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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

Scheme of the cloning procedure used for the selection of homogenous, transgenic tobacco BY-2 cell lines.A – Selection of H6-GFP-DB-CVIL cell line.B – Selection of the double-transformed H6-GFP-DB-CVIL/NLS-mRFP cell line. The selection of primary and secondary calli was performed on antibiotica-supplemented solid plates. As the expression of both fluorescent reporter proteins was inducible, no pre-selection by fluorescence microscopy could be used to eliminate non-fluorescent calli (it was tried by incubation of the calli in presence of the inducers, but no fluorescence could be detected with this method). The selection criteria for cultures used in the cloning procedure were based on the properties optimal for the bioassay (homogenous, bright fluorescence and high ratios of fluorescent to non-fluorescent cells.). Rounded values for statistical evaluation are indicated if available.
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fs1: Scheme of the cloning procedure used for the selection of homogenous, transgenic tobacco BY-2 cell lines.A – Selection of H6-GFP-DB-CVIL cell line.B – Selection of the double-transformed H6-GFP-DB-CVIL/NLS-mRFP cell line. The selection of primary and secondary calli was performed on antibiotica-supplemented solid plates. As the expression of both fluorescent reporter proteins was inducible, no pre-selection by fluorescence microscopy could be used to eliminate non-fluorescent calli (it was tried by incubation of the calli in presence of the inducers, but no fluorescence could be detected with this method). The selection criteria for cultures used in the cloning procedure were based on the properties optimal for the bioassay (homogenous, bright fluorescence and high ratios of fluorescent to non-fluorescent cells.). Rounded values for statistical evaluation are indicated if available.


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)

Scheme of the cloning procedure used for the selection of homogenous, transgenic tobacco BY-2 cell lines.A – Selection of H6-GFP-DB-CVIL cell line.B – Selection of the double-transformed H6-GFP-DB-CVIL/NLS-mRFP cell line. The selection of primary and secondary calli was performed on antibiotica-supplemented solid plates. As the expression of both fluorescent reporter proteins was inducible, no pre-selection by fluorescence microscopy could be used to eliminate non-fluorescent calli (it was tried by incubation of the calli in presence of the inducers, but no fluorescence could be detected with this method). The selection criteria for cultures used in the cloning procedure were based on the properties optimal for the bioassay (homogenous, bright fluorescence and high ratios of fluorescent to non-fluorescent cells.). Rounded values for statistical evaluation are indicated if available.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fs1: Scheme of the cloning procedure used for the selection of homogenous, transgenic tobacco BY-2 cell lines.A – Selection of H6-GFP-DB-CVIL cell line.B – Selection of the double-transformed H6-GFP-DB-CVIL/NLS-mRFP cell line. The selection of primary and secondary calli was performed on antibiotica-supplemented solid plates. As the expression of both fluorescent reporter proteins was inducible, no pre-selection by fluorescence microscopy could be used to eliminate non-fluorescent calli (it was tried by incubation of the calli in presence of the inducers, but no fluorescence could be detected with this method). The selection criteria for cultures used in the cloning procedure were based on the properties optimal for the bioassay (homogenous, bright fluorescence and high ratios of fluorescent to non-fluorescent cells.). Rounded values for statistical evaluation are indicated if available.
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