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1.
FASEB J ; 38(14): e23815, 2024 Jul 31.
Article in English | MEDLINE | ID: mdl-38989587

ABSTRACT

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.


Subject(s)
Aging , Brain , Fatty Acids , Mice, Knockout , Receptors, Adiponectin , Animals , Mice , Brain/metabolism , Fatty Acids/metabolism , Aging/metabolism , Receptors, Adiponectin/metabolism , Receptors, Adiponectin/genetics , Male , Mice, Inbred C57BL , Phosphatidylcholines/metabolism , Phospholipids/metabolism
2.
Nat Commun ; 15(1): 2315, 2024 Mar 14.
Article in English | MEDLINE | ID: mdl-38485951

ABSTRACT

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.


Subject(s)
Membrane Fluidity , Telomere , Animals , Male , Mice , Carrier Proteins/metabolism , Meiosis , Nuclear Envelope/metabolism , Sphingolipids/metabolism , Telomere/genetics , Telomere/metabolism
3.
Diabetes ; 73(4): 585-591, 2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38211571

ABSTRACT

Glucolipotoxicity (GLT), in which elevated levels of glucose and fatty acids have deleterious effects on ß-cell biology, is thought to be one of the major contributors in progression of type 2 diabetes. In search of novel small molecules that protect ß-cells against GLT, we previously discovered KD025, an inhibitor of Rho-associated coiled-coil-containing kinase isoform 2 (ROCK2), as a GLT-protective compound in INS-1E cells and dissociated human islets. To further understand the mechanism of action of KD025, we found that pharmacological and genetic inhibition of ROCK2 was not responsible for the protective effects of KD025 against GLT. Instead, kinase profiling revealed that KD025 potently inhibits catalytic subunits of casein kinase 2 (CK2), a constitutively active serine/threonine kinase. We experimentally verified that the inhibition of one of the catalytic subunits of casein kinase 2, CK2A1, but not CK2A2, improved cell viability when challenged with GLT. We conclude that KD025 inhibits CK2 to protect ß-cells from GLT.


Subject(s)
Diabetes Mellitus, Type 2 , Insulin-Secreting Cells , Humans , Casein Kinase II/pharmacology , Diabetes Mellitus, Type 2/drug therapy , Heterocyclic Compounds, 4 or More Rings/pharmacology
4.
J Biol Chem ; 299(6): 104799, 2023 06.
Article in English | MEDLINE | ID: mdl-37164154

ABSTRACT

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 membrane-fluidizing 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.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Cell Membrane , Fatty Acids , Membrane Fluidity , Receptors, Adiponectin , Animals , Humans , Caenorhabditis elegans/cytology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Membrane/metabolism , Fatty Acids/metabolism , HEK293 Cells , Membrane Fluidity/physiology , Phospholipids/metabolism , Receptors, Adiponectin/metabolism , Protein Binding
5.
Cell Metab ; 35(5): 887-905.e11, 2023 05 02.
Article in English | MEDLINE | ID: mdl-37075753

ABSTRACT

Cellular exposure to free fatty acids (FFAs) is implicated in the pathogenesis of obesity-associated diseases. However, there are no scalable approaches to comprehensively assess the diverse FFAs circulating in human plasma. Furthermore, assessing how FFA-mediated processes interact with genetic risk for disease remains elusive. Here, we report the design and implementation of fatty acid library for comprehensive ontologies (FALCON), an unbiased, scalable, and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids associated with decreased membrane fluidity. Furthermore, we prioritized genes that reflect the combined effects of harmful FFA exposure and genetic risk for type 2 diabetes (T2D). We found that c-MAF-inducing protein (CMIP) protects cells from FFA exposure by modulating Akt signaling. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism.


Subject(s)
Diabetes Mellitus, Type 2 , Fatty Acids, Nonesterified , Humans , Fatty Acids, Nonesterified/metabolism , Fatty Acids , Signal Transduction , Biology
6.
bioRxiv ; 2023 Feb 20.
Article in English | MEDLINE | ID: mdl-36865221

