Cellular neutral lipids, such as triglyceride (TAG), are mainly stored in the form of lipid droplets with a neutral lipid core covered by a phospholipid monolayer coated with various proteins. The size and the number of lipid droplets determine the cellular capacity for lipid storage. In terms of lipolysis, large lipid droplets are relatively inactive compared with small lipid droplets. Under lipid-overload conditions, the formation of large lipid droplets is beneficial to reduce lipotoxicity caused by excess lipids (Farese & Walther, 2009; Walther & Farese, 2012; Krahmer et al, 2013; Gluchowski et al, 2017; Yu & Li, 2017; Chen et al, 2019).
Lipid droplet size is determined by three factors: the neutral lipid content, the composition of the phospholipid monolayer, and the action of lipid droplet-associated proteins. Excess cellular energy, in the form of fatty acids or of glucose, which can be converted to fatty acids through glycolysis and de novo lipogenesis, can be stored through increasing the neutral lipid content and the expansion of lipid droplets (Ruggles et al, 2013; Welte, 2015; Ding et al, 2018). Phosphatidylcholine (PC) and phosphatidic acid (PA) regulate lipid droplet size by affecting the membrane curvature and the coalescence of lipid droplets, respectively (Guo et al, 2008; Fei et al, 2011). Some lipid droplet-associated proteins, such as DGAT2, CCT1, and GPAT4, are able to change the neutral lipid content and the composition of the phospholipid monolayer through local synthesis of phospholipids or TAG (Wilfling et al, 2013). In addition, other lipid droplet-associated proteins, such as Fsp27, PLIN2, and PLIN5, affect lipid droplet size through regulating lipid droplet fusion, lipolysis or lipid droplet contacts with other organelles such as mitochondria or peroxisomes (Listenberger et al, 2007; Guo et al, 2008; Fei et al, 2011; Gong et al, 2011; Wang et al, 2011; Li et al, 2017; Kong et al, 2020). Despite this rapidly growing knowledge, the activities of lipid droplet-associated proteins and the mechanisms that regulate them are far from clear.
Heterogeneity, both intercellular and intracellular, is an important but poorly studied feature of lipid droplets (Rinia et al, 2008; Herms et al, 2013). The variation in size represents just one type of lipid droplet heterogeneity. Lipid droplets may contain different neutral lipids, such as TAG, cholesterol ester (CE), or retinyl ester (RE), and these neutral lipids may also differ in the saturation of their fatty-acyl chains (Blaner et al, 2009; Molenaar et al, 2017; Pacia et al, 2020). The composition of lipid droplet-associated proteins also varies greatly, and this contributes significantly to lipid droplet dynamics (Rinia et al, 2008). Moreover, lipid droplets can even have heterogeneous origins. For example, lipid droplets in Drosophila fat cells can be classified into two subgroups (Diaconeasa et al, 2013; Ugrankar et al, 2019). One subgroup is called peripheral lipid droplets, which are small and near the cell surface. Peripheral lipid droplet formation depends on exogenous lipids transported from the intestine to adipose tissue. The other subgroup is medial lipid droplets, which originate from the peri-nuclear endoplasmic reticulum (ER). Their formation and growth mainly rely on de novo fatty acid synthesis. Although the heterogeneous aspects of lipid droplets are well-recognized, the regulation and coordination of different sub-populations of lipid droplets have not been well studied.
Here, we report a retinol dehydrogenase (RDH)/CG2064-Plin2-Bmm axis which regulates lipid droplet size under high-fat conditions. RDH/CG2064 directly interacts with Plin2. Increasing the level of RDH/CG2064 changes the localization of Plin2 from peripheral lipid droplets to small lipid droplets around the nucleus and also reduces the level and lipid droplet localization of Bmm lipase. The reduction of Bmm results in large lipid droplets under high-fat conditions. Finally, this axis maintains larval survival under prolonged starvation.
