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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

Time course of induction for the NLS-mRFP.In vivo targeting of the NLS-mRFP fusion protein after induction with 6 µM Est (and in the presence of 10 µM Dex – not shown). The red fluorescent signals were examined at different time points after induction.A) Overlay of transmission microscopy images with the RFP channel.B) Same images as inA shown in LSM image browser rainbow mode (red indicates saturated areas). Saturation is already reached after 24 h of induction with both elicitors. All images were acquired with the same microscope settings (red channel, Carl Zeiss LSM510 microscope; EC-Plan-Neofluar, 10x/0,3 M27)
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f7: Time course of induction for the NLS-mRFP.In vivo targeting of the NLS-mRFP fusion protein after induction with 6 µM Est (and in the presence of 10 µM Dex – not shown). The red fluorescent signals were examined at different time points after induction.A) Overlay of transmission microscopy images with the RFP channel.B) Same images as inA shown in LSM image browser rainbow mode (red indicates saturated areas). Saturation is already reached after 24 h of induction with both elicitors. All images were acquired with the same microscope settings (red channel, Carl Zeiss LSM510 microscope; EC-Plan-Neofluar, 10x/0,3 M27)

Mentions: To determine the level of saturation, images were converted to a rainbow scale with the Zeiss LSM image browser. Red signals indicate saturation. As seen inFigure 7[A], nuclear localization of the NLS:mRFP protein could already be observed 3 h after induction. Nevertheless, it took at least 24 h until most of the signal arising from the nuclei was saturated (red dots,Figure 7[B]).


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)

Time course of induction for the NLS-mRFP.In vivo targeting of the NLS-mRFP fusion protein after induction with 6 µM Est (and in the presence of 10 µM Dex – not shown). The red fluorescent signals were examined at different time points after induction.A) Overlay of transmission microscopy images with the RFP channel.B) Same images as inA shown in LSM image browser rainbow mode (red indicates saturated areas). Saturation is already reached after 24 h of induction with both elicitors. All images were acquired with the same microscope settings (red channel, Carl Zeiss LSM510 microscope; EC-Plan-Neofluar, 10x/0,3 M27)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Time course of induction for the NLS-mRFP.In vivo targeting of the NLS-mRFP fusion protein after induction with 6 µM Est (and in the presence of 10 µM Dex – not shown). The red fluorescent signals were examined at different time points after induction.A) Overlay of transmission microscopy images with the RFP channel.B) Same images as inA shown in LSM image browser rainbow mode (red indicates saturated areas). Saturation is already reached after 24 h of induction with both elicitors. All images were acquired with the same microscope settings (red channel, Carl Zeiss LSM510 microscope; EC-Plan-Neofluar, 10x/0,3 M27)
Mentions: To determine the level of saturation, images were converted to a rainbow scale with the Zeiss LSM image browser. Red signals indicate saturation. As seen inFigure 7[A], nuclear localization of the NLS:mRFP protein could already be observed 3 h after induction. Nevertheless, it took at least 24 h until most of the signal arising from the nuclei was saturated (red dots,Figure 7[B]).

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