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. 2020 Mar;8(1):20-30.
doi: 10.1007/s40484-019-0193-6. Epub 2020 Mar 6.

Quantifying E2F1 protein dynamics in single cells

Bernard Mathey-Prevot  1   2 , Bao-Tran Parker  1 , Carolyn Im  1 , Cierra Hong  3 , Peng Dong  1 , Guang Yao  4 , Lingchong You  5   6   7
Affiliations

Quantifying E2F1 protein dynamics in single cells

Bernard Mathey-Prevot et al. Quant Biol. 2020 Mar.

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.

Keywords: E2F1 reporter; cell cycle; protein.

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Conflict of interest statement

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.

Figures

Figure 1.
Figure 1.. E2F1 protein reporter constructs.
(A) Schematics of the E2F1 transcriptional and E2F activity reporters previously published [8,9]. hE2F1p: human E2F1 promoter; E2F1p: short human E2F1 promoter; NLS: SV40 nuclear localization domain; dVenus: destabilized Venus; dmCherry: destabilized monomeric Cherry; XR: drug resistance gene (puromycin or neomycin); orange hatched ovals: E2F consensus binding sites. (B) Schematic of E2F1 protein reporters generated in the pQCXIP (or pQCXIN) expression construct (Takara Bio/Clontech). mE2F1p and hE2F1p: E2F1 mouse and human promoter, respectively [8]; E2: sequence encoding a.a. 1–152 of hE2F1; Venus: sequence coding for the Venus fluorescent protein; F1: sequence encoding a.a. 175–437 of hE2F1; 3′UTR: 3′ untranslated region of hE2F1 gene. (C) Schematics of human E2F1 and E2VF1 proteins. Main functional domains are highlighted. P: phosphorylation sites; Ac: acetylation sites; NLS: nuclear localization domain; Δ: amino acids 153–174 deletion corresponding to leucine zipper in DNA binding domain (LNWAAEVLKVQKRRIYDITNVL); fl1: flexible linker 1; fl2: flexible linker 2.
Figure 2.
Figure 2.. Characterization of the E2VF1 fusion protein.
(A–D) Cell extracts were prepared from cells grown under normal or experimental conditions (as indicated) and used for detection of E2VF1 protein. Top panel: α -E2F1 antibody. Bottom panel: β –actin antibody. (A) a: REF52 (uninfected); b: REF52E2VF1–3′UTR (clone B); c: REF52E2VF1–3′UTR (clone D); d: REF52E2VF1 (clone 8). (B) a: Wi-38 (uninfected); b: WI-38E2VF1–3′UTR (polyclonal); c: REF52E2VF1–3′UTR (clone D). (C) a: HMEE2Fact (clone 1) cells grown in full medium. b: HMEE2Fact Rb+, E2VF1–3′UTR (clone R15) cells were incubated sequentially in starvation medium #1 and #2 for a total of 48 h (see Material and Methods). c: HMEE2Fact Rb+, E2VF1–3′UTR (clone R15), starved for 48 h and grown again with full medium for 24 h (Top panel: α -E2F1 antibody. Bottom panel: β-actin antibody). (D) Extracts from REF52E2VF1 (clone 8) or REF52E2VF1–3′UTR (clone D) grown in the absence or in the presence of increasing amount of cisplatin [μM]. Fold increase in E2vF1 protein levels was determined after normalization against β-actin levels.
Figure 3.
Figure 3.. Live detection and nuclear localization of E2VF1.
Rat or human fibroblasts were grown in 35 mm Mattek optic plates (A and B) for live imaging, and HME cells were grown on glass coverslips (C) placed in regular 35 mm tissue culture dishes before being fixed and permeabilized for immunofluorescence detection. (A) Live REF52E2VF1 (clone 8) cells imaged under DIC and YFP illumination respectively (Olympus VivaView FL microscope, 20×). Red oval highlights nucleus in top cell in DIC panel. (B) Live WI-38E2VF1–3′UTR (polyclonal) cells imaged as in A. Red ovals highlight nuclei in the two cells shown in DIC panel. (C) Confocal images of fixed HMEE2Fact Rb−, E2VF1–3′UTR (polyclonal) cells, taken in the DAPI, EYFP and mcherry channels respectively (Zeiss 780 inverted confocal microscope, 63×/1.4 NA Oil Plan-Apochromat DIC).
Figure 4.
Figure 4.. Time course of E2VF1 protein expression in REF52E2VF1 (clone 8) cells.
Quiescent REF52E2VF1 (clone 8) cells, starved of serum for 48 h, were released back into the cell cycle after addition of 10% serum. Cells were placed into the Vivaview incubator microscope and once the focus was fully stabilized (about 90 min) (t = 0), images were recorded every 30 min in the DIC and YFP modes (20× objective) for 36 h. E2VF1 fluorescent tracings for 4 representative cells are presented. AU: Arbitrary units of fluorescence. Red arrow: time of mitosis. Blue dots: values for each time point. Solid line: fitted curve (ggplot2: geom_point(), stat_smooth(method =“loess”, span = 0.4). Darker grey area: 95% confidence range for the fitted curve.
Figure 5.
Figure 5.. Rb+ and Rb‒ HME cells clones expressing both E2 F activity and protein reporters.
Viral stocks of the E2VF1–3′UTR construct were used to infect a human mammary epithelial cell clone expressing the E2F activity reporter (HMEE2Fact) [9] or a CRISPR-derived subclone harboring an Rb deletion (HMEE2Fact Rb−, clone 0). After selection, single cell clones were derived, and cell extracts were probed for expression of E2VF1, Rb and β-actin respectively. (a) HMEE2Fact Rb+; (b) HMEE2Fact Rb+, E2VF1–3′UTR (polyclonal); (c) HMEE2Fact Rb+, E2VF1–3′UTR (clone R11); (d) HMEE2Fact Rb+, E2VF1–3′ UTR (clone R15); (e) HMEE2Fact Rb− (clone 0); (f) HMEE2Fact Rb−, E2VF1–3′UTR (polyclonal); (g) HMEE2Fact Rb−, E2VF1–3′ UTR (clone H1); (h) HMEE2Fact Rb−, E2VF1–3′UTR (clone H4).
Figure 6.
Figure 6.. Time course of E2VF1 protein expression in HME cells.
HMEE2Fact Rb+, E2VF1–3′UTR (clone R15) or HMEE2Fact Rb−, E2VF1–3′ UTR (clone H4) cells (top 3 and bottom 2 panels respectively) were driven to quiescence by culturing them in minimum medium for 48 h. Cells were then released back into the cell cycle after addition of full growth medium. Cells were imaged under DIC, RFP and YFP modes every 30 min for 40 h with the Vivaview incubator microscope (20× objective) as described under Fig. 5. Representative time course tracings of E2F activity and E2VF1 protein reporters in singles cells. AU: Arbitrary units of fluorescence. Red arrow: time of mitosis. Solid lines: fitted curves for activity (red dots) and E2VF1 (green dots) reporters (ggplot2: geom_point(), stat_smooth(method =“loess”, span = 0.4). Darker grey area: 95% confidence range for fitted curves.
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