Limits...
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

Two different clonal selections of the double-transformed cell line N-20.The most promising cell line N-20 was re-selected in order to obtain a performing cell line. The resulting cell lines (N20–22 and N20-25) are characterized by a high ratio of bright fluorescent cells (>95%). Images were taken as described in the legend toFigure 5. Cells were induced for 24 h with 10 µM Dex and 6 µM Est (final concentrations). White bars represent 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4536634&req=5

f8: Two different clonal selections of the double-transformed cell line N-20.The most promising cell line N-20 was re-selected in order to obtain a performing cell line. The resulting cell lines (N20–22 and N20-25) are characterized by a high ratio of bright fluorescent cells (>95%). Images were taken as described in the legend toFigure 5. Cells were induced for 24 h with 10 µM Dex and 6 µM Est (final concentrations). White bars represent 50 µm.

Mentions: Cloning of transgenic BY-2 cells: generation of a cell line with strongly reduced heterogeneity. Generation of a performing double-transformed cell line was a process of constant engineering of the initial GFP-BD-CVIL BY-2 cell line in order to obtain a clonal selection of cells responding appropriately to different stimuli. In the course of this procedure, cloning of primary heterogeneous suspensions generated secondary homogenous lines. The resulting calli and suspensions derived thereof were both screened and evaluated by fluorescence. In this context it is important to consider that the terms “homogenous” and “heterogeneous” do not describe the intensity of the fluorescence, but rather if a given culture showed well-balanced and stable fluorescence within the cell population. To achieve this goal, the most promising double-fluorescent cell line (N-20) was chosen for a rigorous re-selection process. Two liquid cultures started from these secondary calli showed bright fluorescence in both channels as well as a high ratio of fluorescent cells (>95%) and were maintained for further experiments (Figure 8). Nevertheless, it proved to be a time-consuming challenge, as within cell suspensions of supposedly clonal origin (primary suspensions derived from primary calli), important variations were regularly observed. These variations not only concerned the morphology of the cells, but also more importantly the homogeneity and intensity levels of the fluorescence. Therefore, a major focus was to re-select homogenous transgenic cell lines with high intensity levels of fluorescence. A relatively simple method to generate more homogenous cell lines, derived from secondary calli, was established and constantly improved over time, leading to the final protocol summarized inTable 2 together with a comparison of a procedure recently published by Nocarova and Fischer103.


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)

Two different clonal selections of the double-transformed cell line N-20.The most promising cell line N-20 was re-selected in order to obtain a performing cell line. The resulting cell lines (N20–22 and N20-25) are characterized by a high ratio of bright fluorescent cells (>95%). Images were taken as described in the legend toFigure 5. Cells were induced for 24 h with 10 µM Dex and 6 µM Est (final concentrations). White bars represent 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Two different clonal selections of the double-transformed cell line N-20.The most promising cell line N-20 was re-selected in order to obtain a performing cell line. The resulting cell lines (N20–22 and N20-25) are characterized by a high ratio of bright fluorescent cells (>95%). Images were taken as described in the legend toFigure 5. Cells were induced for 24 h with 10 µM Dex and 6 µM Est (final concentrations). White bars represent 50 µm.
Mentions: Cloning of transgenic BY-2 cells: generation of a cell line with strongly reduced heterogeneity. Generation of a performing double-transformed cell line was a process of constant engineering of the initial GFP-BD-CVIL BY-2 cell line in order to obtain a clonal selection of cells responding appropriately to different stimuli. In the course of this procedure, cloning of primary heterogeneous suspensions generated secondary homogenous lines. The resulting calli and suspensions derived thereof were both screened and evaluated by fluorescence. In this context it is important to consider that the terms “homogenous” and “heterogeneous” do not describe the intensity of the fluorescence, but rather if a given culture showed well-balanced and stable fluorescence within the cell population. To achieve this goal, the most promising double-fluorescent cell line (N-20) was chosen for a rigorous re-selection process. Two liquid cultures started from these secondary calli showed bright fluorescence in both channels as well as a high ratio of fluorescent cells (>95%) and were maintained for further experiments (Figure 8). Nevertheless, it proved to be a time-consuming challenge, as within cell suspensions of supposedly clonal origin (primary suspensions derived from primary calli), important variations were regularly observed. These variations not only concerned the morphology of the cells, but also more importantly the homogeneity and intensity levels of the fluorescence. Therefore, a major focus was to re-select homogenous transgenic cell lines with high intensity levels of fluorescence. A relatively simple method to generate more homogenous cell lines, derived from secondary calli, was established and constantly improved over time, leading to the final protocol summarized inTable 2 together with a comparison of a procedure recently published by Nocarova and Fischer103.

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