In C. elegans, the epidermis and its overlying extracellular matrix form a primary protective barrier, functioning as the first line of defense against environmental factors. To properly develop those cellular boundaries, a tightly controlled interaction of many molecules and pathways is needed. Mutant alleles of paqr-2 and iglr-2 (lipid homeostasis), dpy-21 (membrane trafficking), and sma-1 (actin-binding spectrin) result in hermaphrodite tail tip defects suggesting that this simple 4-cell structure can serve as a sensitive model for the identification of pathways responsible for the establishment of cellular boundaries. With this in mind, we performed a small forward genetics screen of ∼800 ethyl methanesulfonate-mutagenized haploid genomes and identified 21 mutants with a Tail End Defects in the hermaphrodite phenotype. Whole genome sequencing of these mutants identified mutations in genes encoding either structural constituents of the cuticle itself (mostly collagen genes) or protein with regulatory functions. By using CRISPR/Cas9 we confirmed 6 novel alleles of ptr-18, paqr-2, nab-1, ncam-1, vab-9 and efn-4. We further characterized the loss of function allele ptr-18(et70), which encodes a patch domain-containing (PTCHD) protein homologous to human PTCHD1. ptr-18(et70) has a significant effect on growth and development of the worms, while also increasing membrane permeability. Lipidomics analysis revealed no major alterations in membrane lipid composition, implicating cuticle defects as the primary cause of the observed permeability phenotype.

Phosphatidylcholine (PC) is the most abundant phospholipid in eukaryotic membranes and is synthesized in part via the rate-limiting enzyme PCYT1A. In humans, hypomorphic PCYT1A variants cause diverse disorders. To define how graded reductions in PC synthesis affect organismal physiology, we generated and characterized a series of mutant alleles in the Caenorhabditis elegans homolog pcyt-1, including variants corresponding to disease-causing human mutations, as well as an auxin-inducible degradation allele. We identify a clear allelic hierarchy. The V146M variant is embryonic lethal, whereas A97T is largely benign. P154A is temperature-sensitive, and C211Y causes growth delay, reduced brood size, sterility, and lengthened lifespan at standard temperature. Phenotypes of C211Y are rescued by choline, CDP-choline, or PC supplementation, supporting reduced enzymatic function. Lipidomic profiling reveals that decreased PC synthesis consistently increases long-chain polyunsaturated fatty acids (LCPUFAs) in both PCs and phosphatidylethanolamine (PEs) at the expense of shorter saturated species, without markedly altering the PC/PE ratio at 20 °C. At elevated temperature, the P154A variant exhibits protein instability and a decreased PC/PE ratio. Despite significant lipid remodeling, canonical ER, mitochondrial, and metabolic stress GFP-based reporters are not activated; only the oxidative stress response is elevated, consistent with increased peroxidation-prone LCPUFAs in the pcyt-1 mutant. Acute auxin-induced degradation of PCYT-1 in larvae causes developmental arrest, while acute PCYT-1 degradation in adults disrupts oogenesis, demonstrating a continuous requirement for PC synthesis. Together, these findings establish a functional pcyt-1 allelic series and show that limiting PC synthesis drives compensatory remodeling toward LCPUFA-enriched membranes while rendering the germline particularly vulnerable.

In C. elegans , the epidermis and its overlying extracellular matrix form a primary protective barrier, functioning as the first line of defense against environmental factors. To properly develop those cellular boundaries, a tightly controlled interaction of many molecules and pathways is needed. Mutant alleles of paqr-2 and iglr-2 (lipid homeostasis), dpy-21 (membrane trafficking), and sma-1 (actin-binding spectrin) result in hermaphrodite tail tip defects suggesting that this simple four-cell structure can serve as a sensitive model for the identification of pathways responsible for the establishment of cellular boundaries. With this in mind, we performed a small forward genetics screen of ∼800 ethyl methanesulfonate-mutagenized haploid genomes and identified 21 mutants with a tail end defective (Ted) phenotype. Whole genome sequencing of these mutants identified mutations in genes encoding either structural constituents of the cuticle itself (mostly collagen genes) or protein with regulatory functions. By using CRISPR/Cas9 we confirmed six novel alleles of ptr-18, paqr-2, nab-1, ncam-1, vab-9 and efn-4. We further characterized the loss of function allele ptr-18(et70) , which encodes a patch domain-containing (PTCHD) protein homologous to human PTCHD1. ptr-18(et70) has a significant effect on growth and development of the worms, while also increasing membrane permeability. Lipidomics analysis revealed no major alterations in membrane lipid composition, implicating cuticle defects as the primary cause of the observed permeability phenotype. Article summary We performed a forward genetics screen to identify hermaphrodite C. elegans mutants with a tail end defect with the goal to discover membrane and morphogenesis regulators. The screen of 800 haploid genomes revealed 21 tail end defective mutants, including 8 novel alleles of interesting regulator protein. We conclude that the tail tip phenotype can be useful in discovery of new pathways and interactions during development.

