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

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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.