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In vitro inhibition of hepatic stellate cell activation by the autophagy-related lipid droplet protein ATG2A
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Publication date (Electronic):
18 June 2018
Journal:
Scientific Reports
Publisher:
Nature Publishing Group UK
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Abstract
Clinical studies have found that moderate intake of retinol or oleic acid can enlarge the lipid droplets of hepatic stellate cells and suppress their activation. However, the link between lipid droplets and cell activation is unknown. This study compared the dynamics of lipid droplet-associated protein expression between activated and reverted stellate cells. Reversion of the activated human stellate cell line LX-2 and inhibition of primary mouse stellate cell activation were induced by retinol or oleic acid, which resulted in larger lipid droplets and the downregulation of cell activation markers. Quantitative proteomics and immunoblotting were performed to compare lipid-droplet protein profiles between activated and reverted LX-2 cells. Compared to expression in activated cells, 50 lipid-droplet proteins were upregulated, whereas 28 were downregulated upon reversion. ATG2A was significantly enriched in lipid droplets of retinol/oleic acid-treated LX-2 cells and quiescent primary stellate cells. Reduced expression of α-SMA, increased expression of perilipin-3, enlarged lipid droplets, and suppression of autophagic flux were observed in ATG2A-deficient LX2 cells. Lipid-droplet protein profile changes during the reversion of activated stellate cells might provide new insights into the molecular mechanisms linking lipid droplets to liver fibrosis. ATG2A could represent a potential new drug target for hepatic fibrosis.
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Most cited references38
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Expanding roles for lipid droplets.
Michael Welte (2015)
Lipid droplets are the intracellular sites for neutral lipid storage. They are critical for lipid metabolism and energy homeostasis, and their dysfunction has been linked to many diseases. Accumulating evidence suggests that the roles lipid droplets play in biology are significantly broader than previously anticipated. Lipid droplets are the source of molecules important in the nucleus: they can sequester transcription factors and chromatin components and generate the lipid ligands for certain nuclear receptors. Lipid droplets have also emerged as important nodes for fatty acid trafficking, both inside the cell and between cells. In immunity, new roles for droplets, not directly linked to lipid metabolism, have been uncovered, with evidence that they act as assembly platforms for specific viruses and as reservoirs for proteins that fight intracellular pathogens. Until recently, knowledge about droplets in the nervous system has been minimal, but now there are multiple links between lipid droplets and neurodegeneration: many candidate genes for hereditary spastic paraplegia also have central roles in lipid-droplet formation and maintenance, and mitochondrial dysfunction in neurons can lead to transient accumulation of lipid droplets in neighboring glial cells, an event that may, in turn, contribute to neuronal damage. As the cell biology and biochemistry of lipid droplets become increasingly well understood, the next few years should yield many new mechanistic insights into these novel functions of lipid droplets.
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Lipid droplet functions beyond energy storage.
Michael Welte, Alex P. Gould (2017)
Lipid droplets are cytoplasmic organelles that store neutral lipids and are critically important for energy metabolism. Their function in energy storage is firmly established and increasingly well characterized. However, emerging evidence indicates that lipid droplets also play important and diverse roles in the cellular handling of lipids and proteins that may not be directly related to energy homeostasis. Lipid handling roles of droplets include the storage of hydrophobic vitamin and signaling precursors, and the management of endoplasmic reticulum and oxidative stress. Roles of lipid droplets in protein handling encompass functions in the maturation, storage, and turnover of cellular and viral polypeptides. Other potential roles of lipid droplets may be connected with their intracellular motility and, in some cases, their nuclear localization. This diversity highlights that lipid droplets are very adaptable organelles, performing different functions in different biological contexts. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.