Results COP9 signalosome complex regulates lipid droplet size under high-fat conditionsTo study lipid storage regulation, we previously used pumpless-GAL4 (ppl-GAL4)-driven gene overexpression or knockdown in Drosophila 3rd instar salivary gland and fat body (Liu et al, 2014; Fan et al, 2017; Yao et al, 2018). Interestingly, overexpressing DGAT1 (ppl>DGAT1), which encodes a diacylglycerol O-acyltransferase for TAG biosynthesis, did not lead to significantly enlarged lipid droplets in 3rd instar larval fat body. The overall level of TAG is only marginally increased (Fig 1A and B). Besides genetic manipulation, we also reared larvae on a 30% coconut oil-containing high-fat diet. The expression of DGAT1 increases 2.8 fold under high-fat feeding (Fig EV1A). However, similar to DGAT1 overexpression (OE), the high-fat diet did not increase lipid droplet size in 3rd instar larval fat body (Fig 1C). In addition, the combination of high-fat diet and DGAT1 overexpression did not dramatically increase lipid droplet size compared with DGAT1 overexpression alone (Fig EV1B).
Figure 1. The CSN complex regulates lipid droplet size under high-fat conditions
3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm.
A. Top: schematic diagram of the TAG biosynthetic pathway. Bottom: overexpression of DGAT1 (ppl>DGAT1) does not affect lipid droplet size. B. Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. Error bars represent ± SEM. ***P < 0.001; NS: non-significant. C. The lipid droplet size is not increased when larvae are reared on a 30% high-fat diet. D. CSN RNAi alone does not affect lipid droplet size, but CSN RNAi dramatically increases the lipid droplet size when DGAT1 is overexpressed. E. Quantification of the lipid droplet size in (A) and (D). F. Lipid droplet size is increased in CSN7 RNAi larvae fed on a 30% high-fat diet. G. Quantification of the lipid droplet size in (C and F). ND: normal diet; HFD: high-fat diet.Data information: In (E) and (G), data were analyzed by one-way ANOVA with a post hoc Turkey’s multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were analyzed for each fat body. Error bars represent ± SEM. ***P < 0.001; **P < 0.01; and NS: non-significant.
Click here to expand this figure.Figure EV1. CSN-mediated protein degradation regulates lipid droplet size
A. Relative level of DGAT1 mRNA under normal and high-fat diet conditions assayed by Q-RT–PCR. Error bars represent ± SEM. ***P < 0.001. 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bar represents 25 µm. B. DGAT1 overexpression under high-fat feeding does not dramatically increase lipid droplet size compared with DGAT1 overexpression under normal feeding. C. Knocking down Cullin1, 2, 3 or 5 under the high-fat diet condition does not affect lipid droplet size. D. Quantification of the lipid droplet size in (C). E. Knocking down Cullin4 in larvae reared on a high-fat diet increases the lipid droplet size. tub-GAL80ts was used to decrease the RNAi efficiency. F. Quantification of the lipid droplet size in (E). HFD: high-fat diet. G. Knocking down Cand1 in DGAT1-overexpressing larvae (ppl>DGAT1) increases the lipid droplet size. H. Quantification of the lipid droplet size in (G).Data information: In (D, F and H), data were analyzed by one-way ANOVA with a post hoc Tukey’s multiple-comparison test. Each point represents data from one fat body. At least 30 cells were analyzed from each fat body. Error bars represent ± SEM. ***P < 0.001; **P < 0.01; and NS: non-significant.
To investigate why these high-fat conditions did not result in large lipid droplets, we conducted an RNAi screen in the ppl>DGAT1 background. We found that RNAi of CSN3 led to significantly enlarged lipid droplets in 3rd instar larval fat body (Fig 1D and E). Constitutively photomorphogenic 9 (COP9) signalosome (CSN) is an eight-subunit protein complex that modulates the activity of an E3 ubiquitin ligase, Cullin-Ring Ligase (CRL), by removing the ubiquitin-like molecule Nedd8 from Cullin (Craney & Rape, 2013). We then examined whether RNAi of other CSN subunits phenocopies CSN3 RNAi. RNAi knockdown of several other CSNs, including CSN2, CSN5, or CSN7, results in enlarged lipid droplets in ppl>DGAT1 3rd instar larval fat body (Fig 1D and E). Consistent with the increased lipid droplet size, the level of TAG is increased in CSN2 RNAi with DGAT1 OE compared with DGAT1 OE alone (Fig 1B).
