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Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

Mahen R, Koch B, Wachsmuth M, Politi AZ, Perez-Gonzalez A, Mergenthaler J, Cai Y, Ellenberg J - Mol. Biol. Cell (2014)

Bottom Line: Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells.Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B.We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

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Overexpression alters AURKB-GFP biophysical properties on chromatin and in the cytosol. (A) Schematic of FCS-calibrated imaging. Concentration is determined in one location using FCS, and then an image is taken of the same cell. The image is converted to absolute concentrations using the FCS calibration. (B) Absolute concentration maps of AURKB-GFP in metaphase cells. The line profiles were taken in the areas depicted by the white arrows in the image directly above. Scale bar, 7 μm. (C) AURKB-GFP concentration on the metaphase plate. DNA was automatically identified based on a threshold of Hoechst staining of DNA (white line). Scale bar, 10 μm. (D) Fraction of AURKB-GFP proteins bound on chromatin. Horizontal bar shows the mean. (E) Fraction of chromatin bound AURKB-GFP (dots) as a function of the total number of AURKB proteins. The line is the equilibrium solution of a mass action model of reversible AURKB chromatin binding (see Materials and Methods; total number of chromatin-binding sites CT = 147,000, Kd = 5.21 nM). (F) AURKB-GFP diffusion coefficient from FCS data of >56 cells/sample from four experiments. (G) Single-cell-tracking automated FCS and time-lapse microscopy of AURKB-GFP diffusional mobility through the cell cycle as described in Materials and Methods. Plotted are the median and interquartile range from 9–45 cells. Scale bar, 10 μm. Significance testing by Mann–Whitney test.
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Figure 3: Overexpression alters AURKB-GFP biophysical properties on chromatin and in the cytosol. (A) Schematic of FCS-calibrated imaging. Concentration is determined in one location using FCS, and then an image is taken of the same cell. The image is converted to absolute concentrations using the FCS calibration. (B) Absolute concentration maps of AURKB-GFP in metaphase cells. The line profiles were taken in the areas depicted by the white arrows in the image directly above. Scale bar, 7 μm. (C) AURKB-GFP concentration on the metaphase plate. DNA was automatically identified based on a threshold of Hoechst staining of DNA (white line). Scale bar, 10 μm. (D) Fraction of AURKB-GFP proteins bound on chromatin. Horizontal bar shows the mean. (E) Fraction of chromatin bound AURKB-GFP (dots) as a function of the total number of AURKB proteins. The line is the equilibrium solution of a mass action model of reversible AURKB chromatin binding (see Materials and Methods; total number of chromatin-binding sites CT = 147,000, Kd = 5.21 nM). (F) AURKB-GFP diffusion coefficient from FCS data of >56 cells/sample from four experiments. (G) Single-cell-tracking automated FCS and time-lapse microscopy of AURKB-GFP diffusional mobility through the cell cycle as described in Materials and Methods. Plotted are the median and interquartile range from 9–45 cells. Scale bar, 10 μm. Significance testing by Mann–Whitney test.

Mentions: AURKB plays several critical roles in genome segregation via the phosphorylation of substrates on chromatin such as core histones and kinetochore proteins and by forming phosphorylation gradients (Tanaka et al., 2002; Wang et al., 2011; Carmena et al., 2012). The balance between soluble cytoplasmic and chromosome-bound kinase is therefore key to interpreting its function. To determine whether the transgene method would perturb this balance, we quantified the amount of AURKB-GFP localized to chromosomes on the metaphase plate using FCS-calibrated confocal imaging in live, dividing cells. Here first we recorded an FCS trace in the cytoplasm to determine the absolute local concentration of GFP and calibrate the corresponding confocal pixel intensity, which thus allows us to transform the entire confocal image into a concentration map (Figure 3A). Despite the different total cell expression levels, BACs showed a similar chromatin-bound AURKB-GFP concentration to genome-edited cells, whereas cDNAs were very variable (Figure 3, B and C). By combining our concentration measurements with three-dimensional reconstructed volumes of mitotic chromosomes and cytoplasm, we obtained the total number of chromatin-bound and free AURKB-GFP proteins. We found that the fraction of AURKB-GFP bound to chromatin is significantly higher in genome-edited cells than in plasmid-based systems (Figure 3D). Whereas in genome-edited cells, ∼75% of total cellular AURKB-GFP resides on chromatin, only 40-50% does so in plasmid-based systems, with the fraction decreasing as the total amount of AURKB increases. This relationship is in agreement with a mathematical model (solid line in Figure 3E; see Materials and Methods) that has a limited number of high-affinity binding sites on chromatin (∼130,000–150,000 molecules), with a binding Kd of 3.4–5.8 nM. Thus, our results suggest that chromatin-binding sites become saturated in overexpression systems, forcing aberrant accumulation of the fusion protein in the cytosol.


Comparative assessment of fluorescent transgene methods for quantitative imaging in human cells.

