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Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation.

Hartmann M, Gas-Pascual E, Hemmerlin A, Rohmer M, Bach TJ - F1000Res (2015)

Bottom Line: In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization.As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum.Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

View Article: PubMed Central - PubMed

Affiliation: Département "Réseaux Métaboliques, Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357, Université de Strasbourg, 28 rue Goethe, F-67083 Strasbourg, France ; Current address: Department Biologie, Institut für Molekulare Ökophysiologie der Pflanzen, Universität Düsseldorf, Universitätsstr. 1, D-40225, Düsseldorf, Germany.

ABSTRACT
In a preceding study we have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

No MeSH data available.


Related in: MedlinePlus

Double-transformed BY-2 cell line (N-20) showing different intracellular localization of fluorescent fusion proteins (after induction).All channels: tetrade with red and green fluorescence induced at the same time (+ differential interference contrast, DIC).GFP: the GFP-BD-CVIL fusion protein is visualized after induction with 10 µM Dex and is mainly localized to the PM and cytosol.RFP: The monomeric RFP fused to the C-terminus of the SV40 NLS is visualized after induction with 5 µM estradiol and is predominantly found in the nuclear compartment.GFP/RFP: Overlay of the GFP and RFP channels. Induction time with both elicitors was 15 h. Images were acquired using aLSM510 confocal laser scanning microscope equipped with an inverted Carl Zeiss axiovert 100 M microscope. Dual color imaging was performed using dual excitation/emission scanning in the multitracking mode (Carl Zeiss Laser Scanning Microscope software). White bar represents 20 µm.
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f5: Double-transformed BY-2 cell line (N-20) showing different intracellular localization of fluorescent fusion proteins (after induction).All channels: tetrade with red and green fluorescence induced at the same time (+ differential interference contrast, DIC).GFP: the GFP-BD-CVIL fusion protein is visualized after induction with 10 µM Dex and is mainly localized to the PM and cytosol.RFP: The monomeric RFP fused to the C-terminus of the SV40 NLS is visualized after induction with 5 µM estradiol and is predominantly found in the nuclear compartment.GFP/RFP: Overlay of the GFP and RFP channels. Induction time with both elicitors was 15 h. Images were acquired using aLSM510 confocal laser scanning microscope equipped with an inverted Carl Zeiss axiovert 100 M microscope. Dual color imaging was performed using dual excitation/emission scanning in the multitracking mode (Carl Zeiss Laser Scanning Microscope software). White bar represents 20 µm.

Mentions: The most promising cell line (N-20) showed bright fluorescence in both channels set for the visualization of GFP and RFP, respectively, and had a significant ratio of fluorescent positive cells (>50% of total cells), making it a good candidate for first tests and further re-selection efforts to obtain a performing cell line suitable for statistical approaches. InFigure 5 a typical cell tetrade is displayed, after induction by both dexamethasone (Dex, green fluorescence) and estradiol (Est, red fluorescence).


Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation.

Hartmann M, Gas-Pascual E, Hemmerlin A, Rohmer M, Bach TJ - F1000Res (2015)

Double-transformed BY-2 cell line (N-20) showing different intracellular localization of fluorescent fusion proteins (after induction).All channels: tetrade with red and green fluorescence induced at the same time (+ differential interference contrast, DIC).GFP: the GFP-BD-CVIL fusion protein is visualized after induction with 10 µM Dex and is mainly localized to the PM and cytosol.RFP: The monomeric RFP fused to the C-terminus of the SV40 NLS is visualized after induction with 5 µM estradiol and is predominantly found in the nuclear compartment.GFP/RFP: Overlay of the GFP and RFP channels. Induction time with both elicitors was 15 h. Images were acquired using aLSM510 confocal laser scanning microscope equipped with an inverted Carl Zeiss axiovert 100 M microscope. Dual color imaging was performed using dual excitation/emission scanning in the multitracking mode (Carl Zeiss Laser Scanning Microscope software). White bar represents 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4536634&req=5

f5: Double-transformed BY-2 cell line (N-20) showing different intracellular localization of fluorescent fusion proteins (after induction).All channels: tetrade with red and green fluorescence induced at the same time (+ differential interference contrast, DIC).GFP: the GFP-BD-CVIL fusion protein is visualized after induction with 10 µM Dex and is mainly localized to the PM and cytosol.RFP: The monomeric RFP fused to the C-terminus of the SV40 NLS is visualized after induction with 5 µM estradiol and is predominantly found in the nuclear compartment.GFP/RFP: Overlay of the GFP and RFP channels. Induction time with both elicitors was 15 h. Images were acquired using aLSM510 confocal laser scanning microscope equipped with an inverted Carl Zeiss axiovert 100 M microscope. Dual color imaging was performed using dual excitation/emission scanning in the multitracking mode (Carl Zeiss Laser Scanning Microscope software). White bar represents 20 µm.
Mentions: The most promising cell line (N-20) showed bright fluorescence in both channels set for the visualization of GFP and RFP, respectively, and had a significant ratio of fluorescent positive cells (>50% of total cells), making it a good candidate for first tests and further re-selection efforts to obtain a performing cell line suitable for statistical approaches. InFigure 5 a typical cell tetrade is displayed, after induction by both dexamethasone (Dex, green fluorescence) and estradiol (Est, red fluorescence).

Bottom Line: In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization.As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum.Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

View Article: PubMed Central - PubMed

Affiliation: Département "Réseaux Métaboliques, Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357, Université de Strasbourg, 28 rue Goethe, F-67083 Strasbourg, France ; Current address: Department Biologie, Institut für Molekulare Ökophysiologie der Pflanzen, Universität Düsseldorf, Universitätsstr. 1, D-40225, Düsseldorf, Germany.

ABSTRACT
In a preceding study we have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was there demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, in this initial study complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established now new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

No MeSH data available.


Related in: MedlinePlus