Probing protein interactions in living mammalian cells on a microtubule bench

Sci Rep. 2015 Nov 27;5:17304. doi: 10.1038/srep17304.

Abstract

Microtubules are μm-long cylinders of about 25 nm in diameter which are present in the cytoplasm of eukaryotic cells. Here, we have developed a new method which uses these cylindrical structures as platforms to detect protein interactions in cells. The principle is simple: a protein of interest used as bait is brought to microtubules by fusing it to Tau, a microtubule-associated protein. The presence of a protein prey on microtubules then reveals an interaction between bait and prey. This method requires only a conventional optical microscope and straightforward fluorescence image analysis for detection and quantification of protein interactions. To test the reliability of this detection scheme, we used it to probe the interactions among three mRNA-binding proteins in both fixed and living cells and compared the results to those obtained by pull-down assays. We also tested whether the molecular interactions of Cx43, a membrane protein, can be investigated with this system. Altogether, the results indicate that microtubules can be used as platforms to detect protein interactions in mammalian cells, which should provide a basis for investigating pathogenic protein interactions involved in human diseases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Connexin 43 / genetics
  • Connexin 43 / metabolism
  • DNA Helicases
  • Gene Expression
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Microscopy, Fluorescence
  • Microtubules / metabolism*
  • Microtubules / ultrastructure
  • Poly A / metabolism
  • Poly-ADP-Ribose Binding Proteins
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • RNA Helicases
  • RNA Recognition Motif Proteins
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Y-Box-Binding Protein 1 / genetics
  • Y-Box-Binding Protein 1 / metabolism
  • tau Proteins / genetics
  • tau Proteins / metabolism*

Substances

  • Carrier Proteins
  • Connexin 43
  • Poly-ADP-Ribose Binding Proteins
  • RNA Recognition Motif Proteins
  • RNA-Binding Proteins
  • Recombinant Fusion Proteins
  • Y-Box-Binding Protein 1
  • YBX1 protein, human
  • tau Proteins
  • Green Fluorescent Proteins
  • Poly A
  • DNA Helicases
  • G3BP1 protein, human
  • RNA Helicases