Quantifying the Length and Variance of the Eukaryotic Cell Cycle Phases by a Stochastic Model and Dual Nucleoside Pulse Labelling

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 and 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.Author Summary: Among the important characteristics of dividing cell populations is the time necessary for cells to complete each of the cell cycle phases, that is, to increase the cell's mass, to duplicate and repair its genome, to properly segregate its chromosomes, and to make decisions whether to continue dividing or enter a quiescent state. The cycle phase times also determine the maximal rate at which a dividing cell population can grow in size. Cell cycle phase completion times largely differ between cell types, cellular environments as well as metabolic stages, and can thus be considered as part of the phenotype of a given cell. Our article advances the methods to quantitatively characterize this phenotype. We introduce a novel phase-resolved cell cycle progression model and use it to estimate the mean and variance of the cycle phase completion times based on nucleoside analog pulse labelling experiments. This classic workhorse of cell cycle kinetic studies is revamped by our approach to potentially rival in accuracy and precision with modern phase-specific biosensor-based fluorescent imaging, while superseding the latter in its application scope.