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

代谢对细胞分化、存活和增殖起着至关重要的作用。为了充分了解细胞代谢与这些不同反应之间的联系，我们需要具有高时空分辨率的测量代谢通量的方法。Rocheleau实验室长期以来一直对结合定量荧光显微镜和微流体装置（“胰岛芯片”）来测量活胰岛内单个β细胞的代谢感兴趣。胰岛是营养刺激胰岛素分泌的一个有价值的模型系统；同时也为研究组织内原代细胞的可塑性和存活提供了机会。我们之前的NSERC资助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。我们最近还开发了用于高效腺病毒转导（HEAT）的微流体装置，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增殖，并通过清除活性氧支持细胞存活。为了解决组织内的这些细胞反应，我们将开发并进一步开发Apollo-NADP+传感器和胰岛芯片设备。特别是，我们预计光谱多功能阿波罗- nadp +将允许与其他基因编码传感器进行多参数成像，并通过进一步适应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代谢如何最终控制组织表型，包括刺激偶联分泌、增殖和存活。这些研究针对NADPH/NADP+氧化还原状态有三个短期目标：

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.