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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

Automatic image acquisition from multiwell plates.Images from multiple locations can be taken using the “AutofocusScreen for LSM” macro, developed in collaboration between Carl Zeiss MicroImaging GMBH (Jena, Germany) and the group of Dr. Jan Ellenberg at the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany). It is freely downloadable athttp://www.zeiss.de/LSM-Macros. Well positions selected to be scanned can be defined by simply clicking check boxes. Steps X and Y define the distance between two acquisition locations. The exact position (here marked by a red cross) will be defined by the user, at the beginning of the image acquisition procedure. The user also has the option to define multiple tiles (with no or partial overlap to each other) around this position. The size of the scanned image will also be defined by the user and the scanning settings he chooses. Autofocus can be hardware- or cell-based, acquiring the emitted laser light or the emitted light of the sample respectively (the above shown image displays a cell-based scan across the Z-axis, using the green channel). Initial experiments with tobacco- BY-2 cells however showed that at multiple positions in the Z-axis above the glass bottom, a significant amount of cells could be visualized in the respective focal plane. This factor however will have to be adjusted for the scanning of multiple wells, as BY-2 cells will sediment quite fast (within minutes) to the bottom of the well, resulting in a significant change in the conditions and number of cells in the Z-axis.
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f16: Automatic image acquisition from multiwell plates.Images from multiple locations can be taken using the “AutofocusScreen for LSM” macro, developed in collaboration between Carl Zeiss MicroImaging GMBH (Jena, Germany) and the group of Dr. Jan Ellenberg at the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany). It is freely downloadable athttp://www.zeiss.de/LSM-Macros. Well positions selected to be scanned can be defined by simply clicking check boxes. Steps X and Y define the distance between two acquisition locations. The exact position (here marked by a red cross) will be defined by the user, at the beginning of the image acquisition procedure. The user also has the option to define multiple tiles (with no or partial overlap to each other) around this position. The size of the scanned image will also be defined by the user and the scanning settings he chooses. Autofocus can be hardware- or cell-based, acquiring the emitted laser light or the emitted light of the sample respectively (the above shown image displays a cell-based scan across the Z-axis, using the green channel). Initial experiments with tobacco- BY-2 cells however showed that at multiple positions in the Z-axis above the glass bottom, a significant amount of cells could be visualized in the respective focal plane. This factor however will have to be adjusted for the scanning of multiple wells, as BY-2 cells will sediment quite fast (within minutes) to the bottom of the well, resulting in a significant change in the conditions and number of cells in the Z-axis.

Mentions: A prerequisite for an image-based screening system is a certain degree of automatization as far as repetitive tasks are concerned. The use of theAutofocusScreen for LSM macro provided by Zeiss allows the automation of different steps of the image acquisition process. The first tests performed with the 96-well glass bottom plates indicated that all features could be used, including the autofocus routine and the automatic well-readout (Figure 16). However, in order to find the right balance between speed and image quality, the protocol still requires refinement and further validation before reproducible and exploitable data sets may be obtained.


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)

Automatic image acquisition from multiwell plates.Images from multiple locations can be taken using the “AutofocusScreen for LSM” macro, developed in collaboration between Carl Zeiss MicroImaging GMBH (Jena, Germany) and the group of Dr. Jan Ellenberg at the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany). It is freely downloadable athttp://www.zeiss.de/LSM-Macros. Well positions selected to be scanned can be defined by simply clicking check boxes. Steps X and Y define the distance between two acquisition locations. The exact position (here marked by a red cross) will be defined by the user, at the beginning of the image acquisition procedure. The user also has the option to define multiple tiles (with no or partial overlap to each other) around this position. The size of the scanned image will also be defined by the user and the scanning settings he chooses. Autofocus can be hardware- or cell-based, acquiring the emitted laser light or the emitted light of the sample respectively (the above shown image displays a cell-based scan across the Z-axis, using the green channel). Initial experiments with tobacco- BY-2 cells however showed that at multiple positions in the Z-axis above the glass bottom, a significant amount of cells could be visualized in the respective focal plane. This factor however will have to be adjusted for the scanning of multiple wells, as BY-2 cells will sediment quite fast (within minutes) to the bottom of the well, resulting in a significant change in the conditions and number of cells in the Z-axis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f16: Automatic image acquisition from multiwell plates.Images from multiple locations can be taken using the “AutofocusScreen for LSM” macro, developed in collaboration between Carl Zeiss MicroImaging GMBH (Jena, Germany) and the group of Dr. Jan Ellenberg at the European Molecular Biology Laboratory (EMBL, Heidelberg, Germany). It is freely downloadable athttp://www.zeiss.de/LSM-Macros. Well positions selected to be scanned can be defined by simply clicking check boxes. Steps X and Y define the distance between two acquisition locations. The exact position (here marked by a red cross) will be defined by the user, at the beginning of the image acquisition procedure. The user also has the option to define multiple tiles (with no or partial overlap to each other) around this position. The size of the scanned image will also be defined by the user and the scanning settings he chooses. Autofocus can be hardware- or cell-based, acquiring the emitted laser light or the emitted light of the sample respectively (the above shown image displays a cell-based scan across the Z-axis, using the green channel). Initial experiments with tobacco- BY-2 cells however showed that at multiple positions in the Z-axis above the glass bottom, a significant amount of cells could be visualized in the respective focal plane. This factor however will have to be adjusted for the scanning of multiple wells, as BY-2 cells will sediment quite fast (within minutes) to the bottom of the well, resulting in a significant change in the conditions and number of cells in the Z-axis.
Mentions: A prerequisite for an image-based screening system is a certain degree of automatization as far as repetitive tasks are concerned. The use of theAutofocusScreen for LSM macro provided by Zeiss allows the automation of different steps of the image acquisition process. The first tests performed with the 96-well glass bottom plates indicated that all features could be used, including the autofocus routine and the automatic well-readout (Figure 16). However, in order to find the right balance between speed and image quality, the protocol still requires refinement and further validation before reproducible and exploitable data sets may be obtained.

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