A genomic toolkit to investigate kinesin and myosin motor function in cells

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Towards a comprehensive motor transgene collection in HeLa cells.
Figure 2: Localization of motor BAC transgenes in HeLa cells.
Figure 3: KIF23 localization in neuroblastoma and neural progenitor cells.
Figure 4: Composite localization–interaction data for the motor protein interactome.
Figure 5: Summary of validated motor protein interaction partners.
Figure 6: Three kinesins regulate CEP170 targeting to centrosomes.
Figure 7: KIF5B binds KLC heterodimers in HeLa cells.

Accession codes

Accessions

Gene Expression Omnibus

References

  1. 1

    Vale, R. D. & Milligan, R. A. The way things move: looking under the hood of molecular motor proteins. Science 288, 88–95 (2000).

    CAS  Article  Google Scholar 

  2. 2

    Miki, H., Okada, Y. & Hirokawa, N. Analysis of the kinesin superfamily: insights into structure and function. Trends Cell Biol. 15, 467–476 (2005).

    CAS  Article  Google Scholar 

  3. 3

    Berg, J. S., Powell, B. C. & Cheney, R. E. A millennial myosin census. Mol. Biol. Cell 12, 780–794 (2001).

    CAS  Article  Google Scholar 

  4. 4

    Hirokawa, N., Noda, Y., Tanaka, Y. & Niwa, S. Kinesin superfamily motor proteins and intracellular transport. Nat. Rev. Mol. Cell Biol. 10, 682–696 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Hirokawa, N., Niwa, S. & Tanaka, Y. Molecular motors in neurons: transport mechanisms and roles in brain function, development, and disease. Neuron 68, 610–638 (2010).

    CAS  Article  Google Scholar 

  6. 6

    Ohsugi, M. et al. Kid-mediated chromosome compaction ensures proper nuclear envelope formation. Cell 132, 771–782 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Zhu, C. et al. Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. Mol. Biol. Cell 16, 3187–3199 (2005).

    CAS  Article  Google Scholar 

  8. 8

    Goshima, G. & Vale, R. D. The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line. J. Cell Biol. 162, 1003–1016 (2003).

    CAS  Article  Google Scholar 

  9. 9

    Goshima, G. et al. Genes required for mitotic spindle assembly in Drosophila S2 cells. Science 316, 417–421 (2007).

    CAS  Article  Google Scholar 

  10. 10

    Semiz, S. et al. Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules. EMBO J. 22, 2387–2399 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Patino-Lopez, G. et al. Myosin 1G is an abundant class I myosin in lymphocytes whose localization at the plasma membrane depends on its ancient divergent pleckstrin homology (PH) domain (Myo1PH). J. Biol. Chem. 285, 8675–8686.

    CAS  Article  Google Scholar 

  12. 12

    Verhey, K. J. et al. Cargo of kinesin identified as JIP scaffolding proteins and associated signalling molecules. J. Cell Biol. 152, 959–970 (2001).

    CAS  Article  Google Scholar 

  13. 13

    Hutchins, J. R. et al. Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science 328, 593–599 (2010).

    CAS  Article  Google Scholar 

  14. 14

    Poser, I. et al. BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. Nat. Methods 5, 409–415 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Bird, A. W. & Hyman, A. A. Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A. J. Cell Biol. 182, 289–300 (2008).

    CAS  Article  Google Scholar 

  16. 16

    Bird, A. W. E. et al. High-efficiency counterselection recombineering for site-directed mutagenesis in bacterial artificial chromosomes. Nat. Methods 9, 103–109 (2011).

    Article  Google Scholar 

  17. 17

    Kittler, R. et al. RNA interference rescue by bacterial artificial chromosome transgenesis in mammalian tissue culture cells. Proc. Natl Acad. Sci. USA 102, 2396–2401 (2005).

    CAS  Article  Google Scholar 

  18. 18

    Jakobsen, L. et al. Novel asymmetrically localizing components of human centrosomes identified by complementary proteomics methods. EMBO J. 30, 1520–1535 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Vermeulen, M. et al. Quantitative interaction proteomics and genome-wide profiling of epigenetic histone marks and their readers. Cell 142, 967–980 (2010).

