AN INVESTIGATION OF NUCLEAR LIPID DROPLET BIOGENESIS IN CELL CULTURE AND ORGANOID MODELS.

Abstract

Lipid droplets (LDs) are dynamic organelles central to lipid storage and metabolic homeostasis in eukaryotic cells. In lipid-loaded cells, LD biogenesis primarily occurs in the ER membrane yielding cytoplasmic LDs, but nuclear LDs (nLDs) are also present in some cell types and found in a diverse range of eukaryotes. Promyelocytic leukemia (PML) protein isoform II promotes nLD biogenesis in human cells forming lipid associated PML structures (LAPS), but the molecular mechanisms are unclear. While nLDs often form in situ at the inner nuclear membrane (INM), nLD biogenesis in hepatocytes also involves ER luminal LD (eLD) lipoprotein precursors that accumulate in response to ER stress. We tested the hypothesis that nLD biogenesis in the intestine occurs by the same mechanism using cultured cells and mouse organoid models. Contrary to our hypothesis, nLDs did not arise from eLD lipoprotein precursors in intestinal cells. In oleate-treated Caco2 cells, nLDs and LAPS formed in situ at the INM and expanded from a pool of nascent precursors. nLDs were similarly observed in other intestinal cell lines as well as mouse enteroids, and differentiation of Caco2 cells promoted spontaneous nLD biogenesis without oleate addition. To investigate how nLDs are formed, multiple cultured cell types were treated with DGAT inhibitors (DGATi) and oleate to capture nLD associated proteins at early stages of nLD biogenesis at the INM. PML and CCTα translocated to the INM in DGATi and oleate-treated U2OS and Huh7 cells. PML isoform II promoted patches at sites deficient in nuclear lamina proteins, often occurring at sites of nuclear envelope (NE) ruptures. In U2OS cells, CCTα translocation to the INM blocked its proteasomal degradation resulting in a four-fold induction in protein expression and increased PC synthesis. Incubation of U2OS cells with unsaturated fatty acids, farnesol, or fatty alcohols, as well as phosphatidylcholine deficiency cell culture models, similarly promoted PML and CCTα translocation. Collectively, these data show that CCTα and PML localize to the INM in response to stored elastic curvature stress (ECS) caused by packing defects in the membrane. This, in turn, promotes nLD biogenesis in specific cell types to relieve ECS and maintain NE integrity.