| 1. |
- Bodhicharla, Rakesh, et al.
(författare)
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Membrane Fluidity Is Regulated Cell Nonautonomously by Caenorhabditis elegans PAQR-2 and Its Mammalian Homolog AdipoR2
- 2018
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Ingår i: Genetics. - : Oxford University Press (OUP). - 0016-6731 .- 1943-2631. ; 210:1, s. 189-201
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Busayavalasa, Kiran, et al.
(författare)
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Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins
- 2020
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Ingår i: Plos Genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 16:8
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Tidskriftsartikel (refereegranskat)abstract
- TheC.elegansproteins 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. Author summary Cells are enclosed within membranes primarily composed of fat. When membranes contain much saturated fats, they tend to become more rigid, as with butter. Conversely, when membranes are rich in unsaturated fats, they become more fluid, as with vegetable oils. Our goal is to better understand how cells monitor and adjust the composition and properties of their membranes. We focus on a small group of proteins found in all animals, and called AdipoR1 and AdipoR2 in humans, and PAQR-1 and PAQR-2 in the wormCaenorhabditis elegans. We now found a version of PAQR-1 that is more "active", and promotes increased levels of unsaturated fats in membranes. By swapping different parts of the PAQR-1 protein with those of PAQR-2, we were able to determine which protein parts played which roles. We found that it is the transmembrane domains of PAQR-2 that dictate its requirements for another protein called IGLR-2 and that the intracellular domains of PAQR-1 and PAQR-2 play a regulatory role. These studies help understand how AdipoR1 and AdipoR2 regulate membrane composition in human cells, which is a vital function for us to thrive on diets that vary greatly in the types of fats that they contain.
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| 3. |
- Chien, Jason, et al.
(författare)
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Regulation of Axon Guidance by the Wnt Receptor Ror/CAM-1 in the PVT Guidepost Cell in Caenorhabditis elegans
- 2017
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Ingår i: Genetics. - : Oxford University Press (OUP). - 0016-6731 .- 1943-2631. ; 207:4, s. 1533-1545
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Tidskriftsartikel (refereegranskat)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.
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| 4. |
- Devkota, Ranjan, et al.
(författare)
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A genetic titration of membrane composition in Caenorhabditis elegans reveals its importance for multiple cellular and physiological traits
- 2021
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Ingår i: Genetics. - 0016-6731 .- 1943-2631. ; 219:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Devkota, Ranjan, et al.
(författare)
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FRAP: A Powerful Method to Evaluate Membrane Fluidity in Caenorhabditis elegans
- 2018
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Ingår i: Bio-Protocol. ; 8:13
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Tidskriftsartikel (refereegranskat)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.
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| 6. |
- Devkota, Ranjan
(författare)
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Membrane Fluidity Regulation: From C. elegans to mammalian cells
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Biological membranes are primarily an assembly of lipids and proteins. Collectively, these constituents within a phospholipid bilayer determine the physical membrane properties such as fluidity, viscosity, thickness, packing and permeability. Maintenance of membrane properties within acceptable margins, i.e. membrane homeostasis, is fundamentally important for cellular processes. For example, it is a well-established phenomenon that poikilothermic organisms, that cannot control their body temperature, constantly adapt their membrane lipid composition in order to maintain optimal membrane fluidity for membrane functions in spite of variation in ambient temperatures. Regulatory mechanisms must also exist in mammals to maintain membrane lipid heterogeneity across the secretory pathway and to compensate for dietary lipid variation. However, the molecular mechanisms of such an adaptive response in mammals remain poorly understood. Here, using systematic genetics, lipidomics and membrane property assays, we have established that the PAQR-2/IGLR-2 complex in C. elegans and AdipoR2 (a PAQR-2 homolog) in mammalian cells specifically respond to the toxic membrane-rigidifying effects of dietary saturated fatty acids (SFAs) and promote fatty acid desaturation to restore membrane composition and fluidity. In an attempt to understand other mechanisms essential to prevent SFA-mediated cellular toxicity, we also performed an unbiased forward genetic screen in C. elegans. Strikingly, this screen for SFA-tolerance genes led only to the isolation of novel paqr-2 and iglr-2 alleles; this strongly indicates that paqr-2 and iglr-2 are important genes specifically essential to respond to toxic effects of dietary saturated fats. In particular, we noted that in worms and cells that lack PAQR-2/AdipoR2 function, exogenous SFAs becomes rapidly incorporated into membrane phospholipids, leading to membrane rigidification. This was accompanied by an abnormal transcriptional response, impaired mitochondrial respiration and increased ER-UPR as measured in HEK293 cells. Interestingly, we noticed that the toxic effects of exogenous SFAs can be completely mitigated by supplying the cultured cells with small amounts of membrane fluidizing unsaturated fatty acids (UFAs). Consistently, we also found that facilitating the accumulation of UFAs either with mutations in fld-1 in worms or silencing the fld-1 mammalian homologs TLCD1/2, which normally function to limit the incorporation of polyunsaturated fatty acids in membrane phospholipids, is protective and able to attenuate SFA-mediated cellular toxicity. Altogether, these results suggest that maintenance of an optimal SFA/UFA ratio is crucial for normal cellular function and that the PAQR-2/AdipoR2 proteins essentially act as “guardians of membrane homeostasis”.
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| 7. |
- Devkota, Ranjan, et al.
(författare)
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The adiponectin receptor AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 prevent membrane rigidification by exogenous saturated fatty acids.
- 2017
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Ingår i: PLoS genetics. - : Public Library of Science (PLoS). - 1553-7404. ; 13:9
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Tidskriftsartikel (refereegranskat)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.
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| 8. |
- Devkota, Ranjan, et al.
(författare)
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The C. elegans PAQR-2 and IGLR-2 membrane homeostasis proteins are uniquely essential for tolerating dietary saturated fats
- 2021
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Ingår i: Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981. ; 1866:4
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Tidskriftsartikel (refereegranskat)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.
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| 9. |
- Ruiz, Mario, 1984, et al.
(författare)
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AdipoR2 recruits protein interactors to promote fatty acid elongation and membrane fluidity
- 2023
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 299:6
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Tidskriftsartikel (refereegranskat)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.
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| 10. |
- Ruiz, Mario, 1984, et al.
(författare)
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Extensive transcription mis-regulation and membrane defects in AdipoR2-deficient cells challenged with saturated fatty acids
- 2021
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Ingår i: Biochimica Et Biophysica Acta-Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981. ; 1866:4
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Tidskriftsartikel (refereegranskat)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.
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