CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly

Nature News · Feb 25, 2026 · Collected from RSS

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MainLipid droplets (LDs) are the primary lipid storage organelle in cells3,4, providing a safeguard against lipotoxicity and acting as a lipid repository that can be broken down rapidly to release lipids for cellular utilization3,4. LDs are derived from the endoplasmic reticulum (ER) and consist of a core of neutral lipids, such as triacylglycerol (TAG), which is encircled by a phospholipid monolayer decorated with regulatory proteins3,4. In liver hepatocytes, TAG may either be stored in cytoplasmic LDs or packaged into very low density lipoproteins (VLDLs) in the ER lumen for secretion1,5,6. The mechanisms that govern the storage and secretion of neutral lipids in hepatocyte LDs remain incompletely understood, and addressing this gap in knowledge is crucial for the development of new therapeutic strategies targeting hepatic steatosis1,2.Genetic modifiers of lipid storageTo identify genes involved in lipid storage, we performed a genome-wide fluorescence-activated cell sorting (FACS)-based CRISPR–Cas9 screen in Huh7 hepatoma cells using BODIPY 493/503 fluorescence as a reporter of neutral lipid storage (Fig. 1a, Extended Data Fig. 1a–c and Supplementary Table 1). Duplicate follow-up screens were performed using a custom validation of LD and metabolism (VLDM) single guide RNA (sgRNA) library to increase confidence and reduce false positives and negatives (Fig. 1b and Extended Data Fig. 1d,e). Genetic modifiers were associated with glycerolipid metabolism, including neutral lipid synthesis, lipolysis and additional processes that influence lipid metabolism (Fig. 1b, Extended Data Fig. 1f and Supplementary Fig. 1).Fig. 1: Parallel CRISPR–Cas9 screens reveal metabolic state-dependent genetic modifiers and identify CLCC1 as a key regulator of lipid storage.a, Schematic of FACS-based CRISPR–Cas9 screen approach to identify genes that regulate neutral lipid abundance, using BODIPY 493/503 as a neutral lipid reporter. gDNA, genomic DNA; SEQ, sequencing. b, Volcano plot indicating the gene effects (phenotype) and gene scores (confidence) for individual genes from batch retest screens in Huh7 cells. Gene effects and scores are calculated from two biological replicates. Genes of interest that increase (red) and decrease (blue) the amount of neutral lipid when deleted are highlighted. c, Heat map of clustered genes based on gene score across all conditions. Boxes 1–4 indicate clusters of core negative regulators that act to decrease LDs and boxes 5–10 indicate clusters of core positive regulators that act to increase LDs. HBSS, Hanks’ balanced salt solution; LPS, lipopolysaccharide; MASH, metabolic dysfunction-associated steatohepatitis. d, Representative confocal images of LDs labelled with BODIPY 493/503 in control (expressing safe-targeting sgRNA) and CLCC1-KO cells under basal conditions or following treatment with 1 µg ml−1 triacsin C for 24 h. Scale bar, 50 μm.Full size imageTo provide insights into the genetic modifiers of lipid storage under different metabolic states, we performed duplicate FACS-based screens using our VLDM sgRNA library under 11 different metabolic conditions (Extended Data Fig. 1g and Supplementary Table 2). This series of 22 genetic screens provides chemical–genetic interaction data, enabling the clustering of genes with similar functional profiles and facilitating functional predictions for candidate regulators (Fig. 1c and Extended Data Fig. 1h–r). These findings highlight the importance of the glycerolipid metabolic pathways and identify differences in the utilization of members of enzyme families, such as the use of different 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT), lipin and diacylglycerol acyltransferase (DGAT) enzymes under different conditions (Extended Data Fig. 1h–r and Supplementary Table 1). Genes that are known to have similar roles clustered together, such as the genes encoding the LD nucleation factor BSCL2 (also known as seipin) and its binding partner TMEM159 (also known as LDAF1 or promethin)7 (Extended Data Fig. 1k). These data establish a phenotype-rich compendium of genetic modifiers of neutral lipid storage. Screen data were deposited in the CRISPRlipid online data commons8 (https://crisprlipid.org/).CLCC1 controls LD dynamicsCLCC1 emerged as a priority candidate for characterization because: (1) it ranked highly as a core negative regulator (Fig. 1c and Extended Data Fig. 2a); (2) it clustered with known lipolysis regulator genes ABHD5 and PNPLA2 (Fig. 1c); and (3) genetic variants in CLCC1 in humans are associated with serum lipid alterations (Supplementary Fig. 2). Knockout of CLCC1 in Huh7 cells led to increased lipid storage (Extended Data Fig. 2b). CLCC1-knockout (CLCC1-KO) cells accumulated large LDs under multiple metabolic conditions and the LDs were stable following induction of lipolysis with the acyl-CoA synthetase inhibitor triacsin C (Fig. 1d and Extended Data