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Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets
Anoop Velikkakath, Taki Nishimura, Eiko Oita … (2012)
INTRODUCTION Macroautophagy, hereafter referred to simply as autophagy, is an intracellular degradation process accompanied by unique membrane dynamics. An isolation membrane extends to enclose the cytoplasmic contents, resulting in formation of a double-membrane autophagosome. The autophagosome fuses with acidic compartments, endosomes and lysosomes, to degrade the materials inside the autophagosome. Autophagy is important for a wide range of physiological processes such as adaptation to starvation, quality control of intracellular proteins and organelles, embryonic development, elimination of intracellular microbes, and prevention of neurodegeneration and tumor formation (Cecconi and Levine, 2008; Deretic and Levine, 2009; Mizushima and Levine, 2010; Levine et al., 2011; Mizushima and Komatsu, 2011; White et al., 2010). Thirty-five autophagy-related (ATG) genes have been identified in yeast, and among them, ATG1–10, 12–14, 16–18, 29, and 31 are essential for autophagosome formation (Nakatogawa et al., 2009). The products of these genes are classified into six functional groups: the starvation-responsive Atg1 kinase complex (Atg1–Atg13–Atg17–Atg29–Atg31), the class III phosphatidylinositol 3 (PtdIns3)-kinase complex (Atg6–Atg14–Vps15–Vps34), the Atg12—Atg5–Atg16 complex (— indicates covalent attachment), the Atg8—phosphatidylethanolamine (PE) conjugate, the multimembrane-spanning protein Atg9, and the Atg2–Atg18 complex (Suzuki et al., 2007; Nakatogawa et al., 2009; Mizushima et al., 2011). These Atg products and their functional groups are highly conserved in other species, including mammals. It has been suggested that the Atg1 complex, the PtdIns3-kinase complex, and Atg9 appear to be important for nucleation of the isolation membrane and that the Atg12 and Atg8 conjugation systems are important for membrane elongation and/or membrane closure. However, the function of Atg2 has not been well characterized. Yeast Atg2, a ∼200-kDa protein, localizes to a perivacuolar punctate structure termed the preautophagosomal structure (PAS), where most Atg proteins gather and autophagosome formation is initiated (Shintani et al., 2001; Stromhaug et al., 2001; Suzuki et al., 2001, 2007; Wang et al., 2001). Recruitment of Atg2 to the PAS requires the Atg1 complex, the PtdIns3-kinase complex, and Atg9. Atg2 interacts with Atg18, a PtdIns(3)P-binding protein (Suzuki et al., 2001; Dove et al., 2004; Obara et al., 2008), and Atg9 (Wang et al., 2001). Atg2 and Atg18 are required for retrograde transport of Atg9 from the PAS (Reggiori et al., 2004). However, Atg2 is not essential for Atg8—PE conjugate and PAS localization of most of Atg proteins (besides Atg18), suggesting that Atg2 is genetically the most downstream factor in the autophagy pathway (Suzuki et al., 2007). In addition to identification of autophagy-related molecules, the membrane dynamics of autophagosome formation has also been extensively analyzed. Elongating isolation membranes are frequently observed to closely attach to the endoplasmic reticulum (ER; Kovács et al., 2007), and single or multiple connections between the isolation membrane and the ER have been detected (Hayashi-Nishino et al., 2009; Yla-Anttila et al., 2009). Consistent with these observations, autophagosomes appear to be generated at a subdomain of the ER termed the omegasome, which is labeled with another PtdIns(3)P-binding protein, double FYVE-containing protein 1 (DFCP1; Axe et al., 2008). Furthermore, many mammalian Atg proteins are also observed on or close to the ER membrane (Itakura and Mizushima, 2010; Matsunaga et al., 2010). All these findings suggest that the ER plays an important role in autophagosome formation as a membrane source or a platform. The ER is a central organelle in many cellular functions, such as protein biogenesis/degradation, membrane trafficking, biogenesis of organelles and lipid droplets, lipid metabolism, calcium storage/release, stress sensing, and regulation of cell death (Holthuis and Levine, 2005; Levine and Rabouille, 2005; English et al., 2009; Fagone and Jackowski, 2009; Hotamisligil, 2010; Ma et al., 2011). It is therefore reasonable