Research ArticleCELL BIOLOGY

Heritable stress response dynamics revealed by single-cell genealogy

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Science Advances  18 Apr 2018:
Vol. 4, no. 4, e1701775
DOI: 10.1126/sciadv.1701775
  • Fig. 1 Strain background, choice of glucose limitation stress, experimental setup, and single-cell Msn2 localization trajectories.

    (A) Pictorial representation of Msn2 translocation and activation of a synthetic promoter driving CFP in response to stress. (B) Glucose stress concentrations to be applied in lineage experiments. CFP expression of cells from three different glucose concentrations was collected from microscopy experiments, and the boxplots show distribution of CFP from single cells for each glucose concentration. For each boxplot, red line denotes median or 50th percentile; bottom of the blue box denotes the lower quartile or 25th percentile; top of the blue box denotes the upper quartile or 75th percentile of the distribution. *, significant difference (P = 0.03) in CFP expression between 2% glucose and 0.25% glucose; **, significant difference (P = 1.6 × 10−14) in CFP expression between 0.25% glucose and 0.1% glucose; ***, significant difference (P = 9.3 × 10−17) in CFP expression between 2% glucose and 0.1% glucose. Numbers of cells used for analysis in 2, 0.25, and 0.1% glucose concentrations are 36, 28, and 30, respectively. (C) Schematic showing a typical experimental setup of a time-lapse movie. (D) Snapshot from a time-lapse movie of cells showing Msn2 nuclear localization in 0.1% glucose. Nuclear localization of Msn2 has been quantified by an image analysis algorithm, without using a nuclear marker (Supplementary Materials). (E) Msn2 nuclear localization trajectories of three representative cells in 0.1% glucose. Msn2 nuclear localization is a ratio of two quantities (mean Msn2 nuclear localization in nucleus and mean Msn2 localization in whole cell), each having arbitrary unit (a.u.). Hence, the single-cell Msn2 nuclear localization values in our study are unitless. Cell 11 and cell 111 are the daughter and granddaughter cells, respectively, of cell 1. Arrows denote the time of birth of a new cell. Dashed horizontal lines denote the threshold used to quantify nuclear localization burst of Msn2.

  • Fig. 2 Msn2 localization in single cells collected in a lineage-dependent manner in different glucose concentrations.

    (A to C) Lineage maps of Msn2 localization from one experiment each in (A) 2% glucose, (B) 0.25% glucose, and (C) 0.1% glucose. Each row represents Msn2 localization of a single cell. Genealogical position of that cell in the family tree is labeled to the right of a row. Vertical lines denote the time at which a mother cell divides to form a daughter cell. Color bar shows the intensity of Msn2 localization signal.

  • Fig. 3 Modulation of Msn2 burst dynamics in population of cells.

    (A) Localization trajectory of a cell, showing how amplitude, frequency, and duration of Msn2 nuclear localization are quantified. Amplitude of Msn2 nuclear localization in a single cell is unitless. The dashed horizontal line denotes the threshold level above which there would be an Msn2 nuclear localization event. ni denotes the number of above-the-threshold localization events. T denotes the length of time interval used here for the calculation of frequency. (B and C) Mean (B) and CV (C) of Msn2 burst amplitude in different glucose concentrations. (D and E) Mean (D) and CV (E) of Msn2 burst frequency in different glucose concentrations. (F and G) Mean (F) and CV (G) of Msn2 burst duration in different glucose concentrations. Error bars in (B), (D), and (F) represent SEM. Error bars in (C), (E), and (G) were obtained from bootstrapping.

  • Fig. 4 Msn2 burst dynamics as a function of cell generation.

    (A to C) Msn2 burst amplitude (A), frequency (B), and duration (C) as a function of cell generations in different glucose concentrations. Error bars represent SEM. One cell generation is defined as the time interval between the onset of two consecutive buds of a cell. Generation 0 refers to the time interval between the birth of a cell to the emergence of its first bud. Table S5 lists the P values resulting from comparing amplitude, frequency, and duration between 2 and 0.25% glucose concentrations as well as between 2 and 0.1% glucose concentrations across different cell generations.

  • Fig. 5 Single-cell level correlations in Msn2 nuclear localization frequency between the first and second generations of a cell.

    (A to C) Correlations emerge as stress intensity is increased, with the correlation coefficients equal to 0.43 (P = 4.2 × 10−4) and 0.59 (P = 2.25 × 10−7) in 0.25 and 0.1% glucose, respectively. No significant correlation was observed in the case of 2% glucose (P = 0.13). One generation duration for a cell was defined as the time interval between the start of two consecutive S phases in a cell.

  • Fig. 6 Heritability of localization features of Msn2.

