When stimulated, T and B lymphocytes proliferate but can vary their time to divide; their time that the proliferative burst progresses before stopping (division destiny), and their time to die. Previous work has shown division destiny time is inherited through generations and regulated by the production and loss of the oncoprotein Myc. Here we search for molecular regulators of division and death governing analogous timed control mechanisms.
Using quantitative cell-based assays for B and T cell proliferation, we found the rate of D-Cyclin production and loss is unaffected by passage through generations, linked to activation signal strength and correlates with division times. Inhibitors of D-Cyclin/CDK catalytic activity slowed division entry and subsequent division rates. Furthermore, the forced over-expression of Cyclin D3 increased B cell division rates indicating that D-Cyclin protein level and activity regulate and govern division times. Variation in expression level accounts for broad differences in division times by individual cells.
To study the death timer, we analysed CpG stimulated B cells as they die over a precise, predictable period. Induction of pro- and anti-apoptotic molecule expression was observed to rise and fall in B cells prior to, and during, the death phase. A timer model based on stochastic differences in molecular expression, coupled to death if the protein ensemble fell below a critical value was developed. To test this model, we used data from Bax/Bak KO single cell FACS expression, that do not die, to predict death frequency in wild type cells. Weighting the combined effect of BCL2, MCL1, BCLxL and BIM, predicted death and identified the collective ‘threshold’ required for B cell survival, supporting the timer model.
Together these findings support a conceptual model that can link quantitative molecular changes to the control of multiple cell fates in response to complex signal combinations.