    CAS  Article  Google Scholar 

  20. 20

    Hubner, N. C. et al. Quantitative proteomics combined with BAC TransgeneOmics reveals in vivo protein interactions. J. Cell Biol. 189, 739–754 (2010).

    CAS  Article  Google Scholar 

  21. 21

    Anko, M. L., Morales, L., Henry, I., Beyer, A. & Neugebauer, K. M. Global analysis reveals SRp20- and SRp75-specific mRNPs in cycling and neural cells. Nat. Struct. Mol. Biol. 17, 962–970 (2010).

    Article  Google Scholar 

  22. 22

    Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    CAS  Article  Google Scholar 

  23. 23

    Jimbo, T. et al. Identification of a link between the tumour suppressor APC and the kinesin superfamily. Nat. Cell Biol. 4, 323–327 (2002).

    CAS  Article  Google Scholar 

  24. 24

    Levy, S., Hannenhalli, S. & Workman, C. Enrichment of regulatory signals in conserved non-coding genomic sequence. Bioinformatics 17, 871–877 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Midorikawa, R., Takei, Y. & Hirokawa, N. KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity. Cell 125, 371–383 (2006).

    CAS  Article  Google Scholar 

  26. 26

    Jones, W. M., Chao, A. T., Zavortink, M., Saint, R. & Bejsovec, A. Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation. J. Cell Sci. 123, 2179–2189.

  27. 27

    Steigemann, P. et al. Aurora B-mediated abscission checkpoint protects against tetraploidization. Cell 136, 473–484 (2009).

    Article  Google Scholar 

  28. 28

    Yu, W., Sharp, D. J., Kuriyama, R., Mallik, P. & Baas, P. W. Inhibition of a mitotic motor compromises the formation of dendrite-like processes from neuroblastoma cells. J. Cell Biol. 136, 659–668 (1997).

    CAS  Article  Google Scholar 

  29. 29

    Ettinger, A. W. et al. Proliferating versus differentiating stem and cancer cells exhibit distinct midbody-release behaviour. Nat. Commun. 2, 503 (2011).

    Article  Google Scholar 

  30. 30

    Theis, M. et al. Comparative profiling identifies C13orf3 as a component of the Ska complex required for mammalian cell division. EMBO J. 28, 1453–1465 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Matos, J. et al. Dbf4-dependent CDC7 kinase links DNA replication to the segregation of homologous chromosomes in meiosis I. Cell 135, 662–678 (2008).

    CAS  Article  Google Scholar 

  32. 32

    Maffini, S. et al. Motor-independent targeting of CLASPs to kinetochores by CENP-E promotes microtubule turnover and poleward flux. Curr. Biol. 19, 1566–1572 (2009).

    CAS  Article  Google Scholar 

  33. 33

    Manning, G., Whyte, D. B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).

    CAS  Article  Google Scholar 

  34. 34

    Behrends, C., Sowa, M. E., Gygi, S. P. & Harper, J. W. Network organization of the human autophagy system. Nature 466, 68–76 (2010).

    CAS  Article  Google Scholar 

  35. 35

    Nogales-Cadenas, R., Abascal, F., Diez-Perez, J., Carazo, J. M. & Pascual-Montano, A. CentrosomeDB: a human centrosomal proteins database. Nucl. Acids Res. 37, D175–D180 (2009).

    CAS  Article  Google Scholar 

  36. 36

    Guarguaglini, G. et al. The forkhead-associated domain protein Cep170 interacts with Polo-like kinase 1 and serves as a marker for mature centrioles. Mol. Biol. Cell 16, 1095–1107 (2005).

    CAS  Article  Google Scholar 

  37. 37

    Tanenbaum, M. E. et al. Kif15 cooperates with eg5 to promote bipolar spindle assembly. Curr. Biol. 19, 1703–1711 (2009).

    CAS  Article  Google Scholar 

  38. 38

    Hammond, J. W., Griffin, K., Jih, G. T., Stuckey, J. & Verhey, K. J. Co-operative versus independent transport of different cargoes by Kinesin-1. Traffic 9, 725–741 (2008).

