We thank Dr. Rachid Mazroui (Universit&#233; Laval, Qu&#233;bec, Canada) for the critical reading of the manuscript. We thank the Cell Imaging Unit of the Research Center for their technical assistance. M.-&#201;. Huot is a Junior 1 Research Scholar of the Fonds de Recherche du Qu&#233;bec-Sant&#233; (FRQ-S). This work was supported by the Canadian Institutes of Health Research (grant number CIHR, MOP-286437 to M.-&#201;. Huot).

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 conditio...

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+puromycin+action:+fate+of+ribosomes+after+release+of+nascent+protein+chains+from+polysomes.">Mechanism of puromycin action: fate of ribosomes after release of nascent protein chains from polysomes.</a> Proc Natl Acad Sci U S A. 70, 3866-3869 (1973).</li><li>Goodman, C. A., Hornberger, T. 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S., Klann, E., Sonenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+control+of+long-lasting+synaptic+plasticity+and+memory.">Translational control of long-lasting synaptic plasticity and memory.</a> Neuron. 61, 10-26 (2009).</li><li>Du, T. G., Schmid, M., Jansen, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+cells+move+messages:+the+biological+functions+of+mRNA+localization.">Why cells move messages: the biological functions of mRNA localization.</a> Semin Cell Dev Biol. 18, 171-177 (2007).</li><li>Ephrussi, A., St Johnston, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing:+the+bicoid+morphogen+gradient+matures.">Seeing is believing: the bicoid morphogen gradient matures.</a> Cell. 116, 143-152 (2004).</li><li>Mardakheh, F. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Analysis+of+mRNA,+Translation,+and+Protein+Localization:+Local+Translation+Is+a+Key+Regulator+of+Cell+Protrusions.">Global Analysis of mRNA, Translation, and Protein Localization: Local Translation Is a Key Regulator of Cell Protrusions.</a> Dev Cell. 35, 344-357 (2015).</li><li>Lacsina, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+translational+termination+products+are+rapidly+degraded+substrates+for+MHC+class+I+presentation.">Premature translational termination products are rapidly degraded substrates for MHC class I presentation.</a> PLoS One. 7, e51968 (2012).</li><li>Schneider-Poetsch, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+eukaryotic+translation+elongation+by+cycloheximide+and+lactimidomycin.">Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin.</a> Nat Chem Biol. 6, 209-217 (2010).</li><li>Jacobs, J. P., Jones, C. M., Baille, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+a+human+diploid+cell+designated+MRC-5.">Characteristics of a human diploid cell designated MRC-5.</a> Nature. 227, 168-170 (1970).</li><li>Gandin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+fractionation+and+analysis+of+mammalian+translatomes+on+a+genome-wide+scale.">Polysome fractionation and analysis of mammalian translatomes on a genome-wide scale.</a> J Vis Exp. (87), (2014).</li></ol>

The authors have nothing to disclose.

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...

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 synthesis1,2,3,4. 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 metastasis5,6,7,8. 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 mRNA9,11,12.
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 granulation6,13,14,15, morphogen mRNA localization, and translation during development16,17. 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 changes5,7,18. Overall, this method allows for the visualization of localized or controlled translation events in a rapid, precise, and cost-effective way.

