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The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise.

Kempe H, Schwabe A, Crémazy F, Verschure PJ, Bruggeman FJ - Mol. Biol. Cell (2014)

Bottom Line: We compared three cell clones that differ only in the genomic integration site of an identical constitutively expressed reporter gene.We found that the cell-to-cell variability in the mRNA concentration is almost exclusively due to cell-to-cell variation in gene expression activity, whereas the cell-to-cell variation in mRNA number is larger, due to a significant contribution of cell volume variability.We concluded that the precise relationship between transcript number and cell volume sets the biological stochasticity of living cells.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Systems Biology and Nuclear Organization Group, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, The Netherlands.

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Statistics of single-cell mRNA numbers. (A) Schematic overview of the smFISH method applied to our reporter gene mRNA. Colocalization of the mRNA molecules with the DAPI counter staining identified spots as nuclear mRNA (mN); others are cytoplasmic mRNA (mc). (B) Statistics of the mRNA molecules in the cell (m), nucleus (mN), and cytoplasm (mc) for the three different clones (color coded: Clone I is shown in red, Clone II is shown in blue, and Clone III is shown in green). Notation: μ = mean; σ = SD; cv = coefficient of variation; Δσ = the fraction of samples between μ - σ and μ + σ; ρ = correlation between mc and mN; and *p < 0.001 The 95% confidence intervals of the statistics are given in Supplemental Figure S15. (C) For a specific cell volume (V), the mean mRNA number is calculated from the data. This conditional mean displays linear scaling with respect to volume, indicating homeostasis in mRNA concentration. The gray histogram in the background shows the total number of cells per volume bin for all three clones (bin size = 100 〈m/V〉). Higher counts indicate higher reliability of the corresponding determination of σ. A least-squares linear fit is shown for all three clones. The explained fraction of the variance in 〈m/V〉 with this fit is 0.80, 0.77, and 0.84 for clones I, II, and III, respectively. Supplemental Figure S9 shows the single-cell relation between cell volume and mRNA number. The conditional variances of the data are given in Supplemental Figure S10. (D) Representative confocal images of a cell, with Z1 to Z12 corresponding to subsequent optical sections (z-slices) of the cell. The mRNA molecules are shown in red; the DAPI-stained nucleus is shown in blue. Additional images are given in Supplemental Figure S2. (E–G) Scatter plots of mc and mN for the three different clones. Marginal histograms show the distribution of mc (top) and mN (right). The measured number of cells is given by n.
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Figure 1: Statistics of single-cell mRNA numbers. (A) Schematic overview of the smFISH method applied to our reporter gene mRNA. Colocalization of the mRNA molecules with the DAPI counter staining identified spots as nuclear mRNA (mN); others are cytoplasmic mRNA (mc). (B) Statistics of the mRNA molecules in the cell (m), nucleus (mN), and cytoplasm (mc) for the three different clones (color coded: Clone I is shown in red, Clone II is shown in blue, and Clone III is shown in green). Notation: μ = mean; σ = SD; cv = coefficient of variation; Δσ = the fraction of samples between μ - σ and μ + σ; ρ = correlation between mc and mN; and *p < 0.001 The 95% confidence intervals of the statistics are given in Supplemental Figure S15. (C) For a specific cell volume (V), the mean mRNA number is calculated from the data. This conditional mean displays linear scaling with respect to volume, indicating homeostasis in mRNA concentration. The gray histogram in the background shows the total number of cells per volume bin for all three clones (bin size = 100 〈m/V〉). Higher counts indicate higher reliability of the corresponding determination of σ. A least-squares linear fit is shown for all three clones. The explained fraction of the variance in 〈m/V〉 with this fit is 0.80, 0.77, and 0.84 for clones I, II, and III, respectively. Supplemental Figure S9 shows the single-cell relation between cell volume and mRNA number. The conditional variances of the data are given in Supplemental Figure S10. (D) Representative confocal images of a cell, with Z1 to Z12 corresponding to subsequent optical sections (z-slices) of the cell. The mRNA molecules are shown in red; the DAPI-stained nucleus is shown in blue. Additional images are given in Supplemental Figure S2. (E–G) Scatter plots of mc and mN for the three different clones. Marginal histograms show the distribution of mc (top) and mN (right). The measured number of cells is given by n.

Mentions: Early studies on stochastic gene expression relied on fluorescent proteins to assess protein noise by either taking snapshots (Elowitz et al., 2002; Ozbudak et al., 2002) or by using real-time fluorescence imaging (Rosenfeld et al., 2005; Sigal et al., 2006). More recently, single-molecule mRNA counting has been introduced as a method for absolute quantification of mRNA number (Golding et al., 2005; Raj et al., 2006, 2008). The advantage of single-transcript counting with single-molecule RNA fluorescence in situ hybridization (smFISH) is that it does not require genetic engineering (Raj et al., 2006, 2008; Zenklusen et al., 2008; Youk et al., 2010). Specific DNA probes tagged with fluorescent dyes are used to visualize individual mRNA molecules within fixed cells (Figure 1A).


The volumes and transcript counts of single cells reveal concentration homeostasis and capture biological noise.

