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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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Re-inoculation of a recovered single millifluidic droplet of the Sample A WT11+ strain yields a new algal population displaying increased growth-parameters diversity.(a) Statistical distributions of final algal yields in millifluidic drops for freshly subcloned (Sample A) Chlamydomonas wild-type strain WT11+. One drop, originating from fast-growers from the 1400–1600 category (circled in red), was sorted and collected at the end of a millifluidic experiment (i.e. after having grown for 10–11 generations). (b) 200 μL of fresh TAP was added to the drop and the cells were then re-encapsulated for a new millifluidic growth experiment at an occupancy of 0.12 cells/drop. A larger diversity of growth phenotypes is observed for the re-inoculated population when compared to the monoclonal original population, as can be seen by comparing the distribution of final algal yields (compare a and b). A larger variety of growth dynamics is also observed for the re-inoculated sample, when comparing their millifluidic growth curves (compare c and d).
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pone.0118987.g010: Re-inoculation of a recovered single millifluidic droplet of the Sample A WT11+ strain yields a new algal population displaying increased growth-parameters diversity.(a) Statistical distributions of final algal yields in millifluidic drops for freshly subcloned (Sample A) Chlamydomonas wild-type strain WT11+. One drop, originating from fast-growers from the 1400–1600 category (circled in red), was sorted and collected at the end of a millifluidic experiment (i.e. after having grown for 10–11 generations). (b) 200 μL of fresh TAP was added to the drop and the cells were then re-encapsulated for a new millifluidic growth experiment at an occupancy of 0.12 cells/drop. A larger diversity of growth phenotypes is observed for the re-inoculated population when compared to the monoclonal original population, as can be seen by comparing the distribution of final algal yields (compare a and b). A larger variety of growth dynamics is also observed for the re-inoculated sample, when comparing their millifluidic growth curves (compare c and d).

Mentions: The increased heterogeneity in growth dynamics for Sample B of the three wild-type strains, should be due to their propagation on agar plates for 1000 generations. This propagation may lead to the accumulation of spontaneous mutations over time, which would affect growth dynamics of individual mutants (see Discussion section). Therefore we performed an additional millifluidic experiment with Sample A from the WT11+ strain, with a low initial drop occupancy of 0.12 cells per drop (Fig. 10). The behaviour of these Sample A batches of WT11+ (Fig. 10a, 10c and 10e) was nearly identical to the behaviour observed for Sample A batches in previous experiments (Fig. 5b, 5e and 5h), including the clearly-defined subpopulations of slow- and fast-growers, with little to no intermediate growth dynamics (Fig. 10c and 10e). At the end of the millifluidic experiment, we selected one particular drop, containing a population of fast-growers (about 1600 cells as final algal yield, circled in red in Fig. 10a). These cells still should be isogenic, because they originate from a single cell from isogenic Sample A and have been propagated in non-selective conditions for only 10–11 generations (about 100 hours of active growth, Fig. 10c), a much too short period of time for the population to be subjected to significant genetic variations. We recovered the cell content from this drop and used it for re-encapsulation, at a low initial occupancy (0.12 cells per drop). The subsequent analysis of the growth-dynamics in that experiment (Fig. 10b and 10d) disclosed an unexpected heterogeneity in this cell population, recovered originally from a single drop of fast-growers. The final algal yield had a broad distribution with two peaks at about 500 and 1300 cells per drop (Fig. 10b), and the slow-growers had now a much wider distribution than originally observed for Sample A (Fig. 10a). Whereas the numbers of cell divisions in individual droplets showed significant but limited variability (Fig. 10f), the much wider distribution in final algal yields (Fig. 10b) rather originated from a pronounced variety of doubling-times between individual droplets (Fig. 10g). This re-encapsulation experiment thus shows that growing an isogenic population for only 10–11 generations in liquid media is sufficient to generate a wider diversity of growth phenotypes than what was observed for the initial Sample A (Fig. 5a-5c and Fig. 10a and 10c).


