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-2GFP, pRF4], N2;Ex[punc-119GFP, pRF4], edc-3(ok1427);Ex[punc-119GFP, pRF4], N2;Ex[psod-3GFP; pRF4]
1. Maintenance, synchronization and preparation of transgenic nematodes for monitoring de novo protein synthesis
<ol>
	<li>Use a dissecting stereomicroscope to assess the developmental stages and growth of wild type (wt) and mutant transgenic nematodes.</li>
	<li>Day 1: Use a worm pick to select and transfer 10 L4 larvae of wt and mutant transgenic animals carrying the desired fluorescent reporter onto Nematode Growth Media (NGM) plates seeded with Escherichia coli (OP50) (Table 1).
	<ol>
		<li>Make a worm pick by attaching a platinum wire into the tapered end of a glass Pasteur pipette by melting the glass at the contact site on a Bunsen burner. Then, flatten the end of the platinum wire into a spatula shape by using any tool (e.g., pincer or light hammer).</li>
		<li>Inoculate a single colony of E. coli (OP50) bacteria into a flask containing 50 mL of Luria-Bertani (LB) liquid medium and grow for 10 hours at 37 &#176;C in a shaking incubator (200 rpm; Table 1). Then, seed NGM plates with 200 &#956;L of bacterial culture. Incubate the seeded plates at room temperature overnight to allow the growth of the bacterial lawn.</li>
	</ol>
	</li>
	<li>Incubate and grow the nematodes at the standard temperature of 20 &#176;C.</li>
	<li>Day 5: The plates contain a mixed population of transgenic worms. Select and transfer 15 L4 larvae of each strain on freshly seeded OP50-NGM plates.</li>
	<li>Day 6: Perform FRAP assay and monitor protein synthesis rate on day 1 of adulthood.</li>
	<li>Prepare and use cycloheximide containing NGM plates as a positive control: 
	NOTE: Prepare a stock solution of cycloheximide by diluting in water to a concentration of 10 mg/mL. Keep stock solution at 4 &#176;C.
	<ol>
		<li>Kill bacteria by exposing the seeded NGM plates for 15 minutes with UV light (222 &#956;W/cm2 intensity).</li>
		<li>Add cycloheximide on top of bacterial-seeded plates to 500 &#181;g/mL final concentration in the agar volume.</li>
		<li>Allow plates to dry at room temperature for 30 minutes.</li>
		<li>Transfer transgenic nematodes expressing psod-3GFP on vehicle and cycloheximide-containing plates.</li>
		<li>Incubate animals for 2 h at the standard temperature of 20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. FRAP assay using transgenic animals expressing somatic tissue reporters pife-2GFP and psod-3GFP
NOTE: Monitor global protein synthesis rate in the whole animal body. These transcriptional reporters present different expression levels and are ubiquitously expressed in multiple somatic tissues, including intestine, pharynx and body wall muscles.
<ol>
	<li>Pick and transfer 1-day-adult transgenic animals to individual NGM plates seeded with a 20 &#181;L OP50 drop in the center.</li>
	<li>Remove the lid and place each plate under a 20x objective lens of an epifluorescence microscope.</li>
	<li>Focus and capture a reference image before photobleaching (pre-bleach).</li>
	<li>Photobleach each sample for 10 minutes. 
	NOTE: Stop the photobleaching when the fluorescent signal is quenched to 30 &#8211; 50% intensity relative to pre-bleaching image. The light intensity and the duration of the bleaching period should be adjusted accordingly for the specific fluorophore, animal stage and cell or tissue under examination. The appropriate duration of irradiation required to reach an adequate extent of photobleaching, for different specimens should be experimentally determined.</li>
	<li>Capture an image after photobleaching (bleach).</li>
