1. Real-Time Imaging of Translation on Single mRNA Transcripts in Live Cells 
Chong Wang, Boran Han, Ruobo Zhou, Xiaowei Zhuang 
Cell 
Volume 165, Issue 4, Pages (May 2016)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1
Visualization of Translation on Individual mRNA Molecules in Live HeLa Cells
(A) Top: an mRNA construct map used for the detection of translation in live cells. Bottom: scheme showing translation on an mRNA transcript (black) by multiple ribosomes (yellow). The tandem array of V4 peptides in the translation product are shown in orange, scFv-GFP molecules are shown in green, and tdPCP-tdTomato molecules are shown in magenta. Binding of scFv-GFP to the V4 peptide array allows detection of translation and binding of tdPCP-tdtomato to the PP7 hairpin array at the 3′ UTR of the mRNA allows the detection of the mRNA.
(B) Image of individual translation complexes (polysomes) labeled by GFP (left), image of individual mRNA molecules labeled by tdTomato (middle) and their overlay (right). Scale bars, 5 μm. Insets: enlarged view of the boxed region. Some mRNA (tdTomato) foci do not have colocalizing GFP signal because not all mRNA molecules are simultaneously translated.
See also Figure S1.
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4. Figure 2 Probing Translation Sites with Translation Inhibitors
(A) Images of translation foci in the same field of view before and after treatment of 275 μM puromycin (Puro) at 37°C. Insets show the zoom-in of the boxed region. Scale bars, 5 μm. The full time course is shown in Movie S1.
(B) Time courses of the normalized total intensities of the GFP foci in individual cells under different drug treatments. Thin black line: upon 275 μM Puro treatment. Thick black line: upon 200 μM Cycloheximide (CHX) treatment. Medium black line: upon 275 μM Puro treatment in the presence of 50 μM CHX. The curves are normalized to the average of the first 5 data points. Puro was added at the time indicated by the dashed line. The translation signal shows a decay after the Puro or Puro + CHX treatment. A single-exponential fit of the decay region for Puro treatment (red line) and for Puro + CHX treatment (purple line) gives the observed rate constant (kobs) of the foci disappearance after drug treatment.
(C) Plot of kobs versus [Puro] under conditions of Puro treatment only (red) or Puro treatment in the presence of three different concentrations of CHX (purple, green, and blue). Error bars are SEM (n = 5–10 cells for each condition. In each cell, the number of detected polysomes before drug treatment is 30–100. We note that some polysomes were out of focus and hence not detected).
See also Figures S2 and S3 and Movie S1.
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5. Figure 3
Imaging Translational Responses to Unfolded Protein and Oxidative Stresses
(A) Images of translation foci in the same field of view before and after treatment of 1 mM DTT at 37°C. The full time course is shown in Movie S2.
(B and C) Typical time courses of the translation activity measured in single cells in response to DTT treatment (B) and NaAsO2 treatment (C). Each cell contains 50–100 detected (in focus) polysomes at time zero. The translational activity is measured as the total intensity of the translation foci in the cell normalized to the average value of the first 80 s.
(D and E) Averaged time courses of translation activity changes in response to DTT (D) and NaAsO2 (E) under different conditions. The curves are average over 20–30 cells each and the shades represent SEM. For each cell, the translation activity is normalized as described (B and C). Red lines are average time courses in response to DTT (1 mM) or NaAsO2 (0.5 mM) treatment alone. Black, blue, and magenta lines are average DTT or NaAsO2 time courses in the presence of translation inhibitor CHX (200 μM), kinase inhibitor GSK (1 μM), and integrated stress response inhibitor ISRIB (400 nM), respectively.
See also Figure S4 and Movie S2.
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6. Figure 4
Transient Upregulation of Translation for Construct Harboring ATF4 uORFs in Responses to Unfolded Protein and Oxidative Stresses
(A) The translation reporter construct regulated by ATF4 uORFs. The construct contains two uORFs (uORF1 and uORF2) before the open reading frame 3 (ORF3). The stop codon of uORF2 located downstream of the start codon of ORF3 is frame-shifted from ORF3 and thus does not affect translation the V4 peptide array and ODC contained in ORF3.
(B and D) Snapshots of the translation activity of the ATF4 reporter construct in a cell under DTT-induced stress (B) or NaAsO2-induced stress (D) at indicated time points. The full time course of the cell shown in (B) is shown in Movie S3. Scale bars, 5 μm.
