Abstract
Lipid droplets (LDs) are evolutionarily conserved organelles that play critical roles in mammalian lipid storage and metabolism. However, the molecular mechanisms governing the biogenesis and growth of LDs remain poorly understood. Phosphatidic acid (PA) is a precursor of phospholipids and triacylglycerols and substrate of CDP-diacylglycerol (CDP-DAG) synthase 1 (CDS1) and CDS2, which catalyze the formation of CDP-DAG. Here, using siRNA-based gene knockdowns and CRISPR/Cas9-mediated gene knockouts, along with immunological, molecular, and fluorescence microscopy approaches, we examined the role of CDS1 and CDS2 in LD biogenesis and growth. Knockdown of either CDS1 or CDS2 expression resulted in the formation of giant or supersized LDs in cultured mammalian cells. Interestingly, down-regulation of cell death-inducing DFF45-like effector C (CIDEC), encoding a prominent regulator of LD growth in adipocytes, restored LD size in CDS1- but not in CDS2-deficient cells. On the other hand, reducing expression of two enzymes responsible for triacylglycerol synthesis, diacylglycerol O-acyltransferase 2 (DGAT2) and glycerol-3-phosphate acyltransferase 4 (GPAT4), rescued the LD phenotype in CDS2-deficient, but not CDS1-deficient, cells. Moreover, CDS2 deficiency, but not CDS1 deficiency, promoted the LD association of DGAT2 and GPAT4 and impaired initial LD maturation. Finally, although both CDS1 and CDS2 appeared to regulate PA levels on the LD surface, CDS2 had a stronger effect. We conclude that CDS1 and CDS2 regulate LD dynamics through distinct mechanisms.
Highlights
Lipid droplets (LDs) are evolutionarily conserved organelles that play critical roles in mammalian lipid storage and metabolism
LDs can be classified into initial LDs and expanding LDs according to their size and stage in life cycle. iLDs are those LDs of 400 – 800-nm diameter that are formed from the endoplasmic reticulum (ER). eLDs are converted from iLDs through a maturation/ growth process, mediated by specific proteins such as triglyceride synthesis enzymes [12], Arf1/COPI [13], and cell death-inducing DFF45-like effector C (CIDEC)/ Fsp27 [18, 19]
We confirmed our previous findings in CDS1 and CDS2 knockdown (KD) cells (Fig. 1, A–D and H), and here, we further extended those observations in CDS1 and CDS2 knockout (KO) cells generated by the CRISPR/Cas9 system (Fig. 1, E–G), that CDS depletion resulted in supersized LDs (sLDs) formation [15]
Summary
Lipid droplets (LDs) are evolutionarily conserved organelles that play critical roles in mammalian lipid storage and metabolism. The molecular mechanisms of LD biogenesis and expansion remain to be elucidated, the prevailing model of LD growth is that LDs originate from the endoplasmic reticulum (ER) before they expand and mature [5]. This process is regulated by metabolic enzymes, nonbilayer lipids, and LD-associated proteins [2, 5, 9,10,11,12,13,14,15,16,17]. Markers, such as Livedrop and Hpos, have been artificially made to label these pre-LDs [11, 17]
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