Quantitative Immunofluorescence to Measure Global Localized Translation

Summary

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Abstract

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

Introduction

The regulation of translation by different cellular functions has prompted many research teams to develop new tools and methods to determine the subcellular localization of mRNA translation and regulated protein synthesis^1,2,3,4. These recent technological advances allow for an improved understanding of the mechanisms involving translation upregulation or the repression of specific mRNAs during biological processes, such as neuronal development, drug response, and metastasis^5,6,7,8. However, most of these methods require expensive or hazardous reagents and specific equipment that might not be available to most laboratories. As such, a cost-effective method to allow for the rapid assessment of translation events was developed to specifically circumvent these potential issues. This method detects acute translational modulations that occur during specific cellular processes and also allows for the localization of translation using confocal microscopy.

The methods described here were used to monitor localized translation within subcellular compartments called spreading initiation centers (SIC)⁵. SICs are transient structures found in seeded cells that are localized on top of nascent adhesion complexes. Although SICs and adhesion complexes are distinct, their fates are closely linked. Indeed, SICs are known to gradually disappear upon focal adhesion complex maturation into an adhesion site during the initial phase of adhesion. We found that RNA-binding proteins known to specifically control mRNA translation (e.g., Sam68, FMRP, and G3BP1) and polyadenylated RNAs were enriched within these structures⁵. Using the methods described here, we showed that the regulation of SIC-associated mRNA translation acts as a checkpoint allowing seeded cells to consolidate cell adhesion. This method, based on puromycin incorporation, could be considered an adapted version of the surface sensing of translation assay (SunSET). Originally developed to measure global protein synthesis rates using a non-radioactively labeled amino acid, protein puromycilation offers an efficient way to visualize de novo protein synthesis⁹. This method relies on the intrinsic behavior of puromycin, an antibiotic that blocks translation through premature chain termination in the ribosome¹⁰. Indeed, puromycin is structurally analogous to tyrosyl-tRNA, which allows for incorporation into elongating peptide chains via the formation of a peptide bond. However, puromycin binding to a growing peptide chain prevents a new peptide bond from being formed with the next aminoacyl-tRNA, since puromycin has a non-hydrolysable amide bond instead of the hydrolysable ester bond found in tRNAs. Thus, the incorporation of puromycin into elongating polypeptides results in the premature release of numerous truncated puromycilated polypeptides corresponding to actively translated mRNA^9,11,12.

Using this method, it was possible to assess active translation within a short time widow (e.g., 5 min) during cellular adhesion using a specific antibody directed against puromycin on cells that were supplemented with the antibiotic for 5-min periods at different time points during the cell adhesion process⁵. The precision of this assay relies on highly specific antibodies directed against the puromycilated moiety. Immunofluorescent detection of the puromycilated polypeptide provides a general subcellular repartition of the newly translated mRNA, which can also be quantified with great accuracy using confocal imaging systems.

Hence, this method offers a relevant option for a large number of laboratories studying translational regulatory mechanisms involved in processes such as neuronal granulation^6,13,14,15, morphogen mRNA localization, and translation during development^16,17. It is also well suited to studying localized or compartmentalized translation during rapid biological events, such as cell migration, adhesion, or invasion, or to simply assess drug treatments that might induce translational changes^5,7,18. Overall, this method allows for the visualization of localized or controlled translation events in a rapid, precise, and cost-effective way.

Protocol

1. Determination of Puromycilation Conditions

NOTE: This technique describes the method used to assess localized translation during the MRC-5 cell adhesion process⁵. As puromycilation can be done in any cell, it is important to optimize the puromycilation conditions for the specific cell lines to be used, because the treatment conditions are not identical for each cell line in terms of the puromycin concentration and the desired incubation time. To show how these conditions are defined, three example cell lines (i.e., HeLa, MRC-5, and Huh-7) were treated with increasing concentrations of puromycin for different incubation times (Figure 1).

