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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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Expression system determines AURKB-GFP levels. (A) Western blot comparison of AURKB levels in nocodazole-arrested cells. BAC-expressed AURKB-GFP runs at a higher molecular weight due to the presence of an S-peptide in addition to the EGFP tag. The graph shows the mean ± SD of three independent experiments. (B) Flow cytometry analysis of AURKB-GFP intensity in nocodazole-arrested cells. Coefficient of variation is from the whole population. GFP+ was defined as 3 SD above autofluorescence from wild-type cells. (C) Sum intensity confocal images of AURKB-GFP using the same imaging conditions throughout. One metaphase cell is shown per panel. Left to right: ZHZ2, ZHZ1, ZHET1, THET1, THET2, BP1, BC1, BP2, BC2, cDP1, cDC1, cDC2, and cDC3. Scale bar, 7 μm. (D) The amplitude of the FCS autocorrelation function (see Materials and Methods), G(0), is inversely proportional to particle number. Absolute protein concentration, c, is calculated using the measured confocal volume, V. (E) AURKB-GFP FCS autocorrelation curves from single representative cells. Top to bottom: ZHZ2, THET1, ZHET1, cDC3, BP2, BC2, cDC2, and cDC1. (F) AURKB-GFP cytoplasmic concentration calculated by fitting FCS autocorrelation curves with a one-component anomalous model of diffusion as described in Materials and Methods. The box and whiskers plot is from >23 cells/sample from two experiments. The mean is depicted as a diamond and the median as a horizontal line, and the whiskers show the minimum and maximum.
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Figure 2: Expression system determines AURKB-GFP levels. (A) Western blot comparison of AURKB levels in nocodazole-arrested cells. BAC-expressed AURKB-GFP runs at a higher molecular weight due to the presence of an S-peptide in addition to the EGFP tag. The graph shows the mean ± SD of three independent experiments. (B) Flow cytometry analysis of AURKB-GFP intensity in nocodazole-arrested cells. Coefficient of variation is from the whole population. GFP+ was defined as 3 SD above autofluorescence from wild-type cells. (C) Sum intensity confocal images of AURKB-GFP using the same imaging conditions throughout. One metaphase cell is shown per panel. Left to right: ZHZ2, ZHZ1, ZHET1, THET1, THET2, BP1, BC1, BP2, BC2, cDP1, cDC1, cDC2, and cDC3. Scale bar, 7 μm. (D) The amplitude of the FCS autocorrelation function (see Materials and Methods), G(0), is inversely proportional to particle number. Absolute protein concentration, c, is calculated using the measured confocal volume, V. (E) AURKB-GFP FCS autocorrelation curves from single representative cells. Top to bottom: ZHZ2, THET1, ZHET1, cDC3, BP2, BC2, cDC2, and cDC1. (F) AURKB-GFP cytoplasmic concentration calculated by fitting FCS autocorrelation curves with a one-component anomalous model of diffusion as described in Materials and Methods. The box and whiskers plot is from >23 cells/sample from two experiments. The mean is depicted as a diamond and the median as a horizontal line, and the whiskers show the minimum and maximum.

Mentions: Having validated this set of AURKB-GFP cells generated from the same parental HeLa line, we systematically analyzed the behavior of the expressed fusion proteins. First, we examined total expression level of AURKB-GFP by Western blotting of whole-cell extracts with anti-AURKB or anti-GFP antibodies (Figure 2A and Supplemental Figure S2A). All genome-edited clones exhibited AURKB-GFP levels similar to the endogenous protein with relatively little variation and independent of the genome-targeting method used to generate them or their zygosity (∼0.6- to 1.7-fold wild type). By contrast, expression of AURKB-GFP from randomly integrated BACs resulted in clearly visible overexpression in the tested clones (∼2.1- to 3.6-fold wild type), suggesting that the presence of the native promoter is not sufficient to ensure physiological expression levels. As expected for a constitutive heterologous promoter (cytomegalovirus; Boshart et al., 1985), different cDNA clones showed the strongest overexpression and the most variable expression between cell lines (∼0.4– to 5.7-fold). Flow cytometry of the intensity distribution of AURKB-GFP from 20,000 cells for each tagging method showed greatest cell-to-cell homogeneity for the two genome-editing methods (CV = 23 [ZFN], 23 [TALEN]), followed by BACs and cDNAs, which exhibited more variability (Figure 2B; CV = 30 and 112, respectively). Similarly, whereas in genome-edited and BAC systems, nearly all cells expressed the fusion protein (ZFN, 99%; BAC, 92–99%), cDNA clones had a variable number of expressing cells (Figure 2B; 28–89%).


