RESOURCE A genomic toolkit to investigate kinesin and myosin motor function in cells Zoltan Maliga1,8, Magno Junqueira2,7, Yusuke Toyoda1,7, Andreas Ettinger3, Felipe Mora-Bermúdez1, Robin W. Klemm4, Andrej Vasilj1, Elaine Guhr1, Itziar Ibarlucea-Benitez5, Ina Poser1, Ezio Bonifacio6, Wieland B. Huttner1, Andrej Shevchenko1 and Anthony A. Hyman1 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. Actin and microtubules define polarized and dynamic cytoskeletal resulting BAC transgenes can be expressed under their native promoter networks required for cell integrity, intracellular transport and faithful and orthologous transgenes can be used as RNAi-resistant third cell division. An important class of proteins that works together with alleles15,17. Indeed, these methods have successfully generated resources these polymer systems is the structurally related, P-loop motor protein to study the interplay between mitotic genes13, to characterize all superfamily1. The human genome contains 39 myosin and 44 kinesin components of a cellular organelle (the centrosome)18, to generate genes but the exact number varies widely across eukaryotes2,3. Gene a genome-wide histone methylation map19 and to perform targeted targeting in vivo4–6, RNA-mediated interference (RNAi) analysis7–9 studies of individual proteins15,20,21. and biochemical approaches10–12 have provided insights into the Here, we generate a transgeneOmics resource for motor proteins to generic functional roles for a number of motors. However, at present, investigate 71 of the 82 annotated human kinesin and myosin genes. there is no resource available that allows a comprehensive analysis Stable transfection of BACs generated 227 HeLa cell and 15 mouse of motor function in a given cell type or to trace the changes embryonic stem cell (mESC) lines. To further show that this resource of a motor-specific activity across cell-fate transitions. Bacterial is readily adaptable to in vivo experiments, we established a transgenic artificial chromosome (BAC) transgenes provide an ideal platform mouse line expressing recombinant human KIF23. We report for high-throughput analysis of gene function in cultured cells and in localization patterns for previously uncharacterized motor proteins vivo13,14. This is because BACs contain the gene together with many in HeLa cells and define candidate protein–protein interactions cis-regulatory sequences required for native control of gene expression, identified by systematic affinity-purification mass spectrometry the production of relevant isoforms and proper translational control (AP–MS) from transgenic HeLa cell lysates. We illustrate the use of our through the cell cycle. Targeted recombination in Escherichia coli can resource with two discoveries: a mechanism that regulates centrosome be used to insert tagging cassettes or substitute nucleotide residues composition and a previously unknown light-chain organization in in its genomic context15,16. Therefore, once stably integrated, the conventional kinesin. 1Max-Planck Institute for Cell Biology and Genetics (CBG), Dresden 01307, Germany. 2Brazilian Center for Protein Research, Department of Cell Biology, University of Brasilia, 70910-900 Brasilia, DF, Brazil. 3Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, California 94143, USA. 4Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA. 5Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720, USA. 6Center for Regenerative Therapies Dresden, Technische Universität Dresden, Dresden 01307, Germany. 7These authors contributed equally to this work. 8Correspondence should be addressed to Z.M. (e-mail: [email protected]) Received 21 August 2012; accepted 10 January 2013; published online 17 February 2013; DOI: 10.1038/ncb2689 NATURE CELL BIOLOGY VOLUME 15 j NUMBER 3 j MARCH 2013 325 © 2013 Macmillan Publishers Limited. All rights reserved. RESOURCE c KinesinsC N Myosins C N ClassGeneHeLa h m h m Class Gene HeLa h m h m a Kinesin and myosin genes expressed KIF1A MYO1A KIF1B MYO1B KIF1C MYO1D KIF13A MYO1G HeLa cells 3 1 55% of genes KIF13B MYO1C 45 genes KIF16 MYO1H KIF16B MYO1E KIF14 MYO1F KIF12 6 MYO6 Naive CD4+ Post-stimulation 12 KIF15 19 MYO19 T-lymphocytes 43 genes