Sources and Consequences of noise in cell cycle regulation

描述（由申请人提供）： 遗传网络运行中的单细胞变异（噪声）是细胞系统尺寸较小的必然结果，导致分子（基因、mRNA、蛋白质）数量较少，可能导致重要遗传控制回路功能的波动。如果这种噪音是有害的，那么噪音抑制机制很可能已经发展起来；在某些情况下，噪音也可能是有益的，可以突破动态死胡同，或者使人口多样化，以满足不断变化的环境。尽管这个主题很重要，但到目前为止，关于噪声的经验结果很大程度上仅限于简单的合成基因电路。我们建议在更复杂和生理相关的情况下分析单细胞变异：真核生物酿酒酵母细胞周期遍历的稳健性和细胞周期间隔的持续时间。我们开发了一种定量全自动延时荧光显微镜装置，可以在完整的多细胞周期谱系的生成过程中对细胞周期调节基因表达进行半自动分析，并重新定位细胞周期调节因子。这种能力可以与有关芽殖酵母细胞周期调节的大量可用信息、系统地干扰细胞周期的遗传试剂的可用性以及细胞周期引擎的有用的确定性数学模型相结合。我们将检验以下假设：G1 长度的变异是由于细胞周期开始时的双稳态造成的。我们将生成野生型和突变体中细胞和核大小与细胞周期转变时间关系的定量单细胞数据，为核-细胞质大小耦合模型提供约束。我们将使用合成启动子策略与单细胞成像相结合来解决什么启动子结构可能导致不同水平的单细胞基因表达变异的问题。我们已经获得了细胞周期起始中随机分子变异的直接证据；我们将通过遗传和随机建模来追求这一发现。为了更动态地深入探索这些想法，我们开发了微流体方法，当单个受监测细胞从单个细胞增殖成微集落时，该方法允许在它们中产生周期性的基因表达脉冲。通过这项技术，我们将测试细胞周期引擎对调节因子的简短强制表达的响应，并寻找模式锁定和其他信息丰富的动态行为。我们将把这些分析工具扩展到有丝分裂调节，测试有丝分裂振荡器的复杂设计可以抑制噪声的假设。这些结果应该揭示单细胞分辨率下细胞周期引擎的鲁棒性和动力学的基本问题，以及基本复杂遗传电路运行中变异性的优点和缺点。

DESCRIPTION (provided by applicant): Single-cell variation (noise) in the operation of genetic networks is an inevitable consequence of the small size of cellular systems, leading to small numbers of molecules (genes, mRNAs, proteins) that may cause fluctuations in the function of important genetic control circuits. Where such noise is deleterious, it is likely that noise suppression mechanisms have evolved; noise could also be beneficial in some cases, by allowing breakout from dynamical dead-ends or by diversifying a population to meet a changing environment. Despite the significance of this topic, empirical results on noise have so far been largely restricted to simple synthetic gene circuits. We propose analysis of single-cell variation in a much more complex and physiologically relevant situation: the robustness of cell cycle traversal and duration of cell cycle intervals in the eukaryote S. cerevisiae. We have developed a quantitative fully automated time- lapse fluorescent microscopy setup allowing semi-automated analysis of cell-cycle-regulated gene expression and relocation of cell cycle regulators, throughout the generation of complete multi-cell cycle pedigrees. This capability can be integrated with the enormous amount of information available about the regulation of the budding yeast cell cycle, the availability of genetic reagents to systematically perturb the cell cycle, and useful deterministic mathematical models of the cell cycle engine. We will test the hypothesis that variability in G1 length is due to bistability at cell cycle Start. We will generate quantitative single-cell data on the relationship of cell and nuclear size to cell cycle transition times, in wild-type and mutants, to provide constraints for models on nucleo-cytoplasmic size coupling. We will attack the question of what promoter architectures may lead to different levels of single-cell variability in gene expression, using a synthetic promoter strategy combined with single-cell imaging. We have obtained direct evidence for stochastic molecular variation in cell cycle Start; we will pursue this finding genetically and with stochastic modeling. To pursue these ideas in more dynamic depth, we have developed microfluidic methods that allow periodic pulses of gene expression in individual monitored cells as they proliferate from single cells into microcolonies. With this technology we will test the response of the cell cycle engine to brief forced expression of regulators, and looking for mode-locking and other informative dynamic behaviors. We will extend these analytical tools to mitotic regulation, testing the hypothesis that the complex design of the mitotic oscillator functions to suppress noise. The results should shed light on fundamental questions of robustness and dynamics of the cell cycle engine at single-cell resolution, and on the advantages and disadvantages of variability in the operation of a fundamental complex genetic circuit.