    CAS  Article  Google Scholar 

  39. 39

    Cox, R. T. & Spradling, A. C. Milton controls the early acquisition of mitochondria by Drosophila oocytes. Development 133, 3371–3377 (2006).

    CAS  Article  Google Scholar 

  40. 40

    Gyoeva, F. K., Bybikova, E. M. & Minin, A. A. An isoform of kinesin light chain specific for the Golgi complex. J. Cell Sci. 113, 2047–2054 (2000).

    CAS  PubMed  Google Scholar 

  41. 41

    DeBoer, S. R. et al. Conventional kinesin holoenzymes are composed of heavy and light chain homodimers. Biochemistry 47, 4535–4543 (2008).

    CAS  Article  Google Scholar 

  42. 42

    Gyoeva, F. K., Sarkisov, D. V., Khodjakov, A. L. & Minin, A. A. The tetrameric molecule of conventional kinesin contains identical light chains. Biochemistry 43, 13525–13531 (2004).

    CAS  Article  Google Scholar 

  43. 43

    Klemm, R. W. et al. Segregation of sphingolipids and sterols duringformation of secretory vesicles at the trans-Golgi network. J. Cell Biol. 185, 601–612 (2009).

    CAS  Article  Google Scholar 

  44. 44

    Kotzamanis, G. & Huxley, C. Recombining overlapping BACs into a single larger BAC. BMC Biotechnol. 4, 1 (2004).

    Article  Google Scholar 

  45. 45

    Liu, X., Zhou, T., Kuriyama, R. & Erikson, R. L. Molecular interactions of Polo-like-kinase 1 with the mitotic kinesin-like protein CHO1/MKLP-1. J. Cell Sci. 117, 3233–3246 (2004).

    CAS  Article  Google Scholar 

  46. 46

    Mollinari, C. et al. Ablation of PRC1 by small interfering RNA demonstratesthat cytokinetic abscission requires a central spindle bundle in mammaliancells, whereas completion of furrowing does not. Mol. Biol. Cell 16, 1043–1055 (2005).

    CAS  Article  Google Scholar 

  47. 47

    Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W. & Roder, J. C. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl Acad. Sci. USA 90, 8424–8428 (1993).

    CAS  Article  Google Scholar 

  48. 48

    Olmsted, J. B., Carlson, K., Klebe, R., Ruddle, F. & Rosenbaum, J. Isolation of microtubule protein from cultured mouse neuroblastoma cells. Proc. Natl Acad. Sci. USA 65, 129–136 (1970).

    CAS  Article  Google Scholar 

  49. 49

    Whitfield, M. L. et al. Identification of genes periodically expressed in the human cell cycle and their expression in tumours. Mol. Biol. Cell 13, 1977–2000 (2002).

    CAS  Article  Google Scholar 

  50. 50

    Skoufias, D. A. et al. S-trityl-L-cysteine is a reversible, tight binding inhibitor of the human kinesin Eg5 that specifically blocks mitotic progression. J. Biol. Chem. 281, 17559–17569 (2006).

    CAS  Article  Google Scholar 

  51. 51

    Kittler, R. et al. Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies. Nat. Methods 4, 337–344 (2007).

    CAS  Article  Google Scholar 

  52. 52

    Ganem, N. J. & Compton, D. A. The KinI kinesin Kif2a is required for bipolar spindle assembly through a functional relationship with MCAK. J. Cell Biol. 166, 473–478 (2004).

    CAS  Article  Google Scholar 

  53. 53

    Nagaraj, N. et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548 (2011).

    Article  Google Scholar 

  54. 54

    Wu, C. et al. BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol. 10, R130 (2009).

    Article  Google Scholar 

  55. 55

    Barrett, T. & Edgar, R. Gene expression omnibus: microarray data storage, submission, retrieval, and analysis. Methods Enzymol. 411, 352–369 (2006).

    CAS  Article  Google Scholar 

  56. 56

    Cheeseman, I. M. & Desai, A. A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci. STKE 266, pl1 (2005).

