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

Correlation between the focal plane and the strongest signals in the red channel.Images are subsequently taken at different levels of the sample (Z-stack). The optimum sharpness of signals for RFP indicates the focus being set on nuclei. The determination of the sharpest image for the GFP signal provides an integrated optimum for the analysis of doubly transformed BY-2 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f13: Correlation between the focal plane and the strongest signals in the red channel.Images are subsequently taken at different levels of the sample (Z-stack). The optimum sharpness of signals for RFP indicates the focus being set on nuclei. The determination of the sharpest image for the GFP signal provides an integrated optimum for the analysis of doubly transformed BY-2 cells.

Mentions: To confirm these early observations, a series of multichannel images of BY-2 cells (expressing both fluorescent proteins) spanning a total distance of 50 µm in the Z-plane was acquired at different focal planes. Afterwards, each optical slice of this Z-stack was analyzed using ImageJ software (Figure 13). The images of the green channel were analyzed by the edge-finding algorithm of ImageJ, whereas the integrated density was calculated for the red channel. The results clearly show that, for the green channel, the sharpest image (as perceived by a human observer) of the Z-stack (identified by the edge-finding algorithm of ImageJ) is also the image with the highest integrated density in the red channel, which is defined as “the sum of the values of the pixels in an image or selection” (ImageJ online manual). This correlates very well with general observations about fluorescence images that indicate a maximum image contrast at the Z-stage height corresponding to the focal position122,123. Therefore, a fluorescence-based autofocusing approach could use the nucleus-located maximum of red fluorescence to define the plane of focus and acquire additional pictures at an offset from this position (in both directions). This vertical series of images could then be summed up into a single projection or used to choose the best focal plane for each fluorophore. In an optimal scenario, two different images in different focal planes should be taken, when working with different fluorophores/wavelengths, due to the chromatic aberration of optical lenses (objectives), which means that different colors/wavelength of light are focused to different points124.


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)

Correlation between the focal plane and the strongest signals in the red channel.Images are subsequently taken at different levels of the sample (Z-stack). The optimum sharpness of signals for RFP indicates the focus being set on nuclei. The determination of the sharpest image for the GFP signal provides an integrated optimum for the analysis of doubly transformed BY-2 cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f13: Correlation between the focal plane and the strongest signals in the red channel.Images are subsequently taken at different levels of the sample (Z-stack). The optimum sharpness of signals for RFP indicates the focus being set on nuclei. The determination of the sharpest image for the GFP signal provides an integrated optimum for the analysis of doubly transformed BY-2 cells.
Mentions: To confirm these early observations, a series of multichannel images of BY-2 cells (expressing both fluorescent proteins) spanning a total distance of 50 µm in the Z-plane was acquired at different focal planes. Afterwards, each optical slice of this Z-stack was analyzed using ImageJ software (Figure 13). The images of the green channel were analyzed by the edge-finding algorithm of ImageJ, whereas the integrated density was calculated for the red channel. The results clearly show that, for the green channel, the sharpest image (as perceived by a human observer) of the Z-stack (identified by the edge-finding algorithm of ImageJ) is also the image with the highest integrated density in the red channel, which is defined as “the sum of the values of the pixels in an image or selection” (ImageJ online manual). This correlates very well with general observations about fluorescence images that indicate a maximum image contrast at the Z-stage height corresponding to the focal position122,123. Therefore, a fluorescence-based autofocusing approach could use the nucleus-located maximum of red fluorescence to define the plane of focus and acquire additional pictures at an offset from this position (in both directions). This vertical series of images could then be summed up into a single projection or used to choose the best focal plane for each fluorophore. In an optimal scenario, two different images in different focal planes should be taken, when working with different fluorophores/wavelengths, due to the chromatic aberration of optical lenses (objectives), which means that different colors/wavelength of light are focused to different points124.

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