BACKGROUND: Maintaining a proper balance between saturated and unsaturated fatty acids in membrane phospholipids is essential for normal cellular function. The evolutionarily conserved transmembrane protein AdipoR2 plays a central role in this homeostatic process. While the detrimental effects of saturated fatty acids on cells have been previously documented, the associated ultrastructural changes remain less investigated. METHODS: Here, we used transmission electron microscopy to study the consequences of silencing AdipoR2 in the presence or absence of fatty acid supplements. RESULTS: We found that exposure of human cells to palmitic acid (PA)—the most abundant saturated fatty acid in the human body—disrupts the ultrastructure of cytoplasmic membranes and mitochondrial cristae. PA exposure also induces distinctive blebbing between the inner and outer membranes of the nuclear envelope. These membrane abnormalities are exacerbated by AdipoR2 silencing and are partially prevented by supplementation with oleic acid (OA), an unsaturated fatty acid. Furthermore, we observed ectopic localization of the mitophagy marker PINK1 and the fatty acid metabolism enzyme ACSL1 to closely apposed ER membranes, a structure that forms exclusively in PA-treated cells. CONCLUSIONS: Together, these findings reveal that exogenous PA triggers significant membrane defects, worsened in the absence of AdipoR2, and alters protein distribution within the cell.

Polyunsaturated fatty acids (PUFAs) are essential for mammalian health and function as membrane fluidizers and precursors for signaling lipids though the primary essential function of PUFAs within organisms has not been established. Unlike mammals who cannot endogenously synthesize PUFAs, C. elegans can de novo synthesize PUFAs starting with the Δ12 desaturase FAT-2 which introduces a second double bond to monounsaturated fatty acids to generate the PUFA linoleic acid. FAT-2 desaturation is essential for C. elegans survival since fat-2 null mutants are non-viable; the near-null fat-2(wa17) allele synthesizes only small amounts of PUFAs and produces extremely sick worms. Using fluorescence recovery after photobleaching (FRAP), we found that the fat-2(wa17) mutant has rigid membranes and can be efficiently rescued by dietarily providing various PUFAs, but not by fluidizing treatments or mutations. With the aim of identifying mechanisms that compensate for PUFA-deficiency, we performed a forward genetics screen to isolate novel fat-2(wa17) suppressors and identified four internal mutations within fat-2, and six mutations within the HIF-1 pathway. The suppressors increase PUFA levels in fat-2(wa17) mutant worms and additionally suppress the activation of the daf-16, UPRer and UPRmt stress response pathways that are active in fat-2(wa17) worms. We hypothesize that the six HIF-1 pathway mutations, found in egl-9, ftn-2, and hif-1 all converge on raising Fe2+ levels and in this way boost desaturase activity, including that of the fat-2(wa17) allele. We conclude that PUFAs cannot be genetically replaced and that the only genetic mechanism that can alleviate PUFA-deficiency do so by increasing PUFA levels.

To investigate how the fatty acid composition of brain phospholipids influences brain-specific processes, we leveraged the AdipoR2 (adiponectin receptor 2) knockout mouse model in which the brain is enlarged, and cellular membranes are excessively rich in saturated fatty acids. Lipidomics analysis of brains at 2, 7, and 18 months of age showed that phosphatidylcholines, which make up about two-thirds of all cerebrum membrane lipids, contain a gross excess of saturated fatty acids in AdipoR2 knockout mice, and that this is mostly attributed to an excess palmitic acid (C16:0) at the expense of oleic acid (C18:1), consistent with a defect in fatty acid desaturation and elongation in the mutant. Specifically, there was a ~12% increase in the overall saturated fatty acid content within phosphatidylcholines and a ~30% increase in phosphatidylcholines containing two palmitic acids. Phosphatidylethanolamines, sphingomyelins, ceramides, lactosylceramides, and dihydroceramides also showed an excess of saturated fatty acids in the AdipoR2 knockout mice while nervonic acid (C24:1) was enriched at the expense of shorter saturated fatty acids in glyceroceramides. Similar defects were found in the cerebellum and myelin sheaths. Histology showed that cell density is lower in the cerebrum of AdipoR2 knockout mice, but electron microscopy did not detect reproducible defects in the ultrastructure of cerebrum neurons, though proteomics analysis showed an enrichment of electron transport chain proteins in the cerebellum. Behavioral tests showed that older (33 weeks old) AdipoR2 knockout mice are hyperactive and anxious compared to control mice of a similar age. Also, in contrast to control mice, the AdipoR2 knockout mice do not gain weight in old age but do have normal lifespans. We conclude that an excess fatty acid saturation in brain phospholipids is accompanied by hyperactivity but seems otherwise well tolerated.

