Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets 1,2 enclosed in a monolayer of phospholipids and associated proteins 3,4 . These dynamic organelles 5 serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. Here we show, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells that about 1.5% of all genes function in lipiddroplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1-COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to our understanding of human diseases involving excessive lipid storage.We studied lipid-droplet formation in Drosophila Schneider 2 (S2) cells, a proven system for functional genomic studies with efficient gene inactivation by RNAi 6 . We induced lipiddroplet formation by incubation with 1 mM oleate for 24 h. Staining with 4,4-difluoro-1, 3,5,7, showed that droplet size, number and overall volume were increased (Fig. 1a); cellular triacylglycerol content increased sevenfold (Fig. 1b) HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptcorresponded to lipid droplets with a red fluorescent protein mCherry 7 fused with lipid storage droplet-1 (LSD1), which localizes exclusively to the surface of lipid droplets 3 (not shown).Imaging this process by time-lapse microscopy of BODIPY-labelled cells after oleate addition (Supplementary Movie 1) showed that droplet formation occurred in steps (Fig. 1c). First, increased numbers of small droplets formed in dispersed locations throughout the cell. Next, droplets increased in size and finally aggregated into one or several large clusters, resembling grapes. Electron microscopy confirmed the tight clustering of the droplets, which were often near the nucleus (Supplementary Fig. 3).To unravel the molecular mechanisms governing this progression of changes during lipiddroplet formation, we performed a genome-wide RNAi screen in S2 cells (Fig. 2a). Images were acquired and examined by two independent observers, who scored them for alterations in droplet number, size and dispersion. The same data were analysed computationally (Supplementary Methods). From visual screening, both observers identified 847 candidate genes with altered lipid-droplet morphology. To verify these genes and to minimize the misidentification of genes from of...

Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.

Summary Lipid droplets (LDs) are cellular storage organelles for neutral lipids that vary in size and abundance according to cellular needs. Physiological conditions that promote lipid storage rapidly and markedly increase LD volume and surface. How the need for surface phospholipids is sensed and balanced during this process is unknown. Here, we show that phosphatidylcholine (PC) acts as a surfactant to prevent LD coalescence, which otherwise yields large, lipolysis resistant LDs and triglyceride (TG) accumulation. The need for additional PC to coat the enlarging surface during LD expansion is provided by the Kennedy pathway, which is activated by reversible targeting of the rate-limiting enzyme, CTP:phospho-cholin cytidylyltransferase (CCT), to growing LD surfaces. The requirement, targeting, and activation of CCT to growing LDs were similar in cells of Drosophila and mice. Our results reveal a mechanism to maintain PC homeostasis at the expanding LD monolayer through targeted activation of a key PC synthesis enzyme.

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LSD2 appears to represent a new class of regulators, a protein that transduces regulatory signals to a separable core motor machinery. In addition, the demonstration that LSD2 regulates both transport and lipid metabolism suggests a link between lipid-droplet motion and lipid homeostasis.