Sipieter F et al. (2015), Novel Reporter for Faithful Monitoring of ERK2 ...

XB-ART-51454

PLoS One 2015 Oct 20;1010:e0140924. doi: 10.1371/journal.pone.0140924.

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Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms.

Sipieter F , Cappe B , Gonzalez Pisfil M , Spriet C , Bodart JF , Cailliau-Maggio K , Vandenabeele P , Héliot L , Riquet FB .

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Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.

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Species referenced: Xenopus laevis
Genes referenced: fgf4 gnl3 map2k1 mapk1

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	Fig 2. Equimolar co-expression of eGFP-rERK2 and mMEK1 restores cytoplasmic localization of eGFP-rERK2.(A) Schematic representation of all genetically encoded molecular constructs used in this study. The corresponding amino acid sequence of 2A (yellow box) encodes a T2A peptide isolated from plasmid Myr-TdTomato-2A-H2B-eGFP (#1). Amino acids (GSG) and (GGAP) improve cleavage efficiency. The red slash symbol at the peptide C-terminal end indicates the 2A peptide cleavage site. (B) Fluorescence confocal imaging of NIH-3T3 cells after transfection with different plasmids and serum starvation for 24 h: top, transfection with eGFP-rERK2 (#2) and mCherry-mMEK1 (#3) plasmids; bottom, transfection with mCherry-mMEK1-2A-eGFP-rERK2 (#5). Representative images are shown of rERK2 protein distribution (green: a, e), mMEK1 distribution (red: b, f) and the merged image (c, g). Corresponding scatter plots of green and red intensities of each pixel on the whole images are shown (d, h). Co-localized pixels are visualized in yellow. Scale bar: 20 μm. (C) Fluorescence confocal imaging of NIH-3T3 cells transfected with mCherry-mMEK1 (middle, red) and labeled with an anti-ERK2 antibody (left, green) after 24 h of serum starvation. Co-localization of rERK2 and mCherry-mMEK1 is shown in the merged images (right, yellow). White arrows point to nuclei of transfected cells. Scale bar: 20 μm. (D) Western blot analysis of NIH-3T3 cells transfected with rERK2-LOC at the indicated time-points. Cell lysates were analyzed by immunoblotting with the indicated antibodies (left of each blot). The percentage of uncleaved polypeptide (full-length mMEK1-2A-GFP-rERK2, red triangle) was quantified by densitometry. Quantitative comparison of the levels of overexpressed rERK2-LOC and endogenous ERK2 (green triangles, middle panel) is indicated below the blot as IrERK2-LOC/ IrERK2. (E) After 24 h of serum starvation, NIH-3T3 cells transfected with rERK2-LOC were left untreated or were pretreated for 1 h with U0126 or DMSO, and then stimulated with serum or FGF4 for 15 min. Corresponding cell lysates were immunoblotted with the indicated antibodies (left of each blot). Relative phosphorylation levels of rERK2-LOC (green triangles) and endogenous ERK2 (blue triangles) were measured by densitometry. The ratios of phosphorylated protein to total proteins (IpYT-rERK2-LOC / IrERK2-LOC and IpYT-ERK2 / IERK2) are indicated below the top blot. (F) rERK2-LOC—transfected NIH-3T3 cells were serum starved for 24 h and then left untreated or incubated or not with U0126 for 1 h before stimulation with serum or FGF4 for 15 min. Cells lysates were immunoprecipitated with anti-eGFP (top panel) or anti-ERK2 antibodies (middle panel), and ERK1/2 kinase activity was assayed in vitro. The phosphorylated form of MBP (pMBP) was detected by immunoblotting. Unconjugated beads and beads conjugated with anti-HA antibodies were used as a control in the assays. Lysate inputs for immunoprecipitation were probed with anti-β-actin antibody as a loading control. At least two independent experiments and 15 cells were measured from fixed cells. Biochemical data are representative of at least two independent experiments.