	<li>Keep animals in individual NGM plates and let them to recover.</li>
	<li>Image and record the recovery of each fluorescent reporter every 1 hour for at least 6 hours at a fluorescence stereomicroscope.</li>
	<li>Alternatively, perform fluorescent recovery assessment by using an epifluorescence microscope (e.g., AxioImager Z2).</li>
	<li>Carefully evaluate the animal healthspan per animal by monitoring their locomotion, touch sensitivity, reproductive capacity and survival over the next 3 days. Nematodes showing signs of damage after photobleaching were excluded from further analysis.</li>
</ol>
3. Mounting the samples and perform FRAP assay using transgenic nematodes expressing pan-neuronally cytoplasmic GFP, punc-119GFP
NOTE: Monitor pan-neuronal protein synthesis by targeted photobleaching at the head region, where nerve ring is located.
<ol>
	<li>Prepare 2% agarose pads.</li>
	<li>Add a 10 &#181;L drop of M9 buffer to the center of the agarose pad.</li>
	<li>Transfer 5 transgenic nematodes expressing pan-neuronally cytoplasmic GFP into a drop of M9 buffer.</li>
	<li>Use an eyelash to spread the liquid. 
	NOTE: Animals display reduced movements within 2 minutes because of M9 absorbance into the agar.</li>
	<li>Change nematode position to avoid overlapping by using an &#8220;eyelash&#8221; pick.</li>
	<li>Place the sample under a 40x objective lens of an epifluorescence microscope. 
	NOTE: Do not use a coverslip.</li>
	<li>Focus and capture a reference image (pre-bleach).</li>
	<li>Photobleach the targeted area of interest for 90 seconds. 
	NOTE: Stop the photobleaching when the fluorescent signal is quenched to 30 &#8211; 50% intensity relative to pre-bleaching image. The light intensity and the duration of the bleaching period should be adjusted accordingly for the specific fluorophore, animal stage and cell or tissue under examination. The appropriate duration of irradiation required to reach an adequate extent of photobleaching, for different specimens should be experimentally determined.</li>
	<li>Capture an image after photobleaching (bleach).</li>
	<li>Add a 10 &#181;L drop of M9 buffer on photobleached nematodes.</li>
	<li>Let the animals recover for 5 minutes.</li>
	<li>Use the &#8220;eyelash&#8221; pick or a pipette to transfer the nematodes to individual NGM plates seeded with 20 &#181;L OP50 drop in the center.</li>
	<li>Capture an image of each sample every 1 hour under an epifluorescence stereomicroscope. 
	NOTE: Count t=0 for the recovery time after photobleaching.</li>
</ol>
4. Data analysis of images capture during the FRAP assay
<ol>
	<li>Analyze the acquired images using software (e.g., Fiji).</li>
	<li>Open images with the software.</li>
	<li>Select the Split Channels command via the Image and Color drop-down menu. 
	NOTE: Keep the green channel image.</li>
	<li>Use the Freehand Selection tool to manually set the fluorescent region of interest (e.g., whole body, head, intestine).</li>
	<li>Select the Measurement command via the Analyze drop-down menu to measure mean pixel intensity of the area of interest for each time point. 
	NOTE: The percentage of fluorescence recovery is calculated by using the reference images captured after photobleaching.</li>
</ol>
5. Report statistical analysis
<ol>
	<li>Use at least 20 &#8211; 25 nematodes of each strain and/or condition. 
	NOTE: Three biological replicates should be performed.</li>
	<li>Perform student t-test (comparison between two groups) or ANOVA (comparison among multiple groups) for statistical analysis with P &#60; 0.05 as significant using statistical analysis software.</li>
</ol>