KIF3A MYO5A KIF3B 5 MYO5B 28 genes 2 KIF3C MYO5C KIF17 MYO7A 7 Mature KIF4A MYO7B KIF4B 10 MYO10 dendritic cells KIF21A MYO9A Primary 32 genes 4 KIF21B 9 MYO9B KIF7 MYO15 monocytes 15 Activated KIF27 MYO15B 28 genes KIF5A MYO3A macrophages 3 1 KIF5C MYO3B 36 genes KIF5B 16 MYO16 KIF6 MYO18A 9 KIF9 18 MYO18B 5 KIF11 MYH14 KIF25 MYH11 b KIFC1 MYH10 Naive CD4+ 14 KIFC2 MYH9 T-lymphocytes KIFC3 MYH15 7 KIF10 MYH7B KIF20A MYH7 2 1 2 6 KIF20B MYH6 KIF23 MYH3 KIF26A MYH13 11 HeLa 21 21 3 KIF26B MYH1 cells 10 KIF22 MYH4 KIF18A MYH8 2 3 8 KIF18B MYH2 KIF19 KIF2A Primary KIF2B monocytes 13 KIF2C Gene expressed in HeLa cells KIF24 HeLa BAC pool available Figure 1 Towards a comprehensive motor transgene collection in HeLa lines generated in this report superposed over a phylogenetic representation cells. (a) The number of kinesin and myosin genes expressed in HeLa of human kinesin and myosin motors annotated with standardized motor cells or human immunocytes measured by microarray transcription profiling class and gene names, as adapted from refs 2,3. Genes normally expressed in biological triplicate (see Supplementary Table S1). Arrows connecting in HeLa cells are indicated with cyan (see Supplementary Table S1). For two cell types indicate ex vivo differentiation. Curved arrows indicate cell all combinations of N- and C-terminal tagging of human (h)- or mouse proliferation. (b) Venn diagram comparing motor gene expression in HeLa (m)-derived BACs, green indicates that a stably transfected HeLa BAC line cells and two primary human immune cell types. (c) A summary of HeLa BAC is available (see Supplementary Table S2). RESULTS motor proteins to provide uniform tags for immunofluorescence Microarray analysis of motor gene expression in cells microscopy (IFM), live-cell video microscopy and AP–MS (ref. 14). We wanted to determine which motors are expressed in HeLa Murine and human genes were tagged as complementary gene sets cells, and how this expression pattern varies between different cell with a nearly identical gene number for use as fluorescent transgenes types. To address this challenge, we performed a single-platform or heterologous RNAi-resistance rescue factors15,17, depending on the transcription profiling analysis in HeLa cells, a human cell line used species origin of the cultured cell line, or to study motor function in vivo for high-throughput BAC transfection14, and primary human immune in mice. A BAC collection including 59 full-length human and 72 mouse cells before and after stimulation. We find that HeLa cells express more motor genes was tagged by high-throughput recombination in E. coli motor genes than either primary CD4+ T-lymphocytes or monocytes. (see Supplementary Table S2), transfected into HeLa cells and selected Ex vivo T-cell activation increases the number of expressed motor genes for stable incorporation of the transgene, as described previously14. to approximately that of HeLa cells, but we find fewer genes activated The 86% overall success rate produced 227 cell lines (Fig. 1c) that by monocyte differentiation (Fig. 1a and Supplementary Table S1). A we characterized by IFM and western blotting of cell lysates (see comparison of HeLa cells to resting immunocytes identified a core set Supplementary Table S3). In most cases, the cells represent transformed of 21 ubiquitously expressed human motors and another 32 expressed pools, but we also prepared GFP-positive cell pools and clones by in at least one cell type (Fig. 1b and Supplementary Table S1). We fluorescence-activated cell sorting (FACS) for genes of interest, (see enumerate the motor proteins endogenous to HeLa cells and highlight Supplementary Table S3) and found levels of the recombinant human differences in motor gene expression between primary human cells. transgenes similar to the endogenous protein (see Supplementary Fig. S1), as previously noted for other BAC transgenes15,20. Generating a motor BAC transgene and cell line collection To generate a comprehensive transgene resource to study motor BAC transgene localization in cultured cells function in cells, C-terminal LAP or N-terminal NFLAP tags were We confirmed known mitotic localizations of motor proteins (see introduced