(C and E) Corresponding time course of the translation activity of the cell shown in (B) and (D), respectively. The translational activity is measured as the total intensity of the translation foci in the cell normalized to the initial value (average of the first 80 s). 20–30 cells were measured for each treatment (NaAsO2 or DTT), and the ensemble-averaged time courses are shown in Figure S5.
See also Figure S5 and Movie S3.
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7. Figure 5 Tracking the Movements of Individual Polysomes in Live Cells
(A) Left panel: a single snapshot of individual polysomes in a region of a cell. Three specific foci are marked and their time trajectories are shown on the right. Scale bar, 5 μm. Right panels: mobility analysis of the three marked polysomes in the left image. The top panels show movement trajectories of polysomes. Scale bars, 320 nm. The bottom panels show mean squared displacement (MSD) versus time for the corresponding polysomes. Polysomes 1, 2 and 3 are representative examples of stationary, sub-diffusive and diffusive movement, respectively.
(B and C) Distributions of MSD of individual polysomes for the indicated constructs. The MSD value is determined at the 0.5 s time delay. The two histograms in (B) are for cytosolic proteins and the two in (C) are for a secreted protein (sBFP, top) and a transmembrane protein (SMOTM, lower). A P2A sequence that undergoes co-translational cleavage is inserted between the sBFP or SMOTM coding region and the V4 peptide array to avoid (1) secretion of the V4 peptide array facilitated by sBFP, which prevents binding of scFv-GFP and (2) detection of the fully translated SMOTM-V4 protein products, which would also be anchored to ER and thus not rapidly diffusing. Although cleavage at P2A prevents observation of the protein products, in a polysome where multiple ribosomes are translating the mRNA simultaneously, the ribosomes that are translating the sBFP/SMOTM portion can mediate anchorage to ER, while other ribosomes that are translating the V4 peptide array portion allow visualization of the polysome through scFv-GFP binding. The observed fluorescent foci disappear after Puro treatment (Figures S6A and S6B), confirming their identity as translating polysomes. As a control, P2A is also inserted between the cytosolic BFP and the V4 peptide array (B, bottom). For each construct, 40–50 cells are analyzed.
See also Figure S6.
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8. Figure 6
Polysomes Translating Cytosolic Proteins Display a Lower Mobility in the Perinuclear Region
(A) Trajectories of polysomes of the cytosolic protein construct in a typical cell. The movies showing the movement of these polysomes are shown in Movie S4. The contour of the nucleus is shown by red dashed line. The trajectories are color-coded according to their mobility values, quantified by MSD at the 0.5 s time delay, with higher mobility shown in red and lower mobility shown in blue. Scale bar, 5 μm.
(B) Distributions of the MSD values at 0.5 s time delay for polysomes in the perinuclear region (magenta) and for polysomes not in the perinuclear region (blue). See the Supplemental Experimental Procedures for the operational definition of perinuclear and non-perinuclear regions.
(C) Average diffusion coefficients of perinuclear polysomes (left) and non-perinuclear polysomes (right). Error bars are SEM. p value is calculated from unpaired t test (n = 40 cells).
See also Movie S4.
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9. Figure 7
Imaging Local Translation in the Dendrites of Live Hippocampal Neurons
(A) Individual fluorescence loci observed inside neurons disappear after Puro treatment, confirming their identity as translating polysomes. The construct without the Arc 3′ UTR is used here.
(B) Translating polysomes observed in a dendrite (corresponding to the boxed region of the neuron image in the inset) using the construct with the Arc 3′ UTR.
(C) Average number of translation foci observed in 10 μm segments of dendrites at varying distance from the cell body using constructs with (blue) and without (red) the Arc 3′ UTR. Error bars represent SEM (n = 26 cells for construct with the Arc 3′ UTR, n = 21 cells for construct without the Arc 3′ UTR).
(D) Left two panels: image snapshots of a dendritic region showing a polysome undergoing directed movement (indicated by arrow). Right panel: kymograph of the boxed region in the left showing the directed movement of the arrow-indicated polysome. The full time course of the movement of this polysome is shown in Movie S5.
(E) Histogram of the movement speeds of 41 polysomes showing directed movement.
The neurons used in this figure are at 14–16 days in vitro (DIV 14–16). Scale bar, 5 μm for (A), (B), and (D). See also Movie S5.
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10. Figure S1
Specificity of Nascent Peptide and mRNA Labeling and Estimate of mRNA Number and Ribosome Number in Each Translation Foci, Related to Figure 1
(A) The expression of construct that contains the V4 peptide array but not the PP7 array results in only GFP foci, which are sensitive to Puro treatment, but no fluorescence foci in the tdTomato channel.
