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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

Two-channel imaging of the N-20 cell line after various treatments.The cell line was treated with different inhibitors affecting theMVA pathway (mevinolin) and theMEP pathway (fosmidomycin, oxoclomazone). Induction of the cell line with Est and Dex was carried out 24 h before observation. The cell line was treated with specific inhibitors 18 h before observation. Fosmidomycin (FOS) and oxoclomazone (OC) clearly shifted the localization of the GFP fusion protein to the nucleus, whereas mevinolin (MEV) treatment had no visible effect on the cells. In addition, we used two new cell lines with artificial micro interfering (amiRNA) silencing constructs as “biological controls”. (These cells expressed only the GFP reporter protein under the control of the dexamethasone-inducible promoter besides the amiRNA constructs.) For instance,DXR-silenced cells show the same phenotype as those inhibitedin vivo by Fos.HMGR silencing however does not exert any visible effect on the localization of the GFP fusion protein. White bars represent 20 µm.
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f10: Two-channel imaging of the N-20 cell line after various treatments.The cell line was treated with different inhibitors affecting theMVA pathway (mevinolin) and theMEP pathway (fosmidomycin, oxoclomazone). Induction of the cell line with Est and Dex was carried out 24 h before observation. The cell line was treated with specific inhibitors 18 h before observation. Fosmidomycin (FOS) and oxoclomazone (OC) clearly shifted the localization of the GFP fusion protein to the nucleus, whereas mevinolin (MEV) treatment had no visible effect on the cells. In addition, we used two new cell lines with artificial micro interfering (amiRNA) silencing constructs as “biological controls”. (These cells expressed only the GFP reporter protein under the control of the dexamethasone-inducible promoter besides the amiRNA constructs.) For instance,DXR-silenced cells show the same phenotype as those inhibitedin vivo by Fos.HMGR silencing however does not exert any visible effect on the localization of the GFP fusion protein. White bars represent 20 µm.

Mentions: The doubly transformed BY-2 cell line was treated for 18 h with inhibitors like oxoclomazone (OC), fosmidomycin (FOS) and mevinolin (MEV) affecting key enzymes of the MEP and MVA pathways, respectively, as indicated in the legend toFigure 10. Induction with 10 µM Dex and 6 µM Est took place 24 h before observation (Figure 10).


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)

Two-channel imaging of the N-20 cell line after various treatments.The cell line was treated with different inhibitors affecting theMVA pathway (mevinolin) and theMEP pathway (fosmidomycin, oxoclomazone). Induction of the cell line with Est and Dex was carried out 24 h before observation. The cell line was treated with specific inhibitors 18 h before observation. Fosmidomycin (FOS) and oxoclomazone (OC) clearly shifted the localization of the GFP fusion protein to the nucleus, whereas mevinolin (MEV) treatment had no visible effect on the cells. In addition, we used two new cell lines with artificial micro interfering (amiRNA) silencing constructs as “biological controls”. (These cells expressed only the GFP reporter protein under the control of the dexamethasone-inducible promoter besides the amiRNA constructs.) For instance,DXR-silenced cells show the same phenotype as those inhibitedin vivo by Fos.HMGR silencing however does not exert any visible effect on the localization of the GFP fusion protein. White bars represent 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f10: Two-channel imaging of the N-20 cell line after various treatments.The cell line was treated with different inhibitors affecting theMVA pathway (mevinolin) and theMEP pathway (fosmidomycin, oxoclomazone). Induction of the cell line with Est and Dex was carried out 24 h before observation. The cell line was treated with specific inhibitors 18 h before observation. Fosmidomycin (FOS) and oxoclomazone (OC) clearly shifted the localization of the GFP fusion protein to the nucleus, whereas mevinolin (MEV) treatment had no visible effect on the cells. In addition, we used two new cell lines with artificial micro interfering (amiRNA) silencing constructs as “biological controls”. (These cells expressed only the GFP reporter protein under the control of the dexamethasone-inducible promoter besides the amiRNA constructs.) For instance,DXR-silenced cells show the same phenotype as those inhibitedin vivo by Fos.HMGR silencing however does not exert any visible effect on the localization of the GFP fusion protein. White bars represent 20 µm.
Mentions: The doubly transformed BY-2 cell line was treated for 18 h with inhibitors like oxoclomazone (OC), fosmidomycin (FOS) and mevinolin (MEV) affecting key enzymes of the MEP and MVA pathways, respectively, as indicated in the legend toFigure 10. Induction with 10 µM Dex and 6 µM Est took place 24 h before observation (Figure 10).

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