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Tracking protein turnover and degradation by microscopy: photo-switchable versus time-encoded fluorescent proteins.

Knop M, Edgar BA - Open Biol (2014)

Bottom Line: Expanded fluorescent protein techniques employing photo-switchable and fluorescent timer proteins have become important tools in biological research.These tools allow researchers to address a major challenge in cell and developmental biology, namely obtaining kinetic information about the processes that determine the distribution and abundance of proteins in cells and tissues.This knowledge is often essential for the comprehensive understanding of a biological process, and/or required to determine the precise point of interference following an experimental perturbation.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, Deutsches Krebsforschungszentrum, DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
Expanded fluorescent protein techniques employing photo-switchable and fluorescent timer proteins have become important tools in biological research. These tools allow researchers to address a major challenge in cell and developmental biology, namely obtaining kinetic information about the processes that determine the distribution and abundance of proteins in cells and tissues. This knowledge is often essential for the comprehensive understanding of a biological process, and/or required to determine the precise point of interference following an experimental perturbation.

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Related in: MedlinePlus

Application of FPs to quantify protein turnover and degradation.(a) FPs that change their spectral properties as afunction of a light intervention (here generally termed‘switcher’; the example of a green-to-red photoconvertibleFP is given) can be used in pulse-chase types of experiments. Dependingon the properties of the FP, which can be either photoactivatable,photoswitchable or photoconvertable [4], a pool of labelled proteins isgenerated using illumination with light of a specific wavelength andintensity. In the simplest scenario as depicted here(b), the speed with which un-marked proteins replacemarked ones (as observed during the chase period) is quantified.Assuming a steady-state situation, in which protein production anddegradation (k) are constant, the half-life of theprotein (t1/2) can be directly estimated[2]. Theillustration depicts whole cell measurements; however, sub-cellularmeasurements to determine local turnover are possible, limited only bythe number of available fluorophores and their particular brightness andphotobleaching properties. (c) Fluorescent timerproteins can be categorized into two groups: single FPs that changetheir colour as a function of time (owing to subsequent chemicalreactions that lead to changes in the fluorophore), and tFTs. Both typesreport on the average age of a pool of proteins. (d) Inits simplest application under steady-state conditions, the average ageof the proteins directly reports on protein degradation rates(‘fast degrading proteins die young’), independent of theprotein production rates [5]. Tandem FP timers use a fast maturing FP such assuperfolder GFP as a reporter for protein abundance, while a slowmaturing protein, i.e. an RFP, reports on the relative age of the GFPmarked pool. Tuning of the dynamic range here is achieved by choosingRFPs with an appropriate maturation time. (e) Tablelisting properties of different ‘switcher’ FPs and someconsiderations for their application to conduct degradation/turnovermeasurements.
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RSOB140002F1: Application of FPs to quantify protein turnover and degradation.(a) FPs that change their spectral properties as afunction of a light intervention (here generally termed‘switcher’; the example of a green-to-red photoconvertibleFP is given) can be used in pulse-chase types of experiments. Dependingon the properties of the FP, which can be either photoactivatable,photoswitchable or photoconvertable [4], a pool of labelled proteins isgenerated using illumination with light of a specific wavelength andintensity. In the simplest scenario as depicted here(b), the speed with which un-marked proteins replacemarked ones (as observed during the chase period) is quantified.Assuming a steady-state situation, in which protein production anddegradation (k) are constant, the half-life of theprotein (t1/2) can be directly estimated[2]. Theillustration depicts whole cell measurements; however, sub-cellularmeasurements to determine local turnover are possible, limited only bythe number of available fluorophores and their particular brightness andphotobleaching properties. (c) Fluorescent timerproteins can be categorized into two groups: single FPs that changetheir colour as a function of time (owing to subsequent chemicalreactions that lead to changes in the fluorophore), and tFTs. Both typesreport on the average age of a pool of proteins. (d) Inits simplest application under steady-state conditions, the average ageof the proteins directly reports on protein degradation rates(‘fast degrading proteins die young’), independent of theprotein production rates [5]. Tandem FP timers use a fast maturing FP such assuperfolder GFP as a reporter for protein abundance, while a slowmaturing protein, i.e. an RFP, reports on the relative age of the GFPmarked pool. Tuning of the dynamic range here is achieved by choosingRFPs with an appropriate maturation time. (e) Tablelisting properties of different ‘switcher’ FPs and someconsiderations for their application to conduct degradation/turnovermeasurements.

Mentions: To do this, two experimental fluorescent protein (FP)-based toolkits are available.The first of these employs microscopy-based pulse-chase type experiments [1–3] (figure1a,b,e). Here, photo-switchable FPsthat change, acquire or lose fluorescence as a function of an intervention [4,6] are the most practical. A pulse of locallight-irradiation generates a labelled or activated population of a protein species;this population is then followed in time and space. With this‘switcher’ method, the temporal component of a measured turnoverconstant must necessarily be obtained from the time course parameters, upon fittingof measured signal intensities to models that describe the process, thus yieldingturnover estimates. Figure 1.