Kempe H, Schwabe A, Crémazy F, Verschure PJ, Bruggeman FJ - Mol. Biol. Cell (2014)

Statistics of single-cell mRNA numbers. (A) Schematic overview of the smFISH method applied to our reporter gene mRNA. Colocalization of the mRNA molecules with the DAPI counter staining identified spots as nuclear mRNA (mN); others are cytoplasmic mRNA (mc). (B) Statistics of the mRNA molecules in the cell (m), nucleus (mN), and cytoplasm (mc) for the three different clones (color coded: Clone I is shown in red, Clone II is shown in blue, and Clone III is shown in green). Notation: μ = mean; σ = SD; cv = coefficient of variation; Δσ = the fraction of samples between μ - σ and μ + σ; ρ = correlation between mc and mN; and *p < 0.001 The 95% confidence intervals of the statistics are given in Supplemental Figure S15. (C) For a specific cell volume (V), the mean mRNA number is calculated from the data. This conditional mean displays linear scaling with respect to volume, indicating homeostasis in mRNA concentration. The gray histogram in the background shows the total number of cells per volume bin for all three clones (bin size = 100 〈m/V〉). Higher counts indicate higher reliability of the corresponding determination of σ. A least-squares linear fit is shown for all three clones. The explained fraction of the variance in 〈m/V〉 with this fit is 0.80, 0.77, and 0.84 for clones I, II, and III, respectively. Supplemental Figure S9 shows the single-cell relation between cell volume and mRNA number. The conditional variances of the data are given in Supplemental Figure S10. (D) Representative confocal images of a cell, with Z1 to Z12 corresponding to subsequent optical sections (z-slices) of the cell. The mRNA molecules are shown in red; the DAPI-stained nucleus is shown in blue. Additional images are given in Supplemental Figure S2. (E–G) Scatter plots of mc and mN for the three different clones. Marginal histograms show the distribution of mc (top) and mN (right). The measured number of cells is given by n.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Statistics of single-cell mRNA numbers. (A) Schematic overview of the smFISH method applied to our reporter gene mRNA. Colocalization of the mRNA molecules with the DAPI counter staining identified spots as nuclear mRNA (mN); others are cytoplasmic mRNA (mc). (B) Statistics of the mRNA molecules in the cell (m), nucleus (mN), and cytoplasm (mc) for the three different clones (color coded: Clone I is shown in red, Clone II is shown in blue, and Clone III is shown in green). Notation: μ = mean; σ = SD; cv = coefficient of variation; Δσ = the fraction of samples between μ - σ and μ + σ; ρ = correlation between mc and mN; and *p < 0.001 The 95% confidence intervals of the statistics are given in Supplemental Figure S15. (C) For a specific cell volume (V), the mean mRNA number is calculated from the data. This conditional mean displays linear scaling with respect to volume, indicating homeostasis in mRNA concentration. The gray histogram in the background shows the total number of cells per volume bin for all three clones (bin size = 100 〈m/V〉). Higher counts indicate higher reliability of the corresponding determination of σ. A least-squares linear fit is shown for all three clones. The explained fraction of the variance in 〈m/V〉 with this fit is 0.80, 0.77, and 0.84 for clones I, II, and III, respectively. Supplemental Figure S9 shows the single-cell relation between cell volume and mRNA number. The conditional variances of the data are given in Supplemental Figure S10. (D) Representative confocal images of a cell, with Z1 to Z12 corresponding to subsequent optical sections (z-slices) of the cell. The mRNA molecules are shown in red; the DAPI-stained nucleus is shown in blue. Additional images are given in Supplemental Figure S2. (E–G) Scatter plots of mc and mN for the three different clones. Marginal histograms show the distribution of mc (top) and mN (right). The measured number of cells is given by n.
Mentions: Early studies on stochastic gene expression relied on fluorescent proteins to assess protein noise by either taking snapshots (Elowitz et al., 2002; Ozbudak et al., 2002) or by using real-time fluorescence imaging (Rosenfeld et al., 2005; Sigal et al., 2006). More recently, single-molecule mRNA counting has been introduced as a method for absolute quantification of mRNA number (Golding et al., 2005; Raj et al., 2006, 2008). The advantage of single-transcript counting with single-molecule RNA fluorescence in situ hybridization (smFISH) is that it does not require genetic engineering (Raj et al., 2006, 2008; Zenklusen et al., 2008; Youk et al., 2010). Specific DNA probes tagged with fluorescent dyes are used to visualize individual mRNA molecules within fixed cells (Figure 1A).

Bottom Line: We compared three cell clones that differ only in the genomic integration site of an identical constitutively expressed reporter gene.We found that the cell-to-cell variability in the mRNA concentration is almost exclusively due to cell-to-cell variation in gene expression activity, whereas the cell-to-cell variation in mRNA number is larger, due to a significant contribution of cell volume variability.We concluded that the precise relationship between transcript number and cell volume sets the biological stochasticity of living cells.

View Article: PubMed Central - PubMed

Affiliation: Synthetic Systems Biology and Nuclear Organization Group, Swammerdam Institute for Life Sciences, University of Amsterdam, 1098 XH Amsterdam, The Netherlands.

Show MeSH
Related in: MedlinePlus