Discovering the new function of DRP1 on lipid metabolism - ABSTRACT Lipid droplets (LDs) are cytosolic organelles that serve as the major energy reservoir in most cell types. Dynamics LDs are tightly linked to the regulation of metabolic homeostasis. Nascent LDs are generated from the endoplasmic reticulum (ER). However, the key factors that are involved in the biogenesis of the LDs are poorly understood. Interestingly, we recently discovered that dynamin-related-protein1 (DRP1), a well-known mitochondrial fission protein, translocates onto ER and regulates the dissociation of the nascent LDs from the ER. Here, based on our preliminary observations, we hypothesize that DRP1 on ER directly fissions micro-LDs via its GTPase activity. To test the hypothesis, we propose three Specific Aims. In Aim 1, we will define the molecular mechanism(s) governing DRP1-mediated LD budding off from ER. Previously we found that lack of DRP1 leads to the retention of the nascent LDs in ER. To determine the direct fission function of DRP1 on the ER-bound LDs, we will apply both in vitro and in vivo systems that compositionally mimic the tubular topology of ER-LD to analyze DRP1 constricting and severing capacity. We will also determine whether GTPase domain in DRP1 is required for LD budding by applying a GTPase mutant (S38A) of DRP1 in the study. In Aim 2, we will define the cellular mechanism(s) governing the recruitment of DRP1 onto the ER. We will first determine how cAMP- PKA signaling regulates translocalization of DRP1 onto the ER. Specifically, we will analyze the phosphorylation sites in DRP1 by PKA and the function of the identified sites on its ER location. Then, we will study the molecular role of Seipin, a known ER membrane protein in ER-recruitment of DRP1. Specifically, we will determine the interaction between DRP1 and Seipin and detect the impaired ER-recruitment of DRP1 in the lieu of the loss-of-function of Seipin. In Aim 3, we will study the abnormal ER-LD phenotype in the DRP1-deficient adipocytes. First, we will investigate the abnormal changes on the pre-existing cytosol LDs by examining their size, number, morphology as well as the consequential effects on LD degradation and lipid metabolism. Then, we will examine the ER morphological changes, ER stress and hence other associated pathological changes. Collectively, the goal of the study is to achieve fundamental findings about DRP1 novel function on LD biogenesis. Understanding the critical factors, such as DRP1 and the detailed events in LD biogenesis and dynamics will not only help us to dissect the basic cellular function of LDs on lipid metabolism, but also shed light on the novel strategies to treat obesity-related lipid disorders.