ABSTRACTPolyunsaturated fatty acids (PUFAs) are essential for mammalian health and function as membrane fluidizers and precursors for signaling lipids though the primary essential function of PUFAs within organisms has not been established. Unlike mammals who cannot endogenously synthesize PUFAs,C. eleganscande novosynthesize PUFAs starting with the Δ12 desaturase FAT-2 which introduces a second double bond to monounsaturated fatty acids to generate the PUFA linoleic acid. FAT-2 desaturation is essential forC. eleganssurvival sincefat-2null mutants are non-viable; the near-nullfat-2(wa17)allele synthesizes only small amounts of PUFAs and produces extremely sick worms. Using fluorescence recovery after photobleaching (FRAP), we found that thefat-2(wa17)mutant has rigid membranes and can be efficiently rescued by dietarily providing various PUFAs, but not by fluidizing treatments or mutations. With the aim of identifying mechanisms that compensate for PUFA-deficiency, we performed a forward genetics screen to isolate novelfat-2(wa17)suppressors and identified four internal mutations withinfat-2, and six mutations within the HIF-1 pathway. The suppressors increase PUFA levels infat-2(wa17)mutant worms and additionally suppress the activation of thedaf-16, UPRerand UPRmtstress response pathways that are active infat-2(wa17)worms. We hypothesize that the six HIF-1 pathway mutations, found inegl-9,ftn-2, andhif-1all converge on raising Fe2+levels and in this way boost desaturase activity, including that of thefat-2(wa17)allele. We conclude that PUFAs cannot be genetically replaced and that the only genetic mechanism that can alleviate PUFA-deficiency do so by increasing PUFA levels.

The cellular membrane in male meiotic germ cells contains a unique class of phospholipids and sphingolipids that is required for male reproduction. Here, we show that a conserved membrane fluidity sensor, AdipoR2, regulates the meiosis-specific lipidome in mouse testes by promoting the synthesis of sphingolipids containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs). AdipoR2 upregulates the expression of a fatty acid elongase, ELOVL2, both transcriptionally and post-transcriptionally, to synthesize VLC-PUFA. The depletion of VLC-PUFAs and subsequent accumulation of palmitic acid in AdipoR2 knockout testes stiffens the cellular membrane and causes the invagination of the nuclear envelope. This condition impairs the nuclear peripheral distribution of meiotic telomeres, leading to errors in homologous synapsis and recombination. Further, the stiffened membrane impairs the formation of intercellular bridges and the germ cell syncytium, which disrupts the orderly arrangement of cell types within the seminiferous tubules. According to our findings we propose a framework in which the highly-fluid membrane microenvironment shaped by AdipoR2-ELOVL2 underpins meiosis-specific chromosome dynamics in testes.

Polyunsaturated fatty acids (PUFAs) are essential for mammalian health and function as membrane fluidizers and precursors for signaling lipids though the primary essential function of PUFAs within organisms has not been established. Unlike mammals who cannot endogenously synthesize PUFAs, C. elegans can de novo synthesize PUFAs starting with the Δ12 desaturase FAT-2 which introduces a second double bond to monounsaturated fatty acids to generate the PUFA linoleic acid. FAT-2 desaturation is essential for C. elegans survival since fat-2 null mutants are non-viable; the near-null fat-2(wa17) allele synthesizes only small amounts of PUFAs and produces extremely sick worms. Using fluorescence recovery after photobleaching (FRAP), we found that the fat-2(wa17) mutant has rigid membranes and can be efficiently rescued by dietarily providing various PUFAs, but not by fluidizing treatments or mutations. With the aim of identifying mechanisms that compensate for PUFA-deficiency, we performed a forward genetics screen to isolate novel fat-2(wa17) suppressors and identified four internal mutations within fat-2 , and six mutations within the HIF-1 pathway. The suppressors increase PUFA levels in fat-2(wa17) mutant worms and additionally suppress the activation of the daf-16 , UPRer and UPRmt stress response pathways that are active in fat-2(wa17) worms. We hypothesize that the six HIF-1 pathway mutations, found in egl-9 , ftn-2 , and hif-1 all converge on raising Fe2+ levels and in this way boost desaturase activity, including that of the fat-2(wa17) allele. We conclude that PUFAs cannot be genetically replaced and that the only genetic mechanism that can alleviate PUFA-deficiency do so by increasing PUFA levels. ### Competing Interest Statement The authors have declared no competing interest.