to assume that autophagosome formation may share some machinery with the other ER-associated functions. However, such cross-talk has not been analyzed at the molecular level. Here we report the identification and characterization of human Atg2 homologues Atg2A and Atg2B. These Atg2 proteins are essential for autophagosome formation, presumably at a late stage. Atg2A is present on not only autophagic membranes but also lipid droplets. Silencing of both Atg2A and Atg2B increases the size and number of lipid droplets and causes their clustering. These changes are not observed in Atg5-depleted cells. These data suggest that mammalian Atg2A and Atg2B function both in autophagosome formation and regulation of lipid droplet volume and distribution. RESULTS Mammalian Atg2A and Atg2B are required for autophagy Humans have two Atg2 homologues, Atg2A (KIAA0404) and Atg2B (FLJ10242). Atg2A and Atg2B are similar to each other (44.5% of Atg2A residues are identical to those of Atg2B), and Saccharomyces cerevisiae Atg2 shows 15.5 and 15.8% identity to human Atg2A and Atg2B, respectively. We first tested whether human Atg2 homologues are essential for autophagy. In cells treated with small interfering RNA (siRNA) directed against Atg2A, Atg2B, or both (Atg2A/B), target proteins were efficiently depleted (Figure 1A). These results also confirmed that these antibodies recognize and distinguish the Atg2 isoforms. We used four different methods to measure autophagic activity. First, we performed the microtubule-associated protein light chain 3 (LC3) turnover assay to monitor autophagy flux. In control siRNA-treated cells, the amount of LC3-II (LC3—PE) increased during starvation, which further increased as a result of treatment with lysosomal protease inhibitors, indicating that LC3 was degraded by autophagy during starvation (Figure 1B). Although siRNA against Atg2B (siAtg2B) alone did not affect autophagic flux, it was partially inhibited by siRNA against Atg2A (siAtg2A; Figure 1B). Combination of both siRNAs (siAtg2A/B) completely abrogated the increase in LC3-II caused by starvation and lysosomal inhibition. In these cells, LC3-II accumulated even without starvation; this accumulation is often observed following abrupt depletion of autophagy factors such as Atg14, Vps34 (Itakura et al., 2008), and vacuole membrane protein 1 (VMP1; Itakura and Mizushima, 2010). FIGURE 1: Atg2A and Atg2B are essential for autophagy in mammalian cells. (A) HeLa cells stably expressing GFP-LC3 were transfected with the indicated siRNAs twice. Cell lysates were analyzed by immunoblotting with anti-Atg2A, anti-Atg2B, and anti-Hsp90 (internal control) antibodies. (B) HeLa cells stably expressing GFP-LC3 were treated with siRNA directed against Atg2A and/or Atg2B twice for 5 d and then cultured in regular DMEM or starvation medium in the presence or absence of protease inhibitors (E64d and pepstatin A) for 3 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies. (C) HeLa cells stably expressing GFP-LC3 were treated with control siRNA (a–c) and siRNAs against Atg2A (d–f), Atg2B (g–i), and both Atg2A and Atg2B (j–l) for 5 d and then cultured in regular DMEM (a, d, g, and j) or starvation medium for 1 h (b, e, h, and k) or 5 h (c, f, i, and l). GFP-LC3 signals were analyzed by fluorescence microscopy. Scale bar, 50 μm. (D) HeLa cells stably expressing GFP-LC3 were treated with control siRNA or siRNA against Atg2A and Atg2B and cultured as in C in the presence or absence of 0.2 μM wortmannin. Total cellular GFP-LC3 signals were analyzed by flow cytometry. Second, starvation-induced degradation of p62, a selective autophagy substrate, was suppressed in cells treated with siAtg2A/B (Figure 1B). Accordingly, p62 markedly accumulated in these cells, suggesting that autophagy flux is blocked in siAtg2A/B-treated cells. Third, we investigated the distribution of green fluorescent protein (GFP)–tagged LC3 (GFP-LC3), a marker for the autophagosomal membrane. HeLa cells stably expressing GFP-LC3 demonstrated diffuse GFP signals in the cytoplasm and nucleus, as well as a small number of tiny punctate structures, which likely represent autophagosomes (Figure 1C, a). The number of GFP-LC3 puncta increased following starvation for 1 h (Figure 1C, b). Prolonged starvation treatment (5 h) destroyed