(B) The expression of construct that contains the PP7 array but not the V4 peptide array results in only tdTomato foci but no fluorescence foci in the GFP channel.
(C) Distribution of the signals of individual fluorescence foci in the tdTomato channel. The construct that contains both the V4 peptide array and the PP7 hairpin array is used for this quantification. The signal of each fluorescence foci is normalized by the average signal of single purified tdTomato molecules. The average signal of single tdTomato molecules is determined by anchoring individual tdTomato proteins to glass coverslips. The distribution is constructed from more than 1500 detected fluorescence foci in 12 cells.
(D) Distribution of signals of individual fluorescence foci in the GFP channel. The construct that contains both the V4 peptide array and the PP7 hairpin array is used for this quantification. The signal of each fluorescence foci is quantified in terms of the number of ribosomes per foci. The ribosome number is obtained by comparison of the fluorescence signals of the GFP translation foci with the average single-molecule signal of the protein product. The signal of individual protein product is measured by expression of a construct of the V4 peptide array fused to maltose-binding protein (MBP), a widely used monomeric protein to facilitate recombinant protein expression. In addition, a plasma membrane targeting motif, CAAX sequence, is added to the construct (V4-MBP-CAAX) to anchor the protein products to plasma membrane to facilitate single-molecule detection. The measurement is performed in the presence of puromycin to remove polysome signals so that only signals from single protein products are measured. Since the signal intensity for each ribosome depends on its location on mRNA (when ribosomes are translating on the V4 peptide array section, their intensity is less than the fully labeled translation product, while when ribosomes are translating on ODC region, their signal is equal to fully labeled product), a correction factor is needed to estimate the average intensity of an ribosome on the mRNA. The V4 peptide array segment contains 594 amino acids, while the ODC segment contain 473 amino acids. Assuming uniform distribution of ribosomes on the mRNA, 594/( ) = 55.67% of the ribosomes will occupy the V4 peptide array segment with an average signal per ribosome that is equivalent to that of 0.5 translation product. 473/( ) = 44.33% of the ribosomes will occupy the ODC segment with an average signal per ribosome that is equivalent to that of 1 translation product. Thus on average, the average signal of single ribosomes on the mRNA is 0.5 × 55.67%+1 × 44.33% = 72% of that of the translation product. The signals from individual translating GFP foci are then divided by this average signal of a single ribosome to determine the number of ribosomes for each translation foci. The distribution is constructed from more than 2,000 translation foci in 40 cells.
Scale bars: 5μm.
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11. Figure S2
Single Polysomes Time Traces Showing the Disappearance of the GFP Signal upon 275 μM Puro Treatment, Related to Figure 2
Three typical time traces are shown. Puro is added at the time indicated by the red dashed line.
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12. Figure S3
Ribosome Run-Off Measurement by Translation Inhibition Using Homoharringtonine (Homo), Related to Figure 2
(A) A cartoon illustrating the change of the translation signal as the results of Homo inhibition, which stalls the newly loaded ribosomes during the initial rounds of peptide formation but does not perturb the ribosomes that are already in the active elongation phase. After Homo inhibition, the ribosomes that already enter active elongation phase can finish translation and run off the mRNA molecule, and hence the number of ribosomes on the mRNA and the GFP signal associated with the mRNA will decrease. Those stalled ribosomes during the early elongation phase are not shown here because they do not allow binding of scFv and hence are not detectable. As shown in the figure, the region coding the V4 peptide array will be depleted of ribosomes before the region coding ODC. In the coding region for the V4 peptide array, the fluorescence intensity associated with individual ribosomes depends on the location of the ribosome: the ribosomes closer to the start codon (AUG) on the 5′ end are dimmer while those closer to the 3′ are brighter. So, the decay at the beginning of the time course due to depletion of ribosomes in the V4 encoding region should exhibit a gradually increasing rate, while the subsequent decay due to depletion of ribosomes in the ODC encoding region should exhibit a constant rate.
