Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids.

Hanada, Kentaro; Nishijima, Masahiro; Kiso, M.; Hasegawa, Ayaka; Fujita, Shushi; Ogawa, Tomoya; Akamatsu, Yuzuru

doi:10.1016/s0021-9258(18)35871-x

Cited by 141 publications

(18 citation statements)

References 45 publications

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“…To identify pathways regulating cellular responses to sphingolipid depletion, we performed a genome-wide CRISPRi growth screen for genes that modulate sensitivity to myriocin, an inhibitor of de novo sphingolipid synthesis. To insure dependence on de novo lipid synthesis (as opposed to exogenous lipid intake, which can circumvent the need for cellular synthesis 31 ), we cultured human K562 cells in lipid-depleted serum (see Material and Methods). Indeed, we found that myriocin treatment efficiently arrested growth in lipid-depleted media but not in lipid-rich media, while lipid depletion alone did not compromise growth (Fig S1A).…”

Section: Resultsmentioning

confidence: 99%

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

et al. 2020

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Sphingolipids have been shown to play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, where depletion of one of these lipid types resulted in increases in the other, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as GPI anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway. INTRODUCTIONThe maintenance of membrane lipid homeostasis is an energetically expensive yet necessary process in cells. Lipid diversity has evolved together with cell complexity to give rise to thousands of different lipids species with specific functions, many of which are still unexplored 1, 2 . Moreover, different lipid metabolic pathways are interconnected, and cells show a high phenotypic plasticity when adapting to changes in membrane lipid composition, which makes it difficult to disentangle the function of individual lipid species 3 . A systematic analysis of the cellular responses to perturbation of specific synthetic pathways is thus needed to reveal co-regulated lipid networks and uncover new lipid functions. Sphingolipids (SL) are a class of lipids that contain a sphingoid-base backbone, in contrast to the more commonly found glycerol backbone in glycerophospholipids (GPL). These bioactive lipids have been extensively studied in the last decades, revealing distinctive physico-chemical properties and connections to diseases 4,5 . SL have been implicated in diabetes 6 , cancer 7 and inflammation 8 , and mutations in SL synthetic or metabolic enzymes are associated with severe genetic disorders [9][10][11] . SL species sphingosine (So) and ceramide (Cer) can permeabilize membranes 12,13 ; Cer induces the flip-flop of neighbouring lipids 14 and can phase-separate to form membrane platforms important for signalling 15 . The most abundant SL species, sphingomyelin (SM), has been shown to modulate membrane properties and regulate signalling pathways 16,17 . Besides direct phosphorylation of ceramide synthases [18][19][20] , the only direct regulators of sphingolipid synthesis identified are Orm proteins (ORMDL in mammalian cells), that associate with serine palmitoyl transferase (SPT), the first enzyme of the sphingolipi...

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Section: Resultsmentioning

confidence: 99%

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

et al. 2020

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“…To further investigate mTRPA1 activation after disruption of lipid rafts, we treated the cells with sphingomyelinase (SMase). This enzyme hydrolyzes sphingomyelin (SM), which is one of the components of lipid rafts and roughly constitutes 2%-15% of total lipids of cellular membranes [37,38]. SM hydrolysis triggers the formation of ceramide and phosphocholine, inducing an increase in the membrane tension [38,39].…”

Section: Cholesterol Depletion Reduces Mtrpa1 Response To Coldmentioning

confidence: 99%

Lipid Raft Destabilization Impairs Mouse TRPA1 Responses to Cold and Bacterial Lipopolysaccharides

Startek

Talavera

2020

IJMS

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The Transient Receptor Potential ankyrin 1 cation channel (TRPA1) is expressed in nociceptive sensory neurons and epithelial cells, where it plays key roles in the detection of noxious stimuli. Recent reports showed that mouse TRPA1 (mTRPA1) localizes in lipid rafts and that its sensitivity to electrophilic and non-electrophilic agonists is reduced by cholesterol depletion from the plasma membrane. Since effects of manipulating membrane cholesterol levels on other TRP channels are known to vary across different stimuli we here tested whether the disruption of lipid rafts also affects mTRPA1 activation by cold or bacterial lipopolysaccharides (LPS). Cooling to 12 °C, E. coli LPS and allyl isothiocyanate (AITC) induced robust Ca2+ responses in CHO-K1 cells stably transfected with mTRPA1. The amplitudes of the responses to these stimuli were significantly lower in cells treated with the cholesterol scavenger methyl β-cyclodextrin (MCD) or with the sphingolipids hydrolyzer sphingomyelinase (SMase). This effect was more prominent with higher concentrations of the raft destabilizers. Our data also indicate that reduction of cholesterol does not alter the expression of mTRPA1 in the plasma membrane in the CHO-K1 stable expression system, and that the most salient effect is that on the channel gating. Our findings further indicate that the function of mTRPA1 is regulated by the local lipid environment and suggest that targeting lipid-TRPA1 interactions may be a strategy for the treatment of pain and neurogenic inflammation.

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“…To decrease SM content of GM95, cells were cultured in E-RDF medium with various concentrations of either ISP-1 or fumonisin B1 for 2 days. To increase SM of MEB4, cells were cultured in E-RDF medium containing fatty acid-free bovine serum albumin-sphingosine complex (Hanada et al, 1992) for 3 days. Final concentration of sphingosine was 2 mM.…”

Section: Cells and Cell Culturementioning

confidence: 99%

A Lipid-Specific Toxin Reveals Heterogeneity of Sphingomyelin-Containing Membranes

Ishitsuka

Yamaji‐Hasegawa

Makino

et al. 2004

Biophysical Journal

137

140

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Little is known about the heterogenous organization of lipids in biological membranes. Sphingomyelin (SM) is a major plasma membrane lipid that forms lipid domains together with cholesterol and glycolipids. Using SM-specific toxin, lysenin, we showed that in cultured epithelial cells the accessibility of the toxin to SM is different between apical and basolateral membranes. Apical membranes are highly enriched with glycolipids. The inhibitory role of glycolipids in the binding of lysenin to SM was confirmed by comparing the glycolipid-deficient mutant melanoma cell line with its parent cell. Model membrane experiments indicated that glycolipid altered the local density of SM so that the affinity of the lipid for lysenin was decreased. Our results indicate that lysenin recognizes the heterogenous organization of SM in biomembranes and that the organization of SM differs between different cell types and between different membrane domains within the same cell. Isothermal titration calorimetry suggests that lysenin binding to SM is presumably the result of a SM-lysenin complex formation of specific stoichiometry, thus supporting the idea of the existence of small condensed lipid complexes consisting of just a few lipid molecules in living cells.

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Sphingolipids are essential for the growth of Chinese hamster ovary cells. Restoration of the growth of a mutant defective in sphingoid base biosynthesis by exogenous sphingolipids.

Cited by 141 publications

References 45 publications

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway

Lipid Raft Destabilization Impairs Mouse TRPA1 Responses to Cold and Bacterial Lipopolysaccharides

A Lipid-Specific Toxin Reveals Heterogeneity of Sphingomyelin-Containing Membranes

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