A genomic toolkit to investigate kinesin and myosin motor function in cells
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...
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| Veröffentlicht in: | Nature cell biology 2013-03, Vol.15 (3), p.325-334 |
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| creator | Maliga, Zoltan Junqueira, Magno Toyoda, Yusuke Ettinger, Andreas Mora-Bermúdez, Felipe Klemm, Robin W. Vasilj, Andrej Guhr, Elaine Ibarlucea-Benitez, Itziar Poser, Ina Bonifacio, Ezio Huttner, Wieland B. Shevchenko, Andrej Hyman, Anthony A. |
| description | 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.
Maliga and colleagues have produced a library of bacterial artificial chromosome (BAC) transgenes encoding tagged human kinesin and myosin motors, and have generated a collection of BAC-expressing human and mouse cell lines for the study of motor function. |
| doi_str_mv | 10.1038/ncb2689 |
| format | Article |
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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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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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| fulltext | fulltext |
| identifier | ISSN: 1465-7392 |
| ispartof | Nature cell biology, 2013-03, Vol.15 (3), p.325-334 |
| issn | 1465-7392 1476-4679 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_1323807900 |
| source | MEDLINE; Nature; SpringerLink Journals - AutoHoldings |
| subjects | 631/57/343/2280 631/61/212 631/80/128/1675 631/80/128/1923 Animals Artificial chromosomes Biological Transport Biology Biomarkers - metabolism Blotting, Western Cancer Research Cell Biology Cell cycle Cell division Cell Movement - physiology Centrosome - metabolism Chromatography, Affinity Chromosomes, Artificial, Bacterial Developmental Biology Embryonic Stem Cells - cytology Embryonic Stem Cells - metabolism Fluorescent Antibody Technique Gene expression Gene Expression Profiling Genetic aspects Genetic engineering Genomes Genomics Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism HeLa Cells Humans Immunoprecipitation Kinesin Kinesin - genetics Kinesin - metabolism Life Sciences Localization Mass spectrometry Mice Mice, Transgenic Microtubule-Associated Proteins - genetics Microtubule-Associated Proteins - metabolism Microtubules Mitosis - physiology Myosin Myosins - genetics Myosins - metabolism Neuroblastoma - metabolism Neuroblastoma - pathology Neurons - cytology Neurons - metabolism Oligonucleotide Array Sequence Analysis Phosphoproteins - genetics Phosphoproteins - metabolism Phylogeny Physiological aspects Protein Multimerization Proteins Real-Time Polymerase Chain Reaction resource Reverse Transcriptase Polymerase Chain Reaction RNA, Messenger - genetics Scientific imaging Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Stem Cells Stem Cells - cytology Stem Cells - metabolism Systems analysis Transgenes - genetics |
| title | A genomic toolkit to investigate kinesin and myosin motor function in cells |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-28T19%3A43%3A08IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_proqu&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=A%20genomic%20toolkit%20to%20investigate%20kinesin%20and%20myosin%20motor%20function%20in%20cells&rft.jtitle=Nature%20cell%20biology&rft.au=Maliga,%20Zoltan&rft.date=2013-03-01&rft.volume=15&rft.issue=3&rft.spage=325&rft.epage=334&rft.pages=325-334&rft.issn=1465-7392&rft.eissn=1476-4679&rft_id=info:doi/10.1038/ncb2689&rft_dat=%3Cgale_proqu%3EA329898655%3C/gale_proqu%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1313546900&rft_id=info:pmid/23417121&rft_galeid=A329898655&rfr_iscdi=true |