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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

Restoration of RFP fluorescence after treatment with 5-azacytidine.A: Double fluorescent cell line that suddenly lost mRFP expression.B andC: Cells were treated with 10 µM azacytidine for one week. RFP fluorescence could be recovered, but many dead cells were due to overall toxic effects. Even though the concentration was scaled down 10–20x compared to values that are indicated in the literature for treatments, the concentration still seemed to be too high for the use in tobacco BY-2 cells (which have a good uptake rate and fast metabolism). All images are shown as merged images taken in green and red fluorescence, as well as white light mode. White bars = 50 µm.
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f9: Restoration of RFP fluorescence after treatment with 5-azacytidine.A: Double fluorescent cell line that suddenly lost mRFP expression.B andC: Cells were treated with 10 µM azacytidine for one week. RFP fluorescence could be recovered, but many dead cells were due to overall toxic effects. Even though the concentration was scaled down 10–20x compared to values that are indicated in the literature for treatments, the concentration still seemed to be too high for the use in tobacco BY-2 cells (which have a good uptake rate and fast metabolism). All images are shown as merged images taken in green and red fluorescence, as well as white light mode. White bars = 50 µm.

Mentions: After some problems with the maintenance of culture conditions due to a failure of the temperature control system of the growth chamber, we observed a sudden loss of all the mRFP fluorescence in one of the double-transformed cell lines, whereas the level of GFP fluorescence remained completely untouched (Figure 9). After elimination of all evident sources of error (replacing the inducer, subculturing the two-week old line, replacing the medium), the culture was incubated over a whole 7-day-growth cycle in presence of 10 µM 5-azacytidine, a nucleotide analog that cannot be methylated and, remarkably, the mRFP fluorescence could be partially restored (Figure 9). As this result clearly suggested a DNA methylation event, we screened the literature for common sources of such sudden drops in gene expression levels of transgenes in plant cultures. As we had already observed the same phenomenon in the original H6-GFP-BD-CVIL cell lines during the inhibitor tests with fosmidomycin-derived prodrugs, this point was quite important, given all the effort put into the generation of the cell lines. Schmittet al.104 reported that the antibiotics kanamycin, hygromycin and cefotaxim caused a DNA hypermethylation at CpG sites in the genome of tobacco plants grownin vitro, as shown by the SssI methylase accepting assays and genomic sequencing with sodium bisulfite. Interestingly, these methylations occurred in a time and dose-dependent manner and were not reversed when the progeny was not grown anymore in the presence of the antibiotics105.


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)

Restoration of RFP fluorescence after treatment with 5-azacytidine.A: Double fluorescent cell line that suddenly lost mRFP expression.B andC: Cells were treated with 10 µM azacytidine for one week. RFP fluorescence could be recovered, but many dead cells were due to overall toxic effects. Even though the concentration was scaled down 10–20x compared to values that are indicated in the literature for treatments, the concentration still seemed to be too high for the use in tobacco BY-2 cells (which have a good uptake rate and fast metabolism). All images are shown as merged images taken in green and red fluorescence, as well as white light mode. White bars = 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f9: Restoration of RFP fluorescence after treatment with 5-azacytidine.A: Double fluorescent cell line that suddenly lost mRFP expression.B andC: Cells were treated with 10 µM azacytidine for one week. RFP fluorescence could be recovered, but many dead cells were due to overall toxic effects. Even though the concentration was scaled down 10–20x compared to values that are indicated in the literature for treatments, the concentration still seemed to be too high for the use in tobacco BY-2 cells (which have a good uptake rate and fast metabolism). All images are shown as merged images taken in green and red fluorescence, as well as white light mode. White bars = 50 µm.
Mentions: After some problems with the maintenance of culture conditions due to a failure of the temperature control system of the growth chamber, we observed a sudden loss of all the mRFP fluorescence in one of the double-transformed cell lines, whereas the level of GFP fluorescence remained completely untouched (Figure 9). After elimination of all evident sources of error (replacing the inducer, subculturing the two-week old line, replacing the medium), the culture was incubated over a whole 7-day-growth cycle in presence of 10 µM 5-azacytidine, a nucleotide analog that cannot be methylated and, remarkably, the mRFP fluorescence could be partially restored (Figure 9). As this result clearly suggested a DNA methylation event, we screened the literature for common sources of such sudden drops in gene expression levels of transgenes in plant cultures. As we had already observed the same phenomenon in the original H6-GFP-BD-CVIL cell lines during the inhibitor tests with fosmidomycin-derived prodrugs, this point was quite important, given all the effort put into the generation of the cell lines. Schmittet al.104 reported that the antibiotics kanamycin, hygromycin and cefotaxim caused a DNA hypermethylation at CpG sites in the genome of tobacco plants grownin vitro, as shown by the SssI methylase accepting assays and genomic sequencing with sodium bisulfite. Interestingly, these methylations occurred in a time and dose-dependent manner and were not reversed when the progeny was not grown anymore in the presence of the antibiotics105.

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