<ol>
	<li>Syntichaki, P., Troulinaki, K., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=eIF4E+function+in+somatic+cells+modulates+ageing+in+Caenorhabditis+elegans.">eIF4E function in somatic cells modulates ageing in Caenorhabditis elegans.</a> Nature. 445 (7130), 922-926 (2007).</li><li>Charmpilas, N., Daskalaki, I., Papandreou, M. E., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+synthesis+as+an+integral+quality+control+mechanism+during+ageing.">Protein synthesis as an integral quality control mechanism during ageing.</a> Ageing Research Reviews. 23, 75-89 (2015).</li><li>Li, G. W., Burkhardt, D., Gross, C., Weissman, J. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Growing+Toolbox+for+Protein+Synthesis+Studies.">The Growing Toolbox for Protein Synthesis Studies.</a> Trends in Biochemical Sciences. 42 (8), 612-624 (2017).</li><li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+Window+into+Biology:+A+Primer+on+Caenorhabditis+elegans.">A Transparent Window into Biology: A Primer on Caenorhabditis elegans.</a> Genetics. 200 (2), 387-407 (2015).</li><li>Kourtis, N., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-specific+monitoring+of+protein+synthesis+in+vivo.">Cell-specific monitoring of protein synthesis in vivo.</a> PLoS One. 4 (2), 4547 (2009).</li><li>Parker, R., Sheth, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P+bodies+and+the+control+of+mRNA+translation+and+degradation.">P bodies and the control of mRNA translation and degradation.</a> Molecular Cell. 25 (5), 635-646 (2007).</li><li>Rieckher, M., Markaki, M., Princz, A., Schumacher, B., Tavernarakis, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+Proteostasis+by+P+Body-Mediated+Regulation+of+eIF4E+Availability+during+Aging+in+Caenorhabditis+elegans.">Maintenance of Proteostasis by P Body-Mediated Regulation of eIF4E Availability during Aging in Caenorhabditis elegans.</a> Cell Reports. 25 (1), 199-211 (2018).</li><li>Reits, E. A., Neefjes, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+fixed+to+FRAP:+measuring+protein+mobility+and+activity+in+living+cells.">From fixed to FRAP: measuring protein mobility and activity in living cells.</a> Nature Cell Biology. 3 (6), 145-147 (2001).</li><li>Keith, S. A., 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=Graded+Proteasome+Dysfunction+in+Caenorhabditis+elegans+Activates+an+Adaptive+Response+Involving+the+Conserved+SKN-1+and+ELT-2+Transcription+Factors+and+the+Autophagy-Lysosome+Pathway.">Graded Proteasome Dysfunction in Caenorhabditis elegans Activates an Adaptive Response Involving the Conserved SKN-1 and ELT-2 Transcription Factors and the Autophagy-Lysosome Pathway.</a> PLoS Genetics. 12 (2), 1005823 (2016).</li><li>Kumsta, C., 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=The+autophagy+receptor+p62/SQST-1+promotes+proteostasis+and+longevity+in+C.+elegans+by+inducing+autophagy.">The autophagy receptor p62/SQST-1 promotes proteostasis and longevity in C. elegans by inducing autophagy.</a> Nature Communications. 10 (1), 5648 (2019).</li><li>Gregersen, N., Bolund, L., Bross, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+aggregation,+and+degradation+in+disease.">Protein misfolding, aggregation, and degradation in disease.</a> Molecular Biotechnology. 31 (2), 141-150 (2005).</li></ol>

