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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

Reproducibility of results on a population of treated BY-2 cells.Low magnification fluorescence images of large numbers of cloned BY-2 cells expressing H6-GFP-DB-CVIL (cf. Gerberet al., 2009). Images were taken with a Nikon E800 microscope equipped with a DXM11200 CCD color camera (specifications: 20 x 0.45 objective; filters: Ex460–500, DM505, EM510–560).A: Untreated cells;B–D: cells treated with 30 µM oxoclomazone (OC,B), 5 µM mevinolin (MEV,C) and 30 µM OC plus 5 µM MEV (D). The white bar represents 50 µm.
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f1: Reproducibility of results on a population of treated BY-2 cells.Low magnification fluorescence images of large numbers of cloned BY-2 cells expressing H6-GFP-DB-CVIL (cf. Gerberet al., 2009). Images were taken with a Nikon E800 microscope equipped with a DXM11200 CCD color camera (specifications: 20 x 0.45 objective; filters: Ex460–500, DM505, EM510–560).A: Untreated cells;B–D: cells treated with 30 µM oxoclomazone (OC,B), 5 µM mevinolin (MEV,C) and 30 µM OC plus 5 µM MEV (D). The white bar represents 50 µm.

Mentions: In order to demonstrate the feasibility and reproducibility of such an image-based approach, BY-2 cells expressing the H6-GFP-BD-CVIL fusion protein was treated with the MEP- or MVA pathway inhibitors, oxoclomazone (OC, 30 µM, synthesized by Dr. Eilers, Monsanto, St Louis, MO) or mevinolin (MEV, 5 µM, made available by MSD, Rahway NJ) respectively, as well as with a combination of both inhibitors according to standard protocols30. Images were acquired at low magnification using a Nikon E800 microscope. Low magnification fluorescence microscopy uses a resolution of 500 µm to 1 mm and is used to monitor phenotypic effects on entire cell populations. Medium-magnification fluorescence microscopy by definition is applied for subpopulation analysis at a resolution between 10–50 µm, whereas high-magnification fluorescence microscopy focuses on intracellular events and uses resolution of 1 µm or lower56. The phenotypes of the untreated control and the MEV-treated cells were more or less identical, with the majority of fluorescence at the cell periphery, especially at the boundary between cells in the files. In addition, GFP fluorescence was also clearly visible at the peri-nuclear membrane, whereas the GFP fluorescence in OC-treated cells was mostly localized in the nucleus. The combination of both inhibitors gave about the same or even more unequivocal results than cells treated with OC alone (Figure 1, cf.30).


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)

Reproducibility of results on a population of treated BY-2 cells.Low magnification fluorescence images of large numbers of cloned BY-2 cells expressing H6-GFP-DB-CVIL (cf. Gerberet al., 2009). Images were taken with a Nikon E800 microscope equipped with a DXM11200 CCD color camera (specifications: 20 x 0.45 objective; filters: Ex460–500, DM505, EM510–560).A: Untreated cells;B–D: cells treated with 30 µM oxoclomazone (OC,B), 5 µM mevinolin (MEV,C) and 30 µM OC plus 5 µM MEV (D). The white bar represents 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Reproducibility of results on a population of treated BY-2 cells.Low magnification fluorescence images of large numbers of cloned BY-2 cells expressing H6-GFP-DB-CVIL (cf. Gerberet al., 2009). Images were taken with a Nikon E800 microscope equipped with a DXM11200 CCD color camera (specifications: 20 x 0.45 objective; filters: Ex460–500, DM505, EM510–560).A: Untreated cells;B–D: cells treated with 30 µM oxoclomazone (OC,B), 5 µM mevinolin (MEV,C) and 30 µM OC plus 5 µM MEV (D). The white bar represents 50 µm.
Mentions: In order to demonstrate the feasibility and reproducibility of such an image-based approach, BY-2 cells expressing the H6-GFP-BD-CVIL fusion protein was treated with the MEP- or MVA pathway inhibitors, oxoclomazone (OC, 30 µM, synthesized by Dr. Eilers, Monsanto, St Louis, MO) or mevinolin (MEV, 5 µM, made available by MSD, Rahway NJ) respectively, as well as with a combination of both inhibitors according to standard protocols30. Images were acquired at low magnification using a Nikon E800 microscope. Low magnification fluorescence microscopy uses a resolution of 500 µm to 1 mm and is used to monitor phenotypic effects on entire cell populations. Medium-magnification fluorescence microscopy by definition is applied for subpopulation analysis at a resolution between 10–50 µm, whereas high-magnification fluorescence microscopy focuses on intracellular events and uses resolution of 1 µm or lower56. The phenotypes of the untreated control and the MEV-treated cells were more or less identical, with the majority of fluorescence at the cell periphery, especially at the boundary between cells in the files. In addition, GFP fluorescence was also clearly visible at the peri-nuclear membrane, whereas the GFP fluorescence in OC-treated cells was mostly localized in the nucleus. The combination of both inhibitors gave about the same or even more unequivocal results than cells treated with OC alone (Figure 1, cf.30).

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