Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9]
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 calmodu...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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F1000 Research Ltd
2015-08-01
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| Series: | F1000Research |
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| Online Access: | http://f1000research.com/articles/4-14/v2 |
| _version_ | 1829483960196923392 |
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| author | Michael Hartmann Elisabet Gas-Pascual Andrea Hemmerlin Michel Rohmer Thomas J. Bach |
| author_facet | Michael Hartmann Elisabet Gas-Pascual Andrea Hemmerlin Michel Rohmer Thomas J. Bach |
| author_sort | Michael Hartmann |
| collection | DOAJ |
| description | 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. |
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| issn | 2046-1402 |
| language | English |
| last_indexed | 2024-12-14T22:18:10Z |
| publishDate | 2015-08-01 |
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| series | F1000Research |
| spelling | doaj.art-edeb638bcd3d45ffb5d72be4f27e79b32022-12-21T22:45:34ZengF1000 Research LtdF1000Research2046-14022015-08-01410.12688/f1000research.5923.27389Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9]Michael Hartmann0Elisabet Gas-Pascual1Andrea Hemmerlin2Michel Rohmer3Thomas J. Bach4Current address: Department Biologie, Institut für Molekulare Ökophysiologie der Pflanzen, Universität Düsseldorf, Universitätsstr. 1, D-40225, Düsseldorf, GermanyCurrent address: Horticulture and Crop Science, Ohio State University, 208 Williams Hall, 1680 Madison Avenue, Wooster, OH, 44691, USADépartement “Réseaux Métaboliques, Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357, Université de Strasbourg, 28 rue Goethe, F-67083 Strasbourg, FranceUMR 7177 CNRS/Université de Strasbourg, Institut Le Bel, 4 rue Blaise Pascal, F-67070 Strasbourg, FranceDépartement “Réseaux Métaboliques, Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357, Université de Strasbourg, 28 rue Goethe, F-67083 Strasbourg, FranceIn 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.http://f1000research.com/articles/4-14/v2Agriculture & BiotechnologyDrug Discovery & DesignPlant Biochemistry & PhysiologyPlant Cell Biology |
| spellingShingle | Michael Hartmann Elisabet Gas-Pascual Andrea Hemmerlin Michel Rohmer Thomas J. Bach Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9] F1000Research Agriculture & Biotechnology Drug Discovery & Design Plant Biochemistry & Physiology Plant Cell Biology |
| title | Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9] |
| title_full | Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9] |
| title_fullStr | Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9] |
| title_full_unstemmed | Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9] |
| title_short | Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation [v2; ref status: indexed, http://f1000r.es/5p9] |
| title_sort | development of an image based screening system for inhibitors of the plastidial mep pathway and of protein geranylgeranylation v2 ref status indexed http f1000r es 5p9 |
| topic | Agriculture & Biotechnology Drug Discovery & Design Plant Biochemistry & Physiology Plant Cell Biology |
| url | http://f1000research.com/articles/4-14/v2 |
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