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

绘制组织中的细胞代谢图：NADPH/NADP 氧化还原状态对细胞去分化、增殖和存活的调节。

• 批准号: RGPIN-2016-06468
• 负责人: Rocheleau, Jonathan
• 金额: $ 2.77万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=616329
• 关键词: Mapping; cell; metabolism; tissues; NADPH

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.

代谢严格控制细胞分化，生存和增殖。为了充分理解细胞代谢与这些多样反应之间的联系，我们需要测量具有高时间和空间分辨率的代谢通量的方法。 Rocheleau Lab对将定量荧光显微镜和微流体设备（“ iSlet-on-A-A-Chip”）相结合，以测量活胰岛中个体β细胞的代谢。胰岛是一种有价值的胰岛素分泌的宝贵模型系统。但还提供了研究组织中原代细胞的可塑性和存活的机会。我们以前的NSERC资金使我们能够开发出可调的遗传编码传感器（Apollo-NADP+），以测量细胞质NADPH/NADPH/NADP+氧化还原状态。我们最近还开发了微流体设备，用于高效的腺病毒转导（热），我们可以用来在整个小岛上表达传感器。 NADPH/NADP+氧化还原状态已与调节胰岛素分泌有关，但该反应的动态尚不清楚。 NADPH/NADP+氧化还原状态还通过生物合成途径来支持增殖，并通过清除活性氧。为了解决组织内的这些细胞反应，我们将利用并进一步开发Apollo-NADP+传感器和芯片上的胰岛。特别是，我们预计频谱通用的Apollo-NADP+将允许与其他遗传编码的传感器进行多参数成像，并通过进一步适应来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，包括刺激偶联的分泌，增殖和存活。这些研究具有三个针对NADPH/NADP+氧化还原状态的短期目标： 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​​否是增殖的标记。 3）确定NADPH/NADP+氧化还原状态在活性氧物种的产生和清除中的作用。 该研究计划非常依赖于我们在整个组织中使用一种新型的遗传编码传感器转导原代细胞（即具有正常代谢的细胞）的能力。我们对胰岛生物学的关注将揭示对NADPH/NADP+代谢动态的新见解，与糖尿病直接相关，但我们开发的工具将显示出对其他组织和疾病的研究的便捷翻译。最后，我们的研究计划将在传感器设计，细胞生物学，定量荧光显微镜，生物工程，微流体设备设计和生物物理学方面提供HQP高级培训，这些培训都处于现代研究的最前沿以及在学术和工业环境中的最大追捧。