics. Nat Biotechnol 30, 708–714 (2012). https://doi.org/10.1038/nbt.2281

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