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)

Re-inoculation of a recovered single millifluidic droplet of the Sample A WT11+ strain yields a new algal population displaying increased growth-parameters diversity.(a) Statistical distributions of final algal yields in millifluidic drops for freshly subcloned (Sample A) Chlamydomonas wild-type strain WT11+. One drop, originating from fast-growers from the 1400–1600 category (circled in red), was sorted and collected at the end of a millifluidic experiment (i.e. after having grown for 10–11 generations). (b) 200 μL of fresh TAP was added to the drop and the cells were then re-encapsulated for a new millifluidic growth experiment at an occupancy of 0.12 cells/drop. A larger diversity of growth phenotypes is observed for the re-inoculated population when compared to the monoclonal original population, as can be seen by comparing the distribution of final algal yields (compare a and b). A larger variety of growth dynamics is also observed for the re-inoculated sample, when comparing their millifluidic growth curves (compare c and d).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0118987.g010: Re-inoculation of a recovered single millifluidic droplet of the Sample A WT11+ strain yields a new algal population displaying increased growth-parameters diversity.(a) Statistical distributions of final algal yields in millifluidic drops for freshly subcloned (Sample A) Chlamydomonas wild-type strain WT11+. One drop, originating from fast-growers from the 1400–1600 category (circled in red), was sorted and collected at the end of a millifluidic experiment (i.e. after having grown for 10–11 generations). (b) 200 μL of fresh TAP was added to the drop and the cells were then re-encapsulated for a new millifluidic growth experiment at an occupancy of 0.12 cells/drop. A larger diversity of growth phenotypes is observed for the re-inoculated population when compared to the monoclonal original population, as can be seen by comparing the distribution of final algal yields (compare a and b). A larger variety of growth dynamics is also observed for the re-inoculated sample, when comparing their millifluidic growth curves (compare c and d).
Mentions: The increased heterogeneity in growth dynamics for Sample B of the three wild-type strains, should be due to their propagation on agar plates for 1000 generations. This propagation may lead to the accumulation of spontaneous mutations over time, which would affect growth dynamics of individual mutants (see Discussion section). Therefore we performed an additional millifluidic experiment with Sample A from the WT11+ strain, with a low initial drop occupancy of 0.12 cells per drop (Fig. 10). The behaviour of these Sample A batches of WT11+ (Fig. 10a, 10c and 10e) was nearly identical to the behaviour observed for Sample A batches in previous experiments (Fig. 5b, 5e and 5h), including the clearly-defined subpopulations of slow- and fast-growers, with little to no intermediate growth dynamics (Fig. 10c and 10e). At the end of the millifluidic experiment, we selected one particular drop, containing a population of fast-growers (about 1600 cells as final algal yield, circled in red in Fig. 10a). These cells still should be isogenic, because they originate from a single cell from isogenic Sample A and have been propagated in non-selective conditions for only 10–11 generations (about 100 hours of active growth, Fig. 10c), a much too short period of time for the population to be subjected to significant genetic variations. We recovered the cell content from this drop and used it for re-encapsulation, at a low initial occupancy (0.12 cells per drop). The subsequent analysis of the growth-dynamics in that experiment (Fig. 10b and 10d) disclosed an unexpected heterogeneity in this cell population, recovered originally from a single drop of fast-growers. The final algal yield had a broad distribution with two peaks at about 500 and 1300 cells per drop (Fig. 10b), and the slow-growers had now a much wider distribution than originally observed for Sample A (Fig. 10a). Whereas the numbers of cell divisions in individual droplets showed significant but limited variability (Fig. 10f), the much wider distribution in final algal yields (Fig. 10b) rather originated from a pronounced variety of doubling-times between individual droplets (Fig. 10g). This re-encapsulation experiment thus shows that growing an isogenic population for only 10–11 generations in liquid media is sufficient to generate a wider diversity of growth phenotypes than what was observed for the initial Sample A (Fig. 5a-5c and Fig. 10a and 10c).

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
Related in: MedlinePlus