Since we identified CSN3 in the DGAT1 OE background, we then tested whether CSN regulates lipid storage and lipid droplet size under normal conditions. In 3rd instar larval fat body, CSN2 RNAi slightly, but not significantly, increased the TAG level (Fig 1B), and it had no effect on lipid droplet size (Fig 1D and E). Similarly, RNAi of other CSN subunits did not increase the lipid droplet size (Fig 1D and E), which suggests that regulation of lipid droplet size by CSN depends on high-fat conditions. Indeed, CSN3 RNAi also increased the lipid droplet size of 3rd instar larval fat body when the animals were fed on a 30% coconut oil-containing high-fat diet (Fig 1F and G). Together, these results demonstrate that CSN plays an important role in regulating lipid droplet size under high-fat conditions.
CSN-mediated protein degradation regulates lipid droplet sizeCSN regulates CRL-mediated protein degradation by deneddylating Cullin. Since RNAi of the deneddylase CSN5 affects lipid droplet size (Fig 1D and E), we speculated that CSN may regulate lipid droplet size through modulating CRL-mediated protein degradation. Therefore, we examined the lipid droplet phenotype of Cullin knockdowns under high-fat conditions. There are five Cullin genes in Drosophila. RNAi of Cullin1, 2, 3, or 5 did not affect lipid droplet size in 3rd instar fat body of DGAT1 OE larvae (Fig EV1C and D), which suggests that these genes are unlikely to be involved in CSN-mediated lipid droplet size regulation. ppl-GAL4-driven Cullin4 RNAi results in early larval lethality, which prevents us from directly observing the lipid droplet phenotype in 3rd instar larvae. To circumvent the early lethality barrier, we used a repressible binary expression GAL4/GAL80 system to temporally control Cullin4 RNAi. We grew Tub-GAL80ts, ppl>Cullin4 RNAi animals at 18°C to lower the RNAi efficiency and shifted the temperature to 29°C at 96 h after egg laying to activate the RNAi effect. With that manipulation, we found large lipid droplets in 3rd instar fat body of Cullin4 RNAi larvae fed on a high-fat diet (Fig EV1E and F), which suggests that Cullin4 is important for lipid droplet size regulation.
Depletion of CUL4 and its “negative” regulator CSN induce the same phenotype. This apparent conflict is not surprising, because the neddylation-de-neddylation cycle is essential to maintain proper CRL function and disrupting the cycle often leads to a loss-of-function phenotype (Schwechheimer et al, 2001; Pierce et al, 2013; Bagchi et al, 2018). Along the same line, similar to the Cullin4 RNAi/high-fat diet combination, lipid droplet size is increased in 3rd instar fat body of DGAT1 OE larvae with Cand1 RNAi (Fig EV1G and H). Cand1 is a key player in recycling NEDD8 from CRL (Min et al, 2003). Loss of Cand1 impairs CRL-mediated protein degradation and mimics CSN knockdown. Taken together, these results indicate that CSN-mediated protein degradation regulates lipid droplet size under high-fat conditions.
CSN regulates lipid droplet size through degrading CG2064, an RDH homologTo identify downstream target(s) of CSN in this process, we performed comparative quantitative proteomic analysis on fat body samples from animals overexpressing DGAT1 alone (control), and overexpressing DGAT1 with CSN2 RNAi or CSN7 RNAi (Dataset EV1). We identified ~3,000 proteins from the proteomic analysis and we plotted the proteins according to the logarithm-transformed ratios of different samples and P-values. We focused on proteins with a P value below 0.05 and with a fold change of over 1.5 or below 0.67. With this standard, there are 131 up-regulated proteins and 66 down-regulated proteins in CSN2 RNAi compared with control, and 121 up-regulated proteins and 66 down-regulated proteins in CSN7 RNAi compared with control (Fig 2A). There are 90 up-regulated proteins and 45 down-regulated proteins in common between CSN2 RNAi and CSN7 RNAi samples (Fig 2A).