    (A) Left: Example of a lineage tree to the third generation. Three generations down, starting from the ancestor cell, give rise to eight cells in different genealogical positions of the tree. Right: All the M-D cell pairs (orange boxes), GM-GD cell pairs (blue boxes), and reference cell pairs (gray boxes) that can be obtained from the lineage tree of three generations. Boxes arranged diagonally do not correspond to pairs with unique cell labels, and hence do not belong to either of the three categories. (B) Normalized dissimilarity index of Msn2 burst amplitude between M-D, GM-GD, and reference cell pairs for each glucose concentration. (C) Normalized dissimilarity index of Msn2 burst frequency between M-D, GM-GD, and reference cell pairs for each glucose concentration. (D) Normalized dissimilarity index of Msn2 burst duration between M-D, GM-GD, and reference cell pairs for each glucose concentration. Error bars represent SEM. Table S6A gives a list of all the P values obtained by comparing amplitude, frequency, and duration of Msn2 localization between M-D pairs, GM-GD pairs, and reference pairs for each glucose concentration.

  • Fig. 7 Inheritance of dynamical patterns of Msn2 localization.

    (A) Pattern of Msn2 localization spikes between a mother and daughter cell in 2% glucose (top) and 0.1% glucose (bottom). Dashed blue lines denote the time at which the mother cell divides. Dashed black lines denote simultaneous localization spike events between the mother and daughter. P(Y) and P(X) quantifies the probability of exhibiting an Msn2 localization spike, following the birth of the daughter, in a mother cell and daughter cell, respectively. P(X,Y) quantifies the probability that the mother and daughter cell pair shows Msn2 localization spikes simultaneously. (B) Similarity in pattern of Msn2 localization spikes in M-D, GM-GD, GGM-GGD, and reference cell pairs for different glucose concentrations. Here, reference cell pairs are all pairs of cells that are not linked by mother-daughter, mother-granddaughter, or mother–great granddaughter relationships. The metric used to compute the abovementioned similarity provides a quantitative measure of how often mother and daughter exhibit synchronous occurrence of spikes. Error bars were obtained from bootstrapping. Table S8A gives a list of all P values obtained by comparing similarity of Msn2 spikes between M-D, GM-GD, GGM-GGD, and reference pairs for each glucose concentration.

  • Fig. 8 Genealogical gene expression dynamics in single cells and a quantitative model linking Msn2 nuclear localization to downstream gene expression.

    (A)Heat map of CFP expression in single cells collected in a lineage-dependent manner from one experiment in 2% glucose (top), 0.25% glucose (middle), and 0.1% glucose (bottom). Each row represents CFP expression of a single cell. Genealogical position of a cell in the family tree is labeled to the right of a row. Color bar shows the intensity of CFP signal (a.u.). (B) Quantification of relative contribution of the three different Msn2 localization features on CFP expression using Lasso. Amplitude and frequency of Msn2 nuclear localization were found to be the major features affecting CFP expression. (C) Prediction results for CFP expression for 0.25% (top) and 0.1% (bottom) glucose conditions using a linear state-space model. CFP levels were measured in single cells at every 60 min and normalized by the average CFP expression obtained from all cells grown in the 2% glucose condition. Blue square at a given time point represents the average CFP expression across all cells at that time point. Blue shaded area denotes the SEM (n = 8 to 67) calculated from all cells at each time point and extended between 60-min intervals as guide to the eye. The red points denote the CFP expression predicted by the model (at each 60 min) and averaged for all cells at that time point (red lines connecting red points during the 60-min intervals are guide to the eye). Model identification and prediction is discussed in more detail in the Supplementary Materials.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/4/e1701775/DC1

    Supplementary Materials and Methods

    fig. S1. Design of a microfluidic platform and a typical experimental setup.

    fig. S2. Growth of cell populations for cells grown in 2, 0.25, and 0.1% glucose.

    fig. S3. Cumulative distribution function of budding intervals measured from individual cells grown in 2, 0.25, and 0.1% glucose.

    fig. S4. Budding interval durations of spatially separate cells grown in 2, 0.25, and 0.1% glucose.

    fig. S5. Example of erroneous approximation of Msn2 nuclear localization intensity.

    fig. S6. Ratio of nuclear to cellular area as a function of the cell area.

    fig. S7. Performance of algorithm in estimation of nuclear localization of Msn2 from whole cell, in the absence of a nuclear marker.

    fig. S8. Estimation of nuclear localization of Msn2 using the algorithm is not sensitive to the variability in ratio of nuclear to cellular area at the single-cell level.

    fig. S9. No photobleaching or significant drop in Msn2 signal was observed over the course of an experiment.

    fig. S10. Proportion of cells having localization values below a given threshold k as a function of different thresholds k.

    fig. S11. Quantification of Msn2 localization dynamics is robust to the choice of threshold used for localization quantification.

    fig. S12. Heritability analysis of different localization features of Msn2 and analysis of inheritance of dynamical patterns of Msn2 localization is not sensitive to the choice of threshold used for Msn2 localization quantification.

    fig. S13. Lack of correlation in localization amplitude between the first and second generation of the same cell.

    fig. S14. Integral of Msn2 nuclear localization in different stress conditions.

    fig. S15. Dissimilarity analysis of different localization features of Msn2 is not affected by spatial proximity between cell pairs.

    fig. S16. Time dependent analysis of inheritance of Msn2 amplitude.

    fig. S17. Similarity of Msn2 localization spikes as a function of time for M-D cell pairs in different glucose concentrations (related to Fig. 7B).

