Spatiotemporal Regulation of Organelle Interactions and Metabolism

ABSTRACT Metabolic diseases are a pressing public health issue in the United States. With the meteoric rise of insulin resistance, obesity, and atherosclerosis, mechanistic understanding of lipid metabolism could not be more imperative. Many of these metabolic disorders involve improper storage of lipids into cellular lipid storage organelles known as lipid droplets (LDs). These unique organelles, which contain a core of triglycerides (TAGs) and other neutral lipids, accumulate when dietary free fatty acids are abundant but are consumed when there is a high demand for energy. Although the metabolic roles of LDs are well appreciated, the mechanisms employed to regulate the growth and catabolism of individual LDs are poorly understood. It has emerged that protein networks that reside on the surface of LDs play critical roles in controlling the release of the lipids stored within. We have previously found that a protein that drives physical contacts between the ER and LDs, DFCP1, plays a key regulatory role in modulating the activity of adipose triacylglycerol lipase (ATGL) the rate limiting enzyme in LD catabolism. Specifically, we found that DFCP1 recruits and sequesters ATGL on the LD surface - even under conditions that promote the activation of ATGL - such as phosphorylation by energy sensing kinases, AMPK and PKA, or by interacting with cofactor CGI-58. Ablating DFCP1 from cells or forcing DFCP1 to disassemble from LDs leads to a dramatic increase in ATGL dynamics and lipolysis. Under these conditions, LD turnover is greatly accelerated leading to rapid breakdown of LDs and a surplus of free fatty acids in cells. Owing to the importance of this interaction, we found that ablation of DFCP1 from stem cells leads to changes in mitochondrial activity and lipid metabolism that ultimately leads to severe impairment of the ability of cells to differentiate. How DFCP1 specifically, and ER contact sites in general regulate lipid metabolism is not well understood, much less the roles these proteins serve in stem cell metabolism during differentiation. To address these gaps in our understanding of LD lipolysis, we are using bottom-up approaches to biophysically and structurally define how lipolytic complexes are assembled and regulated by protein networks on the LD surface. We will also employ novel optical biosensors to examine how organelle interactions correlate with metabolism and to define how LD contact site proteins regulate LD interactions with other organelles. Finally, we will examine how disruption of these LD contact site proteins and regulators of LD lipolytic complexes impairs the regenerative and differentiation capacity of stem cells. The mechanistic insight obtained by these studies will provide new understanding into how LD metabolism is regulated and helps to maintain cellular homeostasis, as well as how this process can be controlled to improve stem cell health and tissue regeneration.