the GFP-LC3 signals because GFP-LC3 was degraded as an autophagic substrate in the lysosome (Figure 1C, c). By contrast, in siAtg2A/B-treated cells, large GFP-LC3 structures accumulated even before starvation treatment (Figure 1C, j and k). Of note, these larger structures remained after prolonged starvation, suggesting that they represent abnormal structures, which were not delivered to the lysosome (Figure 1C, l). Cells treated with either siAtg2A or siAtg2B were only slightly or not affected (Figure 1C, d–i). These data suggest that normal autophagosome formation was perturbed in siAtg2A/B-treated cells. Finally, we quantified the total GFP-LC3 level by flow cytometry, as it is a good measure of autophagic degradation (Shvets et al., 2008). In control siRNA-treated HeLa cells expressing GFP-LC3, GFP fluorescence was reduced following starvation for 5 h (Figure 1D, left). The fluorescence level was completely restored by addition of wortmannin, a PtdIns 3-kinase inhibitor, to suppress autophagosome formation, confirming that the reduction in the GFP-LC3 signal depends on autophagy. By contrast, such a reduction was not observed in siAtg2A/B-treated cells; a higher level of GFP fluorescence was maintained even after starvation for 5 h (Figure 1D, right). Taken together these results suggest that both Atg2A and Atg2B are required for autophagy and that they have redundant and overlapping functions, although Atg2A may have a dominant role in HeLa cells. Accumulation of unclosed autophagosome-related membranes in Atg2-depleted cells Although Atg2A/B-depleted cells showed defective autophagy, PE conjugation of LC3 (LC3-II formation) and formation of large GFP-LC3 puncta were observed (Figure 1, B and C). In these cells, LC3 might be included in aberrant autophagic structures, or LC3 protein might simply be aggregated. We therefore sought to determine whether other Atg proteins also accumulated in these structures. Punctate structures of endogenous Atg9A, GFP-ULK1 (an Atg1 homologue), GFP-Atg14, GFP–WD-repeat protein interacting with phosphoinoside (WIPI; an Atg18 homologue), and GFP-Atg5 were rarely observed in control siRNA-treated cells under nutrient-rich conditions (Figure 2). However, all these Atg proteins formed several punctate structures in siAtg2A/B cells without starvation treatment, and they colocalized with LC3 (Figure 2). Because these accumulated Atg proteins are representatives of major Atg functional complexes, these results suggest that Atg2A/B play an essential role, probably at a late step of autophagosome formation. FIGURE 2: Autophagy-related proteins accumulate in Atg2A/B-depleted cells. HeLa cells or HeLa cells stably expressing the indicated GFP-fused proteins were treated with control siRNA or siRNA against Atg2A and Atg2B. Cells cultured in regular medium were fixed and stained with the anti-Atg9A, anti-LC3 (CTB-LC3-2-1C; Cosmo Bio), and anti-GFP (A6455; Invitrogen) antibodies. Immunofluorescence images were obtained using a confocal microscope. Signal color is indicated by color of typeface. Scale bar, 5 μm. Next we analyzed the autophagosome-related membranes in siAtg2A/B cells by differential centrifugation. HeLa cells stably expressing GFP-LC3 were homogenized and the postnuclear supernatant (PNS) was fractionated by differential centrifugation. We used GFP-LC3 as a marker of the autophagosome-related membrane and p62 as a representative autophagy substrate (Figure 3A). In control siRNA-treated HeLa cells, under nutrient-rich conditions GFP-LC3 was present mostly in an LC3-I form (cytosolic form), which was recovered in a high-speed supernatant (HSS) fraction (Figure 3B and Supplemental Figure S1). To accumulate autophagosomes, we starved cells in the presence of bafilomycin A1, an inhibitor of V-type ATPase, which inhibits intralysosomal degradation and/or autophagosome–lysosome fusion (Klionsky et al., 2008; Figure 3, A and C). In these cells, GFP-LC3-II (membrane-bound PE-conjugated form) accumulated and was recovered in the low-speed pellet (LSP) fraction together with p62 (Figure 3C). GFP-LC3-II and p62 in the LSP fraction were resistant to proteinase K, but they became sensitive in the presence of Triton X-100 (Figure 3, A and C). LAMP1, a lysosomal marker, was recovered in both LSP and HSP fractions under nutrient-rich