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A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

Damodaran SP, Eberhard S, Boitard L, Rodriguez JG, Wang Y, Bremond N, Baudry J, Bibette J, Wollman FA - PLoS ONE (2015)

Bottom Line: We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers).Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times.We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Colloïdes et Matériaux Divisés, Institute of Chemistry, Biology and Innovation ESPCI ParisTech/CNRS UMR 8231/PSL* Research University, Paris, France.

ABSTRACT
To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

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Growth kinetics for Chlamydomonas cultures in millidroplets inoculated with no more than one cell.(a) Poisson distribution showing the number of droplets that should encapsulate single (32), two (0.5) or more than two (0.06) algal cells, for an overall droplet occupancy of 0.07. Multiple initial occupancy of droplets is hence highly unlikely and growth kinetics relate to the growth dynamics for populations originating from a single cell. (b) Distribution of final algal yield for Sample B from WT222+, showing a wide distribution of growth phenotypes. (c) Millifluidic growth curves for single cell encapsulations of sample B from WT222+, showing a wide variety in both final algal yield and division time. (d) Number of cell divisions in each drop for WT222+ (Sample B) for the single-cell encapsulation experiment.
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pone.0118987.g011: Growth kinetics for Chlamydomonas cultures in millidroplets inoculated with no more than one cell.(a) Poisson distribution showing the number of droplets that should encapsulate single (32), two (0.5) or more than two (0.06) algal cells, for an overall droplet occupancy of 0.07. Multiple initial occupancy of droplets is hence highly unlikely and growth kinetics relate to the growth dynamics for populations originating from a single cell. (b) Distribution of final algal yield for Sample B from WT222+, showing a wide distribution of growth phenotypes. (c) Millifluidic growth curves for single cell encapsulations of sample B from WT222+, showing a wide variety in both final algal yield and division time. (d) Number of cell divisions in each drop for WT222+ (Sample B) for the single-cell encapsulation experiment.

Mentions: In all the experiments described above, the inoculum size ranged from 0.12 to 0.33 cells/drop, which was close to, but did not fully, eliminate the chances of having a few droplets entrapping more than one cell at the start of the experiment. According to a Poisson distribution, only 5–15% of all droplets could have encapsulated two or more cells at the beginning of the experiment. We refrained from further decreasing the initial drop occupancy in the previous experiments, in order to keep a large enough number of algae-containing drops in the millidroplet train, to preserve the statistical significance of our results. However, to fully rule out a contribution of limited variations in initial droplet occupancy to the observed heterogeneity in growth dynamics, we carried out a millifluidic test with sample B from WT222+ at the very low droplet occupancy of only 0.07 inoculated cells per drop. Fig. 11a shows that, according to a Poisson distribution with this occupancy, 32 droplets should encapsulate single cells, while only 0.5 drops would encapsulate two algal cell, and 0.06 drops would have more than 2 initial cells, along with 935 empty droplets along the millifluidic train. Populations derived from this single-cell-encapsulation experiment still showed large heterogeneities in cell growth dynamics (Fig. 11b-11d). A large diversity in doubling-times (Fig. 11c) and in final algal yield (Fig. 11b) is readily observed. Fig. 11c displays a set of growth curves leading to different final algal yields, with contrasting generation times, ranging from 9h to 14h. Fig. 11d shows the distribution of cell-divisions per droplet, ranging from 7 to 10. Thus the contents of a single droplets from this experiment, solely derived from single-cell-encapsulations, still show large heterogeneity in cell growth dynamics.


A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

Damodaran SP, Eberhard S, Boitard L, Rodriguez JG, Wang Y, Bremond N, Baudry J, Bibette J, Wollman FA - PLoS ONE (2015)

Growth kinetics for Chlamydomonas cultures in millidroplets inoculated with no more than one cell.(a) Poisson distribution showing the number of droplets that should encapsulate single (32), two (0.5) or more than two (0.06) algal cells, for an overall droplet occupancy of 0.07. Multiple initial occupancy of droplets is hence highly unlikely and growth kinetics relate to the growth dynamics for populations originating from a single cell. (b) Distribution of final algal yield for Sample B from WT222+, showing a wide distribution of growth phenotypes. (c) Millifluidic growth curves for single cell encapsulations of sample B from WT222+, showing a wide variety in both final algal yield and division time. (d) Number of cell divisions in each drop for WT222+ (Sample B) for the single-cell encapsulation experiment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356620&req=5

pone.0118987.g011: Growth kinetics for Chlamydomonas cultures in millidroplets inoculated with no more than one cell.(a) Poisson distribution showing the number of droplets that should encapsulate single (32), two (0.5) or more than two (0.06) algal cells, for an overall droplet occupancy of 0.07. Multiple initial occupancy of droplets is hence highly unlikely and growth kinetics relate to the growth dynamics for populations originating from a single cell. (b) Distribution of final algal yield for Sample B from WT222+, showing a wide distribution of growth phenotypes. (c) Millifluidic growth curves for single cell encapsulations of sample B from WT222+, showing a wide variety in both final algal yield and division time. (d) Number of cell divisions in each drop for WT222+ (Sample B) for the single-cell encapsulation experiment.
Mentions: In all the experiments described above, the inoculum size ranged from 0.12 to 0.33 cells/drop, which was close to, but did not fully, eliminate the chances of having a few droplets entrapping more than one cell at the start of the experiment. According to a Poisson distribution, only 5–15% of all droplets could have encapsulated two or more cells at the beginning of the experiment. We refrained from further decreasing the initial drop occupancy in the previous experiments, in order to keep a large enough number of algae-containing drops in the millidroplet train, to preserve the statistical significance of our results. However, to fully rule out a contribution of limited variations in initial droplet occupancy to the observed heterogeneity in growth dynamics, we carried out a millifluidic test with sample B from WT222+ at the very low droplet occupancy of only 0.07 inoculated cells per drop. Fig. 11a shows that, according to a Poisson distribution with this occupancy, 32 droplets should encapsulate single cells, while only 0.5 drops would encapsulate two algal cell, and 0.06 drops would have more than 2 initial cells, along with 935 empty droplets along the millifluidic train. Populations derived from this single-cell-encapsulation experiment still showed large heterogeneities in cell growth dynamics (Fig. 11b-11d). A large diversity in doubling-times (Fig. 11c) and in final algal yield (Fig. 11b) is readily observed. Fig. 11c displays a set of growth curves leading to different final algal yields, with contrasting generation times, ranging from 9h to 14h. Fig. 11d shows the distribution of cell-divisions per droplet, ranging from 7 to 10. Thus the contents of a single droplets from this experiment, solely derived from single-cell-encapsulations, still show large heterogeneity in cell growth dynamics.

Bottom Line: We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers).Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times.We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Colloïdes et Matériaux Divisés, Institute of Chemistry, Biology and Innovation ESPCI ParisTech/CNRS UMR 8231/PSL* Research University, Paris, France.

ABSTRACT
To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes.

Show MeSH