Quantifying the length and variance of the eukaryotic cell cycle phases by a stochastic model and dual nucleoside pulse labelling.
Bottom Line:
To overcome this limitation, a redesigned experimental protocol is derived and validated in silico.The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases.The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase-specific biosensor-based fluorescent imaging.
View Article:
PubMed Central - PubMed
Affiliation: Instituto Gulbenkian de Ciência, Oeiras, Portugal; Department of Biology, Humboldt-Universität zu Berlin, Berlin Germany and Research Center ImmunoSciences, Charité - Universitätsmedizin Berlin, Berlin, Germany.
ABSTRACT
Show MeSH
A fundamental property of cell populations is their growth rate as well as the time needed for cell division and its variance. The eukaryotic cell cycle progresses in an ordered sequence through the phases G1, S, G2, and M, and is regulated by environmental cues and by intracellular checkpoints. Reflecting this regulatory complexity, the length of each phase varies considerably in different kinds of cells but also among genetically and morphologically indistinguishable cells. This article addresses the question of how to describe and quantify the mean and variance of the cell cycle phase lengths. A phase-resolved cell cycle model is introduced assuming that phase completion times are distributed as delayed exponential functions, capturing the observations that each realization of a cycle phase is variable in length and requires a minimal time. In this model, the total cell cycle length is distributed as a delayed hypoexponential function that closely reproduces empirical distributions. Analytic solutions are derived for the proportions of cells in each cycle phase in a population growing under balanced growth and under specific non-stationary conditions. These solutions are then adapted to describe conventional cell cycle kinetic assays based on pulse labelling with nucleoside analogs. The model fits well to data obtained with two distinct proliferating cell lines labelled with a single bromodeoxiuridine pulse. However, whereas mean lengths are precisely estimated for all phases, the respective variances remain uncertain. To overcome this limitation, a redesigned experimental protocol is derived and validated in silico. The novelty is the timing of two consecutive pulses with distinct nucleosides that enables accurate and precise estimation of both the mean and the variance of the length of all phases. The proposed methodology to quantify the phase length distributions gives results potentially equivalent to those obtained with modern phase-specific biosensor-based fluorescent imaging. Related in: MedlinePlus |
Related In:
Results -
Collection
License getmorefigures.php?uid=PMC4109856&req=5
Mentions: where and represent the equivalents of and we had previously defined for the case of no cell loss. The former quantities, which now depend on are derived applying to Eq. 5 the same substitution as above. Expressions equivalent to Eq. 10 and Eq. 11 are obtained along the same lines. These become however rather lengthy and are therefore omitted here. Eq. 29 reproduces accurately in simulated BrdU pulse labelling experiments, if death occurs, as specified above (see Fig. 7 A for an example with and ). The differences between the analytical predictions for with 30% death and without death (denoted by ) are, for the parameter sets that we tested, relatively small, and vanish as expected, as tends to zero (see Fig. 7 B for computed at one specific time point ( h) for different values of ). |
View Article: PubMed Central - PubMed
Affiliation: Instituto Gulbenkian de Ciência, Oeiras, Portugal; Department of Biology, Humboldt-Universität zu Berlin, Berlin Germany and Research Center ImmunoSciences, Charité - Universitätsmedizin Berlin, Berlin, Germany.