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High-throughput fluorescence correlation spectroscopy enables analysis of proteome dynamics in living cells

Abstract

To understand the function of cellular protein networks, spatial and temporal context is essential. Fluorescence correlation spectroscopy (FCS) is a single-molecule method to study the abundance, mobility and interactions of fluorescence-labeled biomolecules in living cells. However, manual acquisition and analysis procedures have restricted live-cell FCS to short-term experiments of a few proteins. Here, we present high-throughput (HT)-FCS, which automates screening and time-lapse acquisition of FCS data at specific subcellular locations and subsequent data analysis1,2. We demonstrate its utility by studying the dynamics of 53 nuclear proteins3,4. We made 60,000 measurements in 10,000 living human cells, to obtain biophysical parameters that allowed us to classify proteins according to their chromatin binding and complex formation. We also analyzed the cell-cycle-dependent dynamics of the mitotic kinase complex Aurora B/INCENP5 and showed how a rise in Aurora concentration triggers two-step complex formation. We expect that throughput and robustness will make HT-FCS a broadly applicable technology for characterizing protein network dynamics in cells.

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Figure 1: HT-FCS Workflow.
Figure 2: Screening FCS.
Figure 3: Time-lapse FCS.

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Acknowledgements

We thank F. Sieckmann and W. Knebel of Leica Microsystems for providing microscope hardware and for implementing FCS functionality in the HCS-A module of the LASAF software. We also thank the mechanical and the electronics workshop of EMBL for custom hardware and the Advanced Light Microscopy Facility of EMBL for microscopy support. We gratefully acknowledge S. Trautmann and M. Knop for fruitful discussions, J.-K. Hériché for advice on data analysis, Y. Cai for critically reading the manuscript and B. Krämer of PicoQuant for help with FCS acquisition hardware and software. We thank K. Rippe and D. Boeke for comments on and testing of software and J. Braumann for technical help. We are grateful for support from EMBL and from the MitoSys and the Systems Microscopy projects funded by the European Commission (FP7/2007-2013-241548 and FP7/2007-2013-258068). HeLa Kyoto cells stably expressing the chromatin-targeted FRET biosensor were provided by D. Gerlich (IMBA, Vienna, Austria).

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Authors

Contributions

M.W., C.C., R.P. and J.E. conceived the research. M.W. and C.C. developed and implemented microscopy and analysis software and hardware. M.W., C.C. and J.B. conducted the FCS experiments and the data processing and analysis. M.I. and R.M. performed FRAP and FRET experiments. J.B., B.K. and R.M. performed transfections and generated cell lines. M.W., C.C., R.P. and J.E. wrote the manuscript. All authors commented on the manuscript.

Corresponding authors

Correspondence to Rainer Pepperkok or Jan Ellenberg.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Note (PDF 1989 kb)

41587_2015_BFnbt3146_MOESM450_ESM.mov

HeLa cell expressing AURKB-EGFP and H2B-mCherry followed for 1000 min through the cell cycle with imaging and FCS every 10 min (MOV 642 kb)

41587_2015_BFnbt3146_MOESM451_ESM.mov

HeLa cell expressing AURKB-EGFP and INCENP-mCherry and stained with Hoechst followed for 1220 min through the cell cycle with imaging and FCS every 10 min (MOV 605 kb)

Supplementary List 1

Compressed complete set of HTML files (ZIP 21653 kb)

Supplementary Software 1

LabVIEW source code files and installable executable version (ZIP 96091 kb)

Supplementary Software 2

LabVIEW source code (ZIP 1287 kb)

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Wachsmuth, M., Conrad, C., Bulkescher, J. et al. High-throughput fluorescence correlation spectroscopy enables analysis of proteome dynamics in living cells. Nat Biotechnol 33, 384–389 (2015). https://doi.org/10.1038/nbt.3146

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