ABSTRACT

Cellular exposure to free fatty acids (FFA) is implicated in the pathogenesis of obesity-associated diseases. However, studies to date have assumed that a few select FFAs are representative of broad structural categories, and there are no scalable approaches to comprehensively assess the biological processes induced by exposure to diverse FFAs circulating in human plasma. Furthermore, assessing how these FFA- mediated processes interact with genetic risk for disease remains elusive. Here we report the design and implementation of FALCON (Fatty Acid Library for Comprehensive ONtologies) as an unbiased, scalable and multimodal interrogation of 61 structurally diverse FFAs. We identified a subset of lipotoxic monounsaturated fatty acids (MUFAs) with a distinct lipidomic profile associated with decreased membrane fluidity. Furthermore, we developed a new approach to prioritize genes that reflect the combined effects of exposure to harmful FFAs and genetic risk for type 2 diabetes (T2D). Importantly, we found that c-MAF inducing protein (CMIP) protects cells from exposure to FFAs by modulating Akt signaling and we validated the role of CMIP in human pancreatic beta cells. In sum, FALCON empowers the study of fundamental FFA biology and offers an integrative approach to identify much needed targets for diverse diseases associated with disordered FFA metabolism. Highlights: FALCON (Fatty Acid Library for Comprehensive ONtologies) enables multimodal profiling of 61 free fatty acids (FFAs) to reveal 5 FFA clusters with distinct biological effectsFALCON is applicable to many and diverse cell typesA subset of monounsaturated FAs (MUFAs) equally or more toxic than canonical lipotoxic saturated FAs (SFAs) leads to decreased membrane fluidityNew approach prioritizes genes that represent the combined effects of environmental (FFA) exposure and genetic risk for diseaseC-Maf inducing protein (CMIP) is identified as a suppressor of FFA-induced lipotoxicity via Akt-mediated signaling.

7.
Nat Commun ; 13(1): 7162, 2022 11 22.
Article in English | MEDLINE | ID: mdl-36418331

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans , Receptors, Adiponectin , Humans , Animals , Mice , Receptors, Adiponectin/genetics , Fibroblasts , Lysophospholipids , Homeostasis , Embryonic Development/genetics
8.
Genetics ; 219(1)2021 08 26.
Article in English | MEDLINE | ID: mdl-34125894

ABSTRACT

Communicating editor: B. Grant The composition and biophysical properties of cellular membranes must be tightly regulated to maintain the proper functions of myriad processes within cells. To better understand the importance of membrane homeostasis, we assembled a panel of five Caenorhabditis elegans strains that show a wide span of membrane composition and properties, ranging from excessively rich in saturated fatty acids (SFAs) and rigid to excessively rich in polyunsaturated fatty acids (PUFAs) and fluid. The genotypes of the five strain are, from most rigid to most fluid: paqr-1(tm3262); paqr-2(tm3410), paqr-2(tm3410), N2 (wild-type), mdt-15(et14); nhr-49(et8), and mdt-15(et14); nhr-49(et8); acs-13(et54). We confirmed the excess SFA/rigidity-to-excess PUFA/fluidity gradient using the methods of fluorescence recovery after photobleaching (FRAP) and lipidomics analysis. The five strains were then studied for a variety of cellular and physiological traits and found to exhibit defects in: permeability, lipid peroxidation, growth at different temperatures, tolerance to SFA-rich diets, lifespan, brood size, vitellogenin trafficking, oogenesis, and autophagy during starvation. The excessively rigid strains often exhibited defects in opposite directions compared to the excessively fluid strains. We conclude that deviation from wild-type membrane homeostasis is pleiotropically deleterious for numerous cellular/physiological traits. The strains introduced here should prove useful to further study the cellular and physiological consequences of impaired membrane homeostasis.


Subject(s)
Caenorhabditis elegans , Animals
9.
Article in English | MEDLINE | ID: mdl-33444759

ABSTRACT

How cells maintain vital membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remains largely unknown. Here, we used transcriptomics, lipidomics, growth and respiration assays, and membrane property analyses in human HEK293 cells or human umbilical vein endothelial cells (HUVEC) to show that the function of AdipoR2 is to respond to membrane rigidification by regulating many lipid metabolism genes. We also show that AdipoR2-dependent membrane homeostasis is critical for growth and respiration in cells challenged with saturated fatty acids. Additionally, we found that AdipoR2 deficiency causes transcriptome and cell physiological defects similar to those observed in SREBP-deficient cells upon SFA challenge. Finally, we compared several genes considered important for lipid homeostasis, namely AdipoR2, SCD, FADS2, PEMT and ACSL4, and found that AdipoR2 and SCD are the most important among these to prevent membrane rigidification and excess saturation when human cells are challenged with exogenous SFAs. We conclude that AdipoR2-dependent membrane homeostasis is one of the primary mechanisms that protects against exogenous SFAs.


