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

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

Membrane sculpting by curved DNA origami scaffolds

BAR domain proteins feature a “banana-like” shape which is thought to aid membrane scaffolding and membrane tubulation. Here authors use DNA origami mimicking BAR domains, giant unilamellar vesicles and fluorescence imaging to study how different BAR domain shapes bind and deform membranes

Optical Control of Lipid Rafts with Photoswitchable Ceramides

Ceramide is a pro-apoptotic sphingolipid with unique physical characteristics. Often viewed as a second messenger, its generation can modulate the structure of lipid rafts. We prepared three photoswitchable ceramides, <b>ACe</b>s, which contain an azobenzene photoswitch allowing for optical control over the <i>N</i>-acyl chain. Using combined atomic force and confocal fluorescence microscopy, we demonstrate that the <b>ACe</b>s enable reversible switching of lipid domains in raft-mimicking supported lipid bilayers (SLBs). In the <i>trans</i>-configuration, the <b>ACe</b>s localize into the liquid-ordered (L_o) phase. Photoisomerization to the <i>cis</i>-form triggers a fluidification of the L_o domains, as liquid-disordered (L_d) “lakes” are formed within the rafts. Photoisomerization back to the <i>trans</i>-state with blue light stimulates a rigidification inside the L_d phase, as the formation of small L_o domains. These changes can be repeated over multiple cycles, enabling a dynamic spatiotemporal control of the lipid raft structure with light

Sifuvirtide screens rigid membrane surfaces : establishment of a correlation between efficacy and membrane domain selectivity among HIV fusion inhibitor peptides

© 2008 American Chemical SocietySifuvirtide, a 36 amino acid negatively charged peptide, is a novel and promising HIV fusion inhibitor, presently in clinical trials. Because of the aromatic amino acid residues of the peptide, its behaviour in aqueous solution and the interaction with lipid-membrane model systems (large unilammelar vesicles) were studied by using mainly fluorescence spectroscopy techniques (both steady-state and time-resolved). No significant aggregation of the peptide was observed with aqueous solution. Various biological and nonbiological lipid-membrane compositions were analyzed, and atomic force microscopy was used to visualize phase separation in several of those mixtures. Results showed no significant interaction of the peptide, neither with zwitterionic fluid lipid membranes (liquid-disordered phase), nor with cholesterol-rich membranes (liquid-ordered phase). However, significant partitioning was observed with the positively charged lipid models (Kp = (2.2 ± 0.3) × 10_3), serving as a positive control. Fluorescence quenching using Förster resonance acrylamide and lipophilic probes was carried out to study the location of the peptide in the membrane models. In the gel-phase DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) membrane model, an adsorption of the peptide at the surface of these membranes was observed and confirmed by using Förster resonance energy-transfer experiments. These results indicate a targeting of the peptide to gelphase domains relatively to liquid-disordered or liquid-ordered phase domains. This larger affinity and selectivity toward the more rigid areas of the membranes, where most of the receptors are found, or to viral membrane, may help explain the improved clinical efficiency of sifuvirtide, by providing a local increased concentration of the peptide at the fusion siteFundação para a Ciência e Tecnologia is acknowledged for funding (SFRH/BD/39039/2007 grant to H.G.F. and projects PTDC/QUI/69937/2006 and REEQ/140/BIO/2005)

Cationic liposomes are possible drug-delivery systems for HIV fusion inhibitor sifuvirtide

© The Royal Society of Chemistry 2011HIV-1 inhibitor sifuvirtide presents increased affinity for cationic dipalmitoylethyl-phosphatidylcholine (EDPPC) in contrast to viral/raft-mimicking (VRM) membranes. When associated to sifuvirtide, EDPPC-containing vesicles fuse with VRM vesicles. After fusion, the peptide co-localizes on VRM vesicles, indicating an effective delivery and concentration of sifuvirtide towards the viral and lipid raft vicinity.Fundação para a Ciência e a Tecnologia – Ministério do Ensino e Ciência (FCT-MES; Portugal

Structural diversity of photoswitchable sphingolipids for optodynamic control of lipid microdomains

Sphingolipids are a structurally diverse class of lipids predominantly found in the plasma membrane of eukaryotic cells. These lipids can laterally segregate with other rigid lipids and cholesterol into liquid-ordered domains that act as organizing centers within biomembranes. Owing the vital role of sphingolipids for lipid segregation, controlling their lateral organization 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-blocked sphingosine) that are able to shuttle between liquid-ordered and liquid-disordered 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 photoisomerization, notably in terms of domain area changes, height mismatch, line tension, and membrane piercing. Hereby, we show that the sphingosine-based (Azo-β-Gal-Cer, Azo-SM, Azo-Cer) and phytosphingosine-based (Azo-α-Gal-PhCer, Azo-PhCer) photoswitchable lipids promote a reduction in liquid-ordered microdomain area when in the UV-adapted cis-isoform. In contrast, azo-sphingolipids having tetrahydropyran groups that block H-bonding at the sphingosine backbone (lipids named Azo-THP-SM, Azo-THP-Cer) induce an increase in the liquid-ordered 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 liquid-ordered domains