The authors declare no competing interests.

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, induces oxidative stress and accelerates the aging process1. Moreover, neuron-specific EDC-3, which interacts with mRNA processing bodies, P bodies and increases the rate of mRNA decapping, also accelerates the aging process8. Specifically, EDC-3 suppression allows for IFE-2 sequestration into P bodies, ultimately reducing protein translation levels, delaying neuronal aging9.
FRAP is a technique initially developed to investigate protein dynamics, localization, interactions and activity with non-invasive fluorescent tagging using live imaging in vivo, making it a powerful tool to study protein synthesis rates in real time8,10. However, there are certain critical steps that require attention and troubleshooting. We provide alternative solutions to these potential obstacles or limitations encountered during the experimental procedure.
One issue concerns worm movement as the worm can escape the light source during photobleaching. This issue can be partially overcome by using transgenic animal expressing the rol-6(su1006) allele, which causes animals to move in a sinusoidal manner. This way, animal photobleaching can be performed continuously. Moreover, the experimenter can follow the movement of the worm that moves outside the plane of view much slower. Nevertheless, agar pads can also be used as an alternative to fully immobilize animals.
The optimal duration of photobleaching is an additional critical factor that should be determined and kept stable throughout the experiments. Insufficient photobleaching due to untargeted photobleaching or short duration can be encountered. This can be surpassed by increasing (a) the magnification objective and numerical aperture, (b) the light intensity and/or (c) photobleaching duration.
If no significant fluorescence recovery is detected, there are potential parameters that can be altered. If photobleaching is excessive, reduce photobleaching duration as specimen damage has occurred. Damage to the worm can be assessed by observing life traits such as touch sensitivity, locomotion, egg laying, pharyngeal pumping and reproductive capacity. If kinetics of protein translation recovery are slow, allow longer recovery period. Protein synthesis recovery varies between different protein families and depends on the reporter fusions length7.
Promoter activity could influence kinetics of protein synthesis. If the promoter is inactive during recovery, change the promoter used. Promoter activity might be altered upon stress conditions, such as photodamage. Use stable activity promoters to monitor either global (e.g. let-858, ife-2, sod-3) or tissue-specific (e.g. body wall muscles; myo-3, unc-54; pharynx: myo-2; pan-neuronal: unc-119, rab-3; mechanosensory neurons: mec-4; intestine: vha-6, ges-1) protein translation rates.
If inadequate protein translation inhibition by cycloheximide occurs, increasing pre-incubation time of the nematodes would overcome this problem. A caveat that should be taken into consideration is the use of transgenic worms overexpressing proteins. Using CRISPR/Cas9-generated transgenic nematodes expressing the fluorescent tagged proteins of interest at physiological levels will provide a more accurate picture of protein kinetics and turnover.
Total protein levels detected are the result of their protein degradation and synthesis rates. Protein degradation invariably occurs at basal levels in all eukaryotic cells. Thus, protein fluorescence recovery rates, which are measured in this protocol, are not absolute but relative to the conditions being compared. In essence, the fluorescence recovery in the presence of protein degradation is measured. Therefore, it is assumed that all conditions have equal protein degradation rates after photobleaching. In order to measure absolute protein translation rates, protein degradation-deficient mutant worm strains should be used. Specifically, mutations in genes such as rpn-10 of the proteasome or lgg-2 for autophagy can be used as a control11. Alternatively, RNAi knockdown of proteasomal and autophagic proteins can be performed. Additionally, autophagic substrates such as SQST-1::GFP can be used to detect the contribution of autophagic degradation under the experimental conditions selected12.
In both experimental settings, this FRAP technique is non-radioactive, non-invasive, which detects de novo protein synthesis rates in vivo in different tissues in C. elegans. It allows for live imaging before and during photobleaching in an in vivo setting and subsequent monitoring of the animals for their whole lifetime. Perturbation of translation initiation decelerates aging, hence measuring global protein synthesis rates by FRAP could be used as a direct read-out of the aging process. This protocol can be optimized and used on a larger scale to screen genes and/or chemicals related to the aging process or age-related disease. Alternatively, total or specific protein degradation rates can be measured by photobleaching under different genetic backgrounds using the same protocol in combination with cycloheximide. As protein translation is inhibited by cycloheximide, the rate of protein degradation will be detected. This protocol will aid in better understanding protein degradation pathways in genetic models of protein aggregation diseases13.

