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

Influence of different shaking conditions.Shown is the expression of both fluorescent reporter proteins in transgenic tobacco BY-2 cells incubated over-night in the wells of a 96-well microtiter plate. A 7-day-old culture was diluted tenfold into a fresh culture medium before the inducers Dex (10 µM) and Est (5 µM) were added. Afterwards 200 µl of the cell suspension were added to the wells of a 96-well glass-bottom microtiter plate and shaken under different conditions (150 rpm and 320 rpm, respectively) before being examined by fluorescence microscopy as described in previous Figure legends. White bars = 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f15: Influence of different shaking conditions.Shown is the expression of both fluorescent reporter proteins in transgenic tobacco BY-2 cells incubated over-night in the wells of a 96-well microtiter plate. A 7-day-old culture was diluted tenfold into a fresh culture medium before the inducers Dex (10 µM) and Est (5 µM) were added. Afterwards 200 µl of the cell suspension were added to the wells of a 96-well glass-bottom microtiter plate and shaken under different conditions (150 rpm and 320 rpm, respectively) before being examined by fluorescence microscopy as described in previous Figure legends. White bars = 10 µm.

Mentions: We examined the influence of suboptimal agitation in this small-scale system (volume 100 to 200 µl) on the expression of the reporter proteins in several independent experiments in which different shaking conditions for the BY-2 cultures were tested. Therefore, 7-day-old cells were diluted (1:10) into fresh BY-2 medium and then induced by the addition of 10 µM Dex and 5 µM Est. Then 200 µl of this dilution was transferred into the wells of a 96-well plate (conventional round-shaped wells, with conical bottom) and incubated for 20 h in the dark under permanent shaking (160 rpm or 320 rpm). Cells that were shaken at 160 rpm (which corresponds to the shaking frequency of 6-well plates and culture flasks) showed a normal induction of the GFP fusion protein, whereas the mRFP fusion protein was barely expressed. However, in cells that were cultivated at 320 rpm, the expression of the NLS-mRFP protein could clearly be detected by fluorescence microscopy (Figure 15). The gas-liquid mass transfer properties of shaken 96-well plates have been investigated in detail by Hermannet al.127) and revealed that the oxygen transfer rate (OTR) measured in the wells was strongly influenced by different parameters, such as the surface tension of the medium, the material of the well, the filling volume and the shape of the well. In round-shaped wells, for example, due to the high surface tension, no liquid movement occurred until a critical shaking intensity was reached: for 200 µl of water shaken at shaking diameter of 25 mm, the rate had to exceed 300 rpm. On the other hand, frequencies above 450 rpm could not be used without the risk of the liquid spilling out of the well127. As a general rule, one can say that the OTR increases proportionally with shaking amplitude and frequency due to an increase in the total surface that is available for oxygen (gas) transfer. The same effect was observed by replacing round-shape wells by rectangular or square wells, which can be explained by the increase of the turbulence of the system due to the effect of the corners. A higher fill volume on the other hand decreases the oxygen transfer rate if all other parameters are kept constant127–129. No agitation of the fluid (diluted cells in BY-2 medium) was observed for 250 µl until around 300 rpm (using a Heidolph unimax 1010 shaker, 10 mm). Therefore a frequency of 320 rpm and higher was used. However the limitation for further testing was the maximum speed of the available shaker (500 rpm). In addition, these results were obtained by using wells with a conical bottom. The use of an inverted microscope required 96-wells with a flat-bottom for the imaging process, and we found that the hydrodynamic behavior of a BY-2 culture in a flat-bottomed well differs significantly from a conventional deep-well. Preliminary results indicate that the speed has to exceed 500 rpm to assure an optimum agitation of the cells for a filling volume of 200 µl. This result prompted us to purchase 96-well glass-bottom plates with a square-shaped cross-section area/ground profile. Besides increasing the OTR at lower shaking frequencies compared to round wells, it should also confer an additional advantage to the read-out process by significantly increasing the total surface area of the well.


