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    Quantifying E2F1 protein dynamics in single cells

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    Author
    Mathey-Prevot, Bernard
    Parker, Bao-Tran
    Im, Carolyn
    Hong, Cierra
    Dong, Peng
    Yao, Guang
    You, Lingchong
    Affiliation
    Univ Arizona, Mol & Cellular Biol
    Issue Date
    2020-03
    Keywords
    E2F1 reporter
    cell cycle
    PROTEIN
    
    Metadata
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    Publisher
    HIGHER EDUCATION PRESS
    Citation
    Mathey-Prevot, B., Parker, B., Im, C. et al. Quantifying E2F1 protein dynamics in single cells. Quant Biol 8, 20–30 (2020). https://doi.org/10.1007/s40484-019-0193-6
    Journal
    QUANTITATIVE BIOLOGY
    Rights
    © Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020.
    Collection Information
    This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at repository@u.library.arizona.edu.
    Abstract
    Background: E2F1 protein, a major effector of the Rb/E2F pathway plays a central role in regulating cell-fate decisions involved in proliferation, apoptosis, and differentiation. Its expression is highly dynamic and tightly modulated through a combination of transcriptional, translational and posttranslational controls. However, the mechanisms by which its expression and activity can promote different cellular outcomes remain to be fully elucidated. To better document E2F1 expression in live cells, we have engineered a series of fluorescent E2F1 protein reporters that quantitatively capture E2F1 protein dynamics. Methods: Reporter constructs, under the control of the mouse or human E2F1 proximal promoter, were designed to express an E2F1-Venus fusion protein incapable of binding DNA. In addition, constructs either included or excluded the 3 untranslated region (3UTR) of the E2F1 gene. These constructs were introduced into fibroblasts and epithelial cells, and expression of the fusion reporter protein was validated and quantified in single cells using live imaging. Results: In all cases, expression of the reporter protein effectively recapitulated the behavior of E2F1 under various conditions, including cell cycle progression and genotoxic stress. No or little fluorescent signal of the reporter was detected in G(0), but as the cycle progressed, expression of the reporter protein steadily increased in the nucleus, peaking a few hours before cell division, but declining to baseline 2-3 h prior to the onset of mitosis. The absence of the E2F1 3 ' UTR in the constructs led to considerably higher steady-state levels of the fusion protein, which although normally regulated, exhibited a slightly less complex dynamic profile during the cell cycle or genotoxic stress. Lastly, the presence or absence of Rb failed to impact the overall detection and levels of the reporter proteins. Conclusions: Our validated E2F1 protein reporters complement nicely other reporters of the Rb/E2F pathway and provide a unique tool to follow the complex dynamics of E2F1 expression in real time in single cells.
    Note
    12 month embargo; published online: 6 March 2020
    ISSN
    2095-4689
    PubMed ID
    32542116
    DOI
    10.1007/s40484-019-0193-6
    Version
    Final accepted manuscript
    ae974a485f413a2113503eed53cd6c53
    10.1007/s40484-019-0193-6
    Scopus Count
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    UA Faculty Publications

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