Subject(s)
Cell Membrane/metabolism , Endothelial Cells/metabolism , Fatty Acids/metabolism , Membrane Fluidity , Receptors, Adiponectin/genetics , Cell Membrane/genetics , Endothelial Cells/cytology , Fatty Acids/genetics , Gene Deletion , HEK293 Cells , Humans , Receptors, Adiponectin/metabolism , Transcriptional Activation , Transcriptome
10.
Article in English | MEDLINE | ID: mdl-33444761

ABSTRACT

How cells maintain vital membrane lipid homeostasis while obtaining most of their constituent fatty acids from a varied diet remains largely unknown. Here, we report the first whole-organism (Caenorhabditis elegans) forward genetic screen to identify genes essential for tolerance to dietary saturated fatty acids (SFAs). We found that only the PAQR-2/IGLR-2 pathway, homologous to the human adiponectin receptor 2 (AdipoR2) pathway, is uniquely essential to prevent SFA-mediated toxicity. When provided a SFA-rich diet, worms lacking either protein accumulate an excess of SFAs in their membrane phospholipids, which is accompanied by membrane rigidification. Additionally, we used fluorescence resonance energy transfer (FRET) to show that the interaction between PAQR-2 and IGLR-2 is regulated by membrane fluidity, suggesting a mechanism by which this protein complex senses membrane properties. We also created versions of PAQR-2 that lacked parts of the cytoplasmic N-terminal domain and showed that these were still functional, though still dependent on the interaction with IGLR-2. We conclude that membrane homeostasis via the PAQR-2/IGLR-2 fluidity sensor is the only pathway specifically essential for the non-toxic uptake of dietary SFAs in C. elegans.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Dietary Fats/metabolism , Membrane Proteins/metabolism , Animals , Cell Membrane/metabolism , Homeostasis , Membrane Fluidity , Protein Interaction Maps
11.
PLoS Genet ; 16(8): e1008975, 2020 08.
Article in English | MEDLINE | ID: mdl-32750056

ABSTRACT

The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Membrane Proteins/genetics , Receptors, Adiponectin/genetics , Alleles , Animals , Caenorhabditis elegans/metabolism , Cell Membrane/genetics , Cell Membrane/metabolism , Gain of Function Mutation/genetics , HEK293 Cells , Humans , Membrane Fluidity/genetics , Phenotype , Phospholipids/genetics , Phospholipids/metabolism
12.
Biometals ; 33(2-3): 147-157, 2020 06.
Article in English | MEDLINE | ID: mdl-32506305

ABSTRACT

Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10-15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Molecular Chaperones/metabolism , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/growth & development , Caenorhabditis elegans Proteins/genetics , Cell Movement , Copper/metabolism , Copper Transport Proteins/genetics , Copper Transport Proteins/metabolism , Humans , Molecular Chaperones/genetics
13.
Elife ; 72018 12 04.
Article in English | MEDLINE | ID: mdl-30509349

ABSTRACT

Dietary fatty acids are the main building blocks for cell membranes in animals, and mechanisms must therefore exist that compensate for dietary variations. We isolated C. elegans mutants that improved tolerance to dietary saturated fat in a sensitized genetic background, including eight alleles of the novel gene fld-1 that encodes a homolog of the human TLCD1 and TLCD2 transmembrane proteins. FLD-1 is localized on plasma membranes and acts by limiting the levels of highly membrane-fluidizing long-chain polyunsaturated fatty acid-containing phospholipids. Human TLCD1/2 also regulate membrane fluidity by limiting the levels of polyunsaturated fatty acid-containing membrane phospholipids. FLD-1 and TLCD1/2 do not regulate the synthesis of long-chain polyunsaturated fatty acids but rather limit their incorporation into phospholipids. We conclude that inhibition of FLD-1 or TLCD1/2 prevents lipotoxicity by allowing increased levels of membrane phospholipids that contain fluidizing long-chain polyunsaturated fatty acids. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).


