Differentiating the biological effects of individual fatty acids in single cells

背景。我的研究计划的长期目标是推进我们对脂肪酸（FA）如何促进哺乳动物细胞生物学的基本理解。基于我们的工作，越来越清楚的是，两个基本的知识差距仍然存在：个体FA的作用，以及它们在特定细胞类型中的作用如何不同。因此，拟议的研究结合了现代分子和敲除（KO）小鼠方法，以推进我们关于个体FA生物学作用的基础知识，这是一个由于方法和模型的限制而一直难以捉摸的主题。我的研究项目揭示了FA家族如何影响细胞生物学，包括细胞膜组成，通过脂筏和小泡的信号传导，以及脂肪酸去饱和酶介导的代谢。这项工作也提供了越来越多的证据，证明单个FA具有独特的生物学特性。脂肪酸被认为是一个大家族，包括多不饱和脂肪酸（PUFA）、饱和脂肪酸（SAFA）、单不饱和脂肪酸（MUFA）和反式脂肪酸（TFA），尽管它们的结构存在显著差异（如链长、不饱和脂肪酸）。双键，顺式和反式几何)可能赋予独特的功能性质。同时，我们的工作也引发了一场关键的讨论，即作为一个领域，我们是如何依赖过于简单的方法来研究组织的，而这些方法没有考虑到这些组织中包含的细胞类型的多样性。在试点工作中，我们建立了分离特定成年乳腺细胞的方法，并对n-3 PUFA表现出不同的信号反应。出乎意料的是，我们还观察到n-3 PUFA影响管腔和肌上皮子细胞的比例，这表明n-3 PUFA在干细胞生物学中的作用。因此，虽然在特定细胞类型中研究单个FA在以前是不可能的，但现在存在模型和技术来追求这些问题。因此，我的短期目标是在单个细胞类型的水平上检查主要个体FA的精确影响。目标和方法。使用尖端的体内动物模型系统，我们的短期目标是研究主要个体PUFA， SAFA， MUFA和TFA对成体和干细胞膜脂组成，信号传导和基因表达的影响。这项工作将在能够分离单个脂肪酸影响的小鼠KO模型中进行。作用机制将从分离细胞的细胞膜、细胞信号和基因表达等多个层面进行评估。的意义。拟议的研究将解决我们对单个脂肪酸如何影响单细胞类型的理解中的基本空白。这项工作是在加拿大脂质营养研究的前沿，同时也有助于代谢和生理学领域。该研究项目还将为HQP培训做出重大贡献，为25名学员提供先进的技术和专业技能，使他们在广泛的职业生涯中具有很强的竞争力。

Background. The long-term objective of my research program is to advance our basic understanding of how fatty acids (FA) contribute to mammalian cell biology. Based on our work, it is increasingly clear that two fundamental knowledge gaps remain: the role of individual FA, and how their effects differ across specific cell types. Thus, the proposed research combines modern molecular and knock out (KO) mouse approaches to advance our foundational knowledge regarding the biological role of individual FA, a topic that has historically remained elusive due to limitations in methodologies and models. My research program has revealed how families of FA impact cell biology, including cell membrane composition, signalling through lipid rafts and caveolae, and metabolism mediated by fatty acid desaturases. This work has also provided increasing evidence that individual FA have unique biological properties. FA are viewed as large families that include polyunsaturated fatty acids (PUFA), saturated (SAFA), monounsaturated (MUFA), and trans (TFA) despite marked structural differences (e.g., chain length, no. of double bonds, cis & trans geometry) that likely confer unique functional properties. As well, our work sparked a critical discussion about how, as a field, we have relied on overly simplistic approaches to the study of tissues that do not account for the diversity of cell types contained within these tissues. In pilot work, we established methods to isolate specific adult mammary cells and show distinct signalling responses to n-3 PUFA. Unexpectedly, we also observed that n-3 PUFA influences the ratio of luminal and myoepithelial daughter cells, suggesting a role for n-3 PUFA in stem cell biology. Thus, while studying individual FA within specific cell types was not previously possible, models and technology now exists to pursue these questions. Therefore, my short-term objectives are to examine the precise effects of major individual FA at the level of individual cell types. Objectives and Approach. Using cutting edge in vivo animal model systems, our short-term objectives are to investigate the effect of major individual PUFA, SAFA, MUFA and TFA on membrane lipid composition, signalling and gene expression in adult and stem cells. This work will be conducted in mouse KO models capable of isolating the effects of individual fatty acids. Mechanisms of action will be assessed from isolated cells at multiple levels including the cell membrane, cell signalling and gene expression. Significance. The proposed research will address basic gaps in our understanding of how individual fatty acids affect single cell types. This work is at the forefront of lipid nutrition research in Canada while also contributing to fields of metabolism and physiology. This research program will also significantly contribute to HQP training, providing 25 trainees with advanced technical and professional skills and making them highly competitive for a wide range of careers.