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Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins

Nature Methods volume 3, pages 525–531 (2006)Cite this article

Abstract

We examined cell cycle–dependent changes in the proteome of human cells by systematically measuring protein dynamics in individual living cells. We used time-lapse microscopy to measure the dynamics of a random subset of 20 nuclear proteins, each tagged with yellow fluorescent protein (YFP) at its endogenous chromosomal location. We synchronized the cells in silico by aligning protein dynamics in each cell between consecutive divisions. We observed widespread (40%) cell-cycle dependence of nuclear protein levels and detected previously unknown cell cycle–dependent localization changes. This approach to dynamic proteomics can aid in discovery and accurate quantification of the extensive regulation of protein concentration and localization in individual living cells.

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Figure 1: Creation of a library of CD-tagged proteins.
Figure 2: In silico synchronization of individual cells.
Figure 3: Nuclear accumulation as a function of cell cycle for 20 nuclear proteins.
Figure 4: Cell cycle–dependent translocation of proteins.

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Acknowledgements

We thank Quantomix for plasmid construction; Z. Kam for microscopy guidance; B. Zimmerman for cytoskeleton staining; E. Ariel and A. Sharp from the Weizmann flow cytometry unit for assistance, and M. Springer, P. Bordalo, O. Zuk and members of the Alon lab for discussions of the manuscript. We thank the Kahn Family Foundation and the Israel Science Foundation for support. R.M. thanks the Horowitz Complexity Science Foundation for support. A.E.C. thanks the Novartis Life Science Foundation for support.

Author information

Author notes

  1. Alex Sigal and Ron Milo: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, 76100, Israel

    Alex Sigal, Ron Milo, Ariel Cohen, Naama Geva-Zatorsky, Yael Klein, Inbal Alaluf, Naamah Swerdlin, Natalie Perzov, Tamar Danon, Yuvalal Liron & Uri Alon

  2. Department of Physics of Complex Systems, Weizmann Institute of Science, Rehovot, 76100, Israel

    Ron Milo & Uri Alon

  3. Department of Developmental Biology, Stanford University, Palo Alto, 94305-5439, California, USA

    Tal Raveh

  4. Whitehead Institute for Biomedical Research, Cambridge, 02142, Massachusetts, USA

    Anne E Carpenter

  5. Department of Systems Biology, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Galit Lahav

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Correspondence to Uri Alon.

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Supplementary information

Supplementary Fig. 1

Integration positions of the YFP CD-tag. (PDF 107 kb)

Supplementary Fig. 2

Distribution of cell cycle lengths for 20 nuclear clones. (PDF 61 kb)

Supplementary Fig. 3

Determination of the relative durations of cell cycle phases. (PDF 91 kb)

Supplementary Fig. 4

Automatic segmentation based on fluorescence levels in the nuclei. (PDF 135 kb)

Supplementary Fig. 5

Raw accumulation measurements without any correction. (PDF 84 kb)

Supplementary Fig. 6

Synchrogram before cell centering and rotation, and before elimination of neighboring cells. (PDF 441 kb)

Supplementary Table 1

Localization of CD-tagged clones compared to previous studies. (PDF 108 kb)

Supplementary Video 1

Panel of 9 proteins, shown for one complete division, approximately 20 hours. Protein identities (localizations), first row, left to right: MSN (cytoplasm), BSG (plasma membrane), ATP1A1 (plasma membrane). Second row: FBL (nucleoli), RPL4 (cytoplasm and nucleoli), RTN4 (endoplasmic reticulum). Third row: H2AFV (DNA bound), USP7 (nucleus, nuclear bodies), LMNB1 (nuclear membrane). (MPG 1091 kb)

Supplementary Video 2

Histone H2AFV transmitted light. Duration shown: 42 hours. (MPG 4861 kb)

Supplementary Video 3

Histone H2AFV fluorescence. Duration shown: 42 hours. (MPG 2360 kb)

Supplementary Video 4

USP7 translocation to nuclear bodies. Duration shown: 42 hours. (MPG 5660 kb)

Supplementary Methods (PDF 129 kb)

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Sigal, A., Milo, R., Cohen, A. et al. Dynamic proteomics in individual human cells uncovers widespread cell-cycle dependence of nuclear proteins. Nat Methods 3, 525–531 (2006). https://doi.org/10.1038/nmeth892

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