Figure 2. Elevating the level of RDH/CG2064 in CSN RNAi increases lipid droplet size
A. The number of proteins changed in CSN2 or CSN7 RNAi revealed by quantitative proteomics. Red: the number of up-regulated proteins and green: the number of down-regulated proteins. Candidate proteins from the quantitative proteomics are listed along with protein fold changes in CSN2 or CSN7 RNAi. 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bars represent 25 µm. B. RDH/CG2064 RNAi suppresses the enlarged lipid droplet phenotype in CSN2 RNAi under DGAT1 overexpression (ppl>DGAT1) or high-fat diet conditions. C. Quantification of the lipid droplet size in (B). HFD: high-fat diet. D. In Drosophila S2 cells expressing RDH/CG2064-GFP, treatment with the proteasome inhibitor MG132 increases the RDH/CG2064-GFP protein level detected by anti-GFP antibody in a western blot. E. The RDH/CG2064-GFP protein level is increased in ppl>DGAT1 larvae with CSN2 RNAi, as detected by anti-GFP antibody in a western blot. F. RDH/CG2064 overexpression increases lipid droplet size in ppl>DGAT1 larvae. G. Quantification of the lipid droplet size in (F). H. Relative TAG levels in different genetic backgrounds were measured by glyceride assay kit. For quantification, TAG levels were normalized to protein. I. RDH/CG2064 and CG2065 deletion does not affect lipid droplet size under normal or high-fat diet conditions.Data information: In (C), (G), and (H), data were analyzed by one-way ANOVA with a post hoc Turkey’s multiple-comparison test. Each point represents data from one fat body, and at least 30 cells were examined in each fat body. Error bars represent ± SEM. ***P < 0.001; **P < 0.01; and NS: non-significant.
We further examined the molecular functions of proteins in the overlapping group and performed a literature search for potential lipid links. This led us to four up-regulated proteins (CG2064, CG2065, CG9360, and Arc1) and three down-regulated proteins (Pka-R1, CG18135, and CG4500) (Fig 2A). CG2064, CG2065, and CG9360 belong to the NADP(H)-dependent short-chain dehydrogenase/reductase (SDR) family and are up-regulated more than 3-fold in CSN2 or CSN7 RNAi. SDR is a large protein superfamily and two SDR subfamilies, hydroxysteroid dehydrogenase (HSD) and RDH, have been reported to regulate lipid droplet dynamics (Deisenroth et al, 2011; Adams et al, 2014; Siddiqah et al, 2017; Yang et al, 2018). CG2064 and CG2065 belong to the RDH subfamily. Arc1 is a cytoskeleton protein and the cytoskeleton regulates lipid droplet morphology (Mosher et al, 2015). Pka-R1 is a cAMP-dependent protein kinase (PKA) regulator, and PKA is well known for its function in regulating lipase activity (Greenberg et al, 1991; Miyoshi et al, 2007). CG18135 is a glycerophosphocholine phosphodiesterase and CG4500 is a long-chain fatty acid ligase.
To further evaluate these candidates, we manipulated their expression in different genetic backgrounds and examined the lipid droplet phenotype. Overexpression of Arc1 and RNAi of Pka-R1, CG18135 or CG4500 did not lead to increased lipid droplet size (Fig EV2A). In addition, CG2065 RNAi or CG9360 RNAi did not rescue the enlarged fat body lipid droplet phenotype in 3rd instar DGAT1 OE larvae with CSN2 RNAi (Fig EV2B). These results suggest that these genes do not act downstream of CSN in regulating lipid droplet size. In contrast, CG2064 RNAi suppresses the large lipid droplet phenotype caused by CSN2 RNAi in DGAT1 OE larvae (Fig 2B and C). We observed a similar rescuing effect of CG2064 RNAi on CSN2 RNAi with high-fat feeding (Fig 2B and C). These results suggest that CG2064 acts downstream of CSN to control lipid droplet size under high-fat conditions.
Click here to expand this figure.Figure EV2. Testing candidate CSN downstream targets
3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bar represents 25 µm.