Tracking protein turnover and degradation by microscopy: photo-switchable versus time-encoded fluorescent proteins.

Knop M, Edgar BA - Open Biol (2014)

Application of FPs to quantify protein turnover and degradation.(a) FPs that change their spectral properties as afunction of a light intervention (here generally termed‘switcher’; the example of a green-to-red photoconvertibleFP is given) can be used in pulse-chase types of experiments. Dependingon the properties of the FP, which can be either photoactivatable,photoswitchable or photoconvertable [4], a pool of labelled proteins isgenerated using illumination with light of a specific wavelength andintensity. In the simplest scenario as depicted here(b), the speed with which un-marked proteins replacemarked ones (as observed during the chase period) is quantified.Assuming a steady-state situation, in which protein production anddegradation (k) are constant, the half-life of theprotein (t1/2) can be directly estimated[2]. Theillustration depicts whole cell measurements; however, sub-cellularmeasurements to determine local turnover are possible, limited only bythe number of available fluorophores and their particular brightness andphotobleaching properties. (c) Fluorescent timerproteins can be categorized into two groups: single FPs that changetheir colour as a function of time (owing to subsequent chemicalreactions that lead to changes in the fluorophore), and tFTs. Both typesreport on the average age of a pool of proteins. (d) Inits simplest application under steady-state conditions, the average ageof the proteins directly reports on protein degradation rates(‘fast degrading proteins die young’), independent of theprotein production rates [5]. Tandem FP timers use a fast maturing FP such assuperfolder GFP as a reporter for protein abundance, while a slowmaturing protein, i.e. an RFP, reports on the relative age of the GFPmarked pool. Tuning of the dynamic range here is achieved by choosingRFPs with an appropriate maturation time. (e) Tablelisting properties of different ‘switcher’ FPs and someconsiderations for their application to conduct degradation/turnovermeasurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043113&req=5

RSOB140002F1: Application of FPs to quantify protein turnover and degradation.(a) FPs that change their spectral properties as afunction of a light intervention (here generally termed‘switcher’; the example of a green-to-red photoconvertibleFP is given) can be used in pulse-chase types of experiments. Dependingon the properties of the FP, which can be either photoactivatable,photoswitchable or photoconvertable [4], a pool of labelled proteins isgenerated using illumination with light of a specific wavelength andintensity. In the simplest scenario as depicted here(b), the speed with which un-marked proteins replacemarked ones (as observed during the chase period) is quantified.Assuming a steady-state situation, in which protein production anddegradation (k) are constant, the half-life of theprotein (t1/2) can be directly estimated[2]. Theillustration depicts whole cell measurements; however, sub-cellularmeasurements to determine local turnover are possible, limited only bythe number of available fluorophores and their particular brightness andphotobleaching properties. (c) Fluorescent timerproteins can be categorized into two groups: single FPs that changetheir colour as a function of time (owing to subsequent chemicalreactions that lead to changes in the fluorophore), and tFTs. Both typesreport on the average age of a pool of proteins. (d) Inits simplest application under steady-state conditions, the average ageof the proteins directly reports on protein degradation rates(‘fast degrading proteins die young’), independent of theprotein production rates [5]. Tandem FP timers use a fast maturing FP such assuperfolder GFP as a reporter for protein abundance, while a slowmaturing protein, i.e. an RFP, reports on the relative age of the GFPmarked pool. Tuning of the dynamic range here is achieved by choosingRFPs with an appropriate maturation time. (e) Tablelisting properties of different ‘switcher’ FPs and someconsiderations for their application to conduct degradation/turnovermeasurements.
Mentions: To do this, two experimental fluorescent protein (FP)-based toolkits are available.The first of these employs microscopy-based pulse-chase type experiments [1–3] (figure1a,b,e). Here, photo-switchable FPsthat change, acquire or lose fluorescence as a function of an intervention [4,6] are the most practical. A pulse of locallight-irradiation generates a labelled or activated population of a protein species;this population is then followed in time and space. With this‘switcher’ method, the temporal component of a measured turnoverconstant must necessarily be obtained from the time course parameters, upon fittingof measured signal intensities to models that describe the process, thus yieldingturnover estimates. Figure 1.

Bottom Line: Expanded fluorescent protein techniques employing photo-switchable and fluorescent timer proteins have become important tools in biological research.These tools allow researchers to address a major challenge in cell and developmental biology, namely obtaining kinetic information about the processes that determine the distribution and abundance of proteins in cells and tissues.This knowledge is often essential for the comprehensive understanding of a biological process, and/or required to determine the precise point of interference following an experimental perturbation.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg, Deutsches Krebsforschungszentrum, DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

ABSTRACT
Expanded fluorescent protein techniques employing photo-switchable and fluorescent timer proteins have become important tools in biological research. These tools allow researchers to address a major challenge in cell and developmental biology, namely obtaining kinetic information about the processes that determine the distribution and abundance of proteins in cells and tissues. This knowledge is often essential for the comprehensive understanding of a biological process, and/or required to determine the precise point of interference following an experimental perturbation.

Show MeSH
Related in: MedlinePlus