Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments

Kazumasa Ohashi, Tai Kiuchi, Kazuyasu Shoji, Kaori Sampei, Kensaku Mizuno

    Research output: Contribution to journalArticlepeer-review

    38 Citations (Scopus)

    Abstract

    The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca 2+-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.

    Original languageEnglish
    Pages (from-to)45-50
    Number of pages6
    JournalBioTechniques
    Volume52
    Issue number1
    DOIs
    Publication statusPublished - 2012 Jan

    Keywords

    • Actin
    • Bimolecular fluorescence complementation (BiFC)
    • Calmodulin
    • Cofilin
    • Ras-Raf interaction
    • Split Venus

    ASJC Scopus subject areas

    • Biotechnology
    • Biochemistry, Genetics and Molecular Biology(all)

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