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. 2009;4(2):e4547.
doi: 10.1371/journal.pone.0004547. Epub 2009 Feb 23.

Cell-specific monitoring of protein synthesis in vivo

Nikos Kourtis  1 , Nektarios Tavernarakis
Affiliations

Cell-specific monitoring of protein synthesis in vivo

Nikos Kourtis et al. PLoS One. 2009.

Abstract

Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Transgenic animals expressing pife-2GFP throughout somatic tissues, are subjected to a whole-animal photobleaching session for 8 min that reduces GFP fluorescence down to ∼10% of initial intensity (black line).
Transgenic animals carry the rol-6 (su1006) allele as a co-transformation marker. Fluorescence is measured before photobleaching (Pre-Bleach) as well as immediately following the photobleaching session (Bleach). Subsequent recovery of fluorescence is followed by measurement of average pixel intensity at one-hour time intervals. Error bars represent SEM (4 independent experiments, 10 animals in each experiment). Treatment of animals with the specific protein synthesis blocker cycloheximide (CHX) at 500 µg/ml final concentration, diminishes fluorescence recovery (grey line).
Figure 2
Figure 2. Regression analysis of fluorescence recovery in both wild type and IFE-2 deficient animals expressing pife-2GFP throughout somatic tissues.
Animals were photobleached and fluorescence recovery was followed as described in Materials and Methods. Best-fit lines are generated for average pixel intensity values obtained during the recovery phase for the indicated genetic backgrounds (A, wild type; B, ife-2 (ok306); black lines). The respective equations describing best-fit lines as well as R2 values for each line are also shown. Line slope corresponds to the first derivative of fluorescent change within a time unit (Δf/dt), which is a measure of the recovery rate. Cycloheximide treatment (CHX) results in negligible recovery rate (grey lines).
Figure 3
Figure 3. Representative images of roller, transgenic animals expressing pife-2GFP throughout somatic tissues, before photobleaching, immediately following an 8 min whole-animal photobleaching session, and after a 5 h recovery period, in the absence (A) and presence (B) of cycloheximide at 500 µg/ml final concentration.
Figure 4
Figure 4. Regression analysis of fluorescence recovery in cell and tissue-specific level.
(A) In wild type animals expressing pmec-4GFP in six specific neurons (the touch receptor neurons). Best-fit lines are generated for average pixel intensity values obtained during the recovery phase. Cycloheximide treatment (CHX) results in negligible recovery rate (compare black line: −CHX vs. grey line: +CHX). The respective equations describing best-fit lines as well as R2 values for each line are shown. Line slope corresponds to the first derivative of fluorescent change within a time unit (Δf/dt), which is a measure of the recovery rate. (B) In wild type animals expressing pmyo-2GFP specifically in the pharyngeal muscles. Best-fit lines are generated for average pixel intensity values obtained during the recovery phase. Cycloheximide treatment (CHX) blocks recovery (compare black line: −CHX vs. grey line: +CHX). The respective equations describing best-fit lines as well as R2 values for each line are shown.
Figure 5
Figure 5. Representative images of transgenic animals before photobleaching, immediately following a 10 min whole-animal photobleaching session, and after a 5 h recovery period, in the absence (−CHX) and presence (+CHX) of cycloheximide.
(A) Animals expressing pmec-4GFP specifically in the six touch receptor neurons. (B) Animals expressing pmyo-2GFP specifically in pharyngeal muscle cells.
See this image and copyright information in PMC

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