Mapping cell metabolism in tissues: NADPH/NADP+ redox state in the regulation of cell dedifferentiation, proliferation, and survival.

新陈代谢对细胞的分化、存活和增殖起着至关重要的作用。为了充分了解细胞代谢和这些不同反应之间的联系，我们需要高时间和空间分辨率的方法来测量代谢流量。罗切洛实验室长期以来一直对结合定量荧光显微镜和微流控设备(“芯片上的胰岛”)来测量活的胰岛中单个β细胞的新陈代谢感兴趣。胰岛是营养刺激的胰岛素分泌的一个有价值的模型系统，但也提供了研究组织内原代细胞的可塑性和生存的机会。我们之前的NSERC资金使我们能够开发一种光谱可调的遗传编码传感器(Apollo-NADP+)来测量细胞质NADPH/NADP+氧化还原状态。我们最近还开发了高效的腺病毒转导(HEAT)的微流控设备，我们可以用它来表达整个胰岛的传感器。NADPH/NADP+氧化还原状态与调节胰岛素分泌有关，但这种反应的动力学尚不清楚。NADPH/NADP+氧化还原状态也通过生物合成途径支持细胞增殖，并通过清除活性氧物种支持细胞生存。为了解决组织内的这些细胞反应，我们将开发和进一步开发Apollo-NADP+传感器和单芯片胰岛设备。特别是，我们预计光谱通用的Apollo-NADP+将允许与其他遗传编码传感器进行多参数成像，并通过进一步的适应将用于测量各种细胞器的氧化还原状态。我们的长期目标是揭示单个细胞的新陈代谢如何最终控制组织表型，包括刺激偶联分泌、增殖和生存。这些研究有三个短期目标，重点是NADPH/NADP+氧化还原状态： 1)确定NADPH/NADP+氧化还原状态在调节胰岛素分泌中的作用。 2)确定NADPH/NADP+氧化还原状态是否为细胞增殖的标志。 3)确定NADPH/NADP+氧化还原状态在活性氧产生和清除中的作用。 这项研究计划关键地依赖于我们使用一种新型的基因编码传感器在整个组织中转导原始细胞(即代谢正常的细胞)的能力。我们对胰岛生物学的关注将揭示与糖尿病直接相关的NADPH/NADP+代谢动力学的新见解，而我们开发的工具将显示出轻松转换到其他组织和疾病的研究。最后，我们的研究计划将为HQP提供传感器设计、细胞生物学、定量荧光显微镜、生物工程、微流控设备设计和生物物理学方面的高级培训，所有这些都处于现代研究的前沿，在学术和工业环境中备受欢迎。

Metabolism critically governs cell differentiation, survival, and proliferation. To fully understand the links between cell metabolism and these varied responses, we need methods that measure metabolic flux with high temporal and spatial resolution. The Rocheleau Lab has a long-standing interest in combining quantitative fluorescence microscopy and microfluidic devices ("islet-on-a-chip") to measure the metabolism of individual beta-cells within living pancreatic islets. Pancreatic islets are a valuable model system of nutrient-stimulated insulin secretion; yet also offer the opportunity to study the plasticity and survival of primary cells within a tissue. Our previous NSERC funding allowed us to develop a spectrally tunable genetically encoded sensor (Apollo-NADP+) to measure cytoplasmic NADPH/NADP+ redox state. We also recently developed microfluidic devices for highly efficient adenoviral transduction (HEAT), which we can use to express sensor throughout islets. NADPH/NADP+ redox state has been implicated in modulating insulin secretion yet the dynamics of this response are unclear. NADPH/NADP+ redox state also supports proliferation through biosynthetic pathways, and cell survival through the scavenging of reactive oxygen species. To address these cellular responses within a tissue, we will exploit and further develop the Apollo-NADP+ sensor and islet-on-a-chip devices. In particular, we anticipate the spectrally versatile Apollo-NADP+ will allow multiparametric imaging with other genetically encoded sensors, and by further adaptation be used to measure the redox state of various organelles. Our long-term objective is to reveal how the metabolism of individual cells ultimately controls tissue phenotype including stimulus-coupled secretion, proliferation and survival. These studies have three short-term objectives focused on NADPH/NADP+ redox state: 1) Determine the role of NADPH/NADP+ redox state in regulating insulin secretion. 2) Determine whether NADPH/NADP+ redox state is a marker for proliferation. 3) Determine the role of NADPH/NADP+ redox state in the production and scavenging of reactive oxygen species. This research program critically relies on our ability to transduce primary cells (i.e. cells with normal metabolism) throughout the tissue with a novel genetically encoded sensor. Our focus on islet biology will reveal new insight into the dynamics of NADPH/NADP+ metabolism with direct relevance to diabetes, yet the tools we develop will show facile translation to the study of other tissues and diseases. Finally, our research program will provide HQP advanced training in sensor design, cell biology, quantitative fluorescence microscopy, bioengineering, microfluidic device design, and biophysics, all at the forefront of modern research and highly sought in academic and industrial settings.