Long LD et al. (1984) Worm Breeder's Gazette "a dominant suppressor of dpy-26."

Meyer BJ, Long LD

[Worm Breeder's Gazette, 1984]

We have sought suppressors of dpy-26(n199) as a means of obtaining mutations in genes that interact with dpy-26 or that are involved in general with regulating the expression of X-linked genes. dpy-26(n199) is a maternal-effect, hermaphrodite-specific lethal mutation. XX animals derived from XX animals heterozygous for the dpy mutant allele are slightly Dpy; XX animals derived from homozygous animals are usually dead (occasionally there are escapers). dpy-26 mutations also result in a recessive Him phenotype producing 5% XO animals; these males are essentially wild type. In order to obtain suppressors of n199 we selected for hermaphrodite survivors from animals homozygous for n199. In particular we mutagenized the strain dpy-26(n199) )/nT1 (IV:V) n752 with EMS, picked F1 animals displaying the unc-22 twitcher phenotype and scored their progeny for viable hermaphrodites. In this procedure s7 is used as a visible marker to identify animals homozygous for n199. nT1 balances the right side of chromosome IV. The most frequent class of putative suppressors turned out to be animals which had acquired a new unc-22 mutation on the nT1 chromosome. However, one mutant was isolated which acts as a dominant suppressor of all the dpy-26-associated phenotypes. This mutation has the following additional properties: 1) It also suppresses the closely- linked mutation unc-31(e169). 2) A true breeding suppressed strain cannot be isolated. 3) When suppressed and nonsuppressed hermaphrodites are stained with DAPI and their chromosomes examined a very large free duplication appears in the oocytes of the suppressed hermaphrodites but is absent from the oocytes of the nonsuppressed hermaphrodites. The duplication (yDp1) is probably a breakdown product of nT1 and may contain portions of both chromosomes IV and V. We are presently mapping the chromosomal location and extent of yDp1.

Long LD et al. (1985) Worm Breeder's Gazette "further steps towards a genetic understanding of dosage compensation."

Meyer BJ, Plenefisch JD, Long LD

[Worm Breeder's Gazette, 1985]

Mutations in dpy-21, dpy-27, and dpy-28 all display a strikingly similar phenotype: XX animals are severely affected whereas there is no visible phenotype in XO animals. Mutations in dpy-21 cause recessive dumpiness in XX animals, mutations in the other three cause, in addition to an XX-specific recessive dumpy phenotype, a maternal effect XX-specific lethality. These phenotypes have been interpreted as a disruption of dosage compensation in XX animals,leading to a general increase of X-linked gene expression in XX animals. Biochemical evidence (Meyer and Casson) suggests that this interpretation is valid, at least for the genes dpy-21, nWe have developed a morphological assay to detect changes in X-linked gene expression in these animals. As originally described at the 1985 Cold Spring Harbor C. we used lin-14(n179), an apparently hypomorphic temperature sensitive mutation which leads, at the restrictive temperature, to the precocious production of adult alae during the third larval molt. The assay can be quantitated by scoring the production of alae by the 12 midbody seam cells in hermaphrodites ( 10 in males). A reduction in lin-14(n179) expression would be reflected by an increase in the number of seam cells producing precocious alae, an increase in expression.would be reflected by a reduction in the number of seam cells producing precocious alae. Indeed we can significantly increase the number of seam cells producing alae in isothermally raised XX animals by halving the gene dosage: from 64/240 cells in n179/n179 (2 copy) animals to 228/240 cells in n179/nDf19 (1 copy) animals. We have observed a dramatic suppression of precocious alae formation in XX hermaphrodites homozygous for both lin- 14(n179) and mutations in dpy-21, n[See Figure 1] The non-chauvinistic dumpy mutation dpy-6(e14) mutation has no effect on lin-14(n179) expression in this assay, indicating that the suppression seen is not simply a consequence of being dumpy. Analysis of animals in the L4 molt, reveals that over 95% of the seam cells in all the strains produced alae, showing that suppression is not simply due to an inability of these strains to produce alae. In the corresponding XO animals we see no suppression, and perhaps some enhancement, of the mutant phenotype. [See Figure 2] Thus we show that mutations in dpy-21, ects on lin-14(n179) expression consistent with their predicted involvement in dosage compensation. As an additional characterization of the functioning of these genes we have investigated the interactions between mutations at these loci. We constructed pairwise combinations of these mutations and have scored the resulting strains for viability and phenotype. [See Figure 2] The double mutant strains are no dumpier than the relevant single mutants. This lack of additivity suggests that these genes are involved in a common process, and that a mutation in any one of these genes is sufficient to disrupt this process such that mutations in the others genes have little additional effect.