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 (CaCl&lt;sub&gt;2&lt;/sub&gt;?2H&lt;sub&gt;2&lt;/sub&gt;O)</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>&lt;em&gt;edc-3(ok1427);Ex&lt;/em&gt;[p&lt;sub&gt;&lt;em&gt;unc-119&lt;/em&gt;&lt;/sub&gt;GFP, pRF4]</td><td>Tavernarakis lab</td><td></td><td>Maintain animals at 20 &amp;deg;C</td></tr><tr><td>Epifluorescence microscope</td><td>Zeiss</td><td></td><td>Axio Imager Z2</td></tr><tr><td>&lt;em&gt;Escherichia coli&lt;/em&gt; 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>&lt;em&gt;ife-2(ok306);Ex&lt;/em&gt;[p&lt;sub&gt;&lt;em&gt;ife-2&lt;/em&gt;&lt;/sub&gt;GFP, pRF4]</td><td>Tavernarakis lab</td><td></td><td>Maintain animals at 20 &amp;deg;C</td></tr><tr><td>image analysis software</td><td>Fiji</td><td></td><td>https://fiji.sc</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>EMD Millipore</td><td>1,37,010</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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 (MgSO&lt;sub&gt;4&lt;/sub&gt;)</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>&lt;em&gt;N2;Ex&lt;/em&gt;[p&lt;sub&gt;&lt;em&gt;ife-2&lt;/em&gt;&lt;/sub&gt;GFP; pRF4]</td><td>Tavernarakis lab</td><td></td><td>Maintain animals at 20 &amp;deg;C</td></tr><tr><td>&lt;em&gt;N2;Ex&lt;/em&gt;[p&lt;sub&gt;&lt;em&gt;sod-3&lt;/em&gt;&lt;/sub&gt;GFP; pRF4]</td><td>Tavernarakis lab</td><td></td><td>Maintain animals at 20 &amp;deg;C</td></tr><tr><td>&lt;em&gt;N2;Ex&lt;/em&gt;[p&lt;sub&gt;&lt;em&gt;unc-119&lt;/em&gt;&lt;/sub&gt;GFP; pRF4]</td><td>Tavernarakis lab</td><td></td><td>Maintain animals at 20 &amp;deg;C</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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, immediately after photobleaching and 5 hours post-recovery. Representative images and quantification show that wild type animals fully recover while ife-2 mutants have diminished recovery capacity (Figure 1A). Thus, wild type worms initiate de novo protein biosynthesis after photobleaching while worms lacking mRNA translation initiation factor IFE-2 are unable to do so, indicating that the rate of fluorescence recovery illustrates the rate of protein synthesis in vivo7. Moreover, 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 sod-3 promoter, do not recover their fluorescence upon photobleaching (Figure 1B).
We also applied this method to detect how mRNA processing bodies (P bodies) affect the rate of protein translation8. Wild type and edc-3(ok1427) mutant nematodes expressing GFP pan-neuronally were examined for their recovery capacity upon targeted photobleaching at the head region. Fluorescence recovery is much slower in EDC-3 deficient animals compared to wild type (Figure 1C). This indicates that EDC-3-mediated mRNA turnover hinders novel protein synthesis and ultimately suppresses protein translation initiation9.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61170/61170fig01.jpg" /> 
Figure 1: In vivo assessment of de novo protein synthesis in C. elegans.&#160;(A) FRAP analysis in both wild type and IFE-2 deficient nematodes expressing cytoplasmic GFP under the promoter of ife-2. Transgenic animals are subjected to whole-animal photobleaching. The GFP signal is reduced to 30-50% of initial intensity. Fluorescence is recorded before photobleaching (pre-bleach), following the end of photobleaching period (10 min; bleach) and 5 hours post recovery. (B) Cycloheximide treatment inhibits fluorescence recovery of transgenic animals expressing cytoplasmic GFP under the promoter of sod-3. Transgenic animals are subjected to whole animal photobleaching. The GFP signal is reduced to 30-50% of initial intensity. Fluorescence is recorded before photobleaching (pre-bleach), following the end of photobleaching period (10 min; bleach) and 5 hours post recovery (A, B: AxioImager Z2 microscope; objective lens: 20x, numerical aperture 0.8; high-power light source, HBO 100; 100 Watt mercury arc lamp; excitation/ emission filter sets, Zeiss Set 38 Endow GFP shift free). Scale bars, 500&#956;m. (C) EDC-3 deficient nematodes display decreased de novo protein synthesis rates in neuronal cells. Fluorescence signal of both wild type and edc-3(ok1427) animals expressing pan-neuronally cytoplasmic GFP is measured before photobleaching (pre-bleach) as well as following the photobleaching period (90sec; bleach). Transgenic animals are subjected to photobleaching at head region (nerve ring). The GFP signal is reduced to 30-50% of initial intensity (AxioImager Z2; objective lens: 40x, numerical aperture 0.75; 30% luminescent power of the fluorescent illumination source (FI illumination System X-Cite 120 XL FL PC, 120W metal halogenide lamp) and fully opened iris). Average pixel intensity of fluorescence recovery is measured at one hour time intervals. 