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)

Influence of different shaking conditions.Shown is the expression of both fluorescent reporter proteins in transgenic tobacco BY-2 cells incubated over-night in the wells of a 96-well microtiter plate. A 7-day-old culture was diluted tenfold into a fresh culture medium before the inducers Dex (10 µM) and Est (5 µM) were added. Afterwards 200 µl of the cell suspension were added to the wells of a 96-well glass-bottom microtiter plate and shaken under different conditions (150 rpm and 320 rpm, respectively) before being examined by fluorescence microscopy as described in previous Figure legends. White bars = 10 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f15: Influence of different shaking conditions.Shown is the expression of both fluorescent reporter proteins in transgenic tobacco BY-2 cells incubated over-night in the wells of a 96-well microtiter plate. A 7-day-old culture was diluted tenfold into a fresh culture medium before the inducers Dex (10 µM) and Est (5 µM) were added. Afterwards 200 µl of the cell suspension were added to the wells of a 96-well glass-bottom microtiter plate and shaken under different conditions (150 rpm and 320 rpm, respectively) before being examined by fluorescence microscopy as described in previous Figure legends. White bars = 10 µm.
Mentions: We examined the influence of suboptimal agitation in this small-scale system (volume 100 to 200 µl) on the expression of the reporter proteins in several independent experiments in which different shaking conditions for the BY-2 cultures were tested. Therefore, 7-day-old cells were diluted (1:10) into fresh BY-2 medium and then induced by the addition of 10 µM Dex and 5 µM Est. Then 200 µl of this dilution was transferred into the wells of a 96-well plate (conventional round-shaped wells, with conical bottom) and incubated for 20 h in the dark under permanent shaking (160 rpm or 320 rpm). Cells that were shaken at 160 rpm (which corresponds to the shaking frequency of 6-well plates and culture flasks) showed a normal induction of the GFP fusion protein, whereas the mRFP fusion protein was barely expressed. However, in cells that were cultivated at 320 rpm, the expression of the NLS-mRFP protein could clearly be detected by fluorescence microscopy (Figure 15). The gas-liquid mass transfer properties of shaken 96-well plates have been investigated in detail by Hermannet al.127) and revealed that the oxygen transfer rate (OTR) measured in the wells was strongly influenced by different parameters, such as the surface tension of the medium, the material of the well, the filling volume and the shape of the well. In round-shaped wells, for example, due to the high surface tension, no liquid movement occurred until a critical shaking intensity was reached: for 200 µl of water shaken at shaking diameter of 25 mm, the rate had to exceed 300 rpm. On the other hand, frequencies above 450 rpm could not be used without the risk of the liquid spilling out of the well127. As a general rule, one can say that the OTR increases proportionally with shaking amplitude and frequency due to an increase in the total surface that is available for oxygen (gas) transfer. The same effect was observed by replacing round-shape wells by rectangular or square wells, which can be explained by the increase of the turbulence of the system due to the effect of the corners. A higher fill volume on the other hand decreases the oxygen transfer rate if all other parameters are kept constant127–129. No agitation of the fluid (diluted cells in BY-2 medium) was observed for 250 µl until around 300 rpm (using a Heidolph unimax 1010 shaker, 10 mm). Therefore a frequency of 320 rpm and higher was used. However the limitation for further testing was the maximum speed of the available shaker (500 rpm). In addition, these results were obtained by using wells with a conical bottom. The use of an inverted microscope required 96-wells with a flat-bottom for the imaging process, and we found that the hydrodynamic behavior of a BY-2 culture in a flat-bottomed well differs significantly from a conventional deep-well. Preliminary results indicate that the speed has to exceed 500 rpm to assure an optimum agitation of the cells for a filling volume of 200 µl. This result prompted us to purchase 96-well glass-bottom plates with a square-shaped cross-section area/ground profile. Besides increasing the OTR at lower shaking frequencies compared to round wells, it should also confer an additional advantage to the read-out process by significantly increasing the total surface area of the well.

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