<table>
	<tbody>
		<tr>
			<td>DMEM</td>
			<td>wisent</td>
			<td>319-005-CL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsine</td>
			<td>wisent</td>
			<td>325-043 EL</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher Scientific</td>
			<td>12483020</td>
			<td></td>
		</tr>
		<tr>
			<td>Puroycin antibody 12D10</td>
			<td>EMD millipore</td>
			<td>MABE343</td>
			<td>western blot dilution 1:25000 
			Immunofluoresence dilution 1:10000</td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>cell signaling technology</td>
			<td>7076</td>
			<td>western blot dilution 1:8000</td>
		</tr>
		<tr>
			<td>Western Lightning Plus-ECL</td>
			<td>Perkin Elmer</td>
			<td>NEL104001EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG (H+L), F(ab&#39;)2 Fragment (Alexa Fluor 488 Conjugate)</td>
			<td>cell signaling technology</td>
			<td>4408</td>
			<td>immunofluoresecence dilution 1:400</td>
		</tr>
		<tr>
			<td>CF568 Phalloidin</td>
			<td>biotium</td>
			<td>00044</td>
			<td>immunofluoresecence dilution 1:400</td>
		</tr>
		<tr>
			<td>Cyclohexmide</td>
			<td>Sigma</td>
			<td>C1988-1G</td>
			<td>50&#181;g/ml final concentration</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td>final concentration 1&#181;g/ml</td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>bio-Basic</td>
			<td>PJ593</td>
			<td>2.5&#181;g/ml to 10&#181;g/ml</td>
		</tr>
		<tr>
			<td>Ibidi &#181;-Dish 35 mm, high, ibiTreat</td>
			<td>Ibidi</td>
			<td>81156</td>
			<td></td>
		</tr>
		<tr>
			<td>MRC-5 cells</td>
			<td>ATCC</td>
			<td>CCL-171</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Huh-7 cells</td>
			<td colspan="2">from Dr. Mazroui (Universit&#233; Laval)</td>
			<td></td>
		</tr>
		<tr>
			<td>Fv1000</td>
			<td>olympus</td>
			<td></td>
			<td>confocal imaging system</td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td></td>
			<td></td>
			<td>http://fiji.sc</td>
		</tr>
		<tr>
			<td>PBS (Phosphate BuffeRed Saline)</td>
			<td>bio-Basic</td>
			<td>PD8117</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37% Solution</td>
			<td>bio-Basic</td>
			<td>C5300-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>bio-Basic</td>
			<td>TB0198</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher Bioreagents</td>
			<td>BP9702-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween20</td>
			<td>Fisher Bioreagents</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative immunofluorescence to measure global localized translation

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.

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...

Watch this Scientific Journal Video about Quantitative Immunofluorescence to Measure Global Localized Translation at JoVE.com

Quantitative Immunofluorescence to Measure Global Localized Translation

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.

