Imaging single mRNA and protein molecules to investigate cell survival to environmental stresses

我们建议使用单分子成像方法来研究生物体在环境挑战中生存的分子机制。所有细胞在应对压力时的行为都是一样的。它们关闭了指导自身功能的基因程序，以激活一种被称为热休克反应的生存机制。热休克反应与热休克蛋白（HSPs）或分子伴侣的快速诱导有关。这种有效的生产克服了细胞中一般的基因表达抑制，并实现了高水平的热休克蛋白来处理所有错误折叠的蛋白质。一旦损伤得到修复，细胞就会彻底终止热休克蛋白的产生，恢复正常功能。因此，应激反应的实现需要激活和完成之间的快速和强大的协调：1)由细胞状态控制，2)直接影响编码热休克蛋白的信使RNA （mRNA）分子的生命周期。虽然引发热休克蛋白诱导的第一步的因素已经得到了很好的研究，但对这一生存程序精确执行背后的机制知之甚少。我们假设HSP mrna完全配备了独特的调节器。它们在转录位点的mRNA合成过程中被装载或印迹，并决定其命运以适应细胞的需要。因此，mRNA分子将能够整合其合成和衰变与定位和蛋白质生产。我们已经开发出新的工具，使mrna在细胞中合成时可见并成像它们编码的蛋白质。我们建议可视化和量化这些生物过程，因为它们发生在活细胞中。这些单分子荧光显微镜方法提供了在HSP mRNA命运的广泛调控中建立因果关系的时空分辨率。它们将使我们能够破译影响热休克蛋白mrna命运的因素。这些实验的结果将与适合研究酵母和哺乳动物细胞培养的生化和遗传方法相结合。这些方法将确定哪些途径和调节因子协调这种精确的细胞存活策略。我们预计，使用新的成像技术将解决细胞适应这一基本的生物学问题，并修订其调控的现有教条。我们未来的目标是研究微环境中的细胞，以确定触发应激反应的生理阈值。这一建议将为开发能够询问活的多细胞生物中的单分子并比较它们如何适应应激反应以追求特定功能的技术铺平道路。生物学和技术的协同作用为培养下一代科学家提供了一个跨学科的环境。

We propose to investigate the molecular mechanisms by which organisms survive to environmental challenges using single-molecule imaging approaches. All cells behave alike when coping with stress conditions. They shut down the genetic programs that direct their functions to activate a survival mechanism known as the heat shock response. The heat shock response relates to the fast induction of the heat shock proteins (HSPs) or molecular chaperones. This potent production overcomes general gene expression suppression in the cell and achieves high levels of HSPs to handle all misfolded proteins. Once the damage is repaired, cells drastically terminate the production of HSPs and resume regular functions. Thus, fulfillment of the stress response requires a fast and robust coordination between activation and completion that: 1) is commanded by the status of the cell and 2) directly influences the life-cycle of the messenger RNA (mRNA) molecules encoding the HSPs. Although the factors triggering the first steps in the induction of HSPs are well studied, little is known about the mechanisms behind the precise execution of this survival program. We hypothesize that the HSP mRNAs are fully equipped with unique regulators. They are loaded or imprinted in the mRNA during its synthesis at the transcription site and determine its fate to suit cellular needs. Hence, the mRNA molecules would be capable to integrate their synthesis and decay with localization and protein production. We have developed novel tools to render mRNAs visible and image the proteins they encode as they are being synthesized in the cells. We propose to visualize and quantify these biological processes as they happen in living cells. These single-molecule fluorescence microscopy approaches provide the spatiotemporal resolution to establish a cause-and-effect relationship in the extensive regulation of the HSP mRNA fate. They will allow us to decipher the elements that influence the fate of HSP mRNAs. The outcome from these experiments will be combined with biochemical and genetic methods suitable to study yeast and mammalian cells in cultures. These approaches will identify which pathways and regulatory factors orchestrate such precise strategy of cell survival. We anticipate that the use of new imaging technologies will solve this fundamental biological question on cell adaptation and revise existing dogmas of its regulation Our future goal is to investigate cells in their microenvironment in order to define the physiological thresholds that trigger the stress response. This proposal will pave the way to develop the technologies capable of interrogating single molecules in live multicellular organisms and compare how they have adapted the stress response to pursue specific functions. The synergy of biology and technology provides an interdisciplinary environment to train the next generation of scientists.