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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

DXR and HMGR gene silencing via inducible amiRNA in BY-2 cells.Two Nicotiana tabacum BY-2 lines, independently transformed with inducible amiRNA constructs against DXR (DXR 1.4 and DXR 3.12) or HMGR (HMGR 3 and HMGR 5) were treated 1 day after sub-culture with (+) or without (-) 5 μM estradiol. Control lines were also treated with FOS or MEV. Gene expression levels for DXR (A), HMGR1 and HMGR2 (B) were measured by qPCR after 24 h of treatment. Experiments and measurements were repeated three times. Statistically significant changes are indicated (asterisk, p-values<0.05). Changes in expression in a BY-2 control line (transformed with the empty vector, pER10.1) are also shown.
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f11: DXR and HMGR gene silencing via inducible amiRNA in BY-2 cells.Two Nicotiana tabacum BY-2 lines, independently transformed with inducible amiRNA constructs against DXR (DXR 1.4 and DXR 3.12) or HMGR (HMGR 3 and HMGR 5) were treated 1 day after sub-culture with (+) or without (-) 5 μM estradiol. Control lines were also treated with FOS or MEV. Gene expression levels for DXR (A), HMGR1 and HMGR2 (B) were measured by qPCR after 24 h of treatment. Experiments and measurements were repeated three times. Statistically significant changes are indicated (asterisk, p-values<0.05). Changes in expression in a BY-2 control line (transformed with the empty vector, pER10.1) are also shown.

Mentions: As a further proof of concept, different cell lines were generated, targetingHMGR (MVA pathway) andDXR genes (MEP pathway) with an artificial micro-RNA (amiRNA) strategy118 in order to confirm the effects of the inhibitors on their target proteins on a biological level (Figure 10). DXR-silenced cells exhibited a phenotype similar to FOS and OC treatment, whereas the silencing of HMGR did not have any effect on the localization of GFP-BD-CVIL, similarly to what happens with MEV addition. Expression levels forDXR andHMGR were tested and, as expected, lines treated with FOS showed increased expression ofDXR whereas lines treated with MEV showedHMGR overexpression. When gene expression was assayed in amiRNA lines, in both cases, the levels of silencing reached were not very high (10–15% for DXR and 10–30% for HMGR,Figure 11) and were in the same line as levels achieved when silencing cycloartenol synthase, an enzyme involved in sterol biosynthesis119. These results could be explained by recent observations on the dependency of miRNA formation on the biosynthesis of sterols: Apparently the formation of repressive complexes with ARGONAUTE (AGO) proteins needs a membrane association in which sterols are functional120, as was shown with miRNA action-deficient (mad)Arabidopsis mutants 3 and 4.MAD3 encodes HMGR1, andMAD4 encodes sterol C-8 isomerase, catalyzing an important step in cytoplasmic sterol biosynthesis120. Thus, silencing of a gene coding for a key enzyme like HMGR or downstream in the pathway might be difficult to achieve beyond a rather low degree120.


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)

DXR and HMGR gene silencing via inducible amiRNA in BY-2 cells.Two Nicotiana tabacum BY-2 lines, independently transformed with inducible amiRNA constructs against DXR (DXR 1.4 and DXR 3.12) or HMGR (HMGR 3 and HMGR 5) were treated 1 day after sub-culture with (+) or without (-) 5 μM estradiol. Control lines were also treated with FOS or MEV. Gene expression levels for DXR (A), HMGR1 and HMGR2 (B) were measured by qPCR after 24 h of treatment. Experiments and measurements were repeated three times. Statistically significant changes are indicated (asterisk, p-values<0.05). Changes in expression in a BY-2 control line (transformed with the empty vector, pER10.1) are also shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f11: DXR and HMGR gene silencing via inducible amiRNA in BY-2 cells.Two Nicotiana tabacum BY-2 lines, independently transformed with inducible amiRNA constructs against DXR (DXR 1.4 and DXR 3.12) or HMGR (HMGR 3 and HMGR 5) were treated 1 day after sub-culture with (+) or without (-) 5 μM estradiol. Control lines were also treated with FOS or MEV. Gene expression levels for DXR (A), HMGR1 and HMGR2 (B) were measured by qPCR after 24 h of treatment. Experiments and measurements were repeated three times. Statistically significant changes are indicated (asterisk, p-values<0.05). Changes in expression in a BY-2 control line (transformed with the empty vector, pER10.1) are also shown.
Mentions: As a further proof of concept, different cell lines were generated, targetingHMGR (MVA pathway) andDXR genes (MEP pathway) with an artificial micro-RNA (amiRNA) strategy118 in order to confirm the effects of the inhibitors on their target proteins on a biological level (Figure 10). DXR-silenced cells exhibited a phenotype similar to FOS and OC treatment, whereas the silencing of HMGR did not have any effect on the localization of GFP-BD-CVIL, similarly to what happens with MEV addition. Expression levels forDXR andHMGR were tested and, as expected, lines treated with FOS showed increased expression ofDXR whereas lines treated with MEV showedHMGR overexpression. When gene expression was assayed in amiRNA lines, in both cases, the levels of silencing reached were not very high (10–15% for DXR and 10–30% for HMGR,Figure 11) and were in the same line as levels achieved when silencing cycloartenol synthase, an enzyme involved in sterol biosynthesis119. These results could be explained by recent observations on the dependency of miRNA formation on the biosynthesis of sterols: Apparently the formation of repressive complexes with ARGONAUTE (AGO) proteins needs a membrane association in which sterols are functional120, as was shown with miRNA action-deficient (mad)Arabidopsis mutants 3 and 4.MAD3 encodes HMGR1, andMAD4 encodes sterol C-8 isomerase, catalyzing an important step in cytoplasmic sterol biosynthesis120. Thus, silencing of a gene coding for a key enzyme like HMGR or downstream in the pathway might be difficult to achieve beyond a rather low degree120.

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