Subject(s)
Caenorhabditis elegans Proteins/physiology , Caenorhabditis elegans/metabolism , Membrane Fluidity , Membrane Proteins/metabolism , Sequence Homology, Amino Acid , Alleles , Amino Acid Sequence , Animals , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Cell Membrane/metabolism , Epistasis, Genetic , Genes, Suppressor , Green Fluorescent Proteins/metabolism , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/physiology , Mice , Mutation/genetics , Organ Specificity , Phenotype , Phospholipids/metabolism , Receptors, Adiponectin/metabolism
14.
BMC Res Notes ; 11(1): 630, 2018 Aug 31.
Article in English | MEDLINE | ID: mdl-30170618

ABSTRACT

OBJECTIVES: ESBL-producing isolates of the Enterobacteriaceae occur throughout the world. The objectives of this study were to characterize uropathogenic Escherichia coli isolated at a tertiary care hospital in southern India, and shed light on blaCTX-M sequences of Indian origin. RESULTS: A cohort of 13 urinary isolates of E. coli (obtained from patients at the Sri Sathya Sai Institute of Higher Medical Sciences, Prasanthigram, Andhra Pradesh, India) were characterized and found to be resistant to multiple antibiotics, including extended-spectrum cephalosporins. All 13 isolates contained blaCTX-M-15, and many of them transferred this genotype to at least one laboratory strain of E. coli after conjugation. Analyses of blaCTX-M-15 sequences (n = 141) of Indian origin showed that > 85% of them were obtained from bacteria not associated with the urinary tract, and that E. coli isolates account for majority of all blaCTX-M-15-carrying bacteria reported from India. Other types of blaCTX-M appear to be rare in India, since only six such sequences were reported as of July 2015. The results indicate that 'selection pressure' exerted by extended-spectrum cephalosporins may have stabilized the blaCTX-M-15 genotype among E. coli in India. The rarity of other blaCTX-M suggests that they lack the survival advantage that blaCTX-M-15 may have.


Subject(s)
Cephalosporins/pharmacology , Drug Resistance, Bacterial/genetics , Escherichia coli Proteins/genetics , Uropathogenic Escherichia coli/genetics , Anti-Bacterial Agents/pharmacology , Escherichia coli Infections , Humans , India , Sequence Analysis, DNA , beta-Lactamases
15.
Genetics ; 210(1): 189-201, 2018 09.
Article in English | MEDLINE | ID: mdl-29997234

ABSTRACT

Maintenance of membrane properties is an essential aspect of cellular homeostasis of which the regulatory mechanisms remain mostly uncharacterized. In Caenorhabditis elegans, the PAQR-2 and IGLR-2 proteins act together as a plasma membrane sensor that responds to decreased fluidity by promoting fatty acid desaturation, hence restoring membrane fluidity. Here, we used mosaic analysis for paqr-2 and iglr-2, and tissue-specific paqr-2 expression, to show that membrane homeostasis is achieved cell nonautonomously. Specifically, we found that expression of paqr-2 in the hypodermis, gonad sheath cells, or intestine is sufficient to suppress systemic paqr-2 mutant phenotypes, including tail tip morphology, membrane fluidity in intestinal cells, cold and glucose intolerance, vitellogenin transport to the germline, germ cell development, and brood size. Finally, we show that the cell nonautonomous regulation of membrane homeostasis is conserved in human cells: HEK293 cells that express AdipoR2, a homolog of paqr-2, are able to normalize membrane fluidity in distant cells where AdipoR2 has been silenced. Finally, using C. elegans mutants and small interfering RNA against Δ9 stearoyl-CoA desaturase in HEK293 cells, we show that Δ9 desaturases are essential for the cell nonautonomous maintenance of membrane fluidity. We conclude that cells are able to share membrane components even when they are not in direct contact with each other, and that this contributes to the maintenance of membrane homeostasis in C. elegans and human cells.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Membrane Fluidity/genetics , Membrane Proteins/metabolism , Receptors, Adiponectin/metabolism , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/physiology , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/physiology , Cell Membrane/metabolism , Fatty Acids/metabolism , HEK293 Cells , Homeostasis , Humans , Membrane Fluidity/physiology , Membrane Proteins/genetics , Mutation , Phenotype , RNA, Small Interfering/metabolism , Receptors, Adiponectin/genetics , Stearoyl-CoA Desaturase/metabolism
16.
Bio Protoc ; 8(13): e2913, 2018 Jul 05.
Article in English | MEDLINE | ID: mdl-34395742

ABSTRACT

FRAP (Fluorescence Recovery After Photobleaching) is probably the most direct method to investigate the dynamics of molecules in living cells. Here, we describe FRAP to quantify membrane fluidity in C. elegans. Using FRAP, we have shown that cold, glucose and exogenous saturated fatty acids can decrease the fluidity of cellular membranes in certain mutants.

