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 disadvantage....

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....

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

List of materials used in this article

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