Name: assessment of de novo protein synthesis rates in caenorhabditis elegans
Uploaded: 2020-09-12T13:00:00
Description: Watch this Scientific Journal Video about Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans at JoVE.com

We thank Chaniotakis M. and Kounakis K. for video recording and editing. K.P. is funded by a grant from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT). N.T. is funded by grants from the European Research Council (ERC &#8211; GA695190 &#8211; MANNA), the European Commission Framework Programmes, and the Greek Ministry of Education.

NOTE: Both transcriptional and translational fusions to fluorophores can be used to evaluate de novo protein translation in several tissues. Protein synthesis rates can be assessed for multiple protein families that localized in different cellular compartments, including cytoplasm, mitochondria and nucleus7. The following strains are used to monitor global and neuronal protein synthesis rates, N2;Ex[pife-2GFP, pRF4], ife-2(ok306);Ex[pife-2</e...

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+absolute+protein+synthesis+rates+reveals+principles+underlying+allocation+of+cellular+resources.">Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources.</a> Cell. 157 (3), 624-635 (2014).</li><li>Dermit, M., Dodel, M., Mardakheh, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+monitoring+and+measurement+of+protein+translation+in+time+and+space.">Methods for monitoring and measurement of protein translation in time and space.</a> Molecular Biosystems. 13 (12), 2477-2488 (2017).</li><li>Iwasaki, S., Ingolia, N. 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Protein synthesis modulation is essential for organismal homeostasis. During ageing, global as well as specific protein synthesis is perturbed. Recent studies reveal the fact that protein translation balance directly controls senescence and aging is not merely a byproduct of the aging process. In particular, core components of the translation machinery such as eukaryotic initiation factor 4E (eIF4E), which facilitates mRNA capping during translation initiation, and thus the rate of cap-dependent protein translation, indu...

Protein synthesis and degradation is essential for organismal homeostasis. A multitude of age-related diseases are instigated by defective protein production1,2. In order to measure global protein translation rates, there are biochemical techniques such as ribosomal profiling, which involves deep sequencing of ribosome-protected mRNA fragments to monitor expression as well as novel protein synthesis3. This method, besides being an indirect readout of translational rates, as increased RNA association to ribosomes does not necessarily mean increased translation, has technical disadvantages, such as high cost and a requirement of a large amount of starting material. On the other hand, proteomics-based methods allow for direct protein quantification by pulse metabolic profiling followed by mass spectrometry analysis4,5. However, this is a semi-quantitative approach with limited temporal resolution that cannot be easily used in vivo. Moreover, labelling of the protein can be unequally distributed in the animal/tissue of interest. Importantly, both these methods can conceal tissue-specific or cell-specific variations in protein translation rates in whole animals or tissues, respectively.
Caenorhabditis elegans is an easy-to-use model organism that can be grown in large numbers6. Additionally, its genetic amenability and transparency allow for live imaging in vivo. This protocol describes the methodology to detect protein synthesis rates using fluorescence recovery after photobleaching (FRAP). We take advantage of the transgenic expression of fluorescent proteins, either in the whole organism or in specific tissues/cells. Transgenic animals may either express GFP using the promoter of a gene, which is widely/globally expressed or a tissue-specific promoter to target specific cell types. This technique can be extended to a specific promoter to examine protein synthesis rates of a specific protein.

<table>
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym</td>
			<td>8,40,004</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose pads 2%</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate (CaCl2&#8729;2H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>SERVA Electrophoresis</td>
			<td>17101.01</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C-7698</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting stereomicroscope</td>
			<td>Nikon Corporation</td>
			<td></td>
			<td>SMZ645</td>
		</tr>
		<tr>
			<td>edc-3(ok1427);Ex[punc-119GFP, pRF4]</td>
			<td>Tavernarakis lab</td>
			<td></td>
			<td>Maintain animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>Epifluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Axio Imager Z2</td>
		</tr>
		<tr>
			<td>Escherichia coli OP50 strain</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence dissecting stereomicroscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>SteREO Lumar V12</td>
		</tr>
		<tr>
			<td>Greiner Petri dishes (60 x 15 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>P5237</td>
			<td></td>
		</tr>
		<tr>
			<td>ife-2(ok306);Ex[pife-2GFP, pRF4]</td>
			<td>Tavernarakis lab</td>
			<td></td>
			<td>Maintain animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>image analysis software</td>
			<td>Fiji</td>
			<td></td>
			<td>https://fiji.sc</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>EMD Millipore</td>
			<td>1,37,010</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>EMD Millipore</td>
			<td>1,04,873</td>
			<td></td>
		</tr>
		<tr>
			<td>LB liquid medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>M9 buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Sigma-Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides (75 x 25 x 1 mm)</td>
			<td>Marienfeld, Lauda-Koenigshofen</td>
			<td>10 006 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2011 Excel software package</td>
			<td>Microsoft Corporation, Redmond, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N2;Ex[pife-2GFP; pRF4]</td>
			<td>Tavernarakis lab</td>
			<td></td>
			<td>Maintain animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>N2;Ex[psod-3GFP; pRF4]</td>
			<td>Tavernarakis lab</td>
			<td></td>
			<td>Maintain animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>N2;Ex[punc-119GFP; pRF4]</td>
			<td>Tavernarakis lab</td>
			<td></td>
			<td>Maintain animals at 20 &#176;C</td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>EMD Millipore</td>
			<td>1,06,586</td>
			<td></td>
		</tr>
		<tr>
			<td>Nematode growth medium (NGM) agar plates</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nystatin stock solution</td>
			<td>Sigma-Aldrich</td>
			<td>N3503</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>BD, Bacto</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>EMD Millipore</td>
			<td>1,06,40,41,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard equipment for preparing agar plates (autoclave, Petri dishes, etc.)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Standard equipment for maintaining worms (platinum wire pick, incubators, etc.).</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>statistical analysis software</td>
			<td>GraphPad Software Inc., San Diego, USA</td>
			<td></td>
			<td>GraphPad Prism software package</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>S6501</td>
			<td></td>
		</tr>
		<tr>
			<td>UV Crosslinker</td>
			<td>Vilber Lourmat BIO-LINK BLX 254</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Worm pick</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of de novo protein synthesis rates in caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Using the procedure presented here, wild type and mutant transgenic nematodes expressing the following somatic reporters, pife-2GFP, psod-3GFP and punc-119GFP, were used to assess protein synthesis rates according to the respective protocols. Particularly, wild type and ife-2 mutant worms expressing cytoplasmic GFP throughout their somatic tissues by using the ife-2 promoter were compared before, immediat...

