Relevance of lipid droplet-mitochondria contact sites for brown adipose tissue function

棕色脂肪组织（BAT）是在寒冷环境中通过称为线粒体非颤抖性产热过程维持稳定体温所必需的。为了满足这一过程的高能量需求，有效吸收血液营养素对维持BAT功能至关重要。因此，BAT能够在短时间内在分解代谢状态（产热）和合成代谢状态（脂质储存）之间切换。从循环中吸收的营养素以甘油三酯的形式储存在BAT的脂滴中。这些细胞器与产热线粒体一起是棕色脂肪细胞的主要成分。在非颤抖性产热开始后，储存在甘油三酯中的脂肪酸被释放出来，并作为产热的燃料。然而，高细胞内水平的游离脂肪酸对细胞存活是有害的，需要有效的转移过程进入线粒体。显微镜分析表明线粒体和脂滴之间存在紧密的空间相互作用，但即使是导致接触部位形成的基本触发因素也仍然难以捉摸。此外，这些接触部位对BAT功能的生理作用在很大程度上仍然未知。因此，在目前的建议中，我想检查的假设，介导线粒体和脂滴之间的相互作用的分子复合物形成后，在BAT外源性刺激。此外，我想阐明这些接触部位的生理作用。为了实现这些目标，将采用显微镜相互作用研究来阐明触发脂肪-脂滴接触部位形成的生理条件。在第二个目标中，介导相互作用的分子机制将使用邻近标记方法进行鉴定。在这种实验环境中，接触位点内富集的蛋白质将被特异性标记并通过质谱法检测。然后将采用遗传操作来检查在该筛选中鉴定的蛋白质在接触位点的起始和维持中的作用。在第三个目标中，将使用CRISPR-Cas9技术在原代脂肪细胞和小鼠模型中分析这些蛋白质与BAT功能的相关性。最后，所提出的方法不仅有助于理解细胞器接触部位形成的复杂机制，而且还定义了BAT功能中这些过程的生理作用。

Brown adipose tissue (BAT) is essential for the maintenance of a stable body temperature in cold environments through a process called mitochondrial non-shivering thermogenesis. To meet the high energetic requirements of this process, efficient uptake of blood-borne nutrients is crucial for sustained BAT function. Thus, BAT is capable of switching between catabolic states (thermogenesis) and anabolic conditions (lipid storage) within a short time frame. Nutrients taken up from the circulation are stored as triglycerides within the lipid droplets of BAT. Together with the heat-producing mitochondria, these organelles are the predominant components of brown adipocytes. After initiation of non-shivering thermogenesis, fatty acids stored in triglycerides are liberated and serve as fuel for thermogenesis. However, high intracellular levels of free fatty acids are detrimental for cellular survival, demanding for efficient transfer processes into mitochondria. Microscopical analyses indicate a tight spatial interaction between mitochondria and lipid droplets, yet even the fundamental triggers leading to the initiation of contact site formation remain elusive. Also, the physiological role of these contact sites for BAT function remains largely unknown. In the current proposal, I therefore want to examine the hypothesis that molecular complexes mediating the interaction between mitochondria and lipid droplets are formed in BAT upon exogenous stimulation. Additionally, I want to elucidate the physiological role of these contact sites. To achieve these goals, microscopical interaction studies will be employed to shed light on the physiological conditions triggering mitochondria-lipid droplet contact site formation. In the second aim, the molecular machinery mediating the interaction will be identified using a proximity labelling approach. In such experimental setting, proteins enriched within the contact site will be specifically labelled and detected by mass spectrometry. Genetic manipulation will then be employed to examine the role of the proteins identified in this screen for the initiation and maintenance of contact sites. In the third aim, the relevance of these proteins for BAT function will be analyzed using CRISPR-Cas9 technology in primary adipocytes and mouse models. Conclusively, the proposed approaches will not only help understanding the complex mechanisms involved in organelle contact site formation but also define a physiological role for these processes in BAT function.