1. Grow identical numbers of cells in a 24-well plate in 0.5 mL of the appropriate complete culture medium 16 h before the test. Seed 30,000 MRC-5 cells, 50,000 HeLa cells, or 50,000 HuH-7 cells per well. Make sure to avoid exceeding 60-70% confluence (Figure 1A).
2. Prepare 6 tubes with 1.5 mL of complete cell culture medium with increasing concentrations of puromycin (0, 5, 10, 15, 20, and 30 µg/mL). Pre-warm at 37 °C.
3. For each concentration of puromycin medium (prepared in step 1.2), transfer 0.5 mL from each tube into 6 new tubes and add cycloheximide at a final concentration of 50 µg/mL to all 6 new tubes. Pre-warm at 37 °C.
4. Change the medium with 0.5 mL of complete culture medium (row 1 and 2). For the third row, add 0.5 mL of complete culture medium supplemented with 50 µg/mL of cycloheximide (Figure 1B).
   NOTE: Cycloheximide treatment has been established as the best negative control for protein puromycilation due to its ability to block translation elongation. Because puromycin incorporation requires translation elongation, cycloheximide treatment leads to a loss of puromycilated protein signals^5,18.
5. Incubate for 15 min at 37 °C (5% CO₂).
6. Add 0.5 mL of the puromycin medium prepared in step 1.2 to the second row to obtain final concentrations of 0, 2.5, 5, 7.5, 10, and 15 µg/mL, respectively, in columns A, B, C, D, E, and F. At the same time, add 0.5 mL of puromycin to the cycloheximide-supplemented medium (step 1.3) in the third row (Figure 1C).
7. Incubate for 5 min at 37 °C (5% CO₂).
8. Add 0.5 mL of the puromycin medium prepared in step 1.2 to the first row to obtain final concentrations of 0.0, 2.5, 5, 7.5, 10, and 15 µg/mL (Figure 1C).
9. Incubate for 5 min at 37 °C (5% CO₂).
10. Wash each well from rows 1 to 3 with 1 mL of ice-cold 1X phosphate-buffered saline (PBS) 5 min after the addition of puromycin to wells of the first row (wash twice). Add 75 µL of 1X Laemmli buffer (4% sodium dodecyl sulfate (SDS), 4% 2-mercaptoethanol, 0.120 M Tris HCl pH 6.8, 0.004% bromophenol blue, and 10% glycerol) to obtain whole-cell lysates for each condition.
11. Run 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) using 1/3 of the prepared samples to assess puromycin incorporation.
    1. Determine the level of puromycin incorporation by Western blot analysis using an anti-puromycin antibody (12D10) diluted at a ratio of 1:25,000 in primary antibody incubation buffer (2% bovine serum albumin (BSA)), 430 mM NaCl, 10 mM Tris pH 7.4, and 0.01% sodium azide).
       NOTE: Representative images corresponding to different cell line extracts made as described in step 1 are shown as examples in Figure 2.

2. In Cellulo Localized Translation Visualization

NOTE: The technique described here was used to assess localized translation within SICs in MRC-5 cells during the adhesion process⁵. Although MRC-5 cells are used here, other cell lines can be used with the same methodology described in step 2.1.

1. Cell preparation.
   1. Detach MRC-5 cells using 0.25% trypsin/2.21 mM ethylenediaminetetraacetic acid (EDTA) in Hank's Balanced Salt Solution (HBSS). Pellet the cells by centrifugation (5 min at 200 x g) in a 15-mL conical centrifuge tube and suspend them in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 40,000 cells/mL after counting with a counting chamber.
   2. Incubate the suspended cells at 37 °C under gentle rotation using a tube rotator for 20 min to maintain them in suspension.
      NOTE: This step is critical to allow for the complete dissociation of the focal adhesion complexes.
   3. Plate 2 mL of suspended MRC-5 cells (40,000 cells/mL) in two 35-mm glass-bottom dishes. Use the first 35-mm glass-bottom dish for the puromycilation assay (see step 2.1.5) and use the second as a negative control (see step 2.1.6).
   4. Keep the cells at 37 °C (5% CO₂) for 55 min to allow for cellular adhesion and SIC formation.
   5. Add puromycin to the medium in the 35-mm glass-bottom dishes (assay) using the concentration previously defined in section 1. To treat MRC-5 cells, use 10 µg/mL puromycin for 5 min at 37 °C (5% CO₂).
   6. As a negative control, add cycloheximide to the second 35-mm glass-bottom dish 40 min after seeding the cells to pre-treat them with 50 µg/mL cycloheximide (Figure 2B). Then, 15 min after cycloheximide addition, add puromycin to the medium using the same concentration and incubation time as in step 2.1.5 (e.g., use 10 µg/mL of puromycin for 5 min at 37 °C (5% CO₂) for MRC-5).
   7. Wash each plate twice with 2 mL of ice-cold 1X PBS and fix the cells with 1 mL of 4% formaldehyde (diluted in 1X PBS) for 15 min at room temperature (RT).
      Caution: Paraformaldehyde must be used strictly in a chemical hood.
2. Immunostaining.
   1. Following paraformaldehyde fixation, wash three times with 2 mL of 1X PBS and incubate in 500 µL of PBS-TritonX-100 (0.5%) for 20 min at RT to permeabilize the cells.
   2. To prevent nonspecific antibody binding, block the sample with 500 µL PBS – BSA (1%) for 20 min at RT.
   3. Wash three times with 2 mL of PBS-Tween20 (0.1%) and incubate with 300 µL of anti-puromycin antibody (12D10) diluted 1:12,500 in 1X PBS for 1 h at RT.
   4. Wash 3 times with 2 mL of PBS-Tween20 (0.1%) and incubate with 300 µL of anti-mouse IgG conjugated to a fluorophore (here, 488 nm) diluted in PBS to visualize puromycilated polypeptides and with phalloidin-conjugated to another fluorophore (here, 555 nm) to visualize F-actin. Incubate for 1 h at RT.
   5. Wash three times with 2 mL of PBS-Tween20 (0.1%) and then incubate for 5 min at RT in 300 µL of PBS-Tween20 (0.1%) supplemented with 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI).
   6. Wash three times with 2 mL of PBS-Tween20 (0.1%) and then wash with 2 mL of 1X PBS. Prevent the cells from drying by keeping the sample in 2 mL of 1X PBS during image acquisition.