Supplementary Methods, figures and tables

Recently published work showed that members of the PAQR protein family are activated by cell membrane rigidity and contribute to our ability to eat a wide variety of diets. Cell membranes are primarily composed of phospholipids containing dietarily obtained fatty acids, which poses a challenge to membrane properties because diets can vary greatly in their fatty acid composition and could impart opposite properties to the cellular membranes. In particular, saturated fatty acids (SFAs) can pack tightly and form rigid membranes (like butter at room temperature) while unsaturated fatty acids (UFAs) form more fluid membranes (like vegetable oils). Proteins of the PAQR protein family, characterized by the presence of seven transmembrane domains and a cytosolic N‐terminus, contribute to membrane homeostasis in bacteria, yeasts, and animals. These proteins respond to membrane rigidity by stimulating fatty acid desaturation and incorporation of UFAs into phospholipids and explain the ability of animals to thrive on diets with widely varied fat composition. Also see the video abstract here: https://youtu.be/6ckcvaDdbQg

Cells pre-treated for 3h with 20 µM nelfinavir.

Control cells, treated only with biotin

The human AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 are multipass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labeled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membranefluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified coimmunoprecipitated proteins using mass spectrometry. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely, with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids.

AdipoR2 and its C. elegans homolog PAQR-2 are hydrolases activated by membrane rigidification that signal via S1P to promote desaturase transcription. Separately, AdipoR2 and PAQR-2 can recruit a fatty acid elongation complex associated with ACSL4 that channels unsaturated fatty acids towards incorporation into phospholipids, hence promoting membrane fluidity. The AdipoR2/PAQR-2 proteins are therefore essential for membrane homeostasis in response to rigidifying conditions. Evidence from C. elegans, human and mouse models supporting this view will be presented.

Membrane homeostasis defects likely underly numerous conditions. AdipoR1 and AdipoR2 sense excesses of saturated fatty acids in phospholipids and correct such imbalance by generating sphingosine 1-phospate that promotes fatty acid desaturation via sterol regulatory-element binding protein and peroxisome proliferator– activated receptor gamma (1Ruiz M. Devkota R. Panagaki D. Bergh P.O. Kaper D. Henricsson M. et al.Sphingosine 1-phosphate mediates adiponectin receptor signaling essential for lipid homeostasis and embryogenesis.Nat. Commun. 2022; 13: 7162Crossref PubMed Scopus (9) Google Scholar, 2Ruiz M. Palmgren H. Henricsson M. Devkota R. Jaiswal H. Maresca M. et al.Extensive transcription mis-regulation and membrane defects in AdipoR2-deficient cells challenged with saturated fatty acids.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866158884PubMed Google Scholar, 3Ruiz M. Stahlman M. Boren J. Pilon M. AdipoR1 and AdipoR2 maintain membrane fluidity in most human cell types and independently of adiponectin.J. Lipid Res. 2019; 60: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Mouse embryonic fibroblasts lacking AdipoR1 and AdipoR2 (AdipoR1/2 double KO) and treated with palmitic acid form abnormal endoplasmic reticulum structures: long straight lines visualized by confocal microscopy (white arrows), whereas electron micrographs reveal distinctive spirals of packed membranes (red arrows) that are likely cross-sections of the lines seen with Laurdan staining (1Ruiz M. Devkota R. Panagaki D. Bergh P.O. Kaper D. Henricsson M. et al.Sphingosine 1-phosphate mediates adiponectin receptor signaling essential for lipid homeostasis and embryogenesis.Nat. Commun. 2022; 13: 7162Crossref PubMed Scopus (9) Google Scholar). These endoplasmic reticulum structures are not present in basal conditions (1Ruiz M. Devkota R. Panagaki D. Bergh P.O. Kaper D. Henricsson M. et al.Sphingosine 1-phosphate mediates adiponectin receptor signaling essential for lipid homeostasis and embryogenesis.Nat. Commun. 2022; 13: 7162Crossref PubMed Scopus (9) Google Scholar). EQUIPMENT AND REAGENTS: LSM880 confocal microscope with Airyscan (Zeiss), Laurdan dye, palmitic acid, Wohlwend Compact 3 high pressure freezer, Leica AFS, Tecnai T12 transmission electron microscope, Ceta CMOS 16M camera (1Ruiz M. Devkota R. Panagaki D. Bergh P.O. Kaper D. Henricsson M. et al.Sphingosine 1-phosphate mediates adiponectin receptor signaling essential for lipid homeostasis and embryogenesis.Nat. Commun. 2022; 13: 7162Crossref PubMed Scopus (9) Google Scholar). The authors declare that they have no conflicts of interest with the contents of this article. M. R. conceptualization; M. R. and D. P. investigation; J. L. H. and M. P. resources; M. R. and D. P. writing–original draft; M. R., D. P., J. L. H., and M. P. writing–review & editing; J. L. H. and M. P. supervision. This work was supported by the Swedish Research Council (Vetenskapsrådet Dnr: 2020-03300 [to M. P.] and 2015-00560 to Richard Neutze (D.P. work) and Åke Wiberg Stiftelse (to M. R.).

