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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

GFP and RFP fluorescence in primary and secondary suspensions.A- andC- Heterogeneity of GFP and RFP fluorescence in a suspension of transgenic BY-2 cells derived from primary calli. Arrows indicate cells with missing fluorescence or significant variations in fluorescence intensities (red and green fluorescence).B- andD- Cell suspensions derived from re-selected calli (secondary calli). The fluorescence is strong and homogenous in both channels. Nevertheless some cells (less than 5%) show heterogeneity in fluorescence. Possible reasons are discussed in the main text. White bars indicate 20 μm.
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f17: GFP and RFP fluorescence in primary and secondary suspensions.A- andC- Heterogeneity of GFP and RFP fluorescence in a suspension of transgenic BY-2 cells derived from primary calli. Arrows indicate cells with missing fluorescence or significant variations in fluorescence intensities (red and green fluorescence).B- andD- Cell suspensions derived from re-selected calli (secondary calli). The fluorescence is strong and homogenous in both channels. Nevertheless some cells (less than 5%) show heterogeneity in fluorescence. Possible reasons are discussed in the main text. White bars indicate 20 μm.

Mentions: Given the higher complexity of plants, such as tobacco, compared to bacteria or yeast, variations in the expression levels of endogenous and reporter proteins from one cell to another can easily be imagined. This aspect is particularly interesting in connection with our observations, where transgene expression in individual cells of the newly generated H6-GFP-BD-CVIL BY-2 cell line proved to be unstable and heterogeneous in many cases (Figure 17).


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)

GFP and RFP fluorescence in primary and secondary suspensions.A- andC- Heterogeneity of GFP and RFP fluorescence in a suspension of transgenic BY-2 cells derived from primary calli. Arrows indicate cells with missing fluorescence or significant variations in fluorescence intensities (red and green fluorescence).B- andD- Cell suspensions derived from re-selected calli (secondary calli). The fluorescence is strong and homogenous in both channels. Nevertheless some cells (less than 5%) show heterogeneity in fluorescence. Possible reasons are discussed in the main text. White bars indicate 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f17: GFP and RFP fluorescence in primary and secondary suspensions.A- andC- Heterogeneity of GFP and RFP fluorescence in a suspension of transgenic BY-2 cells derived from primary calli. Arrows indicate cells with missing fluorescence or significant variations in fluorescence intensities (red and green fluorescence).B- andD- Cell suspensions derived from re-selected calli (secondary calli). The fluorescence is strong and homogenous in both channels. Nevertheless some cells (less than 5%) show heterogeneity in fluorescence. Possible reasons are discussed in the main text. White bars indicate 20 μm.
Mentions: Given the higher complexity of plants, such as tobacco, compared to bacteria or yeast, variations in the expression levels of endogenous and reporter proteins from one cell to another can easily be imagined. This aspect is particularly interesting in connection with our observations, where transgene expression in individual cells of the newly generated H6-GFP-BD-CVIL BY-2 cell line proved to be unstable and heterogeneous in many cases (Figure 17).

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