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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.

Show MeSH
BL-induced cell wall expansion is paralleled by changes in the BRI1-GFP fluorescence lifetime.(A) BL-induced cell wall expansion of BRI1-GFP expressing hypocotyl cells from Arabidopsis seedlings before (black circles) and 30 min after (green circles) hormone application. The fluorescence intensity was recorded over a 7.0 µm section as indicated by the white line in the confocal image inlet. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A before (black squares), 10 (red squares), 20 (blue squares) and 30 min (green squares) after addition of 25 nM BL. The minima in the lifetime curves correspond to the maxima in the intensity profiles shown in A as indicated by the dashed lines. (C) Fluorescence lifetimes of BRI1-GFP in root tip cells 0 to 30 min after application of 25 nM BL. Lifetimes were obtained by integrating over all recorded pixels of the confocal image. (D) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of a BRI1-GFP expressing hypocotyl cell before (black squares), 15 min (blue squares) and 30 min (green squares) after onset of mock treatment. (E) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of an aquaporin-GFP expressing hypocotyl cell before (black squares) and 30 min (green squares) after application of 25 nM BL. For the calculation of the lifetime values and error bars see Material and Methods. The experiments in B and C were repeated three times using cells from three independent seedlings. Representative results are shown. The results of the additional BRI1-GFP lifetime measurements along plasmalemma-cell wall sections are presented in Figure S2.
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pone-0005716-g010: BL-induced cell wall expansion is paralleled by changes in the BRI1-GFP fluorescence lifetime.(A) BL-induced cell wall expansion of BRI1-GFP expressing hypocotyl cells from Arabidopsis seedlings before (black circles) and 30 min after (green circles) hormone application. The fluorescence intensity was recorded over a 7.0 µm section as indicated by the white line in the confocal image inlet. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A before (black squares), 10 (red squares), 20 (blue squares) and 30 min (green squares) after addition of 25 nM BL. The minima in the lifetime curves correspond to the maxima in the intensity profiles shown in A as indicated by the dashed lines. (C) Fluorescence lifetimes of BRI1-GFP in root tip cells 0 to 30 min after application of 25 nM BL. Lifetimes were obtained by integrating over all recorded pixels of the confocal image. (D) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of a BRI1-GFP expressing hypocotyl cell before (black squares), 15 min (blue squares) and 30 min (green squares) after onset of mock treatment. (E) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of an aquaporin-GFP expressing hypocotyl cell before (black squares) and 30 min (green squares) after application of 25 nM BL. For the calculation of the lifetime values and error bars see Material and Methods. The experiments in B and C were repeated three times using cells from three independent seedlings. Representative results are shown. The results of the additional BRI1-GFP lifetime measurements along plasmalemma-cell wall sections are presented in Figure S2.

Mentions: (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.


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 cell wall expansion is paralleled by changes in the BRI1-GFP fluorescence lifetime.(A) BL-induced cell wall expansion of BRI1-GFP expressing hypocotyl cells from Arabidopsis seedlings before (black circles) and 30 min after (green circles) hormone application. The fluorescence intensity was recorded over a 7.0 µm section as indicated by the white line in the confocal image inlet. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A before (black squares), 10 (red squares), 20 (blue squares) and 30 min (green squares) after addition of 25 nM BL. The minima in the lifetime curves correspond to the maxima in the intensity profiles shown in A as indicated by the dashed lines. (C) Fluorescence lifetimes of BRI1-GFP in root tip cells 0 to 30 min after application of 25 nM BL. Lifetimes were obtained by integrating over all recorded pixels of the confocal image. (D) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of a BRI1-GFP expressing hypocotyl cell before (black squares), 15 min (blue squares) and 30 min (green squares) after onset of mock treatment. (E) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of an aquaporin-GFP expressing hypocotyl cell before (black squares) and 30 min (green squares) after application of 25 nM BL. For the calculation of the lifetime values and error bars see Material and Methods. The experiments in B and C were repeated three times using cells from three independent seedlings. Representative results are shown. The results of the additional BRI1-GFP lifetime measurements along plasmalemma-cell wall sections are presented in Figure S2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683565&req=5

pone-0005716-g010: BL-induced cell wall expansion is paralleled by changes in the BRI1-GFP fluorescence lifetime.(A) BL-induced cell wall expansion of BRI1-GFP expressing hypocotyl cells from Arabidopsis seedlings before (black circles) and 30 min after (green circles) hormone application. The fluorescence intensity was recorded over a 7.0 µm section as indicated by the white line in the confocal image inlet. (B) Fluorescence lifetimes of BRI1-GFP in the identical plasmalemma-cell wall section shown in A before (black squares), 10 (red squares), 20 (blue squares) and 30 min (green squares) after addition of 25 nM BL. The minima in the lifetime curves correspond to the maxima in the intensity profiles shown in A as indicated by the dashed lines. (C) Fluorescence lifetimes of BRI1-GFP in root tip cells 0 to 30 min after application of 25 nM BL. Lifetimes were obtained by integrating over all recorded pixels of the confocal image. (D) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of a BRI1-GFP expressing hypocotyl cell before (black squares), 15 min (blue squares) and 30 min (green squares) after onset of mock treatment. (E) GFP fluorescence lifetime (filled squares) and the corresponding intensity profile (open circles) recorded over a 3.0 µm plasmalemma-cell wall section of an aquaporin-GFP expressing hypocotyl cell before (black squares) and 30 min (green squares) after application of 25 nM BL. For the calculation of the lifetime values and error bars see Material and Methods. The experiments in B and C were repeated three times using cells from three independent seedlings. Representative results are shown. The results of the additional BRI1-GFP lifetime measurements along plasmalemma-cell wall sections are presented in Figure S2.
Mentions: (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.

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