Stretched cell cycle model for proliferating lymphocytes

Abstract

Stochastic variation in cell cycle time is a consistent feature of otherwise similar cells within a growing population. Classic studies concluded that the bulk of the variation occurs in the G 1 phase, and many mathematical models assume a constant time for traversing the S/G 2 /M phases. By direct observation of transgenic fluorescent fusion proteins that report the onset of S phase, we establish that dividing B and T lymphocytes spend a near-fixed proportion of total division time in S/G 2 /M phases, and this proportion is correlated between sibling cells. This result is inconsistent with models that assume independent times for consecutive phases. Instead, we propose a stretching model for dividing lymphocytes where all parts of the cell cycle are proportional to total division time. Data fitting based on a stretched cell cycle model can significantly improve estimates of cell cycle parameters drawn from DNA labeling data used to monitor immune cell dynamics.he kinetic relationship between phases of the cell cycle first came to attention with the advent of autoradiographic techniques for detecting DNA synthesis in the 1950s (1, 2). It was realized that such data could be used to resolve the dynamics of the proliferating population if combined with an appropriate cell cycle model. However, direct filming of times to divide revealed remarkable variation, even among cloned, presumed identical, cells (3-6), eliminating simple deterministic models as the basis for cell cycle control. Working toward developing a general model, Smith and Martin made the striking observation that plotting the proportion of undivided cells versus time (so-called "alpha plots"), gave curves suggestive of two distinct phases, one relatively constant and another stochastic (7). They proposed that the two phases mapped to discrete states of the cell cycle. A resting "A state," they suggested, was contained within the G 1 phase from which cells could exit with constant probability per unit time (analogous to radioactive decay). The cells then entered the "B phase," which includes that part of G 1 not included in A state, as well as the entirety of S/G 2 /M. In B phase, cells' activities were first described to be "deterministic, and directed towards replication," implying a constant B phase. However, in the same paper, this assumption was relaxed and the duration of B phase was described with a relatively constant random variable (7).Although details of the quantitative relationship and biological interpretation have been debated (7-12), the rule that the bulk of kinetic variation is in G 1 phase, and that time in S/G 2 /M is relatively fixed, is widely accepted. Furthermore, mathematical models adopting this mechanical description (so-called "transition probability" or "compartment" models) remain popular and form the basis of many studies of lymphocyte and cancer kinetics in vitro and in vivo today (13-21).More recently, a molecular description of cell cycle regulation, including the discovery of key regulatory proteins such as cyclins a...

Converse Smith-Martin cell cycle kinetics by transformed B lymphocytes

“…The average times for division in FUCCI-J558 (13.1 h, s.d 1.5 h) and FUCCI-I.29 (13.4 h, s.d 1.9 h) (Figure 2(a)), were similar to published times for stimulated primary B cells at 11.9 h [17]. However, when broken down further to times in G1 and S/G2/M, the period and proportion in G1 phase was notably different to that reported for primary B cells [17] (Figure 2(a)).…”

“…The average times for division in FUCCI-J558 (13.1 h, s.d 1.5 h) and FUCCI-I.29 (13.4 h, s.d 1.9 h) (Figure 2(a)), were similar to published times for stimulated primary B cells at 11.9 h [17]. However, when broken down further to times in G1 and S/G2/M, the period and proportion in G1 phase was notably different to that reported for primary B cells [17] (Figure 2(a)). First, transformed B cells spent shorter times in G1 (FUCCI J558: 1.5 h, 14% of total division, FUCCI-I.29: 2.41 h, 18% of total division) (Figure 2(a)) compared to that previously observed in CpG stimulated primary B cells (G1: 3.3 h, 27% of total division [17]).…”

“…Single clone lines were established that demonstrated stable expression of each FUCCI component up to and exceeding 30 days (Figure 1(a) and S1). Having established FUCCI-J558 and FUCCI-I.29 lines we adapted a single cell imaging system previously used to investigate cell cycle lengths [17,25,26]. Single cells seeded in microgrids were filmed over 60 hours to observe 1–3 division rounds, and we developed an imaging analysis pipeline (described in Methods) to measure the onset of G1 (t redmax ) and S/G2/M (t div -t redmax ) (Figure 1(b)).…”

“…One widely used method introduced by Sakaue-Sawano and colleagues [12], Fluorescent Ubuiqtination-based Cell Cycle Indicator (FUCCI), enables monitoring of cell-cycle at the single cell level, and has revealed lengths of cell cycle phases in cardiomyocytes, melanoma cells, intestinal stem cells and neural stem cells [13–16]. Using this FUCCI system to monitor cell cycle phases in dividing lymphocytes, Dowling and colleagues reported that B and T lymphocytes did not conform to the Smith-Martin model as they did not exhibit an exponential G1 phase [17]. Rather, dividing B and T lymphocytes displayed stretched cell cycles where time spent in G1 and S/G2/M phases was correlated in individual cells, and each phase represented a relatively constant proportion of the length of the total cell cycle phase [17].…”

“…Using this FUCCI system to monitor cell cycle phases in dividing lymphocytes, Dowling and colleagues reported that B and T lymphocytes did not conform to the Smith-Martin model as they did not exhibit an exponential G1 phase [17]. Rather, dividing B and T lymphocytes displayed stretched cell cycles where time spent in G1 and S/G2/M phases was correlated in individual cells, and each phase represented a relatively constant proportion of the length of the total cell cycle phase [17]. As a common feature of transformed cells is the deregulation of their cell cycles [18–22] we sought to examine the cell cycles of transformed B lymphocytes for comparison to healthy cells.…”