Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Summary

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.

Abstract

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.

Introduction

Protein synthesis and degradation is essential for organismal homeostasis. A multitude of age-related diseases are instigated by defective protein production^1,2. 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 analysis^4,5. 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.

Protocol

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[p_ife-2GFP, pRF4], ife-2(ok306);Ex[p_ife-2GFP, pRF4], N2;Ex[p_unc-119GFP, pRF4], edc-3(ok1427);Ex[p_unc-119GFP, pRF4], N2;Ex[p_sod-3GFP; pRF4]

1. Maintenance, synchronization and preparation of transgenic nematodes for monitoring de novo protein synthesis

1. Use a dissecting stereomicroscope to assess the developmental stages and growth of wild type (wt) and mutant transgenic nematodes.
2. 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).
   1. 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).
   2. 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 °C in a shaking incubator (200 rpm; Table 1). Then, seed NGM plates with 200 μL of bacterial culture. Incubate the seeded plates at room temperature overnight to allow the growth of the bacterial lawn.
3. Incubate and grow the nematodes at the standard temperature of 20 °C.
4. 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.
5. Day 6: Perform FRAP assay and monitor protein synthesis rate on day 1 of adulthood.
6. 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 °C.
   1. Kill bacteria by exposing the seeded NGM plates for 15 minutes with UV light (222 μW/cm² intensity).
   2. Add cycloheximide on top of bacterial-seeded plates to 500 µg/mL final concentration in the agar volume.
   3. Allow plates to dry at room temperature for 30 minutes.
   4. Transfer transgenic nematodes expressing p_sod-3GFP on vehicle and cycloheximide-containing plates.
   5. Incubate animals for 2 h at the standard temperature of 20 °C.

2. FRAP assay using transgenic animals expressing somatic tissue reporters p_ife-2GFP and p_sod-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.

1. Pick and transfer 1-day-adult transgenic animals to individual NGM plates seeded with a 20 µL OP50 drop in the center.
2. Remove the lid and place each plate under a 20x objective lens of an epifluorescence microscope.
3. Focus and capture a reference image before photobleaching (pre-bleach).
4. Photobleach each sample for 10 minutes.
   NOTE: Stop the photobleaching when the fluorescent signal is quenched to 30 – 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.
5. Capture an image after photobleaching (bleach).
6. Keep animals in individual NGM plates and let them to recover.
7. Image and record the recovery of each fluorescent reporter every 1 hour for at least 6 hours at a fluorescence stereomicroscope.
8. Alternatively, perform fluorescent recovery assessment by using an epifluorescence microscope (e.g., AxioImager Z2).
9. 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.

3. Mounting the samples and perform FRAP assay using transgenic nematodes expressing pan-neuronally cytoplasmic GFP, p_unc-119GFP

NOTE: Monitor pan-neuronal protein synthesis by targeted photobleaching at the head region, where nerve ring is located.

1. Prepare 2% agarose pads.
2. Add a 10 µL drop of M9 buffer to the center of the agarose pad.
3. Transfer 5 transgenic nematodes expressing pan-neuronally cytoplasmic GFP into a drop of M9 buffer.
4. Use an eyelash to spread the liquid.
   NOTE: Animals display reduced movements within 2 minutes because of M9 absorbance into the agar.
5. Change nematode position to avoid overlapping by using an “eyelash” pick.
6. Place the sample under a 40x objective lens of an epifluorescence microscope.
   NOTE: Do not use a coverslip.
7. Focus and capture a reference image (pre-bleach).
8. Photobleach the targeted area of interest for 90 seconds.
   NOTE: Stop the photobleaching when the fluorescent signal is quenched to 30 – 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.
9. Capture an image after photobleaching (bleach).
10. Add a 10 µL drop of M9 buffer on photobleached nematodes.
11. Let the animals recover for 5 minutes.
12. Use the “eyelash” pick or a pipette to transfer the nematodes to individual NGM plates seeded with 20 µL OP50 drop in the center.
13. Capture an image of each sample every 1 hour under an epifluorescence stereomicroscope.
    NOTE: Count t=0 for the recovery time after photobleaching.

4. Data analysis of images capture during the FRAP assay

1. Analyze the acquired images using software (e.g., Fiji).
2. Open images with the software.
3. Select the Split Channels command via the Image and Color drop-down menu.
   NOTE: Keep the green channel image.
4. Use the Freehand Selection tool to manually set the fluorescent region of interest (e.g., whole body, head, intestine).
5. 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.

5. Report statistical analysis

1. Use at least 20 – 25 nematodes of each strain and/or condition.
   NOTE: Three biological replicates should be performed.
2. Perform student t-test (comparison between two groups) or ANOVA (comparison among multiple groups) for statistical analysis with P < 0.05 as significant using statistical analysis software.

Results

Using the procedure presented here, wild type and mutant transgenic nematodes expressing the following somatic reporters, p_ife-2GFP, p_sod-3GFP and p_unc-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...

Discussion

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...

Materials

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