Mahen R, Koch B, Wachsmuth M, Politi AZ, Perez-Gonzalez A, Mergenthaler J, Cai Y, Ellenberg J - Mol. Biol. Cell (2014)

Overexpression alters AURKB-GFP biophysical properties on chromatin and in the cytosol. (A) Schematic of FCS-calibrated imaging. Concentration is determined in one location using FCS, and then an image is taken of the same cell. The image is converted to absolute concentrations using the FCS calibration. (B) Absolute concentration maps of AURKB-GFP in metaphase cells. The line profiles were taken in the areas depicted by the white arrows in the image directly above. Scale bar, 7 μm. (C) AURKB-GFP concentration on the metaphase plate. DNA was automatically identified based on a threshold of Hoechst staining of DNA (white line). Scale bar, 10 μm. (D) Fraction of AURKB-GFP proteins bound on chromatin. Horizontal bar shows the mean. (E) Fraction of chromatin bound AURKB-GFP (dots) as a function of the total number of AURKB proteins. The line is the equilibrium solution of a mass action model of reversible AURKB chromatin binding (see Materials and Methods; total number of chromatin-binding sites CT = 147,000, Kd = 5.21 nM). (F) AURKB-GFP diffusion coefficient from FCS data of >56 cells/sample from four experiments. (G) Single-cell-tracking automated FCS and time-lapse microscopy of AURKB-GFP diffusional mobility through the cell cycle as described in Materials and Methods. Plotted are the median and interquartile range from 9–45 cells. Scale bar, 10 μm. Significance testing by Mann–Whitney test.
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Related In: Results  -  Collection

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Figure 3: Overexpression alters AURKB-GFP biophysical properties on chromatin and in the cytosol. (A) Schematic of FCS-calibrated imaging. Concentration is determined in one location using FCS, and then an image is taken of the same cell. The image is converted to absolute concentrations using the FCS calibration. (B) Absolute concentration maps of AURKB-GFP in metaphase cells. The line profiles were taken in the areas depicted by the white arrows in the image directly above. Scale bar, 7 μm. (C) AURKB-GFP concentration on the metaphase plate. DNA was automatically identified based on a threshold of Hoechst staining of DNA (white line). Scale bar, 10 μm. (D) Fraction of AURKB-GFP proteins bound on chromatin. Horizontal bar shows the mean. (E) Fraction of chromatin bound AURKB-GFP (dots) as a function of the total number of AURKB proteins. The line is the equilibrium solution of a mass action model of reversible AURKB chromatin binding (see Materials and Methods; total number of chromatin-binding sites CT = 147,000, Kd = 5.21 nM). (F) AURKB-GFP diffusion coefficient from FCS data of >56 cells/sample from four experiments. (G) Single-cell-tracking automated FCS and time-lapse microscopy of AURKB-GFP diffusional mobility through the cell cycle as described in Materials and Methods. Plotted are the median and interquartile range from 9–45 cells. Scale bar, 10 μm. Significance testing by Mann–Whitney test.
Mentions: AURKB plays several critical roles in genome segregation via the phosphorylation of substrates on chromatin such as core histones and kinetochore proteins and by forming phosphorylation gradients (Tanaka et al., 2002; Wang et al., 2011; Carmena et al., 2012). The balance between soluble cytoplasmic and chromosome-bound kinase is therefore key to interpreting its function. To determine whether the transgene method would perturb this balance, we quantified the amount of AURKB-GFP localized to chromosomes on the metaphase plate using FCS-calibrated confocal imaging in live, dividing cells. Here first we recorded an FCS trace in the cytoplasm to determine the absolute local concentration of GFP and calibrate the corresponding confocal pixel intensity, which thus allows us to transform the entire confocal image into a concentration map (Figure 3A). Despite the different total cell expression levels, BACs showed a similar chromatin-bound AURKB-GFP concentration to genome-edited cells, whereas cDNAs were very variable (Figure 3, B and C). By combining our concentration measurements with three-dimensional reconstructed volumes of mitotic chromosomes and cytoplasm, we obtained the total number of chromatin-bound and free AURKB-GFP proteins. We found that the fraction of AURKB-GFP bound to chromatin is significantly higher in genome-edited cells than in plasmid-based systems (Figure 3D). Whereas in genome-edited cells, ∼75% of total cellular AURKB-GFP resides on chromatin, only 40-50% does so in plasmid-based systems, with the fraction decreasing as the total amount of AURKB increases. This relationship is in agreement with a mathematical model (solid line in Figure 3E; see Materials and Methods) that has a limited number of high-affinity binding sites on chromatin (∼130,000–150,000 molecules), with a binding Kd of 3.4–5.8 nM. Thus, our results suggest that chromatin-binding sites become saturated in overexpression systems, forcing aberrant accumulation of the fusion protein in the cytosol.

Bottom Line: Fluorescence tagging of proteins is a widely used tool to study protein function and dynamics in live cells.Here we use quantitative live-cell imaging and single-molecule spectroscopy to analyze how different transgene systems affect imaging of the functional properties of the mitotic kinase Aurora B.We show that the transgene method fundamentally influences level and variability of expression and can severely compromise the ability to report on endogenous binding and localization parameters, providing a guide for quantitative imaging studies in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, 69117 Heidelberg, Germany.

Show MeSH
Related in: MedlinePlus