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

Nat Cell Biol. 2013 Mar;15(3):325-34. doi: 10.1038/ncb2689. Epub 2013 Feb 17.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Transport
  • Biomarkers / metabolism
  • Blotting, Western
  • Cell Movement / physiology*
  • Centrosome / metabolism
  • Chromatography, Affinity
  • Chromosomes, Artificial, Bacterial
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism*
  • Fluorescent Antibody Technique
  • Gene Expression Profiling
  • Genomics*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Immunoprecipitation
  • Kinesin / genetics
  • Kinesin / metabolism*
  • Mice
  • Mice, Transgenic
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Microtubules
  • Mitosis / physiology
  • Myosins / genetics
  • Myosins / metabolism*
  • Neuroblastoma / metabolism
  • Neuroblastoma / pathology
  • Neurons / cytology
  • Neurons / metabolism
  • Oligonucleotide Array Sequence Analysis
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phylogeny
  • Protein Multimerization
  • RNA, Messenger / genetics
  • Real-Time Polymerase Chain Reaction
  • Reverse Transcriptase Polymerase Chain Reaction
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Stem Cells / cytology
  • Stem Cells / metabolism
  • Transgenes / genetics

Substances

  • Biomarkers
  • Cep170 protein, human
  • KIF23 protein, human
  • Microtubule-Associated Proteins
  • Phosphoproteins
  • RNA, Messenger
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • MKLP1 protein, mouse
  • Myosins
  • Kinesin

Associated data

  • GEO/GSE33271
  • GEO/GSE33272
  • GEO/GSE33273