Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides

Kol, Matthijs; Williams, Benjamin M.; Toombs-Ruane, Henry; Franquelim, Henri G.; Korneev, Sergei M.; Schroeer, Christian; Schwille, Petra; Trauner, Dirk; Holthuis, Joost C. M.; Frank, James A.

doi:10.7554/elife.43230

Cited by 34 publications

(36 citation statements)

References 53 publications

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“…They have been shown to modulate many aspects of membrane biophysics, including permeability, fluidity, lipid mobility and domain formation (47–49). Recently, photoswitchable sphingolipids have emerged as potent tools to investigate cellular physiology using optical control, including receptor-mediated signaling and immune function (50–53). We synthesized AzoSM, a C16:0 sphingomyelin derivative containing an isosteric azobenzene photoswitch following a design principle called ‘azologization’.…”

Section: Resultsmentioning

confidence: 99%

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages

Bryan

You

et al. 2021

Preprint

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Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores, whereby macrophages either aid in clearance or serve as a niche for its dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high order plasma membrane domains enriched in cholesterol and sphingomyelin) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages’ ability to phagocytose C. neoformans. We found that cholesterol or sphingomyelin depletion resulted in significantly deficient IgG-mediated phagocytosis of the fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency while a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable sphingomyelin (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis whereas raft-inhibiting conformation (cis-AzoSM) significantly blunted phagocytosis in a reversible manner. We observed that the effect on phagocytosis may be mediated by facilitating Fcγ receptor (FcγR) function, whereby IgG immune complexes cross-link to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or sphingomyelin depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.

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Section: Resultsmentioning

confidence: 99%

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages

Bryan

You

et al. 2021

Preprint

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“…The use of a photosubstrate where one photoisomer is metabolized more rapidly than the other, allows for precision control of the formation of biologically active lipid species. This was first demonstrated using clickable and photoswitchable ceramides, termed caCers [53] . As shown in yeast lysates and mammalian cells, these bifunctional lipids allow for light‐dependent metabolism of caCers by sphingomyelin synthases.…”

Section: Lipid Metabolizing Enzymesmentioning

confidence: 99%

“…This was first demonstrated using clickable and photoswitchable ceramides, termed caCers. [53] As shown in yeast lysates and mammalian cells, these bifunctional lipids allow for light-dependent metabolism of caCers by sphingomyelin synthases. The click function was used for detection/visualization with a clickable fluorophore by azide-alkyne Huisgen cycloaddition.…”

Section: Lipid Metabolizing Enzymesmentioning

confidence: 99%

Photoswitchable Lipids

2020

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Photoswitchable lipids are emerging tools for the precise manipulation and study of lipid function. They can modulate many aspects of membrane biophysics, including permeability, fluidity, lipid mobility and domain formation. They are also very useful in lipid physiology and enable optical control of a wide array of lipid receptors, such as ion channels, G protein-coupled receptors, nuclear hormone receptors, and enzymes that trans-locate to membranes. Enzymes involved in lipid metabolism often process them in a light-dependent fashion. Photoswitchable lipids complement other functionalized lipids widely used in lipid chemical biology, including isotope-labeled lipids (lipidomics), fluorescent lipids (imaging), bifunctional lipids (lipid-protein crosslinking), photocaged lipids (photopharmacology), and other labeled variants.

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“…Next, we incorporated our newly synthesized Azo-β-Gal-Cer, Azo-SM, Azo-PhCer, Azo-THP-SM and Azo-THP-Cer photoswitchable lipids (or simply photolipids), as well as Azo-Cer and Azo-α-Gal-PhCer, into raft-mimicking L d -L o phase-separated mixtures containing DOPC, Chol and SM (18:0-SM) and formed small unilamellar vesicles (SUVs) as previously described [36,48] .…”

Section: Light-responsiveness Of Membrane-embedded Azo-sphingolipidsmentioning

confidence: 99%

Structural Diversity of Photoswitchable Sphingolipids for Optodynamic Control of Lipid Raft Microdomains

Hartrampf

Leitão

Winter

et al. 2021

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Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other saturated lipids and cholesterol into lipid rafts; liquid-ordered (Lo) microdomains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral localization is of utmost significance. Hence, we made use of the light-induced trans-cis isomerization of azobenzene-modified acyl chains, to develop a set of photoswitchable sphingolipids, with different headgroups (hydroxyl, galactosyl, phosphocholine) and backbones (sphingosine, phytosphingosine, tetrahydropyran (THP)-blocked sphingosine), able to shuttle between liquid-ordered (Lo) and liquid-disordered (Ld) regions of model membranes upon irradiation with UV-A (λ = 365 nm) and blue (λ = 470 nm) light, respectively. Using combined high-speed atomic force microscopy, fluorescence microscopy, and force spectroscopy, we investigated how these active sphingolipids laterally remodel supported bilayers upon photo-isomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that all sphingosine- (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photolipids behave similarly, promoting a reduction in Lo domain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having THP groups that block H-bonding at the sphingosine backbone (Azo-THP-SM, Azo-THP-Cer) induce an increase in the Lo domain area when in cis, accompanied by a major rise in height mismatch and line tension. These changes were fully reversible upon blue light-triggered isomerization of the various lipids back to trans, pinpointing the role of interfacial interactions for the formation of stable Lo lipid raft domains.

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Optical manipulation of sphingolipid biosynthesis using photoswitchable ceramides

Cited by 34 publications

References 53 publications

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages

Cholesterol and sphingomyelin are critical for Fcγ receptor-mediated phagocytosis of Cryptococcus neoformans by macrophages

Photoswitchable Lipids

Structural Diversity of Photoswitchable Sphingolipids for Optodynamic Control of Lipid Raft Microdomains

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