(819.2) CTP:phosphocholine cytidylyltransferase alpha regulates nLD biogenesis in Caco2 cells

Poster Board Number: A426

Michael McPhee (Dalhousie University), Jonghwa Lee (Dalhousie University), Graham Dellaire (Dalhousie University, Dalhousie University), Steven Pelech (University of British Columbia), Neale Ridgway (Dalhousie University, Dalhousie University)

Recent investigations reveal that nuclear lipid droplets (nLDs) stimulate lipid metabolic enzymes in response to excess fatty acid loading of cells. For example, in oleate-treated hepatocytes the rate-limiting enzyme in the CDP-choline pathway, CTP:phosphocholine cytidylyltransferase alpha (CCT alpha), is recruited to the surface of nLDs and stimulates the synthesis of phosphatidylcholine (PC). Hepatocyte nLDs derive from ApoB-free LDs in the lumen of the endoplasmic reticulum that migrate into the nucleoplasm through type I nucleoplasmic reticulum invaginations of the inner nuclear membrane (INM). Once formed inside the nucleus, nLDs recruit proteins CCT alpha, Lipin1 and promyelocytic leukemia (PML) to their surfaces. Endothelial cells of the colon also express ApoB lipoproteins for chylomicron assembly and secretion, so we hypothesized that these cells might also form nLDs in response to excess lipid loading. Indeed, using immunofluorescence confocal microscopy we found that oleate-treated Caco2 cells formed between 5 to 10 nLDs per cell that recruited both CCT alpha and PML to their surface. CRISPR/Cas9-mediated CCT alpha knockout Caco2 (CCT alphaKO) cells had significantly fewer but larger cLDs and nLDs compared to wild-type cells. The percentage of PML-positive nLDs increased in CCT alphaKO cells, suggesting that CCT alpha and PML compete for access to the nLD surface following oleate treatment. To determine if phosphorylation of CCT alpha regulates translocation to nLDs, phospho-mimetic and phospho-null S315/S319 and Y359/S362 mutants were expressed in CCT alphaKO cells and localization to nLDs was measured. In vitro phosphorylation assays of recombinant, dephosphorylated CCT alpha indicated that AMP-dependent kinase phosphorylated CCT alpha S319, which could be involved in regulating CCT alpha activity on nLDs. In summary, we have demonstrated that oleate treatment induces nLD biogenesis in Caco2 cells and that CCT alpha activity regulates the biogenesis of both nLDs and cLDs.

This research was supported by a grant from the Canadian Institutes of Health Research and an IWK Health Centre Graduate Studentship award.