	Fig 3. rERK2-LOC expression faithfully reports localization of ERK2.(A) Non-transfected (top row) and transfected NIH-3T3 cells overexpressing either eGFP-rERK2 (middle row) or rERK2-LOC (bottom row) were serum starved for 24 h, and then were left untreated or were treated with U0126 or DMSO for 1 h. Next, they were stimulated with serum or FGF4 for 15 min, or were left unstimulated (baseline). All cells were fixed and non-transfected cells were processed for immunofluorescence using the anti-ERK1/2 antibody (top row) and all cells were imaged by confocal microscopy. Shown are representative images of ERK2 localization under the different treatments. Scale bars: 20 μm. (B) Quantitative comparison of the nucleo-cytoplasmic concentration index (CI) of ERK2 between endogenous ERK1/2 (blue bars), overexpressed eGFP-rERK2 (red bars) and rERK2-LOC (green bars). CI values were normalized between 0 and 1 (CI¯ values), where 0 and 1 are respectively the minimal and maximal CI values obtained. Bsln: baseline, S: serum, F: FGF4, U0: U0126, V: vehicle (DMSO). (C) Non-transfected (top row) and transfected NIH-3T3 cells overexpressing rERK2-LOC (bottom row) were serum starved for 24 h and then stimulated with serum or FGF4 for 1 or 2 h, or left untreated (baseline). All cells were fixed and non-transfected cells were processed for immunofluorescence using the anti-ERK1/2 antibody (top row) and all cells were imaged by confocal microscopy. Shown are representative images of ERK2 localization under the different treatments. Scale bar: 20 μm. (D) Quantitative comparison of nucleo-cytoplasmic concentration index (CI) of ERK2 at the indicated time-points between endogenous ERK1/2 (blue bars) and overexpressed rERK2-LOC (green bars). CI values were normalized between 0 (minimum obtained) and 1 (maximum obtained) (CI¯ values). Bsln: baseline; 1S and 1F: 1 h serum and 1 h FGF4; 2S and 2F: 2 h serum and 2 h FGF4. (E) Monitoring of the subcellular distribution of rERK2-LOC in (24h) serum-starved NIH-3T3 cells by time-lapse confocal microscopy every 2 min for 10 min (baseline) and after FGF4 stimulation (100 ng/mL) for 30 min. (F) Nuclear and cytoplasmic intensities of each rERK2-LOC transfected cell were measured with Volocity software for each time-point to calculate the concentration index values (CI). Vertical error bars represent the average ± SEM. Two-way ANOVA test, accepting p ≤ 0.05 as significant, was performed to compare CI¯ values differences between endogenous ERK1/2, eGFP-rERK2 and rERK2-LOC for a same treatment. One-way ANOVA test, accepting p ≤ 0.05 as significant, was performed to compare CI¯ values among all the treatments (Tables 2 and 3). At least two independent experiments were performed. The number of cells per condition (n) from fixed cells is indicated in Tables 2 and 3 for statistical analysis; at least 80 cells were measured for time-lapse microscopy.
	Fig 4. Mobility of rERK2-LOC measured by high-speed FRAP.(A) eGFP-transfected NIH-3T3 cells were fixed. Individual living cells were imaged as described in the Materials and Methods section. Image sequences before (left) and after (right) photobleaching are shown. Scale bars: 10 μm. (B) NIH-3T3 cells were transfected with eGFP (left), eGFP-rERK2 (middle) or rERK2-LOC (right) and then serum starved for 24 h. Cells overexpressing rERK2-LOC were stimulated with serum to trigger its nuclear translocation. Bleached ROI correspond to the red lines drawn across the nuclei. Scale bars: 10 μm. (C) Representative kymograms (xt) of fluorescence intensity measured along the line (both red and white) across the selected cells for each experimental condition over-time are shown, indicating the FRAP measurement sequence: pre-bleach of 1 s (broken dark line), bleach of 150 ms (red lines) and post-bleach of 2 s (solid dark line). Correction for overall bleaching effects was applied. Nucl: nucleus (green line), Cyto: cytoplasm (purple line), BG: background (yellow line). (D-E) Curves of cumulative fluorescence recovery over time for fixed eGFP (grey curve), free eGFP (blue curve), overexpressed eGFP-rERK2 (red curve) and rERK2-LOC after serum stimulation (green curve, 8 min after serum stimulation) were normalized (D) and fitted (E). (F-G) Average half-life of recovery (t1/2) and immobile fraction (IF) calculation for cells serum-starved for 24 h and overexpressing free eGFP (blue symbol) or eGFP-rERK2 (red symbol), and serum-stimulated cells overexpressing rERK2-LOC (green symbol, 8 min after serum stimulation). At least two independent experiments were performed. The number of individual cells used for each condition is indicated above each symbol. Statistical significance was determined by a two-tailed unpaired t-test (ns, no significant; , ≤ 0.05; ***, ≤ 0.0001).