20 animals were quantified per strain in each of three independent experiments. Data represent mean S.E.M., ***P &#60; 0.05, unpaired t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61170/61170fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td colspan="2">Recipe</td>
		</tr>
		<tr>
			<td rowspan="9">2% Agarose pads</td>
			<td colspan="2">1.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Weigh 0.5 g o fagarose in a cylindrical glass beaker</td>
		</tr>
		<tr>
			<td colspan="2">2.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Add 25 mL of M9 buffer</td>
		</tr>
		<tr>
			<td colspan="2">3.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Heat in a microwave until close to boiling. Take out, stir with a pipette tip and boil again. Repeat until the agarose is dissolved.</td>
		</tr>
		<tr>
			<td colspan="2">4.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Place an empty microscope slide on the bench.</td>
		</tr>
		<tr>
			<td colspan="2">5.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Put a drop (~ 50 &#181;L) of fresh 2% agarose solution in the middle of the slide.</td>
		</tr>
		<tr>
			<td colspan="2">6.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Take a second microscope slide and place it on top of the agarose drop. Gently press down to flatten the drop.</td>
		</tr>
		<tr>
			<td colspan="2">7.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Let the agarose harden for 30 seconds and remove gently the top microscope slide.</td>
		</tr>
		<tr>
			<td colspan="2">8.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Immediately proceed with the sample preparation, since the agarose pads will start drying within approximately 5 minutes.</td>
		</tr>
		<tr>
			<td colspan="2">Tip: Leave the top microscope slide as a cover to preserve the humidity longer (~ 1 hour). Thus, several agarose pads can be prepared and used swiftly during the experiments.</td>
		</tr>
		<tr>
			<td>LB liquid medium</td>
			<td colspan="2">1.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 10 g of Bactotryptone, 5 g of Bacto Yeast Extract, 5 g of NaCl in 1 L distilled water and autoclave.</td>
		</tr>
		<tr>
			<td rowspan="3">M9 buffer</td>
			<td colspan="2">1.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Dissolve 3 g of KH2PO4, 6 g of Na2HPO4, and 5 g NaCl in 1 L distilled water and autoclave.</td>
		</tr>
		<tr>
			<td colspan="2">2.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Let cool and add 1 mL of 1 M MgSO4 (sterile).</td>
		</tr>
		<tr>
			<td colspan="2">3.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Store M9 buffer at 4 &#176;C.</td>
		</tr>
		<tr>
			<td rowspan="5">Nematode growth medium (NGM) agar plates</td>
			<td colspan="2">1.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Mix 3 g of NaCl, 2.5 g of bactopeptone, 0.2 g of streptomycin, 17 g of agar and add 900 mL of distilled water. Autoclave.</td>
		</tr>
		<tr>
			<td colspan="2">2.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Let cool to 55-60 &#176;C.</td>
		</tr>
		<tr>
			<td colspan="2">3.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Add 1 mL of cholesterol stock solution, 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 1 mL of nystatin stock solution, 25 mL of sterile 1 M phosphate buffer, pH 6.0, and distilled sterile water up to 1 L.</td>
		</tr>
		<tr>
			<td colspan="2">4.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Pipette 10 mL of medium per Petri dish and leave to solidify.</td>
		</tr>
		<tr>
			<td colspan="2">5.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Store the plates at 4 &#176;C until used.</td>
		</tr>
	</tbody>
</table>
Table 1: Recommended recipes for reagents used. All the reagents recipes used in the presented protocol are outlined here.

