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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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ocFLM of BRI1-GFP and aquaporin-GFP reveals gradients in their subcellular environment.(A) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 5.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent BRI1-GFP expressing Arabidopsis seedlings. (B) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 4.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent aquaporin-GFP expressing Arabidopsis seedlings. (C) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) over a 6.0 µm plasmalemma-cell wall area of a hypocotyl cell from a BRI1-GFP expressing Arabidopsis seedling. The white line in the confocal image inlet shows the recorded section. For the calculation of the fluorescence lifetime values and error bars see Material and Methods. Additional BRI1-GFP and aquaporin-GFP fluorescence lifetime measurements are presented in Fig. 10D and E.
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pone-0005716-g009: ocFLM of BRI1-GFP and aquaporin-GFP reveals gradients in their subcellular environment.(A) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 5.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent BRI1-GFP expressing Arabidopsis seedlings. (B) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 4.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent aquaporin-GFP expressing Arabidopsis seedlings. (C) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) over a 6.0 µm plasmalemma-cell wall area of a hypocotyl cell from a BRI1-GFP expressing Arabidopsis seedling. The white line in the confocal image inlet shows the recorded section. For the calculation of the fluorescence lifetime values and error bars see Material and Methods. Additional BRI1-GFP and aquaporin-GFP fluorescence lifetime measurements are presented in Fig. 10D and E.

Mentions: The lifetime of GFP fluorescence provides information about the physical and chemical environment of the fluorophore and, thus, the GFP fusion protein [29]–[30]. In addition to fluorescence intensity, we recorded GFP fluorescence lifetime decay traces across plasmalemmata-cell wall sections in hypocotyl cells of aquaporin-GFP and BRI1-GFP expressing seedlings at up to 200 nm intervals. The measurements were based on a different but sufficient number of fluorescence counts (Table S1), which is reflected in the error calculation [31]. We observed significant differences in GFP fluorescence lifetime across plasmalemma-cell wall sections and regularly also between adjacent cells for both aquaporin-GFP and BRI1-GFP (Fig. 9A and B). A detailed subcellular analysis revealed that the most significant differences in BRI1-GFP lifetime existed within the cell between the cytoplasm, plasmalemma and cell wall (Fig. 9C).


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)

ocFLM of BRI1-GFP and aquaporin-GFP reveals gradients in their subcellular environment.(A) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 5.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent BRI1-GFP expressing Arabidopsis seedlings. (B) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 4.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent aquaporin-GFP expressing Arabidopsis seedlings. (C) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) over a 6.0 µm plasmalemma-cell wall area of a hypocotyl cell from a BRI1-GFP expressing Arabidopsis seedling. The white line in the confocal image inlet shows the recorded section. For the calculation of the fluorescence lifetime values and error bars see Material and Methods. Additional BRI1-GFP and aquaporin-GFP fluorescence lifetime measurements are presented in Fig. 10D and E.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683565&req=5

pone-0005716-g009: ocFLM of BRI1-GFP and aquaporin-GFP reveals gradients in their subcellular environment.(A) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 5.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent BRI1-GFP expressing Arabidopsis seedlings. (B) GFP fluorescence lifetimes (filled squares) and the corresponding intensity profiles (open circles) recorded over 4.0 µm plasmalemma-cell wall sections of two different hypocotyl cells from two independent aquaporin-GFP expressing Arabidopsis seedlings. (C) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) over a 6.0 µm plasmalemma-cell wall area of a hypocotyl cell from a BRI1-GFP expressing Arabidopsis seedling. The white line in the confocal image inlet shows the recorded section. For the calculation of the fluorescence lifetime values and error bars see Material and Methods. Additional BRI1-GFP and aquaporin-GFP fluorescence lifetime measurements are presented in Fig. 10D and E.
Mentions: The lifetime of GFP fluorescence provides information about the physical and chemical environment of the fluorophore and, thus, the GFP fusion protein [29]–[30]. In addition to fluorescence intensity, we recorded GFP fluorescence lifetime decay traces across plasmalemmata-cell wall sections in hypocotyl cells of aquaporin-GFP and BRI1-GFP expressing seedlings at up to 200 nm intervals. The measurements were based on a different but sufficient number of fluorescence counts (Table S1), which is reflected in the error calculation [31]. We observed significant differences in GFP fluorescence lifetime across plasmalemma-cell wall sections and regularly also between adjacent cells for both aquaporin-GFP and BRI1-GFP (Fig. 9A and B). A detailed subcellular analysis revealed that the most significant differences in BRI1-GFP lifetime existed within the cell between the cytoplasm, plasmalemma and cell wall (Fig. 9C).

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