conditions but mainly in the LSP fraction following starvation treatment (Supplemental Figure S1). These data suggest that GFP-LC3-II and p62 collected in the LSP fraction were enclosed by autophagosomes/autolysosomes. FIGURE 3: Unsealed autophagosomes accumulate in Atg2A/B-depleted cells. (A) Schematic drawing of autophagosome formation and protease protection assay. (B–E) HeLa cells expressing GFP-LC3 were treated with control siRNA (B, C) or a mixture of siRNAs against Atg2A and Atg2B (D, E), and cultured in regular DMEM (B, D) or starvation medium containing 0.2 μM bafilomycin A1 (Baf) for 2 h (C, E). The PNS was separated into LSP, HSP, and HSS fractions and then analyzed by SDS–PAGE and immunoblotting using anti-GFP, anti-p62, and anti-Hsp90 antibodies. The subfractions were treated with proteinase K (ProK) with or without Triton X-100. In siAtg2A/B-treated cells, GFP-LC3-II and p62 were collected mainly in the HSP fraction under nutrient-rich conditions (Figure 3D) and both in the LSP and HSP fractions under conditions of starvation with and without bafilomycin A1 (Figure 3E and Supplemental Figure S1). These data suggest that LC3-II–positive structures accumulate in the absence of Atg2A/B. However, GFP-LC3-II and p62 collected in the LSP and HSP fractions were mostly sensitive to proteinase K, suggesting that complete closure of the autophagosome was impaired. Starvation-induced fraction shift of LAMP1 was less apparent in Atg2-depleted cells (Supplemental Figure S1). GFP-LC3-II in the LSP and HSP fractions in siAtg2A/B-treated cells was membrane bound, not an insoluble protein aggregate, because it was solubilized by Triton X-100 (Supplemental Figure S1). We further characterized the autophagy-related membrane structures generated in siAtg2A/B-treated cells by membrane floatation assay on OptiPrep density gradients (Figure 4). PNS fractions were subjected to equilibrium density gradient centrifugation, and membrane fractions were floated up to the top fractions. When the control cells were cultured in starvation medium containing bafilomycin A1, GFP-LC3-II and p62 were floated up to two peaks: fractions 8 and 9 and fractions 11 and 12 (Figure 4C). GFP-LC3-II and p62 in fractions 11 and 12 were highly resistant to proteinase K, whereas they were more sensitive to proteinase K in fractions 8 and 9 (Figure 4D). These results suggest that complete autophagosomes were mostly recovered in fractions 11 and 12 and partially in fractions 8 and 9. On the other hand, only small amounts of GFP-LC3 and p62 were floated up in control cells cultured under nutrient-rich and starvation conditions (Figure 4, A and B). In Atg2A/B-depleted cells, GFP-LC3-II was floated up to the two-peak fractions (8 and 9; 11 and 12) even under nutrient-rich conditions (Figure 4E). Following starvation, the peak around fractions 11 and 12 appeared to shift to a new peak at fractions 13–15, suggesting that aberrant autophagosome-related membranes were induced by starvation (Figure 4F). The amounts of p62 floated up with GFP-LC3 were less than those in control cells, suggesting that p62 cannot remain on these aberrant autophagic structures (Figure 4, C–F). All these morphological and biochemical experiments suggest that unclosed autophagosome-related membranes accumulate in Atg2A/B-depleted cells. FIGURE 4: Aberrant membrane structures accumulate in Atg2A/B-depleted cells. HeLa cells expressing GFP-LC3 were treated with control siRNA (A–D) or mixture of siRNAs against Atg2A and Atg2B (E–F) and cultured in regular DMEM (A, E), starvation medium (B, F), or starvation medium with bafilomycin A1 (C, D). PNS fractions were layered at the bottom of the OptiPrep density gradient and fractionated by centrifugation as illustrated in G. Fractions in C were treated with proteinase K after fractionation (D). The fractions were analyzed by SDS–PAGE and immunoblotting using anti-GFP and anti-p62 antibodies. N and P, nonfloat and PNS fractions, respectively. Atg2A localizes to the isolation membrane and lipid droplet Having demonstrated that Atg2 is essential for autophagy, we next determined the intracellular localization of Atg2A in HeLa cells. Exogenously expressed GFP-Atg2A forms many small punctate structures even under nutrient-rich conditions, with the number increasing only