Abstract

Coordination of multiple kinesin and myosin motors is required for intracellular transport, cell motility and mitosis. However, comprehensive resources that allow systems analysis of the localization and interplay between motors in living cells do not exist. Here, we generated a library of 243 amino- and carboxy-terminally tagged mouse and human bacterial artificial chromosome transgenes to establish 227 stably transfected HeLa cell lines, 15 mouse embryonic stem cell lines and 1 transgenic mouse line. The cells were characterized by expression and localization analyses and further investigated by affinity-purification mass spectrometry, identifying 191 candidate protein–protein interactions. We illustrate the power of this resource in two ways. First, by characterizing a network of interactions that targets CEP170 to centrosomes, and second, by showing that kinesin light-chain heterodimers bind conventional kinesin in cells. Our work provides a set of validated resources and candidate molecular pathways to investigate motor protein function across cell lineages.

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Acknowledgements

Most of this project was funded by the European Commission through the Sixth Framework Programme Integrated Project MitoCheck (LSHG-CT-2004-503464). A.A.H. received support from the Max Planck Society and from Bundesministerium fuer Bildung und Forschung grants NGFN-2 SMP-RNAi (01GR0402) and NGFN-Plus (01GS0859). Y.T. was supported by a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science. F.M-B. was supported by EMBO (ALTF 1080-2007). A.E. was a member of the International Max Planck Research School for Molecular Cell Biology and Bioengineering and a Technische Universität Dresden doctoral student. W.B.H. was supported by grants from the DFG (SFB 655, A2; TRR 83, Tp6) and the ERC (250197), by the DFG-funded Center for Regenerative Therapies Dresden, and by the Fonds der Chemischen Industrie. E.B. and E.G. were supported by the DFG Research Center and Cluster of Excellence—Center for Regenerative Therapies Dresden (FZ 111). We thank J. Jarrells and B. Schilling (MPI-CBG Microarray Facility) for processing microarray samples. M. Biesold, M. Augsburg, A. Ssykor, S. Bastidas and N. Berger provided assistance with cell culture and transfection. I. Nuesslein (MPI-CBG FACS Facility) performed FACS of transgenic HeLa pools. M. Theis and F. Buchholz helped produce esiRNAs. The Trangsgenic Core Facility and Biomedical Services at the MPI-CBG provided technical assistance in generating and maintaining the KIF23 transgenic mouse strain.

Author information

Author notes

    • Magno Junqueira
    •  & Yusuke Toyoda

    These authors contributed equally to this work

Affiliations

  1. Max-Planck Institute for Cell Biology and Genetics (CBG), Dresden 01307, Germany

    • Zoltan Maliga
    • , Yusuke Toyoda
    • , Felipe Mora-Bermúdez
    • , Andrej Vasilj
    • , Elaine Guhr
    • , Ina Poser
    • , Wieland B. Huttner
    • , Andrej Shevchenko
    •  & Anthony A. Hyman
  2. Brazilian Center for Protein Research, Department of Cell Biology, University of Brasilia, 70910-900 Brasilia, DF, Brazil

    • Magno Junqueira
  3. Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, California 94143, USA

    • Andreas Ettinger
  4. Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Robin W. Klemm
  5. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA

    • Itziar Ibarlucea-Benitez
  6. Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden 01307, Germany

    • Ezio Bonifacio

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Contributions

The project was conceived and the paper was written by Z.M. and A.A.H. E.G. and E.B. designed and executed immunology experiments. I.P. and Z.M. performed BAC tagging and generated BAC cell pools. I.I-B. performed IFM of BAC cells. F.M-B. and W.B.H. generated the KIF23–EGFP mouse strain. A.E. and F.M-B. performed IFM of tissues. M.J. and A.S. analysed all AP samples by MS and A.V. calculated peptide intensity scores. R.W.K. isolated vesicles from BAC cells. Y.T. characterized CEP170 interactions. Z.M. performed all other experiments.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Zoltan Maliga.

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    Time-lapse (5 h, 2 frames per hour) GFP fluorescence imaging of mouse KIF22-LAP expressed in a stable HeLa BAC line.

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    Time-lapse (15 h, 3 frames per hour) GFP fluorescence imaging of human KIF3C-LAP expressed in a stable HeLa BAC line.

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    Mouse embryonic stem cell BAC line expressing mouse KIF3A-NFLAP.

    Time-lapse (2 min, 20 frames per min) GFP fluorescence imaging of human KIF3A-NFLAP expressed in stably transfected mouse embryonic stem cells.

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    HeLa BAC line expressing mouse CEP170-LAP.

    Time-lapse (16 h, 3 frames per hour) GFP fluorescence imaging of mouse CEP170-LAP expressed in a stable HeLa BAC line.

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DOI

https://doi.org/10.1038/ncb2689

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