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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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Related in: MedlinePlus

BL-induced expansion of the GFP fluorescence signal in BRI1-GFP expressing root tip cells.(A) Confocal image of root tip tissue before (left) and 30 min after addition of 10 nM BL (right). Fluorescence intensity curves were recorded over the plasmalemmata-cell wall sections indicated by the white, alphabetically numbered lines and the FWHM values of their Gaussian fits calculated before and 30 min after addition of BL. (B–J) FWHM values of the plasmalemmata-cell wall sections indicated in A before (0 min) and 30 min after addition of 10 nM BL. For the determination of the FWHM error in B to J see Material and Methods. For the statistical analysis of the BRI1-GFP fluorescence measurements see Table S2.
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pone-0005716-g005: BL-induced expansion of the GFP fluorescence signal in BRI1-GFP expressing root tip cells.(A) Confocal image of root tip tissue before (left) and 30 min after addition of 10 nM BL (right). Fluorescence intensity curves were recorded over the plasmalemmata-cell wall sections indicated by the white, alphabetically numbered lines and the FWHM values of their Gaussian fits calculated before and 30 min after addition of BL. (B–J) FWHM values of the plasmalemmata-cell wall sections indicated in A before (0 min) and 30 min after addition of 10 nM BL. For the determination of the FWHM error in B to J see Material and Methods. For the statistical analysis of the BRI1-GFP fluorescence measurements see Table S2.

Mentions: We next addressed the problem of real-time measurement of possible physiological effects of BRI1-GFP at subcellular level in living plant cells. Former studies have shown that BL does not alter the fluorescence intensity, the number of vesicles in the endosomal pool and the intracellular distribution of BRI1-GFP in root cells [22]. This is in agreement with our observations (Fig. 4A to C, Fig. 5 and data not shown). However, the local fluorescence intensity of BRI1-GFP as a function of BL, time and a defined subcellular area was not yet studied. We therefore, recorded fluorescence intensity profiles over selected plasmalemmata-cell wall sections before and after treating the cells for 15 to 30 min with 10 nM BL. The intensity profiles enabled us to discriminate between the fluorescence signal of the proper plasmalemmata-cell wall section and membrane vesicles, which budded from the plasmalemmata during the incubation time. As shown in Fig. 4A–D and Fig. 5, the treatment of seedlings with BL induced an expansion of the BRI1-GFP signal in the plasmalemma-cell wall section of the root cells within 30 min. This response was also observed in hypocotyl cells, where BL induced an expansion of the BRI1-GFP signal of two neighboring cells, so that the plasmalemmata became optically distinguishable (Fig. 4E and F). The degree of expansion did not only depend on the cell type but also on the position of the measured section along the cell wall of individual cells (Fig. 5). The Gaussian fitting of the GFP intensity profiles of more than 30 independent sections measured in several individual cells from 5 seedlings revealed that the fluorescence signals expanded around 34±22% (n = 31, p = 0,0003) in response to BL treatment (see Table S2 for the single measurement values).


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 expansion of the GFP fluorescence signal in BRI1-GFP expressing root tip cells.(A) Confocal image of root tip tissue before (left) and 30 min after addition of 10 nM BL (right). Fluorescence intensity curves were recorded over the plasmalemmata-cell wall sections indicated by the white, alphabetically numbered lines and the FWHM values of their Gaussian fits calculated before and 30 min after addition of BL. (B–J) FWHM values of the plasmalemmata-cell wall sections indicated in A before (0 min) and 30 min after addition of 10 nM BL. For the determination of the FWHM error in B to J see Material and Methods. For the statistical analysis of the BRI1-GFP fluorescence measurements see Table S2.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683565&req=5

pone-0005716-g005: BL-induced expansion of the GFP fluorescence signal in BRI1-GFP expressing root tip cells.(A) Confocal image of root tip tissue before (left) and 30 min after addition of 10 nM BL (right). Fluorescence intensity curves were recorded over the plasmalemmata-cell wall sections indicated by the white, alphabetically numbered lines and the FWHM values of their Gaussian fits calculated before and 30 min after addition of BL. (B–J) FWHM values of the plasmalemmata-cell wall sections indicated in A before (0 min) and 30 min after addition of 10 nM BL. For the determination of the FWHM error in B to J see Material and Methods. For the statistical analysis of the BRI1-GFP fluorescence measurements see Table S2.
Mentions: We next addressed the problem of real-time measurement of possible physiological effects of BRI1-GFP at subcellular level in living plant cells. Former studies have shown that BL does not alter the fluorescence intensity, the number of vesicles in the endosomal pool and the intracellular distribution of BRI1-GFP in root cells [22]. This is in agreement with our observations (Fig. 4A to C, Fig. 5 and data not shown). However, the local fluorescence intensity of BRI1-GFP as a function of BL, time and a defined subcellular area was not yet studied. We therefore, recorded fluorescence intensity profiles over selected plasmalemmata-cell wall sections before and after treating the cells for 15 to 30 min with 10 nM BL. The intensity profiles enabled us to discriminate between the fluorescence signal of the proper plasmalemmata-cell wall section and membrane vesicles, which budded from the plasmalemmata during the incubation time. As shown in Fig. 4A–D and Fig. 5, the treatment of seedlings with BL induced an expansion of the BRI1-GFP signal in the plasmalemma-cell wall section of the root cells within 30 min. This response was also observed in hypocotyl cells, where BL induced an expansion of the BRI1-GFP signal of two neighboring cells, so that the plasmalemmata became optically distinguishable (Fig. 4E and F). The degree of expansion did not only depend on the cell type but also on the position of the measured section along the cell wall of individual cells (Fig. 5). The Gaussian fitting of the GFP intensity profiles of more than 30 independent sections measured in several individual cells from 5 seedlings revealed that the fluorescence signals expanded around 34±22% (n = 31, p = 0,0003) in response to BL treatment (see Table S2 for the single measurement values).

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
Related in: MedlinePlus