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)

Expression system determines AURKB-GFP levels. (A) Western blot comparison of AURKB levels in nocodazole-arrested cells. BAC-expressed AURKB-GFP runs at a higher molecular weight due to the presence of an S-peptide in addition to the EGFP tag. The graph shows the mean ± SD of three independent experiments. (B) Flow cytometry analysis of AURKB-GFP intensity in nocodazole-arrested cells. Coefficient of variation is from the whole population. GFP+ was defined as 3 SD above autofluorescence from wild-type cells. (C) Sum intensity confocal images of AURKB-GFP using the same imaging conditions throughout. One metaphase cell is shown per panel. Left to right: ZHZ2, ZHZ1, ZHET1, THET1, THET2, BP1, BC1, BP2, BC2, cDP1, cDC1, cDC2, and cDC3. Scale bar, 7 μm. (D) The amplitude of the FCS autocorrelation function (see Materials and Methods), G(0), is inversely proportional to particle number. Absolute protein concentration, c, is calculated using the measured confocal volume, V. (E) AURKB-GFP FCS autocorrelation curves from single representative cells. Top to bottom: ZHZ2, THET1, ZHET1, cDC3, BP2, BC2, cDC2, and cDC1. (F) AURKB-GFP cytoplasmic concentration calculated by fitting FCS autocorrelation curves with a one-component anomalous model of diffusion as described in Materials and Methods. The box and whiskers plot is from >23 cells/sample from two experiments. The mean is depicted as a diamond and the median as a horizontal line, and the whiskers show the minimum and maximum.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 2: Expression system determines AURKB-GFP levels. (A) Western blot comparison of AURKB levels in nocodazole-arrested cells. BAC-expressed AURKB-GFP runs at a higher molecular weight due to the presence of an S-peptide in addition to the EGFP tag. The graph shows the mean ± SD of three independent experiments. (B) Flow cytometry analysis of AURKB-GFP intensity in nocodazole-arrested cells. Coefficient of variation is from the whole population. GFP+ was defined as 3 SD above autofluorescence from wild-type cells. (C) Sum intensity confocal images of AURKB-GFP using the same imaging conditions throughout. One metaphase cell is shown per panel. Left to right: ZHZ2, ZHZ1, ZHET1, THET1, THET2, BP1, BC1, BP2, BC2, cDP1, cDC1, cDC2, and cDC3. Scale bar, 7 μm. (D) The amplitude of the FCS autocorrelation function (see Materials and Methods), G(0), is inversely proportional to particle number. Absolute protein concentration, c, is calculated using the measured confocal volume, V. (E) AURKB-GFP FCS autocorrelation curves from single representative cells. Top to bottom: ZHZ2, THET1, ZHET1, cDC3, BP2, BC2, cDC2, and cDC1. (F) AURKB-GFP cytoplasmic concentration calculated by fitting FCS autocorrelation curves with a one-component anomalous model of diffusion as described in Materials and Methods. The box and whiskers plot is from >23 cells/sample from two experiments. The mean is depicted as a diamond and the median as a horizontal line, and the whiskers show the minimum and maximum.
Mentions: Having validated this set of AURKB-GFP cells generated from the same parental HeLa line, we systematically analyzed the behavior of the expressed fusion proteins. First, we examined total expression level of AURKB-GFP by Western blotting of whole-cell extracts with anti-AURKB or anti-GFP antibodies (Figure 2A and Supplemental Figure S2A). All genome-edited clones exhibited AURKB-GFP levels similar to the endogenous protein with relatively little variation and independent of the genome-targeting method used to generate them or their zygosity (∼0.6- to 1.7-fold wild type). By contrast, expression of AURKB-GFP from randomly integrated BACs resulted in clearly visible overexpression in the tested clones (∼2.1- to 3.6-fold wild type), suggesting that the presence of the native promoter is not sufficient to ensure physiological expression levels. As expected for a constitutive heterologous promoter (cytomegalovirus; Boshart et al., 1985), different cDNA clones showed the strongest overexpression and the most variable expression between cell lines (∼0.4– to 5.7-fold). Flow cytometry of the intensity distribution of AURKB-GFP from 20,000 cells for each tagging method showed greatest cell-to-cell homogeneity for the two genome-editing methods (CV = 23 [ZFN], 23 [TALEN]), followed by BACs and cDNAs, which exhibited more variability (Figure 2B; CV = 30 and 112, respectively). Similarly, whereas in genome-edited and BAC systems, nearly all cells expressed the fusion protein (ZFN, 99%; BAC, 92–99%), cDNA clones had a variable number of expressing cells (Figure 2B; 28–89%).

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