Li S et al. (2016) G3 (Bethesda) "A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans."

Wu M, Zhang X, Zhou S, Ma Y, Li S, Wu S, Chen L, Xu S, Cui Q, Zhang SO, Kong Y, Feng Y, Yu J

[G3 (Bethesda), 2016]

To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 m in diameter. From a saturated forward genetic screen of 6.7 x 10(5) mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 m. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22 & prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 & drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6 & drop-7, are up-regulated in LD growth, resistant to LD hydrolysis, but not defective in peroxisome import. Class III mutants drop-1 & drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion.

Xu N et al. (2012) J Cell Biol "The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface."

Zhang SO, Farese RV, Mak HY, Guo F, Haas JT, McKinney SA, Cole RA, Xu N, Bobba S

[J Cell Biol, 2012]

At the subcellular level, fat storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs), which are closely associated with the endoplasmic reticulum (ER). However, the molecular mechanisms that enable ER-LD interaction and facilitate neutral lipid loading into LDs are poorly understood. In this paper, we present evidence that FATP1/acyl-CoA synthetase and DGAT2/diacylglycerol acyltransferase are components of a triglyceride synthesis complex that facilitates LD expansion. A loss of FATP1 or DGAT2 function blocked LD expansion in Caenorhabditis elegans. FATP1 preferentially associated with DGAT2, and they acted synergistically to promote LD expansion in mammalian cells. Live imaging indicated that FATP1 and DGAT2 are ER and LD resident proteins, respectively, and electron microscopy revealed FATP1 and DGAT2 foci close to the LD surface. Furthermore, DGAT2 that was retained in the ER failed to support LD expansion. We propose that the evolutionarily conserved FATP1-DGAT2 complex acts at the ER-LD interface and couples the synthesis and deposition of triglycerides into LDs both physically and functionally.

Fu L et al. (2024) J Cell Biol "LET-767 determines lipid droplet protein targeting and lipid homeostasis."

He Y, Zhang J, Liu J, Wu H, Li C, Wu Y, Li Z, Liang B, Xu X, Li J, Wang Y, Wang H, Zou X, Jiang X, Liu P, Fu L

[J Cell Biol, 2024]

Lipid droplets (LDs) are composed of a core of neutral lipids wrapped by a phospholipid (PL) monolayer containing several hundred proteins that vary between different cells or organisms. How LD proteins target to LDs is still largely unknown. Here, we show that RNAi knockdown or gene mutation of let-767, encoding a member of hydroxysteroid dehydrogenase (HSD), displaced the LD localization of three well-known LD proteins: DHS-3 (dehydrogenase/reductase), PLIN-1 (perilipin), and DGAT-2 (diacylglycerol O-acyltransferase 2), and also prevented LD growth in Caenorhabditis elegans. LET-767 interacts with ARF-1 (ADP-ribosylation factor 1) to prevent ARF-1 LD translocation for appropriate LD protein targeting and lipid homeostasis. Deficiency of LET-767 leads to the release of ARF-1, which further recruits and promotes translocation of ATGL-1 (adipose triglyceride lipase) to LDs for lipolysis. The displacement of LD proteins caused by LET-767 deficiency could be reversed by inhibition of either ARF-1 or ATGL-1. Our work uncovers a unique LET-767 for determining LD protein targeting and maintaining lipid homeostasis.

Xu, Ningyi et al. (2011) International Worm Meeting "Local triglyceride synthesis by DGAT-2 promotes lipid droplet expansion."

Guo, Fengli, McKinney, Sean, Xu, Ningyi, Zhang, Shaobing, Cole, Ronald, Mak, HoYi

[International Worm Meeting, 2011]