A. Overexpressing Arc1 or knocking down Pka-R1, CG18135 and CG4500 does not result in large lipid droplets in DGAT1-overexpressing larvae (ppl>DGAT1). B. CG2065 RNAi or CG9360 RNAi does not rescue the increased lipid droplet size phenotype caused by CSN2 RNAi in DGAT1-overexpressing larvae. C, D. The number and diameter of lipid droplets in different genetic backgrounds were quantified.We therefore focused on CG2064. Since CG2064 has not been functionally studied before, we refer to it hereafter as RDH/CG2064. We expressed a C-terminal GFP-tagged RDH/CG2064 in Drosophila S2 cultured cells. Treatment with the proteasome inhibitor MG132 dramatically increased the level of RDH/CG2064-GFP in S2 cells (Fig 2D), which indicates that RDH/CG2064 can be degraded through the proteasome pathway. We also generated an RDH/CG2064-GFP knockin Drosophila reporter line and compared the level of RDH/CG2064-GFP by western blot analysis of 3rd instar fat body lysates of DGAT1 OE larvae with and without CSN2 RNAi. As expected, CSN2 RNAi dramatically increases the level of RDH/CG2064-GFP (Fig 2E), which supports the hypothesis that CSN regulates RDH/CG2064 proteasomal degradation. Consistent with the post-transcriptional regulation of RDH/CG2064 by CSN, the mRNA level of RDH/CG2064 in 3rd instar larval fat body is not significantly changed by CSN2 RNAi (Fig EV3A). Together, these results indicate that CSN regulates lipid droplet size through proteasomal degradation of RDH/CG2064.
Click here to expand this figure.Figure EV3. The level of RDH/CG2064 and Plin2 in different genetic backgrounds
The relative level of RDH/CG2064 mRNA detected by quantitative RT–PCR in different genotypes. NS: non-significant. Fat bodies from 3rd instar larvae raised on a high-fat diet were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bar represents 25 µm. In larvae consuming a high-fat diet, RDH/CG2064 overexpression induces a large lipid droplet phenotype, which is suppressed by DGAT1 RNAi. The relative level of RDH/CG2064 mRNA in larval fat body cells detected by quantitative RT–PCR in different genotypes. ***P < 0.001. The RDH/CG2064-GFP protein level in larval fat cells is decreased in RDH/CG2064 RNAi, as detected by anti-GFP antibody in a western blot. Tubulin was used as loading control. 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Scale bar represents 25 µm. RDH/CG2064 RNAi does not affect lipid droplet size. The level of Plin2-GFP detected by anti-GFP antibody in a western blot. RDH/CG2064 overexpression increases lipid droplet size under high-fat feedingSince CSN RNAi increased the RDH/CG2064 level and the lipid droplet size under high-fat conditions, we next investigated whether RDH/CG2064 overexpression is sufficient to increase lipid droplet size. In 3rd instar larval fat body, RDH/CG2064 overexpression did not change the lipid droplet size under normal conditions, but it led to large lipid droplets with DGAT1 OE (Figs 2F and G, and EV2C and D). TAG measurement yielded a similar result (Fig 2H). In addition, in animals fed on a high-fat diet, DGAT1 RNAi suppressed the large lipid droplet phenotype induced by RDH/CG2064 OE (Fig EV3B). This suggests that RDH/CG2064 is likely a main target of CSN in regulating lipid droplet size under high-fat conditions.
We also examined the phenotype of RDH/CG2064 RNAi and mutants. The RNAi efficiency was verified by Q-RT–PCR (Fig EV3C). In addition, the protein level of RDH/CG2064-GFP was reduced upon RDH/CG2064 knockdown (Fig EV3D). We found that knockdown of RDH/CG2064 did not change the lipid droplet size in the fat body of wild-type and DGAT1 OE 3rd instar larvae (Fig EV3E). Considering that RDH/CG2064 and CG2065 are neighboring genes and both RDH/CG2064 and CG2065 levels are increased in CSN2/CSN7 RNAi, we generated RDH/CG2064 and CG2065 double deletion mutants to further examine the loss-of-function phenotype. We found that the lipid droplet size was not dramatically changed in the double mutants compared with controls under normal diet or high-fat diet conditions (Fig 2I). This suggests that RDH/CG2064 is not required to maintain lipid droplet size under normal conditions.
