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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

Confocal images indicating the unusual distribution of H6-GFP-BD-CVIL in response to a prenylated anthracenoid derived from an African medicinal plant.On the left side as controls cells without and with oxoclomazone (OC) treatment showing plasma membrane (PM) localization or its mislocalization to the nucleus (N), especially in nucleoli (Nu). On the right side cells are depicted that have been treated with an inhibitor that has shown a high efficiency in killing protozoanLeishmania cells, responsible of leishmaniasis. The H6-GFP-BD-CVIL is mainly accumulated in the cytoplasm (Cyt) that is for instance surrounding the nucleus. At the higher magnification some punctuate structures become visible. That might result from coagulated cytoplasm typical of cell death. White bars in the combined images represent 20 µm.
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f18: Confocal images indicating the unusual distribution of H6-GFP-BD-CVIL in response to a prenylated anthracenoid derived from an African medicinal plant.On the left side as controls cells without and with oxoclomazone (OC) treatment showing plasma membrane (PM) localization or its mislocalization to the nucleus (N), especially in nucleoli (Nu). On the right side cells are depicted that have been treated with an inhibitor that has shown a high efficiency in killing protozoanLeishmania cells, responsible of leishmaniasis. The H6-GFP-BD-CVIL is mainly accumulated in the cytoplasm (Cyt) that is for instance surrounding the nucleus. At the higher magnification some punctuate structures become visible. That might result from coagulated cytoplasm typical of cell death. White bars in the combined images represent 20 µm.

Mentions: The bioassay may also be very useful to screen unknown chemical compounds with cytotoxic properties. By testing potential inhibitors derived from plant extracts of theClusiaceae family that are traditionally used for the treatment of parasitic diseases in Cameroon, we made an interesting observation (Figure 18). Whereas none of the compounds was efficiently inducing a mislocalization of the fusion protein to the nucleus (compare OC treatment), several treatments triggered toxic effects. One compound in particular displayed an unusual phenotype, with barely detectable signals from the plasma membrane (PM) and nucleus (N/Nu); instead, the GFP fluorescence seemed to be trapped in the cytoplasm (Cyt). Interestingly, this compound was identified as a prenylated anthracenoid, isolated fromPsorospermum glaberrium (Clusiaceae) found in Cameroon, and shown to have the highest activity in tests against leishmaniasis169, further validating the experimental system as a tool to detect cytotoxic effects. This could be an indication that the transport of H6-GFP-BD-CVIL might be mediated by protein-protein interactions, which are impaired by the prenylated compound.


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)

Confocal images indicating the unusual distribution of H6-GFP-BD-CVIL in response to a prenylated anthracenoid derived from an African medicinal plant.On the left side as controls cells without and with oxoclomazone (OC) treatment showing plasma membrane (PM) localization or its mislocalization to the nucleus (N), especially in nucleoli (Nu). On the right side cells are depicted that have been treated with an inhibitor that has shown a high efficiency in killing protozoanLeishmania cells, responsible of leishmaniasis. The H6-GFP-BD-CVIL is mainly accumulated in the cytoplasm (Cyt) that is for instance surrounding the nucleus. At the higher magnification some punctuate structures become visible. That might result from coagulated cytoplasm typical of cell death. White bars in the combined images represent 20 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f18: Confocal images indicating the unusual distribution of H6-GFP-BD-CVIL in response to a prenylated anthracenoid derived from an African medicinal plant.On the left side as controls cells without and with oxoclomazone (OC) treatment showing plasma membrane (PM) localization or its mislocalization to the nucleus (N), especially in nucleoli (Nu). On the right side cells are depicted that have been treated with an inhibitor that has shown a high efficiency in killing protozoanLeishmania cells, responsible of leishmaniasis. The H6-GFP-BD-CVIL is mainly accumulated in the cytoplasm (Cyt) that is for instance surrounding the nucleus. At the higher magnification some punctuate structures become visible. That might result from coagulated cytoplasm typical of cell death. White bars in the combined images represent 20 µm.
Mentions: The bioassay may also be very useful to screen unknown chemical compounds with cytotoxic properties. By testing potential inhibitors derived from plant extracts of theClusiaceae family that are traditionally used for the treatment of parasitic diseases in Cameroon, we made an interesting observation (Figure 18). Whereas none of the compounds was efficiently inducing a mislocalization of the fusion protein to the nucleus (compare OC treatment), several treatments triggered toxic effects. One compound in particular displayed an unusual phenotype, with barely detectable signals from the plasma membrane (PM) and nucleus (N/Nu); instead, the GFP fluorescence seemed to be trapped in the cytoplasm (Cyt). Interestingly, this compound was identified as a prenylated anthracenoid, isolated fromPsorospermum glaberrium (Clusiaceae) found in Cameroon, and shown to have the highest activity in tests against leishmaniasis169, further validating the experimental system as a tool to detect cytotoxic effects. This could be an indication that the transport of H6-GFP-BD-CVIL might be mediated by protein-protein interactions, which are impaired by the prenylated compound.

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