Lipid droplets (LDs) are evolutionarily conserved organelles for cellular fat storage. We have previously shown that a loss of DAF-22 function, which attenuates peroxisomal fatty acid beta-oxidation, causes LD expansion and an increase in triglyceride levels. In order to identify genes that are required for LD expansion, we performed a genetic suppressor screen in the daf-22 mutant background and recovered multiple alleles that fell into at least 5 complementation groups. We report here the molecular cloning of dgat-2, which encodes a conserved diacylglycerol acyltransferase, the terminal enzyme for triglyceride synthesis. Loss of dgat-2 function inhibits LD expansion and reduced triglyceride levels of daf-22 mutant animals while over-expression of DGAT-2 is sufficient to promote LD expansion in wild-type animals. Photo-bleaching demonstrates that DGAT-2 is a LD resident protein. Electron microscopy reveals that DGAT-2 clusters at discreet foci on the LD surface. We engineered a mutant DGAT-2 that was tethered to the ER. This mutant DGAT-2 failed to support LD expansion, indicating that LD localization of DGAT-2 is necessary for its proper function. Our results demonstrate that local synthesis and deposition of triglyceride is critical for LD expansion. We are currently searching for proteins that act in concert with DGAT-2 using genetic and proteomic approaches.

Zhu X et al. (2018) J Genet Genomics "Whole-genome RNAi screen identifies methylation-related genes influencing lipid metabolism in Caenorhabditis elegans."

Liu Y, Zhu X, Liu P, Zhang H

[J Genet Genomics, 2018]

Lipid droplets (LDs) are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegans. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mthf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C.elegans were involved in the lipid metabolic change of mthf-1(RNAi) worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol (TAG) hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C.elegans, with implications for the study of lipid metabolic diseases.

Na H et al. (2015) Biochim Biophys Acta "Identification of lipid droplet structure-like/resident proteins in Caenorhabditis elegans."

Liu P, Wang MC, Zhu X, Zhang P, Liu Y, Xu N, Na H, Yu Y, Xie K, Cichello S, Chen Y, Zhang H, Mak HY, Yang F

[Biochim Biophys Acta, 2015]

The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans.

Klemm RW et al. (2013) Cell Rep "A conserved role for atlastin GTPases in regulating lipid droplet size."

Shibata Y, Guo Y, Liu TY, Farese RV, Mak HY, Lu H, Li CS, Boye-Doe A, Klemm RW, Jin D, Norton JP, Li L, Blackstone C, Cole RA, Crane MM, Rapoport TA, Park SH

[Cell Rep, 2013]

Lipid droplets (LDs) are the major fat storage organelles in eukaryotic cells, but how their size is regulatedis unknown. Using genetic screens in C.elegans for LD morphology defects in intestinal cells, we found that mutations in atlastin, a GTPase required for homotypic fusion of endoplasmic reticulum (ER) membranes, cause not only ER morphology defects, but also a reduction in LD size. Similar results were obtained after depletion of atlastin or expression of a dominant-negative mutant, whereas overexpression of atlastin had the opposite effect. Atlastin depletion in Drosophila fat bodies also reduced LD size and decreased triglycerides in whole animals, sensitizing them to starvation. In mammalian cells, co-overexpression of atlastin-1 and REEP1, a paralog of the ER tubule-shaping protein DP1/REEP5, generates large LDs. The effect of atlastin-1 on LD size correlates with its activity to promote membrane fusion invitro. Our results indicate that atlastin-mediated fusion of ER membranes is important for LD size regulation.

Zhang J et al. (2021) J Cell Biol "mmBCFA C17iso ensures endoplasmic reticulum integrity for lipid droplet growth."

Liang B, Zhang J, Wang Y, Xu X, Wu Y, Xu J, Fu L, Liu P, Jiang X, Yang R, Hu Y, Zou X, Zhang L, Li C

[J Cell Biol, 2021]

In eukaryote cells, lipid droplets (LDs) are key intracellular organelles that dynamically regulate cellular energy homeostasis. LDs originate from the ER and continuously contact the ER during their growth. How the ER affects LD growth is largely unknown. Here, we show that RNAi knockdown of acs-1, encoding an acyl-CoA synthetase required for the biosynthesis of monomethyl branched-chain fatty acids C15iso and C17iso, remarkably prevented LD growth in Caenorhabditis elegans. Dietary C17iso, or complex lipids with C17iso including phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol, could fully restore the LD growth in the acs-1RNAi worms. Mechanistically, C17iso may incorporate into phospholipids to ensure the membrane integrity of the ER so as to maintain the function of ER-resident enzymes such as SCD/stearoyl-CoA desaturase and DGAT2/diacylglycerol acyltransferase for appropriate lipid synthesis and LD growth. Collectively, our work uncovers a unique fatty acid, C17iso, as the side chain of phospholipids for determining the ER homeostasis for LD growth in an intact organism, C. elegans.