Watch this Scientific Journal Video about Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans at JoVE.com

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

The rate of protein synthesis is perturbed during disease and aging. Fluorescence recovery after photobleaching, or FRAP, allows the measurement of rate of protein synthesis in vivo. We use transparent C.elegans worms, which express GFP, as a model to monitor the novel protein synthesis after photobleaching.We provide practical guidelines to measure fluorescence recovery by using transgenic worms, which express GFP under different promoters, either widely expressed or in specific cells or tissues. Also, this method offers protein synthesis speed monitoring in real time. Use a dissecting stereomicroscope to assess the developmental stages and growth of wild-type and mutant transgenic nematodes.Pick 10 L4 larvae of wild-type and mutant transgenic animals carrying the desired fluorescent reporter onto nematode growth media plates seeded with E.coli. Incubate and grow the nematodes at the standard temperature of 20 degrees Celsius. Four days later, the plate contains a mixed population of transgenic worms.Select and transfer 15 L4 larvae of each strain on freshly seeded OP50 NGM plates. Perform FRAP assay, and monitor protein synthesis rate on day one of adulthood. The next day, prepare and use cycloheximide-containing NGM plates as a positive control.Kill bacteria by exposing the seeded NGM plates for 15 minutes with UV light. Add cycloheximide on the top of bacterial-seeded plates too 500 microgram per mL final concentration in the agar volume. Allow plates to dry.Transfer transgenic nematodes expressing GFP on vehicle and cycloheximide-containing plates. Incubate animals for two hours at the standard temperature of 20 degrees Celsius. Pick and transfer one-day adult transgenic animals to individual NGM plates seeded with a 20 microliters OP50 drop in the center.Remove the lid and place each plate under a 20H objective lens of an epifluorescent microscope. Focus and capture a reference image before photobleaching. Photobleach each sample for 10 minutes.Capture an image after photobleaching. Keep animals in individual NGM plates, and let them to recover. Image and record the recovery of each fluorescent reporter every one hour for at least six hours at an epifluorescent stereomicroscope.Prepare 2%agarose pads, and add 10 microliters drop of M9 buffer at the center of the agarose pad. Transfer five transgenic nematodes expressing pan-neuronal cytoplasmic GFP into a drop of M9 buffer. Use an eyelash to spread the liquid.Animals display reduced movements within two minutes because of M9 absorbance into the agar. Change nematode's position by using an eyelash pick. Place the sample at the 40H objective lens of an epifluorescent microscope without using a cover slip.Focus and capture a reference image. Photobleach a targeted area of interest for 90 seconds. Capture an image after photobleaching.Add a 10 microliters drop of M9 buffer on photobleached nematodes. Let the animals to recover for five minutes. Use the eyelash pick or a pipette to transfer the nematodes to individual NGM plates seeded with 20 microliters OP50 drop in the center.Capture an image of each sample every one hour under an epifluorescent stereomicroscope. Wild-type and fe-2 mutant worms expressing cytoplasmic GFP throughout their somatic tissues by using the fe-2 promoter were compared before, immediately after photobleaching, and five hours post-recovery. Wild-type animals fully recovered while fe-2 mutants had the minis recovery capacity.Thus wild type worms initiate the normal protein synthesis after photobleaching, while worms lacking mRNA translation initiation factor fe-2 are unable to do so, indicating that the rate of fluorescent recovery illustrates the rate of protein synthesis in vivo. Cycloheximide, a specific inhibitor of mRNA translation can be used as a positive control for protein translation inhibition. Indeed, cycloheximide-treated transgenic animals expressing cytoplasmic GFP under the promoter do not recover their fluorescence upon photobleaching.mRNA processing bodies affect the rate of protein translation. Wild-type and edc-3 mutant nematodes expressing GFP pan-neuronally were examined for their recovery capacity upon targeted photobleaching at the head region. Fluorescent recovery is much slower in edc-3-deficient animals compared to wild-type worms.Protein synthesis modulation is essential for organismal homeostasis. During aging, global as well as specific protein synthesis is perturbed. Protein translation balance directly controls senescence and aging.Specifically, core components of the translation machinery as well as P-body interacting components accelerate the aging process. Perturbation of translation initiation decelerates aging. Hence, measuring global protein synthesis rates by FRAP is a direct readout of the aging process.

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