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 ...

assessment-de-novo-protein-synthesis-rates-caenorhabditis

Nanyang Technological University

Research

JoVE Journal

Biology

5.2K Views.  Foundation for Research and Technology-Hellas, Greece. De snelheid van eiwitsynthese wordt verstoord tijdens ziekte en veroudering. Fluorescentie herstel na fotobleaching, of FRAP, maakt het mogelijk de snelheid van eiwitsynthese in vivo. We gebruiken transparante C.elegans wormen, die GFP uitdrukken, als een model om de nieuwe eiwitsynthese te monitoren na het fotoblelen.We bieden praktische richtlijnen om fluorescentieherstel te meten met behulp van transgene wormen, die GFP uitdrukken onder verschillende promotors, hetzij op grote schaa...

Video: Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Genetics

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many historical and contemporary studies have identified thousands of lifespan-altering paradigms, including genetic mutations, transgenic gene expression, and hormesis, a beneficial, low-grade exposure to stress. With its many advantages, including a short lifespan, easy and low-cost maintenance, and fully sequenced genome with homology to almost two-thirds of all human genes, C. elegans has quickly been adopted as an outstanding model for stress and aging biology. Here, several standardized methods are surveyed for measuring lifespan and healthspan that can be easily adapted into almost any research environment, especially those with limited equipment and funds. The incredible utility of C. elegans is featured, highlighting the capacity to perform powerful genetic analyses in aging biology without the necessity of extensive infrastructure. Finally, the limitations of each analysis and alternative approaches are discussed for consideration.

Surveying Low-Cost Methods to Measure Lifespan and Healthspan in Caenorhabditis elegans

Caenorhabditis elegans serve as an excellent model system with robust and low-cost methods for surveying healthspan, lifespan, and resilience to stress.

The discovery and development of Caenorhabditis elegans as a model organism was influential in biology, particularly in the field of aging. Many ...

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis elegans is a well-established model organism that has been widely used in the field to study protein aggregation and toxicity. Its optical transparency enables the direct visualization of protein aggregation by fluorescence microscopy. Moreover, the fast reproductive cycle and short lifespan make the nematode a suitable model to screen for genes and molecules that modulate this process.
However, the quantification of aggregate load in living animals is poorly standardized, typically performed by manual inclusion counting under a fluorescence dissection microscope at a single time point. This approach can result in high variability between observers and limits the understanding of the aggregation process. In contrast, amyloid-like protein aggregation in vitro is routinely monitored by thioflavin T fluorescence in a highly quantitative and time-resolved fashion.
Here, an analogous method is presented for the unbiased analysis of aggregation kinetics in living C. elegans, using a high-throughput confocal microscope combined with custom-made image analysis and data fitting. The applicability of this method is demonstrated by monitoring inclusion formation of a fluorescently labeled polyglutamine (polyQ) protein in the body wall muscle cells. The image analysis workflow allows the determination of the numbers of inclusions at different timepoints, which are fitted to a mathematical model based on independent nucleation events in individual muscle cells. The method described here may prove useful to assess the effects of proteostasis factors and potential therapeutics for protein aggregation diseases in a living animal in a robust and quantitative manner.

Monitoring Protein Aggregation Kinetics In Vivo using Automated Inclusion Counting in Caenorhabditis elegans

Here, a method is presented for the analysis of protein aggregation kinetics in the nematode Caenorhabditis elegans. Animals from an age-synchronized population are imaged at different time points, followed by semiautomated inclusion counting in CellProfiler and fitting to a mathematical model in AmyloFit.

