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

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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.