3. Immunofluorescent Image Acquisition

NOTE: The following methodology can be used with any commercially available confocal imaging system.

1. Determine the appropriate settings for each fluorophore to maximize the dynamic range for quantification.
   NOTE: Representative quantification can only be obtained if pixel saturation is avoided and the signal threshold is adjusted properly. These settings can be attained by carefully adjusting the laser power, high voltage (HV), gain, and offset.
   1. Adjust the zoom factor (5X) and the number of pixels (512 x 512) to optimize the pixel size.
      NOTE: This should be at least 2.3-times smaller than the optical resolution, according to the Nyquist theorem.
   2. Decrease the scan speed (12.5-20 µs/pixel) and use averaging (2-3 times) to improve the signal-to-noise ratio according to the settings mentioned above.
2. Manually determine the appropriate top and bottom focal planes along the Z-axis with the optimal step size (0.4 µm/slice).
   NOTE: The step size plane is determined by the axial resolution divided by 2.3, according to the Nyquist theorem. Typically, 20-25 layers are necessary to cover the entire cell depth of adherent cells.
3. Acquire a confocal image of an entire single cell with a 60X Plan Apo oil immersion objective (1.42 numerical aperture).

4. Immunofluorescent Image Quantification

1. Determination of enrichment.
   1. Open an image corresponding to the z-plane layer of interest from the acquired cell data file using the ImageJ software (freeware available at http://fiji.sc).
   2. Draw a line using the line drawing tool (Tool bar → Straight) through the regions of the cell where quantification is desired. Modify the line width by double-clicking on "straight;" the intensity values will be averaged through the width of the line (set at 8 in Figure 3).
      NOTE: A thinner line is preferred to avoid signal overlap from different structures.
   3. After the line is properly set, profile the signal density along the determined axis (menu bar → Analyze → Plot Profile).
      NOTE: A new window will open that shows a profile corresponding to the signal intensity for each pixel of the selected channel (specific fluorophore) along the line for the selected z-plane section.
      1. Repeat the process for each channel corresponding to the fluorophore used (i.e., one quantification for 488, one for 568, and one for 405).
         NOTE: The signal intensity value will always be ordered following the orientation of the drawn line.
   4. To obtain the numerical gray-scaled values for each pixel of the profile corresponding to the quantifications of the selected z-plane layer, copy the profile data (menu bar in image window → Copy) and paste it in any spreadsheet software.
   5. Repeat steps 4.1.1-4.1.4 for each z-plane section of the acquired confocal image and each channel where quantification is desired.
      NOTE: The quantification of different z-plane layers can be pooled to obtain a general assessment of the special enrichment of the signal by calculating the sum value of each pixel along the line for each z-plane layer. This type of pooling can only be achieved if the line drawn is identical for each z-plane layer included in the mean values.
   6. As an alternative method for the whole-cell quantification of different channels with a large number of layers, use the macro called StackprofileData (see the Supplemental File).
      NOTE: This macro is accessible freely at https://imagej.nih.gov/ij/macros/StackProfileData.txt and can be used as follows:
      1. Paste the macro into a text file and save it.
      2. Open the image file that includes all of the confocal layers of the cell.
      3. Draw a line, as described in step 4.1.2, open a new window to import the macro (menu bar → Plugins → Macros→ Run), choose the previously saved text file corresponding to the StackprofileData macro, and click "Open."
         NOTE: Following macro importation, a new window named "Results" will open and all of the quantification along the determined axis will be listed for each z-plane layer and channel present in the image files.
