Article

Tandem Fluorescent Protein Timers for In Vitro Analysis of Protein Dynamics

Center for Molecular Biology of the University of Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
Nature Biotechnology (Impact Factor: 41.51). 06/2012; 30(7):708-14. DOI: 10.1038/nbt.2281
Source: PubMed

ABSTRACT

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

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Available from: Balca R Mardin, Aug 06, 2014
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    • "To investigate tandem timer behaviour we used an ordinary differential equation model that models the production, degradation and fluorophore maturation kinetics of a pool of proteins tagged with the timer (Khmelinskii et al., 2012). We denote the Development • Advance article faster maturing fluorescent protein as FP1 (maturation rate m 1 , maturation time t 1 = log(2)/m 1 ) and the slower maturing fluorescent protein as FP2 (maturation rate m 2 , maturation time t 2 = log(2)/m 2 ). "
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    ABSTRACT: Studies on signalling dynamics in living embryos have been limited by a scarcity of in vivo reporters. Tandem fluorescent protein timers provide a generic method for detecting changes in protein population age and thus provide readouts for signalling events that lead to changes in protein stability or location. When imaged with quantitative dual-colour fluorescence microscopy, tandem timers offer detailed 'snapshot' readouts of signalling activity from subcellular to organismal scales, and therefore have the potential to revolutionize studies in developing embryos. Here we use computer modelling and embryo experiments to explore the behaviour of tandem timers in developing systems. We present a mathematical model of timer kinetics and provide software tools that will allow experimentalists to select the most appropriate timer designs for their biological question, and guide interpretation of the obtained readouts. Through the generation of a series of novel zebrafish reporter lines, we confirm experimentally that our quantitative model can accurately predict different timer responses in developing embryos and explain some less expected findings. For example, increasing the FRET efficiency of a tandem timer actually increases the ability of the timer to detect differences in protein half-life. Finally, while previous studies have used timers to monitor changes in protein turnover, our model shows that timers can also be used to facilitate the monitoring of gene expression kinetics in vivo.
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    • "As a first step in testing our hypothesis, we analyzed Pma1 protein localization. There are conflicting reports on Pma1 asymmetry (Smardon et al., 2013; Khmelinskii et al., 2012; Malínská et al., 2003), however we found that Pma1 was asymmetric between mother and daughter cells. Pma1 levels at the plasma membrane were higher in mother cells than daughter cells as indicated by indirect immunofluorescence with antibody to Pma1 (Figure 2A). "
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    ABSTRACT: Replicative aging in yeast is asymmetric–mother cells age but their daughter cells are rejuvenated. Here we identify an asymmetry in pH between mother and daughter cells that underlies aging and rejuvenation. Cytosolic pH increases in aging mother cells, but is more acidic in daughter cells. This is due to the asymmetric distribution of the major regulator of cytosolic pH, the plasma membrane proton ATPase (Pma1). Pma1 accumulates in aging mother cells, but is largely absent from nascent daughter cells. We previously found that acidity of the vacuole declines in aging mother cells and limits lifespan, but that daughter cell vacuoles re-acidify. We find that Pma1 activity antagonizes mother cell vacuole acidity by reducing cytosolic protons. However, the inherent asymmetry of Pma1 increases cytosolic proton availability in daughter cells and facilitates vacuole re-acidification and rejuvenation. DOI: http://dx.doi.org/10.7554/eLife.03504.001
    Full-text · Article · Sep 2014 · eLife Sciences
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    • "In addition, protein synthesis and degradation are firmly related and therefore represent an important control factor for metabolic regulation (Schwanhäusser et al., 2013). Individual protein turnover rates have been determined in mammalian cells, yeast and bacteria by measuring the incorporation rate of fluorescent tags (Khmelinskii et al., 2012), affinity tags (Belle et al., 2006) or isotopic labels into the proteome (methods reviewed by Hughes & Krijgsveld, 2012; Trötschel et al., 2013). The metabolic incorporation of isotopic labels is currently the most widely used method for cell cultures and conducted using either labelled ammonium (Helbig et al., 2011; Martin et al., 2012), carbon (Cargile et al., 2004) or amino acids (Gerth et al., 2008; Maier et al., 2011; Schwanhäusser et al., 2011). "
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