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

The overall goal of this experiment is to visualize and quantify compartmentalized translation events taking place during the initial steps of cell adhesion. This method has broad application. Use it whenever rapid specific translation response might be assay such as when analyzing cell migration, adhesion, or early drug response.The main advantage of this technique are that, it is easy, rapid, and cost-effective. It only requires puromycin, antibodies directed against puromycin, and the standard confocal scan imaging system. To begin, make a suspension of MRC-5 cells.Use Trypsinization, followed by centrifugation, removal of the supernatant, then resuspension in DMEM with ten percent FBS, at 40 thousand cells per milliliter. Incubate the suspension with gentle rotation for 20 minutes to completely dissociate the focal adhesion complexes. This is critical.Next, into two 35 millimeter glass-bottom dishes, add two milliliter aliquots suspension, and incubate them to allow for cellular adhesion and SiC formation. After 40 minutes, treat one plate with cycloheximide, and return it to the incubator. This plate will function as the negative control.After 55 minutes, apply puromycin to both plates at the predetermined concentration, and incubate the plates for five more minutes. After one hour of seeding and five minutes of puromycin in cooperation, wash each plate twice with two milliliters of ice-cold PBS. Then fix the cells with one milliliter of four percent formaldehyde in PBS for 15 minutes at room temperature.After the fixation, wash the cells three times with PBS, then permeabelize the cells using 500 microliters of 0.5 percent triton X-100 in PBS for 20 minutes at room temperature. Next, wash the plates three times with PBS, and then apply 500 microliters of one percent BSA in PBS. Continue the room temperature incubation for 20 more minutes.To remove excess BSA, wash the cells three times with 0.1 percent Tween-20 in PBS. Now, incubate the cells with 300 microliters of anti-puromycin antibody. Remove the unbound antibody using three washes with 0.1 percent Tween-20.Next, apply three hundred microliters of a mixture of two fluorophore-conjugated secondary antibodies to visualize puromycolated polypeptides and de factem. Incubate the cells for an hour at room temperature in the dark. After an hour, wash the plates three times to remove the unbound antibodies and then apply three hundred microliters of 0.1 percent Tween-20 solution, supplemented with DAPI.Allow the DAPI to stay in the cells for five minutes at room temperature. Finally, wash the cells four more times, finishing with a wash in pure PBS. Then, store the cells in two milliliters of PBS for imaging.Using any commercially available confocal imaging system, first determine the appropriate settings that maximize the dynamic range for quantification. Focus on quantification can only be obtained if peak size saturation is avoid and the concentration is adjusted properly. The setting can be attained by carefully adjusting the laser power advantage and offsets.First, adjust the zoom factor and the number of pixels to optimize the pixel size. Do so according to the Nyquist theorem. Next, decrease the scan speed and use averaging to improve the signal to noise ratio.Then, manually determine the appropriate top and bottom focal planes along the Z axis, and set the StepSize by calculating the axial resolution and dividing this distance by 2.3. The typical result is 20 to 25 image layers. Once the settings are tweaked, acquire a confocal image of an entire single cell using a 60x Plan Apo oil-immersion objective with a 1.42 numerical aperture.To quantify and analyze the flurophore signals, first open an image corresponding to the z plane layer of interest in image J.Then, using the drawing tool, make a line through the cell where quantification is desired. Next, modify the line whiff by double-clicking on straight. The intensity values will be averaged through the width of the line, so use a thinner line to avoid signal overlap from different structures.Then, profile the signal density along the line. Now, repeat the process of collecting the signal density along the same line in the channel that corresponds to a flurophore. The values will always be ordered using the orientation of the line.Now, copy the profile data and paste it in a spreadsheet for analysis. Repeat this process of collecting data from every Z plane image of interest before proceeding with the statistical analysis. Alternatively, instead of collecting signal along an axis, signal can be collected from an area of the image.First use the geometrical form function to select a region of interest. Next, adjust the threshold level to avoid any background. In the histogram of pixel intensities, move the slider to adjust which pixels will be included in the quantification.Set the threshold to eliminate all the red pixels outside the cell. Now, define the measurement parameters needed to measure the signal of interest and not background signal. Toggle the limit to threshold and integrated density options.Next, use the measure command. This will open a new window with the mean gray values and pixel count in the selected area. Now repeat the process, applying the same threshold to determine the signal intensity in the entire cell.Then use this data to calculate the ratio of signal in the selected area to that of the whole cell. For accurate measurement of translation events using purmycin-incorporation, the optimal conditions for an axial measurement were first assessed. A spectrum of concentrations versus a five or ten minute treatment was compared.The acceptable puromycin concentration for MRC-5 and HeLa cells was a little higher than what is needed for Huh-7 cells. Cells treated for ten minutes with high puromycin concentrations generated mostly smaller puromycolated polypeptides. The smaller polypeptides defused more rapidly, and are not desirable.The shorter incubation time was thus deemed more desirable. Cycloheximide treatment is an effective and important negative control. In MRC-5 cells, only a weak puromycin signal was detected after treatment with cycloheximide.Using axial signal quantification, the general localization of puromycolated polypeptides corresponding to newly-synthesized proteins could be assessed at different levels within the cell. A quantitative distribution of the signal throughout the cell was also feasible. For either technique, it was important to quantify the signal for every Z plane in the image stack to accurate assess translation events in a whole cell.After watching this video, you should have a good understanding on how to evaluate translation event in sub-cellular compartment. Once mastered, this technique can be done within a day, if performed properly. When one thinks this procedure, it's important to remember that the concentration of puromycin and its incubation time is important.To limit the diffusion, it is preferable to limit the incubation time. It is also important to recognize that the quality of the image acquisition type is a critical to obtain good results. Don't forget that working with formaldehyde can be extremely dangerous and precautions such as working in a chemical hood should always be taken while performing this procedure.

quantitative-immunofluorescence-to-measure-global-localized

Research

JoVE Journal

Biochemistry

9.7K Görüntüleme.  Université Laval. The overall goal of this experiment is to visualize and quantify compartmentalized translation events taking place during the initial steps of cell adhesion. This method has broad application. Use it whenever rapid specific translation response might be assay such as when analyzing cell migration, adhesion, or early drug response.The main advantage of this technique are that, it is easy, rapid, and cost-effective. It only requires puromycin, antibodies directed against puromycin, and t...