(B) Normalized total intensity of the translation foci per field of view as a function of time after addition of 200 μM Homo for different constructs indicated in the box. Error bars represent SEM (n = ∼40 cells each). For each curve, the baseline corresponding to the intensity of the last time point is shown as a blue line. At the early phases of the decay, the curve follows the predicted behavior showing a gradually increasing decay rate before entering a linear decay region with a constant decay rate. Near the end of the decay curve, instead of approaching baseline in a linear manner, the decay gradually slowed down, probably reflecting the heterogeneity of the translation rates among different polysomes or cells. For the V4-ODC construct, the length of the region encoding V4 peptide array is 594 amino acids (55.67% of the whole sequence), while the length of the ODC encoding region is 473 amino acid (44.33% of the whole sequence). Assuming ribosomes have an even distribution on the mRNA, 55.67% of the ribosomes will occupy the V4 peptide array segment with an average signal per ribosome that is equivalent to that of 0.5 translation product and 44.33% of the ribosomes will occupy the ODC segment with a signal per ribosome that is equivalent to that of 1 translation product. First the number of ribosomes on the V4 segment will decrease during the run-off, which is then followed by the decrease in the number of the ribosomes in the ODC region (See Figure S3A). The time point at which V4 region is fully depleted of ribosomes but the ODC regions is still fully occupied is point when the signal decay will become linear. At this point, the signal should be 44.33%/(55.67%/ %) = 64.1% for the original signal. Similarly, the corresponding values for V4-BFP-ODC and V4-MBP-ODC construct are 70.5% and 74.0%, respectively. We thus conservatively chose 60% as the starting point of linear fitting for all three constructs (black dashed line). Below 20%–30%, the curves deviate from linear decay due to translation rate heterogeneity. We thus used 40% as a conservative estimate of the end point for linear fitting in all three cases (black dashed line). Extrapolating the linear fit line (red) to the baseline (blue) gives an estimate of the average time required for a ribosome to translate the entire mRNA transcript, which we term Ttrans.
(C) Plot of the average Ttrans as a function of the mRNA construct length. Error bars represent SEM (n = 6 group of cells, each group contains 6-8 cells). Linear fit of the data points (blue line) allows us to determine a translation elongation rate of 4 amino acids/s.
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13. Figure S4
A Scheme of Stress Response Pathways and Measurement of eIF2α Phosphorylation during Stresses, Related to Figure 3
(A) The diagram shows a simplified scheme of the pathways and molecular factors involved in stress response. Translation initiation depends on the GTP-bound form of eIF2 (eIF2-GTP). After initiation of translation, the GTP bound to eIF2 is hydrolyzed into GDP. The recycling of eIF2-GDP back to eIF2-GTP is catalyzed by eIF2B. During DTT-induced unfolded protein stress and NaAsO2-induced oxidative stress, the PERK and HRI kinases are activated, respectively. These kinases phosphorylate the α subunit of eIF2 (eIF2α), and phosphorylated eIF2α (eIF2α(P)) will bind stably to eIF2B, acting as a competitive inhibitor that prevents efficient GDP-to-GTP exchange of eIF2. This leads to a decrease in the level of eIF2-GTP, thereby causing a global reduction in translation. GSK is a kinase inhibitor that inhibits PERK with a stronger potency than its inhibition potency on HRI. The integrated stress response inhibitor ISRIB counteracts eIF2α(P)’s inhibition effect on eIF2B.
(B) Western blot images of phosphorylated eIF2α and total eIF2α at different time points after DTT (1 mM) and NaAsO2 (0.5 mM) treatment.
(C) The ratio of phosphorylated eIF2α signal to total eIF2α signal at different time points after DTT and NaAsO2 treatments normalized to the ratio value at time zero. Error bars: SEM (n = 4 independent replicates).
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14. Figure S5
Average Response Curves of the Pulse-like Translation Upregulation of the Reporter Construct Containing ATF4 uORFs under DTT or NaAsO2 Treatment, Related to Figure 4
The curves are average over 21 cells in the DTT case (red) and 23 cells in the NaAsO2 case (blue). For each cell, the translation activity is normalized as described in Figures 4C and 4E. The shades represent SEM.
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15. Figure S6
Mobility Analysis of Polysomes Translating ER-Targeting Proteins, Related to Figure 5
(A and B) Typical image showing fluorescent translation foci of a construct containing the secreted form of BFP (sBFP) (A) or smoothened receptor transmembrane domain, SMOTM (B) upstream of the V4 peptide array before and after Puro treatment. The foci disappear after Puro treatment, confirming their identity as translating polysomes.
(C) Comparison of cell-averaged diffusion coefficients of polysomes for indicated constructs. Error bars are SEM. P-values are calculated by unpaired t test, n = 42, 42, and 44 cells for V4-ODC, sBFP-P2A-V4-ODC and SMOTM-P2A-V4-ODC, respectively.
(D) The percentage of polysomes classified as stationary, sub-diffusive and diffusive for the three different constructs, V4-ODC (red), sBFP-P2A-V4-ODC (green) and SMOTM-P2A-V4-ODC (blue). Error bars are SEM (n = cells).
Cell  , DOI: ( /j.cell )
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