Fig. 2c–f). CLCC1-KO cells exhibited an increased amount of TAG (Extended Data Fig. 2g) as a result of both increased TAG biosynthesis and decreased TAG breakdown (Supplementary Fig. 3a,b), suggesting alterations of multiple aspects of neutral lipid metabolism. Clcc1 loss disrupts hepatic lipid homeostasisClcc1 deletion is embryonic lethal in mice9. A spontaneous recessive mutation in mouse Clcc1 and conditional knockout of mouse Clcc1 cause ER stress and neurodegeneration10,11. However, the role of Clcc1 in hepatocytes is unknown. Floxed Clcc1 mice were injected with AAV8-TBG-Cre to delete Clcc1 in hepatocytes (Clcc1-HepKO) (Extended Data Fig. 2h). Hepatic loss of Clcc1 resulted in liver steatosis characterized by enlarged, whitened livers (Fig. 2a–c) with an increase in TAG and cholesteryl esters (Fig. 2d,e). Histology and electron microscopy revealed LD accumulation without evidence of fibrosis (Fig. 2f and Extended Data Fig. 2i). Analysis of plasma indicated a decrease in TAG and lipoproteins in the Clcc1-HepKO mice and a near complete abolishment of plasma apolipoprotein B (apoB)-containing lipoproteins (Fig. 2g–j and Extended Data Fig. 2j–l). However, plasma albumin levels were unchanged (Fig. 2i), indicating that not all secretion was impaired. Serum levels of aspartate aminotransferase (AST), a marker of liver damage, were also increased in the Clcc1-HepKO mice (Fig. 2k and Supplementary Fig. 3c). These findings demonstrate that CLCC1 has an important role in the regulation of hepatic lipid storage and secretion, and thereby protects hepatocytes from lipotoxicity.Fig. 2: Liver-specific deletion of CLCC1 results in hepatic steatosis and reduced lipoprotein secretion.a, Representative images of livers from control and Clcc1-HepKO mice. b, Change in liver mass normalized to body mass for control and Clcc1-HepKO mice. n > 8. c, Body mass of the indicated control and Clcc1-HepKO mice. n > 8. d,e, Quantification of TAG (d) and cholesteryl ester (CE) (e) normalized to phospholipid (PL) content using thin layer chromatography (TLC). Data represent mean ± s.d. of six mice. f, Representative haematoxylin and eosin (H&E), oil red O, Masson’s trichrome and picrosirius red-stained liver sections from control and Clcc1-HepKO mice. Scale bar, 100 μm. g,h, Plasma fractionation by fast protein liquid chromatography (FPLC) from female control and Clcc1-HepKO mice measuring cholesterol (g) and TAG (h) in all fractions. LDL, low density lipoprotein. i, Western blot analysis of apoB and albumin in plasma from four female control mice and four Clcc1-HepKO mice. j, Quantification of TAG in plasma using TAG-Glo Assay (Promega, J3160). Data represent mean ± s.d. of six mice. k, Quantification of AST in serum of wild-type and Clcc1-HepKO mice. Data represent mean ± s.d. of more than four mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; NS, not significant (P ≥ 0.05) by one-way ANOVA with Dunnett’s multiple comparisons test.Source dataFull size imageCLCC1 loss traps LDs in the ERIn a pairwise comparison of the previously published PLIN2–GFP8 and current BODIPY 493/503 screens, CLCC1 was an outlier; CLCC1 knockout was linked to an increase in neutral lipids but a counterintuitive decrease in PLIN2–GFP levels (Extended Data Fig. 3a). This was surprising because members of the perilipin family of LD ‘coat’ proteins are constitutively present on LDs12 and the amount of PLIN2 generally correlates with LD abundance8,13. PLIN2 protein was undetectable in CLCC1-KO cells (Fig. 3a). Incubation with the proteasome inhibitor MG132 rescued PLIN2 expression, indicating that despite apparent high amounts of LDs, PLIN2 is degraded post-translationally by the proteasome in CLCC1-KO cells (Extended Data Fig. 3b). Immunofluorescence staining of PLIN2 also revealed a strong reduction in PLIN2-positive LDs in the CLCC1-KO cells, with large LDs appearing to be completely devoid of any PLIN2 staining (Fig. 3b). Overexpression of CLCC1 rescued PLIN2–GFP localization to LDs (Extended Data Fig. 3c) and the reduction in PLIN2–GFP levels (Extended Data Fig. 6d) in CLCC1-KO cells, consistent with these phenotypes reflecting on-target depletion of CLCC1. Proteomics analyses of LD-enriched buoyant fractions validated the decrease in PLIN2 on LDs and revealed the decrease in many well known LD proteins (Fig. 3c and Supplementary Table 3). These results indicate that although CLCC1-KO cells appear to accumulate large LDs, they lack canonical LD proteins.Fig. 3: CLCC1-KO cells accumulate aberrant lipoproteins in the ER lumen.a, Immunoblot of PLIN2 in the indicated Huh7 cell lines. b, Fluorescence microscopy images of indicated Huh7 cell lines. PLIN2 was labelled with anti-PLIN2 and LDs were stained with 500 nM Lipi-Blue. Scale bar, 20 µm. c, Raw abundance values (in arbitrary units) for selected LD proteins obtained using proteomic analyses of buoyant LD-enriched fractions. Two technical replicates. d, Transmission electron microscopy of indicated negative-stained Huh7 cell lines. Scale bar, 200 nm. e, Fluorescence microscopy imag