In vivo assay and modelling of protein and mitochondrial turnover during aging

Figures & data

Figure 1. Overview of methods for calculating protein half-life in cells and organisms

Common to each approach is marking a pool of protein to enable assay of decay rate. Half-life is calculated by fitting the data to the equation for exponential decay. In each plot, time is indicated on the X-axis and protein abundance on the Y-axis, in arbitrary units. (A) Cycloheximide block of protein synthesis. (B) Heavy-isotope labelling (continuous). (C) Photobleach of fluorescent protein. (D) Green-to-red photoconversion. (E) SNAP-tag labelling. (F) Conditional gene expression. In this example, conditional gene expression is activated by a drug, administered for a brief time interval as indicated below the plot. The red curve indicates the rate of production of the protein, and the blue curve indicates the abundance of the protein.

Figure 2. Fluorescent protein synthesis and turnover in free-moving adult Drosophila. (a) Neuronal expression of eGFP. The tissue-general Actin-Gene-Switch[255B] driver strain [87] was crossed to the multi-copy UAS-eGFP strain (‘ultra-GFP’ [29]), and six male progenies were placed in a glass vial with media supplemented with 160 μg/ml mifepristone (RU486). GFP fluorescence was quantified continuously using video and FluoreScore software, and is expressed in relative units (figure adapted from [111]). Light green indicates average GFP intensity per minute and dark green is the LOESS curve. (b) Decay rate of GFP targeted to the mitochondrial matrix. mitoGFP is a mitochondria-targeted form of eGFP created by fusing the human COX VIII mitochondrial targeting signal to the N terminus of eGFP [113]. In a proof-of-principle experiment, the tissue-general Actin-Gene-Switch[255B] driver strain was crossed to the UAS–mitoGFP[AP3] strain [113], and male progeny (1–5 days of age) were placed in vials with media supplemented with 160 μg/ml mifepristone (RU486) or on control media. After 48 hours, all flies were transferred to glass culture vials with control media, at 6 flies per vial, using three replicate vials for drug treated flies and three replicate vials for controls (time 0). The flies were then assayed for GFP fluorescence at the indicated time points using 4 minute videos and FluoreScore software, and the average and SD of the three vials is plotted for each of (-) drug and (+) drug groups. The data suggests an in vivo half-life for mitoGFP of ~3 days

Ford D, Hoe N, Landis GN, et al. Alteration of Drosophila life span using conditional, tissue-specific expression of transgenes triggered by doxycyline or RU486/Mifepristone. Exp Gerontol. 2007;42(6):483–497.

 PubMed Web of Science ®Google Scholar

Yang J, Tower J. Expression of hsp22 and hsp70 transgenes is partially predictive of Drosophila survival under normal and stress conditions. J Gerontol A Biol Sci Med Sci. 2009;20(8):828–838.

 Google Scholar

Ardekani R, Huang YM, Sancheti P, et al. Using GFP video to track 3D movement and conditional gene expression in free-moving flies. PLoS One. 2012;7(7):e40506.

 PubMed Web of Science ®Google Scholar

Cox RT, Spradling AC. A Balbiani body and the fusome mediate mitochondrial inheritance during Drosophila oogenesis. Development. 2003;130(8):1579–1590.

 PubMed Web of Science ®Google Scholar

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.