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Systematic characterization of maturation time of fluorescent proteins in living cells.

ABSTRACT: The slow maturation time of fluorescent proteins (FPs) limits the temporal accuracy of measurements of rapid processes such as gene expression dynamics and effectively reduces fluorescence signal in growing cells. We used high-precision time-lapse microscopy to characterize the maturation kinetics of 50 FPs that span the visible spectrum at two different temperatures in Escherichia coli cells. We identified fast-maturing FPs from this set that yielded the highest signal-to-noise ratio and temporal resolution in individual growing cells.

SUBMITTER: Balleza E

PROVIDER: S-EPMC5765880 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Systematic characterization of maturation time of fluorescent proteins in living cells.

Balleza Enrique E Kim J Mark JM Cluzel Philippe P

Nature methods 20171120 1

The slow maturation time of fluorescent proteins (FPs) limits the temporal accuracy of measurements of rapid processes such as gene expression dynamics and effectively reduces fluorescence signal in growing cells. We used high-precision time-lapse microscopy to characterize the maturation kinetics of 50 FPs that span the visible spectrum at two different temperatures in Escherichia coli cells. We identified fast-maturing FPs from this set that yielded the highest signal-to-noise ratio and tempor ...[more]

PMID: 29320486

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Project description:BackgroundFluorescence imaging at high spectral resolution allows the simultaneous recording of multiple fluorophores without switching optical filters, which is especially useful for time-lapse analysis of living cells. The collected emission spectra can be used to distinguish fluorophores by a computation analysis called linear unmixing. The availability of accurate reference spectra for different fluorophores is crucial for this type of analysis. The reference spectra used by plant cell biologists are in most cases derived from the analysis of fluorescent proteins in solution or produced in animal cells, although these spectra are influenced by both the cellular environment and the components of the optical system. For instance, plant cells contain various autofluorescent compounds, such as cell wall polymers and chlorophyll, that affect the spectral detection of some fluorophores. Therefore, it is important to acquire both reference and experimental spectra under the same biological conditions and through the same imaging systems.ResultsEntry clones (pENTR) of fluorescent proteins (FPs) were constructed in order to create C- or N-terminal protein fusions with the MultiSite Gateway recombination technology. The emission spectra for eight FPs, fused C-terminally to the A- or B-type cyclin dependent kinases (CDKA;1 and CDKB1;1) and transiently expressed in epidermal cells of tobacco (Nicotiana benthamiana), were determined by using the Olympus FluoView™ FV1000 Confocal Laser Scanning Microscope. These experimental spectra were then used in unmixing experiments in order to separate the emission of fluorophores with overlapping spectral properties in living plant cells.ConclusionsSpectral imaging and linear unmixing have a great potential for efficient multicolor detection in living plant cells. The emission spectra for eight of the most commonly used FPs were obtained in epidermal cells of tobacco leaves and used in unmixing experiments. The generated set of FP Gateway entry vectors represents a valuable resource for plant cell biologists.

Project description:Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)-namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4-11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.

Dataset Information

Systematic characterization of maturation time of fluorescent proteins in living cells.

Publications

Systematic characterization of maturation time of fluorescent proteins in living cells.

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