      4. Copy the data for each channel (menu bar → Edit → Copy) to obtain the graphical profile representation corresponding to the signal quantifications. Paste it into any spreadsheet software. Calculate the sum value of each channel for each pixel along the line for each z-plane layer. Use these values to create a graphical representation similar to the one presented in Figure 3.
2. Quantification of signal in a designated cellular area or volume.
   1. Open the image file with ImageJ software.
   2. Draw an area to denote the area of interest using the geometrical form function (tool bar →"rectangle," "oval," or "polygon").
      NOTE: The quantification value will be determined for the area corresponding to the drawn form.
   3. Adjust the threshold level to avoid any background signal (menu bar → Image Adjust → Threshold); a new window will appear to represent the pixel intensities in histogram form. Change the values to include/exclude pixels in the quantification using the slider. Select a threshold that eliminates red pixels outside the cell, as pixels highlighted in red will be included in the quantification.
   4. Define the measurement parameters to quantify the pixel signal within the selected area, without background signal (menu bar → Analyze → Set Measurements), and make sure to check the "Limit to Threshold" and the "Integrated Density" boxes.
   5. Measure the quantitative values for the selected area (menu bar → Analyze → Measure).
      NOTE: Signal intensity quantification will be presented in a new window under the "IntDen" identification, which represents the product of the mean gray values and the number of pixels within the selected area.
   6. Draw an area that includes the entire cell using a geometrical form (tool bar →"rectangle," "oval," or "polygon") and proceed with steps 4.2.3-4.2.5 to obtain a value corresponding to the total signal. Calculate the ratio of the signal within the selected area over the whole signal to obtain the percent of the signal within the area of interest.
   7. Repeat steps 4.2.2-4.2.6 to obtain the quantification of cell volume for each z-plane. Adapt the geometrical form as needed.
   8. Copy/paste the data obtained from the "Results" windows for each z-plane layer into a spreadsheet for graphical depiction and to find a mean value.

Results

To accurately observe translation events using puromycin incorporation, it is critical to determine the optimal conditions for each cell line because each shows different puromycin incorporation kinetics (Figure 1)^9,11,12,18. Hence, to validate puromycin incorporation, it is necessary to treat the desired cell line with a standardiz...

Discussion

Recent technological advances have allowed for a better understanding of the mechanisms involved in translational upregulation or the repression of specific mRNAs in biological processes, such as neuronal development, drug response, and metastasis. The cost-effective methodology described here allows translation events to be visualized in cells to study how RNA-binding proteins regulate metastatic processes, such as cellular adhesion, migration, and invasion.

Although numerous methods to asses...

Materials

Name	Company	Catalog Number	Comments
DMEM	wisent	319-005-CL
Trypsine	wisent	325-043 EL
FBS	Thermo Fisher Scientific	12483020
Puroycin antibody 12D10	EMD millipore	MABE343	western blot dilution 1:25,000 Immunofluoresence dilution 1:10,000
Anti-mouse IgG, HRP-linked Antibody	cell signaling technology	7076	western blot dilution 1:8,000
Western Lightning Plus-ECL	Perkin Elmer	NEL104001EA
Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor 488 Conjugate)	cell signaling technology	4408	immunofluoresecence dilution 1:400
CF568 Phalloidin	biotium	00044	immunofluoresecence dilution 1:400
Cyclohexmide	Sigma	C1988-1G	50µg/ml final concentration
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Invitrogen	D1306	final concentration 1µg/ml
Puromycin	bio-Basic	PJ593	2.5µg/ml to 10µg/ml
Ibidi µ-Dish 35 mm, high, ibiTreat	Ibidi	81156
MRC-5 cells	ATCC	CCL-171
HeLa cells	ATCC	CCL-2
Huh-7 cells	from Dr. Mazroui (Université Laval)
Fv1000	olympus		confocal imaging system
Fiji software			http://fiji.sc
PBS (Phosphate BuffeRed Saline)	bio-Basic	PD8117
Formaldehyde 37% Solution	bio-Basic	C5300-1
Triton X-100	bio-Basic	TB0198
BSA	Fisher Bioreagents	BP9702-100
Tween20	Fisher Bioreagents	BP337-500