Lipid droplets are ubiquitous fat storage organelles and play central roles in lipid metabolism in both health and disease. These complex, dynamic organelles have been implicated in cellular functions well beyond lipid homeostasis. In particular, they have been proposed to serve as temporary storage sites for toxic or unstable proteins, as assembly platforms for macromolecular complexes, or as vehicles to deliver proteins intracellularly. Although lipid droplets clearly play an important role in the life cycle f several viruses and likely promote viral assembly, whether they act as transient protein sequestration site for endogenous proteins remains unresolved. A long list of proteins from other cellular compartments has been shown to relocate to lipid droplets in a regulated manner, but whether these proteins perform novel functions at the droplet surface or are indeed regulated by droplet association is unclear. A critical test of the sequestration model has important implications for our understanding of basic cellular and developmental processes as well as for the management of human diseases resulting from aberrant fat storage, including lipodystrophies and obesity. The goal of this project is to resolve this issue for one particularly provocative case of proposed protein sequestration, histone storage on lipid droplets in early Drosophila embryos. In this model system, it is possible to combine genetics, biochemistry and live imaging to address this general question. In these embryos, massive amounts of certain histones associate transiently with lipid droplets; this association is mediated by the novel protein Jabba, the putative histone anchor on droplets. In the absence of Jabba, histone levels in embryos are dramatically reduced and - when new histone synthesis is mildly compromised - embryos die with phenotypes indicating lack of sufficient histone supplies. These observations support a model that histones are stored on lipid droplets for later use in development. The goal of this project is to test central predictions of this model. Using a structure-function approach, the domains of Jabba that mediate droplet and histone binding will be identified and the physical state of histones on the droplets will be determined. This information will be employed to generate Jabba mutants lacking specific interactions or mislocalized to other cellular compartments; these mutants will be tested for their ability to rescue high histone levels and normal development. Live imaging and droplet transplantation will address how quickly histones are transferred from lipid droplets to nuclei and whether Jabba remains stably behind. Finally, histone overexpression will be employed to address if droplets normally buffer the histone supply and protect against histone overabundance. If successful, the proposed studies will provide novel insights into both histone metabolism and lipid-droplet function. Most importantly, these studies will provide a paradigm for how and why proteins from other compartments are transiently sequestered on lipid droplets.
Aberrant function of lipid droplets, the sites of cellular fat storage, has been linked to a host of human diseases, from obesity, cardiovascular disease, and diabetes to liver problems, wasting diseases, and propagation of viruses. Previously, lipid-droplet studies focused on their role in fat metabolism. This project explores a new function of droplets in regulated protein sequestration, a function that potentially contributes to the origin r severity of these diseases.