RDH/CG2064 regulates lipid droplet size independent of its enzymatic activityWe next investigated the mechanism by which RDH/CG2064 regulates lipid droplet size. RDH/CG2064 has a transmembrane (TM) domain at its N-terminus followed by a large dehydrogenase/reductase domain (Fig 3A). By BLAST, RDH/CG2064 is close to the mammalian RDH11/12 family. RDH12 is localized on the ER and its activity modulates retinol homeostasis (Keller & Adamski, 2007). ENV9, the yeast homolog of RDH12, increases lipid droplet size in a reductase activity-dependent manner (Siddiqah et al, 2017). Therefore, we first examined whether the enzymatic activity of RDH/CG2064 is required for lipid droplet size regulation. The SDR proteins have a YXXXK motif, which is the reductase catalytic sequence (Jornvall et al, 1995). We generated two mutations in the enzymatic domain YXXXK motif, Y204F and K208A, and an upstream active site mutation, N151L (Fig 3A). Surprisingly, overexpressing these putative reductase-dead forms of RDH/CG2064 also increased the lipid droplet size in the fat body of 3rd instar DGAT1 OE larvae (Fig 3B and C), which suggests that RDH/CG2064 regulates lipid droplet size independent of its enzymatic activity.
Figure 3. The membrane protein RDH/CG2064 regulates lipid droplet size independent of its enzymatic activity
The protein domain structure of RDH/CG2064. Grey box: predicted transmembrane region; Pink box: short-chain dehydrogenase domain. The key enzymatic motif (YXXXK) and active site (NXXG) are shown. 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Overexpressing wild-type or site-mutated RDH/CG2064 increases the lipid droplet size in larvae with DGAT1 overexpression (ppl>DGAT1). Scale bar represents 25 µm. Quantification of the lipid droplet size in (B). Immunofluorescent staining of RDH/CG2064-GFP with anti-GFP antibody in 3rd instar larval fat body. DAPI (blue) stains nuclei. RDH/CG2064 is enriched in the peri-nuclear region. Scale bar represents 25 µm. Immunofluorescence imaging of RDH/CG2064-GFP, detected with anti-GFP antibody, and the ER protein marker RFP-KDEL in 3rd instar larval fat body. RDH/CG2064 co-localizes with RFP-KDEL. Scale bar represents 10 µm. Whole lysates and cytosolic and membranous fractions were prepared from fat body samples of 3rd instar larvae expressing an RDH/CG2064-GFP knockin reporter. RDH/CG2064-GFP protein was detected by anti-GFP antibody in a western blot. RDH/CG2064-GFP is mainly present in the membranous fraction.We then examined the sub-cellular location of RDH/CG2064 with the help of the RDH/CG2064-GFP knockin reporter. The fluorescent signal of RDH/CG2064-GFP in 3rd instar larval fat body is very weak, so we boosted the signal by anti-GFP antibody staining. RDH/CG2064-GFP is not enriched on lipid droplets. Instead, the signal is smeared and enriched around the nucleus, likely reflecting the peri-nuclear ER region (Fig 3D). We further examined the sub-cellular location of RDH/CG2064-GFP by co-labeling with the ER protein marker RFP-KDEL. High-resolution confocal microscopy revealed strong co-localization between the RDH/CG2064-GFP signal and the ER marker signal (Fig 3E). To further examine the distribution of RDH/CG2064-GFP, we separated whole lysates of 3rd instar larval fat body into cytosolic and membranous fractions through super-speed centrifugation. By western blot analysis, the majority of the RDH/CG2064-GFP was found in the membranous fraction (Fig 3F), further supporting the ER localization of RDH/CG2064-GFP.