    fig. S18. Correlation analysis between Msn2 localization features and cellular growth rate.

    fig. S19. Mean squared error for Lasso solution as a function of different values of regularization or shrinkage parameter.

    fig. S20. Lasso analysis is not sensitive to the choice of threshold used for Msn2 nuclear localization quantification.

    fig. S21. System and measurement noise for different stress environments.

    fig. S22. Block diagram of system identification and prediction steps.

    fig. S23. Predicting CFP expression levels using cross-validation.

    fig. S24. Sample polynomial fits for single-cell CFP trajectories.

    table S1. Population doubling times of cells in different glucose concentrations obtained from fig. S2 (A to C).

    table S2. Population doubling times calculated from OD600 measurements of cells grown in batch, using a shaker-incubator.

    table S3. Values of parameters obtained from fitting data in fig. S6 to a sigmoidal function.

    table S4. The P values comparing duration of Msn2 nuclear localization between 2 and 0.25% glucose, as well as 2 and 0.1% glucose across all threshold levels.

    table S5. The P values comparing amplitude (A), frequency (B), and duration (C) of Msn2 nuclear localization between 2 and 0.25% glucose, as well as 2 and 0.1% glucose across different cell generations.

    table S6. The P values obtained from Mann-Whitney U test.

    table S7. The P values obtained from Mann-Whitney U test.

    table S8. The P values obtained from Mann-Whitney U test.

    table S9. Parameter values extracted from the linear state-space model’s application to the data obtained from 0.25 and 0.1% glucose experiments.

    References (4253)

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • fig. S1. Design of a microfluidic platform and a typical experimental setup.
    • fig. S2. Growth of cell populations for cells grown in 2, 0.25, and 0.1% glucose.
    • fig. S3. Cumulative distribution function of budding intervals measured from individual cells grown in 2, 0.25, and 0.1% glucose.
    • fig. S4. Budding interval durations of spatially separate cells grown in 2, 0.25, and 0.1% glucose.
    • fig. S5. Example of erroneous approximation of Msn2 nuclear localization intensity.
    • fig. S6. Ratio of nuclear to cellular area as a function of the cell area.
    • fig. S7. Performance of algorithm in estimation of nuclear localization of Msn2 from whole cell, in the absence of a nuclear marker.
    • fig. S8. Estimation of nuclear localization of Msn2 using the algorithm is not sensitive to the variability in ratio of nuclear to cellular area at the single-cell level.
    • fig. S9. No photobleaching or significant drop in Msn2 signal was observed over the course of an experiment.
    • fig. S10. Proportion of cells having localization values below a given threshold k as a function of different thresholds k.
    • fig. S11. Quantification of Msn2 localization dynamics is robust to the choice of threshold used for localization quantification.
    • fig. S12. Heritability analysis of different localization features of Msn2 and analysis of inheritance of dynamical patterns of Msn2 localization is not sensitive to the choice of threshold used for Msn2 localization quantification.
    • fig. S13. Lack of correlation in localization amplitude between the first and second generation of the same cell.
    • fig. S14. Integral of Msn2 nuclear localization in different stress conditions.
    • fig. S15. Dissimilarity analysis of different localization features of Msn2 is not affected by spatial proximity between cell pairs.
    • fig. S16. Time dependent analysis of inheritance of Msn2 amplitude.
    • fig. S17. Similarity of Msn2 localization spikes as a function of time for M-D cell pairs in different glucose concentrations (related to Fig. 7B).
    • fig. S18. Correlation analysis between Msn2 localization features and cellular growth rate.
    • fig. S19. Mean squared error for Lasso solution as a function of different values of regularization or shrinkage parameter.
    • fig. S20. Lasso analysis is not sensitive to the choice of threshold used for Msn2 nuclear localization quantification.
    • fig. S21. System and measurement noise for different stress environments.
    • fig. S22. Block diagram of system identification and prediction steps.
    • fig. S23. Predicting CFP expression levels using cross-validation.
    • fig. S24. Sample polynomial fits for single-cell CFP trajectories.
    • table S1. Population doubling times of cells in different glucose concentrations obtained from fig. S2 (A to C).
    • table S2. Population doubling times calculated from OD600 measurements of cells grown in batch, using a shaker-incubator.
    • table S3. Values of parameters obtained from fitting data in fig. S6 to a sigmoidal function.
    • table S4. The P values comparing duration of Msn2 nuclear localization between 2 and 0.25% glucose, as well as 2 and 0.1% glucose across all threshold levels.
    • table S5. The P values comparing amplitude (A), frequency (B), and duration (C) of Msn2 nuclear localization between 2 and 0.25% glucose, as well as 2 and 0.1% glucose across different cell generations.
    • table S6. The P values obtained from Mann-Whitney U test.
    • table S7. The P values obtained from Mann-Whitney U test.
    • table S8. The P values obtained from Mann-Whitney U test.
    • table S9. Parameter values extracted from the linear state-space model’s application to the data obtained from 0.25 and 0.1% glucose experiments.
    • References (42–53)

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