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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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.
The authors declare no competing financial interests.
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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)
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)
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)
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)
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)
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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
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