RDH/CG2064 interacts with the lipid droplet protein Plin2To reveal the molecular mechanism by which RDH/CG2064 regulates lipid droplet size, we next used anti-GFP antibody and performed immunoprecipitation-mass spectrometry (IP-MS) of RDH/CG2064-GFP with GFP as control to identify the interacting protein partners of RDH/CG2064. Among the potential RDH/CG2064-interacting proteins, the lipid droplet-associated Perilipin protein Plin2 is the most obvious candidate (Table EV1). Drosophila has two Perilipin proteins, Plin1 and Plin2 (Blanchette-Mackie et al, 1995; Miura et al, 2002; Welte et al, 2005). Plin2 genetically antagonizes brummer lipase (bmm), the sole Drosophila homolog of ATGL lipase (Zimmermann et al, 2004; Gronke et al, 2005), and overexpression of Plin2 increases lipid droplet size (Gronke et al, 2003; Bi et al, 2012). To validate the interaction of RDH/CG2064 and Plin2, we expressed a N-terminal Flag-tagged RDH/CG2064 in Plin1-GFP, Plin2-GFP, or Bmm-GFP functional knockin Drosophila reporter lines. Immunoprecipitations with anti-Flag antibody show that RDH/CG2064 pulls down Plin2, but not Plin1 or Bmm (Fig 4A). Furthermore, the physical interaction of RDH/CG2064 and Plin2 is also robust when DGAT1 is overexpressed (Fig 4B).
Figure 4. RDH/CG2064 interacts with Plin2
A, B. Lysates of 3rd instar larval fat body samples of different genetic backgrounds were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin2, but not Plin1 or Bmm, is specifically coimmunoprecipitated with RDH/CG2064 (A). Plin2 was coimmunoprecipitated with RDH/CG2064 in samples from larvae overexpressing DGAT1 (B). C. Schematic diagram of full-length RDH/CG2064 and TM domain-deleted RDH/CG2064 (∆TM). Lysates of Drosophila S2 cells expressing the different proteins were immunoprecipitated with anti-GFP antibody and detected by anti-GFP or anti-Flag antibody in a western blot. Both full-length and ∆TM RDH/CG2064 were coimmunoprecipitated with Plin2. D. Schematic diagram of Plin1, Plin2, and the Plin2-Plin1-C chimeric protein. Lysates of Drosophila S2 cells expressing the different proteins were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Neither Plin1 nor Plin2-Plin1-C chimeric protein coimmunoprecipitated with RDH/CG2064. E. Lysates of Drosophila S2 cells expressing full-length Plin1 or Plin1 with a C-terminal deletion (Plin1ΔC) were immunoprecipitated with anti-Flag antibody and then probed by anti-GFP or anti-Flag antibody in a western blot. Plin1ΔC coimmunoprecipitated with RDH/CG2064. F. 3rd instar larval fat bodies were stained by BODIPY (green) for lipid droplets and DAPI (blue) for nuclei. Under high-fat conditions, RDH/CG2064 overexpression increases the lipid droplet size, and Plin2 RNAi suppresses the enlargement induced by RDH/CG2064 overexpression. Scale bar represents 25 µm.We investigated the interaction of RDH/CG2064 and Plin2 further. RDH/CG2064 contains a TM domain and a dehydrogenase/reductase domain (Fig 3A). We expressed Plin2-GFP with Flag-tagged full-length RDH/CG2064 or TM domain-deleted RDH/CG2064 in Drosophila S2 cells. Both versions of RDH/CG2064 interact with Plin2 when immunoprecipitated with anti-GFP antibody, indicating that the TM domain of RDH/CG2064 is not required for its interaction with Plin2 (Fig 4C). Plin1, the other Drosophila PAT domain protein, has an extended C-terminal region compared with Plin2 (Fig 4D). Previous study showed that this C-terminal region is essential for the sub-cellular location of Plin1 and determines the functional difference between Plin1 and Plin2 (Bi et al, 2012). Considering that Plin2, but not Plin1, is able to interact with RDH/CG2064, we hypothesized that the C-terminal region of Plin1 may prevent its interaction with RDH/CG2064. Therefore, we expressed a chimeric Plin2-Plin1-C-terminal fusion protein along with Flag-tagged RDH/CG2064 in S2 cells. This fusion protein does not bind to RDH/CG2064 when immunoprecipitated with anti-Flag antibody (Fig 4D). Furthermore, although full-length Plin1 does not interact with RDH/CG2064, Plin1 with a C-terminal deletion does indeed interact with RDH/CG2064 (Fig 4E). Together, these results show that RDH/CG2064 interacts with Plin2, but not Plin1, and the C-terminal region of Plin1 may prevent its association with RDH/CG2064. Moreover, genetic analysis results suggest that Plin2 acts downstream of RDH/CG2064. The large lipid droplet phenotype of RDH/CG2064 OE animals on a high-fat diet is suppressed upon Plin2 knockdown (Fig 4F).
