Visualization of intracellular PP1 targeting through transiently and stably expressed fluorescent protein fusions

Laura Trinkle-Mulcahy (Lead / Corresponding author), Janet Chusainow, Yun Wah Lam, Sam Swift, Angus Lamond

    Research output: Chapter in Book/Report/Conference proceedingChapter

    14 Citations (Scopus)

    Abstract

    Protein phosphatase 1 (PP1) is a ubiquitous serine/threonine phosphatase that regulates many cellular processes, including cell division, signaling, differentiation, and metabolism. It is expressed in mammalian cells as three closely related isoforms: α, β/δ, and γ1. These isoforms differ in their relative affinities for proteins, termed targeting subunits, that mediate their intracellular localization and substrate specificity. Because of the dynamic nature of these interactions, it is important to find experimental approaches that permit direct analyses of PP1 localization and PP1-targeting subunit interactions in live cells. When transiently or stably expressed as fluorescent protein (FP) fusions, the three isoforms are active phosphatases with distinct localization patterns and can interact with both endogenous and exogenous targeting subunits. Their changing spatio-temporal distributions can be monitored both throughout the cell cycle and following cellular perturbations by time-lapse fluorescence microscopy, and turnover rates of intracellular pools of the protein calculated by fluorescence recovery after photobleaching (FRAP). Interactions with targeting subunits can be visualized in vivo by fluorescence resonance energy transfer (FRET), using techniques such as sensitized emission, acceptor photobleaching, or fluorescence lifetime imaging.

    Original languageEnglish
    Title of host publicationProtein Phosphatase Protocols
    EditorsGreg Moorhead
    PublisherSpringer
    Pages133-154
    Number of pages22
    ISBN (Electronic)9781597452670
    ISBN (Print)9781588297112
    DOIs
    Publication statusPublished - 1 Dec 2007

    Publication series

    NameMethods in Molecular Biology
    Volume365
    ISSN (Print)1064-3745

    Keywords

    • FLIM
    • fluorescence
    • FRAP
    • FRET
    • GFP
    • isoforms
    • localization
    • microscopy
    • PP1
    • targeting

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  • Cite this

    Trinkle-Mulcahy, L., Chusainow, J., Lam, Y. W., Swift, S., & Lamond, A. (2007). Visualization of intracellular PP1 targeting through transiently and stably expressed fluorescent protein fusions. In G. Moorhead (Ed.), Protein Phosphatase Protocols (pp. 133-154). (Methods in Molecular Biology; Vol. 365). Springer . https://doi.org/10.1385/1-59745-267-X:133