Fluorescent biosensors for metabolite imaging in live cells

描述（由申请人提供）：所有细胞都需要能量和化学成分来生长、增殖和响应环境信号。因此，包括糖尿病、癌症、发育障碍和传染病在内的病症在病因上都与中枢代谢有关。解释这些病理和发展新的治疗干预需要理解生物体生理的系统水平调节，其基础是酶活性和代谢产物的稳态水平。不幸的是，在活细胞中测量这些特性通常超出了当前技术的能力。代谢组学的进步使测量数百种代谢物的浓度和通量成为可能，而光谱技术使同位素标记化合物的低分辨率成像成为可能。然而，所有这些技术都受到侵入性、通用性、成像分辨率和吞吐量的限制。为此，本提案的目标是使具有大动态范围的遗传编码荧光生物传感器能够快速发展，从而能够测量活细胞的代谢状态。最近的研究表明，高度工程化的绿色荧光蛋白变体可以作为细胞内代谢物浓度的报告者。建造这些传感器在历史上

DESCRIPTION (provided by applicant): All cells require energy and chemical building blocks to grow, proliferate, and respond to environmental cues. As a consequence, pathologies including diabetes, cancer, developmental disorders and infectious diseases are etiologically linked to central metabolism. Interpreting these pathologies and developing novel therapeutic interventions requires understanding the systems-level regulation of organismal physiology that has its basis in enzymatic activities and steady-state levels of metabolites. Unfortunately, measuring these properties in living cells is generally beyond the capability of current technology. Advances in metabolomics have enabled measuring the concentration and fluxes for hundreds of metabolites while spectroscopic techniques allow for the low resolution imaging of isotopically labeled compounds. However, all such techniques are inherently limited by a combination of invasiveness, generality, imaging resolution, and throughput. To this end, the goal of this proposal is to enable the rapid development of genetically-encoded fluorescent biosensors with a large dynamic range capable of measuring the metabolic state of living cells. Recent work has shown that highly engineered variants of the green fluorescent protein can act as reporters of intracellular metabolite concentration. Constructing these sensors has historically been a slow, error-prone exercise in rational protein engineering, and thus there are only a handful of known useful biosensors. The goal of our work is to accelerate this process by several orders of magnitude. My laboratory is developing a multiplex directed evolution approach for the rapid assay of large biosensor libraries, enabling us to simultaneously optimize for superior spectral properties while also constructing many different sensors in parallel. As a proof of principle, we are applying this strategy for the construction of biosensors to measure metabolites associated with cellular proliferation, including glucose, glutamine, and lactate. In related work, we propose to use these sensors as genetic screening tools for the in vitro study of pathway regulation and as in vivo imaging probes in a C. elegans cancer model. If successful, these studies will enable the high-throughput measurement and microscopic imaging of metabolic function in living cells and open up new avenues of physiologic research at the cellular, tissue, and whole-animal level.