Howard Hughes Medical Institute and Division of Biology and Department of Applied Physics, California Institute of Technology, Pasadena, California, United States of America.
PLoS Biol. 2012 Jan;10(1):e1001252. doi: 10.1371/journal.pbio.1001252. Epub 2012 Jan 31.
Environmental signals induce diverse cellular differentiation programs. In certain systems, cells defer differentiation for extended time periods after the signal appears, proliferating through multiple rounds of cell division before committing to a new fate. How can cells set a deferral time much longer than the cell cycle? Here we study Bacillus subtilis cells that respond to sudden nutrient limitation with multiple rounds of growth and division before differentiating into spores. A well-characterized genetic circuit controls the concentration and phosphorylation of the master regulator Spo0A, which rises to a critical concentration to initiate sporulation. However, it remains unclear how this circuit enables cells to defer sporulation for multiple cell cycles. Using quantitative time-lapse fluorescence microscopy of Spo0A dynamics in individual cells, we observed pulses of Spo0A phosphorylation at a characteristic cell cycle phase. Pulse amplitudes grew systematically and cell-autonomously over multiple cell cycles leading up to sporulation. This pulse growth required a key positive feedback loop involving the sporulation kinases, without which the deferral of sporulation became ultrasensitive to kinase expression. Thus, deferral is controlled by a pulsed positive feedback loop in which kinase expression is activated by pulses of Spo0A phosphorylation. This pulsed positive feedback architecture provides a more robust mechanism for setting deferral times than constitutive kinase expression. Finally, using mathematical modeling, we show how pulsing and time delays together enable "polyphasic" positive feedback, in which different parts of a feedback loop are active at different times. Polyphasic feedback can enable more accurate tuning of long deferral times. Together, these results suggest that Bacillus subtilis uses a pulsed positive feedback loop to implement a "timer" that operates over timescales much longer than a cell cycle.
环境信号诱导多种细胞分化程序。在某些系统中，细胞在信号出现后会延迟分化很长时间，在决定新命运之前，通过多次细胞分裂进行增殖。细胞如何将延迟时间设定得比细胞周期长得多？在这里，我们研究了枯草芽孢杆菌细胞，它们在分化为孢子之前，对突然的营养限制做出反应，进行多轮生长和分裂。一个特征明确的遗传回路控制着主调控因子 Spo0A 的浓度和磷酸化，Spo0A 浓度上升到临界浓度以启动孢子形成。然而，目前尚不清楚该回路如何使细胞能够延迟多个细胞周期的孢子形成。通过对单个细胞中 Spo0A 动力学的定量延时荧光显微镜观察，我们观察到 Spo0A 磷酸化在特征性细胞周期阶段的脉冲。脉冲幅度在多个细胞周期中系统地和细胞自主地增长，直到孢子形成。这种脉冲增长需要一个关键的正反馈回路，涉及孢子形成激酶，没有这个回路，孢子形成的延迟对激酶表达变得非常敏感。因此，延迟是通过一个脉冲正反馈回路来控制的，其中激酶表达被 Spo0A 磷酸化的脉冲激活。这种脉冲正反馈结构为设定延迟时间提供了比组成型激酶表达更稳健的机制。最后，我们使用数学建模表明，脉冲和时间延迟如何共同实现“多相”正反馈，其中反馈回路的不同部分在不同时间活跃。多相反馈可以更精确地调整长延迟时间。总之，这些结果表明，枯草芽孢杆菌利用脉冲正反馈回路来实现一个“定时器”，该定时器的操作时间远远超过一个细胞周期。
