Biophysics Graduate Program, University of Michigan, Ann Arbor, Michigan.
Program in Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, New York.
Biophys J. 2019 Mar 5;116(5):962-973. doi: 10.1016/j.bpj.2019.01.022. Epub 2019 Jan 30.
Mammalian cells respond in a variable manner when provided with physiological pulses of ligand, such as low concentrations of acetylcholine present for just tens of seconds or TNFα for just tens of minutes. For a two-pulse stimulation, some cells respond to both pulses, some do not respond, and yet others respond to only one or the other pulse. Are these different response patterns the result of the small number of ligands being able to only stochastically activate the pathway at random times or an output pattern from a deterministic algorithm responding differently to different stimulation intervals? If the response is deterministic in nature, what parameters determine whether a response is generated or skipped? To answer these questions, we developed a two-pulse test that utilizes different rest periods between stimulation pulses. This "rest-period test" revealed that cells skip responses predictably as the rest period is shortened. By combining these experimental results with a mathematical model of the pathway, we further obtained mechanistic insight into potential sources of response variability. Our analysis indicates that in both intracellular calcium and NFκB signaling, response variability is consistent with extrinsic noise (cell-to-cell variability in protein levels), a short-term memory of stimulation, and high Hill coefficient processes. Furthermore, these results support recent works that have emphasized the role of deterministic processes for explaining apparently stochastic cellular response variability and indicate that even weak stimulations likely guide mammalian cells to appropriate fates rather than leaving outcomes to chance. We envision that the rest-period test can be applied to other signaling pathways to extract mechanistic insight.
哺乳动物细胞在受到生理脉冲配体（如仅存在数十秒的低浓度乙酰胆碱或仅数十分钟的 TNFα）刺激时会以不同的方式做出反应。对于双脉冲刺激，一些细胞对两个脉冲都有反应，一些则没有反应，还有一些只对一个或另一个脉冲有反应。这些不同的反应模式是由于少量配体只能随机地在不同时间激活途径的结果，还是对不同刺激间隔做出不同反应的确定性算法的输出模式？如果反应具有确定性，那么哪些参数决定是否产生或跳过反应？为了回答这些问题，我们开发了一种双脉冲测试，该测试利用刺激脉冲之间的不同休息期。这种“休息期测试”表明，随着休息期的缩短，细胞有规律地跳过反应。通过将这些实验结果与途径的数学模型相结合，我们进一步深入了解了潜在的反应变异性来源。我们的分析表明，在细胞内钙和 NFκB 信号转导中，反应变异性与外在噪声（蛋白质水平的细胞间变异性）、刺激的短期记忆和高 Hill 系数过程一致。此外，这些结果支持了最近的研究工作，这些工作强调了确定性过程对于解释明显随机的细胞反应变异性的作用，并表明即使是微弱的刺激也可能引导哺乳动物细胞走向适当的命运，而不是将结果留给机会。我们设想，休息期测试可以应用于其他信号通路，以提取机制见解。
