Differentiating the biological effects of individual fatty acids in single cells

背景资料。我的研究计划的长期目标是促进我们对脂肪酸(FA)如何对哺乳动物细胞生物学做出贡献的基本理解。基于我们的工作，越来越清楚的是，仍然存在两个基本的知识差距：单个FA的作用，以及它们的影响在特定细胞类型中是如何不同的。因此，这项拟议的研究结合了现代分子和敲除(KO)小鼠的方法来推进我们关于个体FA的生物学作用的基础知识，这一主题由于方法和模型的限制而历来难以捉摸。我的研究计划揭示了FA家族如何影响细胞生物学，包括细胞膜组成、通过脂筏和小窝的信号传递以及由脂肪酸去饱和酶介导的新陈代谢。这项工作还提供了越来越多的证据表明，单个FA具有独特的生物学特性。脂肪酸被视为大家族，包括多不饱和脂肪酸(PUFA)、饱和脂肪酸(SAFA)、单不饱和脂肪酸(MUFA)和反式不饱和脂肪酸(TFA)，尽管存在显著的结构差异(如链长、NO.双键、顺式和反式几何构型)可能赋予独特的功能性质。此外，我们的工作引发了一场批判性的讨论，即作为一个领域，我们如何依赖过于简单化的方法来研究组织，而这些组织中包含的细胞类型的多样性没有得到考虑。在试点工作中，我们建立了分离特定成人乳腺细胞的方法，并对n-3PUFA显示出不同的信号反应。出乎意料的是，我们还观察到n-3PUFA影响腔上皮子细胞和肌上皮子细胞的比例，提示n-3PUFA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个体FA以前是不可能的，但现在已经有了模型和技术来探索这些问题。因此，我的短期目标是研究主要的个体FA在个体细胞类型水平上的准确影响。目标和方法。利用先进的活体动物模型系统，我们的短期目标是研究主要个体PUFA、SAFA、MUFA和TFA对成体和干细胞的膜脂组成、信号转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。将从多个层面对分离的细胞的作用机制进行评估，包括细胞膜、细胞信号和基因表达。意义重大。这项拟议的研究将解决我们在理解单个脂肪酸如何影响单细胞类型方面的基本空白。这项工作是加拿大脂肪营养研究的前沿，同时也对新陈代谢和生理学领域做出了贡献。这项研究计划还将对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领域具有极强的竞争力。

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.