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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

Expression of fusion proteins is tightly regulated by their specific inducers.No cross-induction of fusion gene expression was visible. Induction time for dexamethasone (Dex, 10 µM final) and estradiol (Est, 6 µM final) was 15 h in all experiments.+ Dex / + Est: control experiments with both inducers with the GFP fusion protein targeted to the PM (G) and the mFRP fusion protein located in the nucleus (R).+Dex (+OC) / + FM4-64: Dex alone is only inducing the expression of the GFP fusion protein (G). The negative control with FM4–64 (5 µg/ml; membrane stain) shows no signals from the nucleus (R). For a better understanding, cells were treated with 50 µM oxoclomazone (OC) 3 h before induction, to indicate the position of the nucleus. Fluorescein diacetate (FDA, 7.5 µM final) is used as negative control, also indicating that the cells were alive during the imaging process.G andR indicate the green and the red channel, respectively. Bars = 20 µm.
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f6: Expression of fusion proteins is tightly regulated by their specific inducers.No cross-induction of fusion gene expression was visible. Induction time for dexamethasone (Dex, 10 µM final) and estradiol (Est, 6 µM final) was 15 h in all experiments.+ Dex / + Est: control experiments with both inducers with the GFP fusion protein targeted to the PM (G) and the mFRP fusion protein located in the nucleus (R).+Dex (+OC) / + FM4-64: Dex alone is only inducing the expression of the GFP fusion protein (G). The negative control with FM4–64 (5 µg/ml; membrane stain) shows no signals from the nucleus (R). For a better understanding, cells were treated with 50 µM oxoclomazone (OC) 3 h before induction, to indicate the position of the nucleus. Fluorescein diacetate (FDA, 7.5 µM final) is used as negative control, also indicating that the cells were alive during the imaging process.G andR indicate the green and the red channel, respectively. Bars = 20 µm.

Mentions: The newly created double fluorescent cell line NLS-mRFP/H6-GFP-BD-CVIL shows no visible cross-induction of fluorescence after treatment with estradiol or dexamethasone. In order to establish a reliable visual test system, it was necessary to verify that the two co-existing induction systems in the NLS-mRFP (Est) and H6-GFP-BD-CVIL (Dex) lines only respond to their specific inducers. As both chemical-inducible systems are based on a similar principle of induction - the action of chimerictrans-activators whose transcriptional activities are regulated by specific hormones and/or structurally related compounds86) - the newly generated cell line was separately treated with both inducers under standard conditions. In addition, fluorescent cell dyes were used in parallel as negative controls (Figure 6). Simultaneous treatment of the cell line with both inducers (24 h induction, 10 µM Dex, 6 µM Est) resulted in a phenotype with green fluorescence predominantly located at the peripheral membrane region and red fluorescence mainly located in the nuclear compartment.


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)

Expression of fusion proteins is tightly regulated by their specific inducers.No cross-induction of fusion gene expression was visible. Induction time for dexamethasone (Dex, 10 µM final) and estradiol (Est, 6 µM final) was 15 h in all experiments.+ Dex / + Est: control experiments with both inducers with the GFP fusion protein targeted to the PM (G) and the mFRP fusion protein located in the nucleus (R).+Dex (+OC) / + FM4-64: Dex alone is only inducing the expression of the GFP fusion protein (G). The negative control with FM4–64 (5 µg/ml; membrane stain) shows no signals from the nucleus (R). For a better understanding, cells were treated with 50 µM oxoclomazone (OC) 3 h before induction, to indicate the position of the nucleus. Fluorescein diacetate (FDA, 7.5 µM final) is used as negative control, also indicating that the cells were alive during the imaging process.G andR indicate the green and the red channel, respectively. Bars = 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Expression of fusion proteins is tightly regulated by their specific inducers.No cross-induction of fusion gene expression was visible. Induction time for dexamethasone (Dex, 10 µM final) and estradiol (Est, 6 µM final) was 15 h in all experiments.+ Dex / + Est: control experiments with both inducers with the GFP fusion protein targeted to the PM (G) and the mFRP fusion protein located in the nucleus (R).+Dex (+OC) / + FM4-64: Dex alone is only inducing the expression of the GFP fusion protein (G). The negative control with FM4–64 (5 µg/ml; membrane stain) shows no signals from the nucleus (R). For a better understanding, cells were treated with 50 µM oxoclomazone (OC) 3 h before induction, to indicate the position of the nucleus. Fluorescein diacetate (FDA, 7.5 µM final) is used as negative control, also indicating that the cells were alive during the imaging process.G andR indicate the green and the red channel, respectively. Bars = 20 µm.
Mentions: The newly created double fluorescent cell line NLS-mRFP/H6-GFP-BD-CVIL shows no visible cross-induction of fluorescence after treatment with estradiol or dexamethasone. In order to establish a reliable visual test system, it was necessary to verify that the two co-existing induction systems in the NLS-mRFP (Est) and H6-GFP-BD-CVIL (Dex) lines only respond to their specific inducers. As both chemical-inducible systems are based on a similar principle of induction - the action of chimerictrans-activators whose transcriptional activities are regulated by specific hormones and/or structurally related compounds86) - the newly generated cell line was separately treated with both inducers under standard conditions. In addition, fluorescent cell dyes were used in parallel as negative controls (Figure 6). Simultaneous treatment of the cell line with both inducers (24 h induction, 10 µM Dex, 6 µM Est) resulted in a phenotype with green fluorescence predominantly located at the peripheral membrane region and red fluorescence mainly located in the nuclear compartment.

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