Quantifying E2F1 protein dynamics in single cells

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 (3′UTR) 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 G₀, 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.

Graphical abstract

Keywords

protein / E2F1 reporter / cell cycle

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SUPPLEMENTARY MATERIALS

The supplementary materials can be found online with this article at https://doi.org/10.1007/s40484-019-0193-6.

ACKNOWLEDGEMENTS

We thank Y. Gao for his help with time-lapse and confocal microscopy at the Duke Light Microscopy Core Facility. This research was supported by a grant from NIH (1R01-GM106107) (L.Y and B.M-P.), and funds from the School of Medicine at Duke University (B.M-P).

AUTHOR CONTRIBUTIONS

B.M-P., P.D., G.Y. and L.Y. developed the concept of the paper. B.M.-P. designed the research approach and performed experiments with the help of P.D., B-T.P., C.I., C.H. B.M-P. analyzed the data; B.M-P., P.D, G.Y. and L.Y. interpreted the results and wrote the manuscript with contributions from C.I.

COMPLIANCE WITH ETHICS GUIDELINES

The authors Bernard Mathey-Prevot, Bao-Tran Parker, Carolyn Im, Cierra Hong, Peng Dong, Guang Yao and Lingchong You declare that they have no conflict of interests.ƒAll procedures performed in studies were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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