Protein aggregation into insoluble inclusions is a hallmark of a variety of human diseases, many of which are age-related. The nematode Caenorhabditis ...

Biochemistry

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate&#160;C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.

Protein Extract Preparation and Co-immunoprecipitation from Caenorhabditis elegans

This method describes a protocol for high-throughput protein extract preparation from Caenorhabditis elegans samples and subsequent co-immunoprecipitation.

Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or ...

Developmental Biology

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture a full picture of the multiple levels of molecular processing and changes between different conditions. This protocol presents the method of isolating DNA, RNA, and protein from the same sample of the nematode Caenorhabditis elegans to remove the variation introduced when these biomolecules are isolated from replicates of similarly treated but ultimately different samples. Nucleic acids and protein are extracted from the nematode using the acid guanidinium thiocyanate-phenol-chloroform extraction method, with subsequent precipitation, washing, and solubilization of each. We show the successful isolation of RNA, DNA, and protein from a single sample from three strains of nematode and HeLa cells, with better protein isolation results in adult animals. Additionally, guanidinium thiocyanate-phenol-chloroform-extracted protein from nematodes improves the resolution of larger proteins, with enhanced detectable levels as observed by immunoblotting, compared to the traditional RIPA extraction of protein.
The method presented here is useful when investigating samples using a multiomic approach, specifically for the exploration of the proteome and transcriptome. Techniques that simultaneously assess multiomics are appealing because molecular signaling underlying complex biological phenomena is thought to occur at complementary levels; however, it has become increasingly common to see that changes in mRNA levels do not always reflect the same change in protein levels and that the time of collection is relevant in the context of circadian regulations. This method removes any intersample variation when assaying different contents within the same sample (intrasample.)

Combined Nucleotide and Protein Extractions in Caenorhabditis elegans

Here, we present a protocol for the isolation of RNA, DNA, and protein from the same sample, in an effort to reduce variation, improve reproducibility, and facilitate interpretations.

A single biological sample holds a plethora of information, and it is now common practice to simultaneously investigate several macromolecules to capture ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble proteins over 40% of the time results in an extension of the lifespan in C. elegans, suggesting that many of these proteins are key determinants of the aging process. Isolation and quantitative identification of these insoluble proteins are crucial to understand key biological processes that occur during aging. Here, we present a modified and improved protocol that details how to extract and isolate the SDS-insoluble proteins (insolublome) from C. elegans more efficiently to streamline mass spectrometric workflows via a novel label-free quantitative proteomics analysis. This improved protocol utilizes a highly efficient sonicator for worm lysis that greatly increases efficiency for protein extraction and allows us to use significantly less starting material (approximately 3,000 worms) than in previous protocols (typically using at least 40,000 worms). Subsequent quantitative proteomic analysis of the insolublome was performed using data-dependent acquisition (DDA) for protein discovery and identification and data-independent acquisition (DIA) for comprehensive and more accurate protein quantification. Bioinformatic analysis of quantified proteins provides potential candidates that can be easily followed up with other molecular methods in C. elegans. With this workflow, we routinely identify more than 1000 proteins and quantify more than 500 proteins. This new protocol enables efficient compound screening with C. elegans. Here, we validated and applied this improved protocol to wild-type C. elegans N2-Bristol strain and confirmed that aged day-10 N2 worms showed greater accumulation of the insolublome than day-2 young worms.

Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

This novel workflow efficiently extracts and isolates SDS-insoluble proteins (insolublome) from Caenorhabditis elegans with minimal starting material for quantitative differential proteomic analysis. The protocol uses a comprehensive data-independent acquisition mass spectrometry analysis to quantify the insolublome and bioinformatic analysis to gain biological insights into aging mechanisms and pathologies.

We and others have shown that the aging process results in a proteome-wide accumulation of insoluble proteins. Knocking down genes encoding the insoluble ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Samenvatting

Meer video's verkennen