slightly during starvation (Figure 5A). Staining of endogenous LC3 showed that only a small population of GFP-Atg2A puncta colocalized with LC3, and majority of GFP-Atg2A structures were negative for LC3. We therefore tested various organelle markers and realized that most GFP-Atg2A puncta colocalized with lipid droplets. When we incubated cells with oleic acid, lipid droplets, which were stained with labeled fatty acids (BODIPY 558/568-C12), increased both in size and number, and GFP-Atg2A was observed on the surface of these lipid droplets both under nutrient-rich and starvation condition (Figure 5A). The shape of Atg2A signals was irregular, but it seemed to be affected during fixation and antibody staining; when we took live images of these cells without fixation and antibody staining, GFP-Atg2A smoothly surrounded the surface of lipid droplets (Figure 5B). Endogenous Atg2A was also detected on the surface of lipid droplets as small patches (Figure 5C). Similar to GFP-Atg2A, endogenous Atg2A partially colocalized with LC3 (Figure 5C). Costaining of Atg2A with LC3 or lipid droplets was specific because siAtg2A abolished these costaining signals (we still observed many small puncta in siAtg2A-treated cells, indicating that these are nonspecific reactions; Supplemental Figure S2). FIGURE 5: Atg2A is present on both autophagic structures and lipid droplets. (A, B) HeLa cells stably expressing GFP-Atg2A were cultured in the presence or absence of oleic acid-BSA (OA) for 16 h and then incubated in regular DMEM or starvation medium without oleic acid for 2 h. Cells were incubated with BODIPY 558/568-C12 during the last 1 h of culture. Cells were fixed, permeabilized, stained with anti-GFP and anti-LC3 antibodies, and examined by confocal microscopy (A). Cells were also directly observed by fluorescence microscopy without fixation and antibody staining (B). (C) HeLa cells were cultured in the presence of oleic acid-BSA for 24 h and then starved for 2 h. Cells were stained with BODIPY 558/568-C12 as in A. Endogenous Atg2A and LC3 were detected with anti-Atg2A and anti-LC3 antibodies. Signal color is indicated by color of typeface. Scale bars, 5 μm, and 1 μm in inset (A–C). Next we determined the stage of autophagosome formation at which Atg2A is recruited to the autophagic structures. Approximately 40% of LC3 puncta were positive for Atg2A, indicating that Atg2A is not always present together with LC3 (Figure 6, A and B). About 14% of the Atg2A+LC3+ structures were positive for Atg5, a typical isolation membrane marker (Mizushima et al., 2001), suggesting that Atg2A remains on the isolation membrane or autophagosome even after Atg5 dissociates from the structures (Figure 6, A and B). By contrast, we observed that ∼20% of the Atg5-positive puncta were negative for both Atg2 and LC3, and ∼50% of the Atg5- and Atg2-double positive puncta were negative for LC3 (Figure 6, A and C), suggesting that Atg2 is recruited to the autophagosome formation site later than Atg5 but before LC3. Autophagosome formation seems not to be directly related to lipid droplets because Atg2A–LC3–lipid droplet triple colocalization was rarely observed (Figure 5C). Taken together these data show that Atg2A is a unique Atg protein that localizes to both isolation membranes (possibly also to early autophagosomes) and lipid droplets. FIGURE 6: Atg2 is recruited to the autophagosome formation site later than Atg5 but before LC3. HeLa cells stably expressing GFP-Atg5 were cultured in starvation medium for 2 h. Cells were stained with anti-GFP, anti-Atg2A, and anti-LC3 antibodies (A). The ratios (%) of Atg2−Atg5 − , Atg2+Atg5−, Atg2 − Atg5+, and Atg2+Atg5+ populations to the total LC3-positive structures (B) and of Atg2 − LC3−, Atg2+LC3−, Atg2−LC3+, and Atg2+LC3+ populations to the total Atg5-positive structures (C) were calculated from 10 cells. Data represent mean ± SE of three independent cultures. Signal color is indicated by color of typeface. Scale bars, 5 μm, and 1 μm in inset. We previously reported the genetic hierarchy of mammalian Atg proteins in terms of recruitment to the autophagosome formation site (Itakura and Mizushima, 2010). We performed similar analysis for Atg2A. Atg2A colocalized with ULK1, an upstream Atg protein, in Atg5-knockout (KO) mouse embryonic fibroblasts (MEFs) under starvation conditions (Figure 7A). As we previously reported, inhibition of PtdIns 3-kinase with wortmannin did not affect ULK1 puncta formation, but it inhibited recruitment of Atg2A to the ULK1 structures (Figure 7, A and B). Thus recruitment of Atg2A to the autophagosome formation depends on PtdIns 3-kinase but not on Atg5. This hierarchical position of Atg2A is exactly the same as that of WIPI1 in mammals (Itakura and Mizushima, 2010) and Atg18 in yeast (Suzuki et al., 2007), suggesting that the WIPI proteins could be important for Atg2 recruitment. FIGURE 7: Targeting of Atg2A to autophagic structures depends on PI3K activity but not on Atg5, and that to lipid droplets depends on neither PI3K activity nor Atg5. Atg5 KO MEFs stably expressing GFP-Atg2A and HA-ULK1 were cultured in starvation medium containing BODIPY 558/568-C12 with or without 0.2 μM wortmannin for 1 h. Cells were subjected to immunofluorescence microscopy using anti-GFP and anti-HA antibodies. Arrows indicate ULK1-positive structures, and arrowheads indicate lipid droplets. Signal color is indicated by color of typeface (A). Atg2A-positive ratio of total ULK1 structures (B) and that of total lipid droplets (C) were quantified from 10 cells. Data represent mean ± SE of three independent experiments. Scale bars, 5 μm and 1 μm in inset. In contrast, localization of Atg2A to lipid droplets was not affected by 1-h and overnight wortmannin treatment (Figure 7, A and B, and data not shown) and did not depend on other Atg proteins such as focal adhesion kinase interacting protein of 200 kDa (FIP200) and Atg5 (Supplemental Figure S3). Amino acids 1723–1829 of Atg2A are essential for localization to both lipid droplets and autophagosomes and are required for autophagy We determined which region of Atg2A is required for targeting lipid droplets using Atg2-deletion mutants. A short fragment containing amino acids 1723–1829 was both required and sufficient for association with lipid droplets (Figure 8, A and C, and Supplemental Figure S4). This region has no sequence similarity with known lipid droplet–binding proteins but is highly conserved in Atg2B (Figure 8B). Consistently, the corresponding region of Atg2B (1863–1982) also has affinity to lipid droplets (Supplemental Figure S4). This region also shows relatively high similarity to Atg2 homologues in other organisms (Figure 8B), suggesting that this region should have some evolutionarily conserved function. In fact, this region is also important for localization to the autophagic membrane; an Atg2A mutant lacking this region—GFP-Atg2AΔ(1723–1829)—was able to colocalize with neither lipid droplets nor LC3 structures (Figure 8C). FIGURE 8: Amino acids 1723–1829 of Atg2A are essential for localization to both lipid droplets and autophagosomes. (A) GFP-tagged Atg2A fragments were expressed in HeLa cells, and their colocalization with lipid droplets was examined. Representative images are shown in Supplemental Figure S4. (B) Amino acid alignment of human Atg2A (NP_055919) with human Atg2B (NP_060506), Drosophila melanogaster Atg2 (NP_647748), Schizosaccharomyces pombe Atg2 (XP_001713120), and S. cerevisiae Atg2 (NP_014157). The alignment was generated using CLUSTAL W. Identical residues are indicated with filled boxes. The black line above the human Atg2A sequence shows the minimal region required for lipid droplet binding (1723–1829). (C) HeLa cells stably expressing wild-type GFP-Atg2A or its mutant (GFP-Atg2A Δ1723–1829) were cultured in regular medium containing oleic acid-BSA for 24 h and then in starvation medium containing BODIPY 558/568-C12) for 1 h. Cells were fixed, stained with anti-GFP and anti-LC3 antibodies, and subjected to confocal microscopy. Signal color is indicated by color of typeface. Scale bars 5 μm, and 2 μm in inset. We therefore determined whether the lipid droplet–targeting domain of Atg2 is essential for autophagy. As shown in Figures 1C and 2, silencing of both Atg2A and Atg2B caused accumulation of large LC3 aggregates, and it was suppressed by reexpression of GFP-Atg2A (Figure 9, A and B). However, reexpression of GFP-Atg2AΔ(1723–1829) showed no effect. Similarly, reexpression of GFP-Atg2A, but not of GFP-Atg2AΔ(1723–1829), restored autophagic flux in siAtg2A/B-treated cells (Figure 9C). Thus the Atg2A