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Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.

Elgass K, Caesar K, Schleifenbaum F, Stierhof YD, Meixner AJ, Harter K - PLoS ONE (2009)

Bottom Line: However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo.Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physical and Theoretical Chemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.

Principal findings: Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes.

Significance: One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.

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BL-induced wall expansion and changes in BRI1-GFP fluorescence lifetime require a functional intracellular trafficking system.(A) FWHM values of GFP intensity profiles recorded over a 4.0 µm plasmalemmata-cell wall section of hypocotyl cells from BRI1-GFP expressing Arabidopsis seedlings in the presence of 50 µM BFA before (0 min) and 30 min after application of 10 nM BL. For the determination of the FWHM error see Material and Methods. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A in the presence of 50 µM BFA before (black squares) and 30 min (green squares) after addition of 25 nM BL. For the calculation of the lifetime values and error see Material and Methods. (C) Confocal images of hypocotyl cells treated with 50 µM BFA. The BFA compartments are indicated by white arrows. The white bars represent 6 µm (left) and 10 µm (right). The experiments in A and B were repeated four times using cells from four independent seedlings. One representative result is presented. The results of additional measurements are shown in Figure S3.
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pone-0005716-g011: BL-induced wall expansion and changes in BRI1-GFP fluorescence lifetime require a functional intracellular trafficking system.(A) FWHM values of GFP intensity profiles recorded over a 4.0 µm plasmalemmata-cell wall section of hypocotyl cells from BRI1-GFP expressing Arabidopsis seedlings in the presence of 50 µM BFA before (0 min) and 30 min after application of 10 nM BL. For the determination of the FWHM error see Material and Methods. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A in the presence of 50 µM BFA before (black squares) and 30 min (green squares) after addition of 25 nM BL. For the calculation of the lifetime values and error see Material and Methods. (C) Confocal images of hypocotyl cells treated with 50 µM BFA. The BFA compartments are indicated by white arrows. The white bars represent 6 µm (left) and 10 µm (right). The experiments in A and B were repeated four times using cells from four independent seedlings. One representative result is presented. The results of additional measurements are shown in Figure S3.

Mentions: Recent studies suggested that the endosomal pool of BRI1 is critical for the signaling and regulation of BL-responsive genes in Arabidopsis as shown by treatment with Brefeldin A (BFA) [22]–[23]. BFA inhibits the function of ARF-GTPases by interacting with their associated GEFs and, thus, has strong effects on the integrity of subcellular compartments and the endosomal vesicle pool by inhibiting intracellular trafficking pathways [32]–[33]. We, therefore, addressed the question whether BFA treatment also interferes with BL-induced cell wall expansion and BRI-GFP fluorescence lifetime. In the presence of the inhibitor BFA compartments appeared and both processes were strongly inhibited, suggesting that an intact intracellular trafficking system is required for their execution (Fig. 11 and Figure S3).


Novel application of fluorescence lifetime and fluorescence microscopy enables quantitative access to subcellular dynamics in plant cells.

Elgass K, Caesar K, Schleifenbaum F, Stierhof YD, Meixner AJ, Harter K - PLoS ONE (2009)

BL-induced wall expansion and changes in BRI1-GFP fluorescence lifetime require a functional intracellular trafficking system.(A) FWHM values of GFP intensity profiles recorded over a 4.0 µm plasmalemmata-cell wall section of hypocotyl cells from BRI1-GFP expressing Arabidopsis seedlings in the presence of 50 µM BFA before (0 min) and 30 min after application of 10 nM BL. For the determination of the FWHM error see Material and Methods. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A in the presence of 50 µM BFA before (black squares) and 30 min (green squares) after addition of 25 nM BL. For the calculation of the lifetime values and error see Material and Methods. (C) Confocal images of hypocotyl cells treated with 50 µM BFA. The BFA compartments are indicated by white arrows. The white bars represent 6 µm (left) and 10 µm (right). The experiments in A and B were repeated four times using cells from four independent seedlings. One representative result is presented. The results of additional measurements are shown in Figure S3.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683565&req=5

pone-0005716-g011: BL-induced wall expansion and changes in BRI1-GFP fluorescence lifetime require a functional intracellular trafficking system.(A) FWHM values of GFP intensity profiles recorded over a 4.0 µm plasmalemmata-cell wall section of hypocotyl cells from BRI1-GFP expressing Arabidopsis seedlings in the presence of 50 µM BFA before (0 min) and 30 min after application of 10 nM BL. For the determination of the FWHM error see Material and Methods. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A in the presence of 50 µM BFA before (black squares) and 30 min (green squares) after addition of 25 nM BL. For the calculation of the lifetime values and error see Material and Methods. (C) Confocal images of hypocotyl cells treated with 50 µM BFA. The BFA compartments are indicated by white arrows. The white bars represent 6 µm (left) and 10 µm (right). The experiments in A and B were repeated four times using cells from four independent seedlings. One representative result is presented. The results of additional measurements are shown in Figure S3.
Mentions: Recent studies suggested that the endosomal pool of BRI1 is critical for the signaling and regulation of BL-responsive genes in Arabidopsis as shown by treatment with Brefeldin A (BFA) [22]–[23]. BFA inhibits the function of ARF-GTPases by interacting with their associated GEFs and, thus, has strong effects on the integrity of subcellular compartments and the endosomal vesicle pool by inhibiting intracellular trafficking pathways [32]–[33]. We, therefore, addressed the question whether BFA treatment also interferes with BL-induced cell wall expansion and BRI-GFP fluorescence lifetime. In the presence of the inhibitor BFA compartments appeared and both processes were strongly inhibited, suggesting that an intact intracellular trafficking system is required for their execution (Fig. 11 and Figure S3).

Bottom Line: However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo.Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes.

View Article: PubMed Central - PubMed

Affiliation: Institute for Physical and Theoretical Chemistry, University of Tübingen, Tübingen, Germany.

ABSTRACT

Background: Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences.

Principal findings: Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes.

Significance: One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy.

Show MeSH