    Google Scholar 

  57. 57

    Hales, C. N. & Woodhead, J. S. Labeled antibodies and their use in the immunoradiometric assay. Methods Enzymol. 70, 334–355 (1980).

    CAS  Article  Google Scholar 

  58. 58

    SPCTools, S.R.-S.a.S.R.-. http://tools.proteomecenter.org/wiki/index.php?title=Software:ReAdW.

  59. 59

    Keller, A., Eng, J., Zhang, N., Li, X. J. & Aebersold, R. A uniform proteomics MS/MS analysis platform using open XML file formats. Mol. Syst. Biol. 1, 20050017 (2005).

    Article  Google Scholar 

  60. 60

    Mueller, L. N. et al. SuperHirn—a novel tool for high resolution LC-MS-based peptide/protein profiling. Proteomics 7, 3470–3480 (2007).

    CAS  Article  Google Scholar 

  61. 61

    Stark, C. et al. The BioGRID Interaction Database: 2011 update. Nucl. Acids Res. 39, D698-D704 (2011).

    Article  Google Scholar 

Download references

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

Affiliations

Authors

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.

Corresponding author

Correspondence to Zoltan Maliga.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 487 kb)

Supplementary Table 1

Supplementary Information (XLS 60 kb)

Supplementary Table 2

Supplementary Information (XLS 151 kb)

Supplementary Table 3

Supplementary Information (XLS 85 kb)

Supplementary Table 4

Supplementary Information (XLS 55 kb)

Supplementary Table 5

Supplementary Information (XLS 73 kb)

Supplementary Table 6

Supplementary Information (XLS 26 kb)

Supplementary Table 7

Supplementary Information (XLSX 57 kb)

Supplementary Table 8

Supplementary Information (XLS 48 kb)

41556_2013_BFncb2689_MOESM35_ESM.avi

Time-lapse (36 h, 2 frames per hour) GFP fluorescence imaging of human KIF22-LAP expressed in a stable HeLa BAC line. (AVI 557 kb)

HeLa BAC line expressing human KIF22-LAP.

Time-lapse (36 h, 2 frames per hour) GFP fluorescence imaging of human KIF22-LAP expressed in a stable HeLa BAC line. (AVI 557 kb)

41556_2013_BFncb2689_MOESM36_ESM.avi

Time-lapse (5 h, 2 frames per hour) GFP fluorescence imaging of mouse KIF22-LAP expressed in a stable HeLa BAC line. (AVI 254 kb)

HeLa BAC line expressing mouse KIF22-LAP.

Time-lapse (5 h, 2 frames per hour) GFP fluorescence imaging of mouse KIF22-LAP expressed in a stable HeLa BAC line. (AVI 254 kb)

41556_2013_BFncb2689_MOESM37_ESM.avi

Time-lapse (15 h, 3 frames per hour) GFP fluorescence imaging of human KIF3C-LAP expressed in a stable HeLa BAC line. (AVI 1224 kb)

HeLa BAC line expressing human KIF3C-LAP.

Time-lapse (15 h, 3 frames per hour) GFP fluorescence imaging of human KIF3C-LAP expressed in a stable HeLa BAC line. (AVI 1224 kb)

41556_2013_BFncb2689_MOESM38_ESM.avi

Time-lapse (2 min, 20 frames per min) GFP fluorescence imaging of human KIF3A-NFLAP expressed in stably transfected mouse embryonic stem cells. (AVI 771 kb)

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. (AVI 771 kb)

41556_2013_BFncb2689_MOESM39_ESM.avi

Time-lapse (16 h, 3 frames per hour) GFP fluorescence imaging of mouse CEP170-LAP expressed in a stable HeLa BAC line. (AVI 2058 kb)

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. (AVI 2058 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Maliga, Z., Junqueira, M., Toyoda, Y. et al. A genomic toolkit to investigate kinesin and myosin motor function in cells. Nat Cell Biol 15, 325–334 (2013). https://doi.org/10.1038/ncb2689

Download citation

Further reading