region containing amino acids 1723–1829 is important not only for targeting to lipid droplets, but also for autophagy. FIGURE 9: Amino acids 1723–1829 of Atg2A are required for autophagy. (A) HeLa cells stably expressing either siRNA-resistant wild-type GFP-Atg2A or GFP-Atg2A Δ1723–1829 were treated with control siRNA or a mixture of siRNAs against Atg2A and Atg2B. Cells grown in regular medium were fixed and subjected to immunofluorescence microscopy using anti-LC3 and anti-GFP antibodies. Scale bar, 5 μm. (B) Ratio of cells containing large LC3 punctate structures was quantified. Data represent mean ± SE of three independent treatments with siRNA. (C) HeLa cells used in A were cultured in regular DMEM or starvation medium in the presence or absence of 20 μM of chloroquine for 2 h. Cell lysates were analyzed by immunoblotting using the indicated antibodies. Depletion of Atg2A/B causes aggregation of enlarged lipid droplets Because mammalian Atg2 was detected on lipid droplets, we analyzed the role of Atg2 in lipid droplet formation and turnover. In control siRNA-treated cells, small lipid droplets were observed, which increased both in number and size following 16 h of treatment with oleic acid (Figure 10, A–C). On the other hand, in siAtg2A/B-treated cells, the size and number of lipid droplets increased even before oleic acid treatment (total pixel area of lipid droplets was approximately twofold higher than that of control cells; Figure 10, A–C). Some lipid droplets were markedly enlarged (Figure 10, A and D). Both total volume and number of lipid droplets further increased following oleic acid treatment (Figure 10, A–C). Of note, the distribution of lipid droplets was also altered. Lipid droplets are usually dispersed in control cells, but they tended to stick to each other and formed clusters (Figure 10, A and E, and Supplemental Movies 1 and 2). Successful knockdown of Atg2A/B was confirmed by immunoblotting (Supplemental Figure S5) and accumulation of large LC3 structures (Figure 10A). FIGURE 10: Silencing of Atg2A/B causes aggregation of enlarged lipid droplets. (A–D) HeLa cells were treated with the indicated siRNAs for 5 d and cultured without (0 h) or with oleic acid (16 h). Cells were fixed, stained with Sudan III (2 mg/ml), and immediately analyzed by fluorescence microscopy (B–D), or stained with anti-LC3 antibody and Sudan III and analyzed by confocal microscopy (A). Total lipid droplet pixel area (B) and the number of lipid droplets (C) were quantified in 20 randomly selected cells. Histogram analysis of size variation of 1000 lipid droplets in each group is shown (D). Data represent mean ± SE of three independent treatments with siRNA and oleic acid. Statistical difference determined by one-way analysis of variance (ANOVA) with Bonferroni–Dunn posttest (*p 3 lipid droplets in a circular region 2 μm in diameter in the absence of exogenous oleic acid or 2) accumulation of >10 lipid droplets in a circular region 5 μm in diameter following 16-h- oleic acid treatment. Cells having these lipid droplet clusters were counted. Statistical analysis Differences were statistically analyzed using analysis of variance with Bonferroni–Dunn posttest. Supplementary Material Supplemental Materials
Author and article information
Contributors
Yun Hong: yunhong@zju.edu.cn
Journal
Journal ID (nlm-ta): Sci Rep
Journal ID (iso-abbrev): Sci Rep
Title:
Scientific Reports
Publisher:
Nature Publishing Group UK
(London
)
ISSN
(Electronic):
2045-2322
Publication date
(Electronic):
18
June
2018
Publication date PMC-release: 18
June
2018
Publication date Collection: 2018
Volume: 8
Electronic Location Identifier: 9232
Affiliations
[1
]ISNI 0000 0004 1759 700X, GRID grid.13402.34, First Affiliated Hospital, , School of Medicine, Zhejiang University, ; Hangzhou, China
Article
Publisher ID: 27686DOI: 10.1038/s41598-018-27686-6
PMC ID: 6006255
PubMed ID: 29915313
SO-VID: 734cddfa-19b9-4b7f-b7bc-e1c8b0ffd191
Copyright © © The Author(s) 2018
License:
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
History
Date
received
: 10
January
2018
Date
accepted
: 18
May
2018
Categories
Subject:
Article
Custom metadata
issue-copyright-statement © The Author(s) 2018
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Data availability:
ScienceOpen disciplines: Uncategorized