17.
Genetics ; 207(4): 1533-1545, 2017 12.
Article in English | MEDLINE | ID: mdl-28993416

ABSTRACT

The Caenorhabditis elegans ventral nerve cord (VNC) consists of two asymmetric bundles of neurons and axons that are separated by the midline. How the axons are guided to stay on the correct sides of the midline remains poorly understood. Here we provide evidence that the conserved Wnt signaling pathway along with the Netrin and Robo pathways constitute a combinatorial code for midline guidance of PVP and PVQ axons that extend into the VNC. Combined loss of the Wnts CWN-1, CWN-2, and EGL-20 or loss of the Wnt receptor CAM-1 caused >70% of PVP and PVQ axons to inappropriately cross over from the left side to the right side. Loss of the Frizzled receptor LIN-17 or the planar cell polarity (PCP) protein VANG-1 also caused cross over defects that did not enhance those in the cam-1 mutant, indicating that the proteins function together in midline guidance. Strong cam-1 expression can be detected in the PVQs and the guidepost cell PVT that is located on the midline. However, only when cam-1 is expressed in PVT are the crossover defects of PVP and PVQ rescued, showing that CAM-1 functions nonautonomously in PVT to prevent axons from crossing the midline.


Subject(s)
Axon Guidance/genetics , Caenorhabditis elegans Proteins/genetics , Phosphoproteins/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptors, G-Protein-Coupled/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Cell Movement/genetics , Cell Polarity/genetics , Gene Expression Regulation, Developmental , Motor Neurons/metabolism , Netrins/genetics , Wnt Proteins/genetics , Wnt Signaling Pathway/genetics
18.
PLoS Genet ; 13(9): e1007004, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28886012

ABSTRACT

Dietary fatty acids can be incorporated directly into phospholipids. This poses a specific challenge to cellular membranes since their composition, hence properties, could greatly vary with different diets. That vast variations in diets are tolerated therefore implies the existence of regulatory mechanisms that monitor and regulate membrane compositions. Here we show that the adiponectin receptor AdipoR2, and its C. elegans homolog PAQR-2, are essential to counter the membrane rigidifying effects of exogenously provided saturated fatty acids. In particular, we use dietary supplements or mutated E. coli as food, together with direct measurements of membrane fluidity and composition, to show that diets containing a high ratio of saturated to monounsaturated fatty acids cause membrane rigidity and lethality in the paqr-2 mutant. We also show that mammalian cells in which AdipoR2 has been knocked-down by siRNA are unable to prevent the membrane-rigidifying effects of palmitic acid. We conclude that the PAQR-2 and AdipoR2 proteins share an evolutionarily conserved function that maintains membrane fluidity in the presence of exogenous saturated fatty acids.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Cell Membrane/genetics , Membrane Fluidity/genetics , Membrane Proteins/genetics , Receptors, Adiponectin/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Fatty Acids/chemistry , Fatty Acids/metabolism , HEK293 Cells , Humans , Membrane Proteins/metabolism , Phospholipids/chemistry , Phospholipids/genetics , RNA, Small Interfering , Receptors, Adiponectin/metabolism
20.
PLoS Genet ; 12(4): e1005982, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27082444

ABSTRACT

In spite of the worldwide impact of diabetes on human health, the mechanisms behind glucose toxicity remain elusive. Here we show that C. elegans mutants lacking paqr-2, the worm homolog of the adiponectin receptors AdipoR1/2, or its newly identified functional partner iglr-2, are glucose intolerant and die in the presence of as little as 20 mM glucose. Using FRAP (Fluorescence Recovery After Photobleaching) on living worms, we found that cultivation in the presence of glucose causes a decrease in membrane fluidity in paqr-2 and iglr-2 mutants and that genetic suppressors of this sensitivity act to restore membrane fluidity by promoting fatty acid desaturation. The essential roles of paqr-2 and iglr-2 in the presence of glucose are completely independent from daf-2 and daf-16, the C. elegans homologs of the insulin receptor and its downstream target FoxO, respectively. Using bimolecular fluorescence complementation, we also show that PAQR-2 and IGLR-2 interact on plasma membranes and thus may act together as a fluidity sensor that controls membrane lipid composition.


Subject(s)
Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans/genetics , Glucose/toxicity , Membrane Fluidity/genetics , Membrane Lipids/metabolism , Membrane Proteins/genetics , Animals , Caenorhabditis elegans Proteins/metabolism , Fluorescence Recovery After Photobleaching , Forkhead Transcription Factors/genetics , Membrane Fluidity/physiology , Membrane Proteins/metabolism , Receptor, Insulin/genetics
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