Boundary region between coexisting lipid phases as initial binding sites for Escherichia coli alpha-hemolysin: A real-time study

α-Hemolysin (HlyA) is a protein toxin, a member of the pore-forming Repeat in Toxin (RTX) family, secreted by some pathogenic strands of Escherichia coli. The mechanism of action of this toxin seems to involve three stages that ultimately lead to cell lysis: binding, insertion, and oligomerization of the toxin within the membrane. Since the influence of phase segregation on HlyA binding and insertion in lipid membranes is not clearly understood, we explored at the meso- and nanoscale—both in situ and in real-time—the interaction of HlyA with lipid monolayers and bilayers. Our results demonstrate that HlyA could insert into monolayers of dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/16:0SM/Cho) and DOPC/24:1SM/Cho. The time course for HlyA insertion was similar in both lipidic mixtures. HlyA insertion into DOPC/16:0SM/Cho monolayers, visualized by Brewster-angle microscopy (BAM), suggest an integration of the toxin into both the liquid-ordered and liquid-expanded phases. Atomic-force-microscopy imaging reported that phase boundaries favor the initial binding of the toxin, whereas after a longer time period the HlyA becomes localized into the liquid-disordered (Ld) phases of supported planar bilayers composed of DOPC/16:0SM/Cho. Our AFM images, however, showed that the HlyA interaction does not appear to match the general strategy described for other invasive proteins. We discuss these results in terms of the mechanism of action of HlyA.     

GUV preparation and imaging: Minimizing artifacts

The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the “raft model” of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies.     

Hemagglutinin of Influenza Virus Partitions into the Nonraft Domain of Model Membranes

The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.     

Three-dimensional dynamic structure of the liquid-ordered domain in lipid membranes as examined by pulse-EPR oxygen probing

Membranes made of dimyristoylphosphatidylcholine and cholesterol, one of the simplest paradigms for the study of liquid ordered-disordered phase separation, were investigated using a pulse-EPR spin-labeling method in which bimolecular collision of molecular oxygen with the nitroxide spin label is measured. This method allowed discrimination of liquid-ordered, liquid-disordered, and solid-ordered domains because the collision rates (OTP) differ in these domains. Furthermore, the oxygen transport parameter (OTP) profile across the bilayer provides unique information about the three-dimensional dynamic organization of the membrane domains. First, the OTP in the bilayer center in the liquid-ordered domain was comparable to that in the liquid-disordered domain without cholesterol, but the OTP near the membrane surface (up to carbon 9) was substantially smaller in the ordered domain, i.e., the cholesterol-based liquid-ordered domain is ordered only near the membrane surface, still retaining high levels of disorder in the bilayer center. This property may facilitate lateral mobility in ordered domains. Second, in the liquid-disordered domain, the domains with ~5 mol % cholesterol exhibited higher OTP than those without cholesterol, everywhere across the membrane. Third, the transmembrane OTP profile in the liquid-ordered domain that contained 50 mol % cholesterol dramatically differed from that which contained 27 mol % cholesterol.     

Fluorinated cholesterol retains domain-forming activity in sphingomyelin bilayers

Lipid rafts are cholesterol (Chol)-rich microdomains floating in a sea of lipid bilayers. Chol is thought to interact preferentially with sphingolipids such as sphingomyelin (SM) rather than with glycerophospholipids, and this putative SM–Chol interaction is generally recognized as a requirement for raft formation. However, the presence of the specific interaction is still controversial, primarily because of the lack of useful molecular probes for scrutinizing this interaction. Recently, we reported that the dynamic properties of 6-F-Chol in DMPC bilayers are similar to those of unmodified Chol. Hence, in the present study, we first compared the roles of 6-F-Chol and Chol in SM bilayers through detergent insolubility, fluorescence polarization, and ²H NMR experiments. The results demonstrated that 6-F-Chol and Chol behave similarly in SM bilayers, whereas, in SM–DOPC membranes, 6-F-Chol is less effective in domain formation. Then, we analyzed the molecular orientation of 6-F-Chol in SM bilayers using solid-state NMR, and found that the dynamics and orientation of 6-F-Chol in SM bilayers are almost identical to those in DMPC bilayers. This supports the notion of the lack of a putative specific interaction between SM and Chol. Thus, this study demonstrates the utility of 6-F-Chol as a molecular probe for understanding molecular recognition in lipid rafts.     

A calorimetric and spectroscopic comparison of the effects of ergosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We performed comparative DSC and FTIR spectroscopic measurements of the effects of cholesterol (Chol) and ergosterol (Erg) on the thermotropic phase behavior and organization of DPPC bilayers. Ergosterol is the major sterol in the biological membranes of yeasts, fungi and many protozoa. It differs from Chol in having two additional double bonds, one in the steroid nucleus at C7-8 and another in the alkyl chain at C22-23. Erg also has an additional methyl group in the alkyl chain at C24. Our DSC studies indicate that the incorporation of Erg is more effective than Chol is in reducing the enthalpy of the pretransition. At lower concentrations Erg is also more effective than Chol in reducing the enthalpies of both the sharp and broad components of main phase transition. However, at sterol concentrations from 30 to 50 mol%, Erg is generally less effective at reducing the enthalpy of the broad components and does not completely abolish the cooperative hydrocarbon chain-melting phase transition at 50 mol%, as does Chol. Nevertheless, in this higher ergosterol concentration range, there is no evidence of the formation of ergosterol crystallites. Our FTIR spectroscopic studies demonstrate that Erg incorporation produces a similar ordering of liquid-crystalline DPPC bilayers as does Chol, but an increased degree of hydrogen bonding of the fatty acyl carbonyl groups in the glycerol backbone region of the DPPC bilayer. These and other results indicate that Erg is less miscible in DPPC bilayers at higher concentrations than is Chol. Finally, we provide a tentative molecular explanation for the comparative experimental and computation results obtained for Erg and Chol in phospholipid bilayers, emphasizing the dynamic conformational differences between these two sterols.     

Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.     

Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells

Background

The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake.

Methods

These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C₂₈) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent.

Results

This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in β₂ integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound.

Conclusions

All these features confirm that the dual lipid modification (C₂₈) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties.

General significance

Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C₂₈ dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.     

Lipid rafts and membrane traffic

Membrane rafts are regions of increased lipid acyl chain order that differ in their lipid and protein composition from the surrounding membrane. By providing an additional level of compartmentalization they have been proposed to serve many functions in cellular signal transduction and trafficking. We will review their potential involvement in different forms of membrane traffic, explicitly excluding signalling, and discuss select aspects of the raft hypothesis in its current form.