RDH/CG2064 changes the sub-cellular localization of Plin2Next, we investigated whether the CSN-RDH/CG2064 axis affects Plin2. We first examined the protein level of Plin2-GFP by western blot. In fat body samples from 3rd instar DGAT1 OE larvae with ppl>CSN2 RNAi or ppl>RDH/CG2064 overexpression, there is no evident difference in Plin2-GFP level compared with the DGAT1 OE control (Fig EV3F). This suggests that the CSN-RDH/CG2064 axis does not affect the Plin2 protein level.
We then explored whether RDH/CG2064 affects the subcellular localization of Plin2. Previous protein overexpression studies show that Plin2 is usually found in small lipid droplets, in particular lipid droplets in the cortical region of larval fat cells (Bi et al, 2012), which are also called peripheral lipid droplets (Diaconeasa et al, 2013). Consistent with previous overexpression results, the Plin2-GFP signal from the endogenous knockin reporter is also prone to localize on small cortical lipid droplets close to the plasma membrane in wild-type 3rd instar larval fat body. In CSN2 RNAi or RDH/CG2064 overexpression conditions, the peripheral Plin2-GFP signal is greatly reduced and the GFP signal is increased in medial lipid droplets (Fig 5A and B). In addition, the localization of Plin2 is significantly increased on lipid droplets around the nucleus (Fig 5A and B). This finding is in line with the peri-nuclear ER localization of RDH/CG2064. Therefore, although elevated expression of RDH/CG2064 in wild type is not sufficient to form large lipid droplets, it promotes the localization of more Plin2 to peri-nuclear and medial lipid droplets. Such a localization change may prime the lipid droplets so that they are ready to expand.
Figure 5. The CSN-RDH/CG2064 axis changes the sub-cellular localization of Plin2
Images of Plin2-GFP in 3rd instar larval fat body cells of different genotypes. Scale bar represents 25 µm.
A. CSN2 RNAi or RDH/CG2064 overexpression reduces the peripheral localization of Plin2-GFP and increases the localization of Plin2-GFP on small peri-nuclear lipid droplets (boxed). DAPI (blue) stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. B. The peripheral localization of Plin2-GFP is greatly reduced in fat body cells from larvae with CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells. C. In fat body cells from ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, the localization of Plin2-GFP is greatly reduced at the periphery and is dramatically increased on large medial lipid droplets. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. D. Fluorescence intensity line plots of the images in (C). GFP intensity along the line across a lipid droplet was measured by ImageJ. In ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression, Plin2-GFP localization on large medial lipid droplets is represented by two peaks. More than 30 lipid droplets were measured for each genotype. The plot shown is a typical image curve for the indicated genotype. E. The peripheral localization of Plin2-GFP is greatly reduced in fat body cells from ppl>DGAT1 larvae with CSN2 RNAi or RDH/CG2064 overexpression. Images were taken by focusing on the periphery of fat cells. F. The localization of Plin2-GFP is greatly reduced in peripheral lipid droplets and increased in medial lipid droplets in CSN2 RNAi larvae fed on a 30% high-fat diet. DAPI (blue) stains nuclei. White arrows indicate peripheral lipid droplet. Red arrows indicate medial lipid droplets. G. The peripheral lipid droplets in larval fat body cells from different genetic backgrounds were stained by BODIPY (green). CSN3 RNAi or RDH/CG2064 overexpression reduces the number of peripheral lipid droplets in both wild-type and ppl>DGAT1 larvae.We further investigated the effect of the CSN-RDH/CG2064 axis on Plin2 localization under high-fat conditions. The peripheral Plin2 localization is reduced in the fat body of 3rd instar DGAT1 OE larvae with CSN2 RNAi or RDH/CG2064 overexpression (Fig 5C–E). In these animals, more Plin2-GFP signal is present in large medial lipid droplets (Fig 5C–E). Similar changes of Plin2 localization were also observed in 3
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