Sources and Consequences of noise in cell cycle regulation

描述（由申请人提供）： 遗传网络运作中的单细胞变异（噪音）是细胞系统小规模的必然结果，导致分子（基因、mRNA、蛋白质）数量少，可能导致重要遗传控制回路功能的波动。在这种噪音有害的地方，很可能是噪音抑制机制已经进化;噪音在某些情况下也可能是有益的，通过允许从动态死胡同中突围或通过使种群多样化以适应不断变化的环境。尽管这一主题意义重大，但迄今为止，关于噪声的经验结果主要限于简单的合成基因回路。我们提出了一个更复杂和生理相关的情况下的单细胞变异分析：在真核细胞S细胞周期遍历和细胞周期间隔的持续时间的鲁棒性。啤酒。我们已经开发了一种定量的全自动延时荧光显微镜设置，允许在整个完整的多细胞周期谱系的生成过程中半自动分析细胞周期调节基因表达和细胞周期调节因子的重新定位。这种能力可以与大量的信息整合，可利用的芽殖酵母细胞周期的调节，遗传试剂的可用性，系统地扰乱细胞周期，和有用的确定性的数学模型的细胞周期引擎。我们将检验G1期长度的变异性是由于细胞周期开始时的双稳态的假设。我们将产生定量的单细胞数据的细胞和细胞核的大小与细胞周期的转换时间的关系，在野生型和突变体，提供核质大小耦合模型的约束。我们将攻击的问题是什么启动子架构可能会导致不同水平的单细胞基因表达的变异性，使用合成启动子策略结合单细胞成像。我们已经获得了细胞周期开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。为了在更动态的深度上追求这些想法，我们开发了微流体方法，当它们从单细胞增殖成小菌落时，允许在单个监测细胞中周期性地脉冲基因表达。有了这项技术，我们将测试细胞周期引擎的反应，以短暂的强制表达的监管机构，并寻找模式锁定和其他信息的动态行为。我们将这些分析工具扩展到有丝分裂调控，测试有丝分裂振荡器功能的复杂设计抑制噪声的假设。这些结果应该揭示在单细胞分辨率的细胞周期引擎的鲁棒性和动力学的基本问题，并在一个基本的复杂的遗传电路的操作的可变性的优点和缺点。

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.