RESUMEN
Cells need to reliably control their proteome composition to maintain homeostasis and regulate growth. How protein synthesis and degradation interplay to control protein expression levels remains unclear. Here, we combined a tandem fluorescent timer and pulse-chase protein labeling to disentangle how protein synthesis and degradation control protein homeostasis in single live mouse embryonic stem cells. We discovered substantial cell-cycle dependence in protein synthesis rates and stabilization of a large number of proteins around cytokinesis. Protein degradation rates were highly variable between cells, co-varied within individual cells for different proteins, and were positively correlated with synthesis rates. This suggests variability in proteasome activity as an important source of global extrinsic noise in gene expression. Our approach paves the way toward understanding the complex interplay of synthesis and degradation processes in determining protein levels of individual mammalian cells.
Asunto(s)
Imagen Óptica/métodos , Proteostasis/fisiología , Animales , Ciclo Celular/fisiología , Células Madre Embrionarias/metabolismo , Ratones , Biosíntesis de Proteínas/fisiología , Proteolisis , Proteoma/metabolismo , Proteómica/métodos , Análisis de la Célula Individual/métodosRESUMEN
The transcription factors BMAL1 and CLOCK drive the circadian transcription of clock and clock-controlled genes, such as Dbp. To investigate the kinetics of BMAL1 binding to target genes in real time, we generated a cell line harboring tandem arrays of Dbp repeats and monitored the binding of a fluorescent BMAL1 fusion protein to these arrays by time-lapse microscopy. BMAL1 occupancy at the Dbp locus was highly circadian and strictly dependent on CLOCK. Moreover, BMAL1-CLOCK associations with Dbp were extremely unstable and displayed stochastic, proteasome-dependent fluctuations. Proteasome inhibition prolonged the residence time of BMAL1-CLOCK but resulted in an immediate attenuation of Dbp transcription. In cells harboring a single Dbp-luciferase reporter gene copy, this silencing was shown to be caused by a decrease in both the frequencies and sizes of transcriptional bursts. Thus, BMAL1 and CLOCK may act as Kamikaze activators, in that they are rapidly degraded once bound to Dbp chromatin.
Asunto(s)
Factores de Transcripción ARNTL , Proteínas CLOCK , Proteínas de Unión al ADN , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Acetilación , Animales , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Cromatina/genética , Cromatina/metabolismo , Relojes Circadianos/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Ratones , Células 3T3 NIH , Unión Proteica , Análisis de la Célula Individual , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Protein expression levels depend on the balance between their synthesis and degradation rates. Even quiescent (G0) cells display a continuous turnover of proteins, despite protein levels remaining largely constant over time. In cycling cells, global protein levels need to be precisely doubled at each cell division in order to maintain cellular homeostasis, but we still lack a quantitative understanding of how this is achieved. Recent studies have shed light on cell cycle-dependent changes in protein synthesis and degradation rates. Here we discuss current population-based and single cell approaches used to assess protein synthesis and degradation, and review the insights they have provided into the dynamics of protein turnover in different cell cycle phases.
Asunto(s)
Puntos de Control del Ciclo Celular/fisiología , División Celular/fisiología , Biosíntesis de Proteínas/fisiología , Proteolisis , Animales , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Homeostasis/fisiología , Humanos , Cinética , Mamíferos/metabolismo , Proteínas/metabolismo , ARN Mensajero/metabolismo , Análisis de la Célula IndividualRESUMEN
Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.