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: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=687150
• 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.**

代谢决定细胞的分化、存活和增殖。为了充分了解细胞代谢和这些不同的反应之间的联系，我们需要高时间和空间分辨率的方法来测量代谢通量。Rocheleau实验室长期以来一直致力于将定量荧光显微镜和微流体装置（“芯片上的胰岛”）结合起来，以测量活胰岛内单个β细胞的代谢。胰岛是营养素刺激胰岛素分泌的有价值的模型系统;还提供了研究组织内原代细胞的可塑性和存活的机会。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，我们可以使用该装置在整个胰岛中表达传感器。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+氧化还原状态还通过生物合成途径支持增殖，并通过清除活性氧物质支持细胞存活。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+传感器和芯片上胰岛设备。特别是，我们预计光谱多功能的阿波罗NADP+将允许与其他基因编码的传感器进行多参数成像，并通过进一步的调整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。这些研究有三个短期目标，重点关注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+氧化还原状态是否是增殖的标志物。* 3)确定NADPH/NADP+氧化还原状态在活性氧物质的产生和清除中的作用。*这项研究计划严重依赖于我们用一种新的遗传编码传感器在整个组织中识别原代细胞（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与糖尿病直接相关的NADPH/NADP+代谢动力学的新见解，但我们开发的工具将显示出对其他组织和疾病研究的简单转化。最后，我们的研究计划将提供传感器设计，细胞生物学，定量荧光显微镜，生物工程，微流体装置设计和生物物理学方面的HQP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追捧。