Recently published work showed that members of the PAQR protein family are activated by cell membrane rigidity and contribute to our ability to eat a wide variety of diets. Cell membranes are primarily composed of phospholipids containing dietarily obtained fatty acids, which poses a challenge to membrane properties because diets can vary greatly in their fatty acid composition and could impart opposite properties to the cellular membranes. In particular, saturated fatty acids (SFAs) can pack tightly and form rigid membranes (like butter at room temperature) while unsaturated fatty acids (UFAs) form more fluid membranes (like vegetable oils). Proteins of the PAQR protein family, characterized by the presence of seven transmembrane domains and a cytosolic N-terminus, contribute to membrane homeostasis in bacteria, yeasts, and animals. These proteins respond to membrane rigidity by stimulating fatty acid desaturation and incorporation of UFAs into phospholipids and explain the ability of animals to thrive on diets with widely varied fat composition. Also see the video abstract here: https://youtu.be/6ckcvaDdbQg.

All identified binding partners of nelfinavir across multiple cell lines

The fatty acid composition of phosphatidylethanolamine (PE) determines cellular metabolism, oxidative stress, and inflammation. However, our understanding of how cells regulate PE composition is limited. Here, we identify a genetic locus on mouse chromosome 11, containing two poorly characterized genes Tlcd1 and Tlcd2 , that strongly influences PE composition. We generated Tlcd1/2 double-knockout (DKO) mice and found that they have reduced levels of hepatic monounsaturated fatty acid (MUFA)-containing PE species. Mechanistically, TLCD1/2 proteins act cell intrinsically to promote the incorporation of MUFAs into PEs. Furthermore, TLCD1/2 interact with the mitochondria in an evolutionarily conserved manner and regulate mitochondrial PE composition. Lastly, we demonstrate the biological relevance of our findings in dietary models of metabolic disease, where Tlcd1/2 DKO mice display attenuated development of non-alcoholic steatohepatitis compared to controls. Overall, we identify TLCD1/2 proteins as key regulators of cellular PE composition, with our findings having broad implications in understanding and treating disease.

Cells and organisms require proper membrane composition to function and develop. Phospholipids are the major component of membranes and are primarily acquired through the diet. Given great variability in diet composition, cells must be able to deploy mechanisms that correct deviations from optimal membrane composition and properties. Here, using lipidomics and unbiased proteomics, we found that the embryonic lethality in mice lacking the fluidity regulators Adiponectin Receptors 1 and 2 (AdipoR1/2) is associated with aberrant high saturation of the membrane phospholipids. Using mouse embryonic fibroblasts (MEFs) derived from AdipoR1/2-KO embryos, human cell lines and the model organism C. elegans we found that, mechanistically, AdipoR1/2-derived sphingosine 1-phosphate (S1P) signals in parallel through S1PR3-SREBP1 and PPARγ to sustain the expression of the fatty acid desaturase SCD and maintain membrane properties. Thus, our work identifies an evolutionary conserved pathway by which cells and organisms achieve membrane homeostasis and adapt to a variable environment.