	Fig 5. Spatiotemporal subcellular distribution of xERK2-LOC in living Xenopus laevis embryo.(A) Embryos were injected with 500 ng of Myr-TdTomato-T2A-Histone2B-GFP mRNA. Maximum-intensity projection of a z-stack of 40 confocal images with a z-step of 0.59 μm is shown at 40X (left, scale bar: 10 μm) and at 63X magnification (right, scale bar: 20 μm). Pictures were merged to visualize Myr-mCherry at the plasma membrane and H2B-eGFP in the nucleus. (B) xERK2-LOC subcellular distribution was visualized at several stages of Xenopus laevis development (stage 9 blastula, stage 12 blastula and stage 32 tadpole). Scale bar: 200 μm. (C) Stage 9 embryos were fixed and processed for immunofluorescence with antibody against total ERK2 (green) and stained for DNA with Hoechst (blue) as described in the Materials and Methods section. Scale bar: 100 μm. (D) Protein extracts were prepared from uninjected (WT, lane 1), H2O injected (lane 2) and xERL2-LOC overexpressing embryos (lane 3) and immunoblotted with antibodies against GFP (top panel), ERK2 (middle panel) and β-actin (bottom panel). The percentage of uncleaved xERK2-LOC was measured by densitometry and is shown below the top panel in lane 3. The levels of overexpressed xERK2-LOC relative to endogenous ERK2 (green triangles, middle) are indicated below the blot as IxERK2-LOC/ IxERK2. (E) Embryos were injected with 500 ng of eGFP-xERK2, 2A-eGFP-xERK2 (control) or xERK2-LOC mRNA. Projections of at least 60 confocal 0.7-μm sections of animal cells at stage 9 blastula are shown. Higher magnification images of representative subcellular distributions of xERK2 are shown in white squares (top left). Scale bar: 150 μm. (F) Monitoring of xERK2-LOC subcellular distribution in the cells of the dorsal blastoporal lip (left panel) and the yolk plug (right panel) in stage-12 gastrula. Projection and 3D reconstruction of z-series of 108 confocal 1.50-μm sections (left panel) and 86 confocal 1.00-μm sections (right panel) are shown. White arrows indicate the trajectories of the cells leading to a progressive internalization of the yolk plug. Scale bar: 150 μm. (G-H) Several images from different viewpoints were recorded and combined to create a whole image of the developing embryos expressing xERK2-LOC, head to the left, at stage 32 (G) and stage 38 (H) tadpoles. The spatiotemporal localization of xERK2-LOC (green) in the embryonic structures, enhanced by autofluorescence of embryo and yolk (red signal), corresponds to notochord (nc), neural tube (nt) (both white arrows), otic vesicle (ov) and branchial arch region (bar) (both white asterisks) (G). Higher magnification of the forebrain-midbrain boundary (fmb) and para-axial structures (pax) are shown in yellow squares (H). Small white arrows indicate xERK2-LOC nuclear accumulation in several cells of the forebrain-midbrain boundary. Scale bar: 500 μm. At least two independent experiments were performed from animal caps, fixed embryos, or live embryos, and at least ten embryos were imaged. Biochemical data are representative of at least two independent experiments.
	Fig 1. Overexpression of eGFP-rERK2 induces nuclear accumulation of eGFP-rERK2. (A) NIH3T3 cells were serum-starved for 24 h (a) and then stimulated with 10% serum (b) or 100 ng/mL FGF4 (f). In other conditions, cells were pretreated with 20 μM U0126 (c, e, g) or vehicle DMSO (d, h) for 30 min before stimulation with 10% serum (c, d) or FGF4 (g, h). Cells were fixed, processed for double immunofluorescence with antibodies against total ERK1/2 and activated di-phosphorylated YT-ERK1/2, and then imaged by confocal microscopy. A maximum-intensity projection of a 5-μm thick z-stack (step size: 0.3 μm) for each overlapping image is shown. (B) NIH-3T3 cells were transiently transfected with increasing amounts of eGFP-rERK2 plasmid as indicated on the top left of each image, serum-starved for 24 h, fixed, and then imaged by confocal microscopy. The total amount of DNA was kept at 500 ng/mL of medium in all conditions. Higher magnification images of representative eGFP-rERK2 localization are shown in white squares (bottom right). Scale bars: 50 μm. (C) Relationship between the concentration of eGFP-rERK2 plasmid and the concentration index (CI). Higher CI values reflect greater accumulation of eGFP-ERK2 in the nucleus. Average CI was determined by examination of at least 30 randomly selected cells for each of the transfected conditions from two independent experiments. Average CI value for endogenous ERK1/2 in serum-starved NIH-3T3 is also shown (green dotted line). (D) NIH-3T3 cells transfected with 25, 125 or 500 ng/mL of eGFP-rERK2 were observed under severe imaging conditions to visualize cells that express very low level of eGFP-rERK2 protein (upper panel, white squares). Higher magnification images of these cells exhibiting mainly cytoplasmic localization of eGFP-rERK2 are also shown (bottom panel, white arrows). Scale bars: 50 μm.

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