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Nicoletta Kahya

Publications and source records attributed to Nicoletta Kahya.

17 recordsLinked to original sources
PubMed2006

Combined AFM and two-focus SFCS study of raft-exhibiting model membranes.

Dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/cholesterol) model membranes exhibit liquid-liquid phase separation and therefore provide a physical model for the putative liquid-ordered domains present in cells. Here we present a combination of atomic force microscopy (AFM) imaging, force measurements, confocal fluorescence imaging and two-focus scanning fluorescence correlation spectroscopy (two-focus SFCS) to obtain structural and dynamical information about this model membrane system. Partition coefficients and diffusion coefficients in the different phases were measured with two-focus SFCS for numerous fluorescent lipid analogues and proteins, while being directly related to the lateral organization of the membrane and its mechanical properties probed by AFM. Moreover we show how the combination of these different approaches is effective in reducing artifacts resulting from the use of a single technique.

Biophysics↗
PubMed2006

How phospholipid-cholesterol interactions modulate lipid lateral diffusion, as revealed by fluorescence correlation spectroscopy.

Cholesterol is a key player in regulating physico-chemical properties of cellular membranes and, thereby, ensuring cell viability. In particular, lipid-cholesterol interactions may provide important information on the spatio-temporal organization of membrane components. Here, we apply confocal imaging and Fluorescence Correlation Spectroscopy (FCS) to Giant Unilamellar Vesicles (GUVs) composed of binary mixtures of lipids and cholesterol. The effect of cholesterol on lipid dynamics and molecular packing order of unsaturated, monounsaturated, fully saturated (with both low and high phase transition temperatures, Tm) glycero-phospholipids and sphingomyelin has been investigated. We show that, for unsaturated glycerophospholipids, the decrease of the lipid diffusion coefficient as a result of the interaction with cholesterol does not depend on the fatty acid chain length. However, the values of the diffusion coefficient change as a function of chain length. The monounsaturated phospholipid palmitoyl-oleoyl-phosphatidylcholine (POPC) exhibits a dynamic behavior very similar to the unsaturated dioleoyl-phosphatidylcholine (DOPC). By contrast, for saturated (low Tm) glycero-phospholipids, cholesterol causes a decrease of lipid mobility in a chain length-dependent manner. FCS can be employed as a valuable tool to study lipid-sterol interactions and their effect on lipid dynamics, molecular packing and degree of conformational order.

Cholesterol↗
PubMed2006

Targeting membrane proteins to liquid-ordered phases: molecular self-organization explored by fluorescence correlation spectroscopy.

The complex and dynamic architecture of biological membranes comprises of various heterogeneities, some of which may include lipid-based and/or protein-based microdomains called "rafts". Due to interactions among membrane components, several types of domains can form with different characteristics and mechanisms of formation. Model membranes, such as giant unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and lipid-protein interactions, which are potentially relevant to raft formation, by (single-molecule) optical microscopy. Here, we review studies of combined confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid dynamics and organization in domains assembled in GUVs, prepared from various lipid mixtures, which are relevant to the problem of raft formation. Finally, we summarize the results on lipid-protein interactions, which govern the targeting of several putative raft- and non-raft-associated membrane proteins to domain-exhibiting GUVs.

Cholesterol↗
PubMed2006

Effects of ceramide on liquid-ordered domains investigated by simultaneous AFM and FCS.

The sphingolipid ceramides are known to influence lipid lateral organization in biological membranes. In particular, ceramide-induced alterations of microdomains can be involved in several cell functions, ranging from apoptosis to immune response. We used a combined approach of atomic force microscopy, fluorescence correlation spectroscopy, and confocal fluorescence imaging to investigate the effects of ceramides in model membranes of biological relevance. Our results show that physiological quantities of ceramide in sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers lead to a significant rearrangement of lipid lateral organization. Our experimental setup allowed a simultaneous characterization of both structural and dynamic modification of membrane microdomains, induced by the presence of ceramide. Formation of similar ceramide-enriched domains and, more general, alterations of lipid-lipid interactions can be of crucial importance for the biological function of cell membranes.

Ceramides↗
PubMed2005

Lipids as modulators of proteolytic activity of BACE: involvement of cholesterol, glycosphingolipids, and anionic phospholipids in vitro.

The beta-secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic beta-amyloid peptide (Abeta). Previous results have suggested that the regulation of beta-secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10-150 mum) unilamellar vesicles, and found approximately 20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large ( approximately 100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol).

Amyloid Precursor Protein Secretases↗
PubMed2005

Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances.

Atomic force microscopy (AFM) has been applied to characterize hydrated sphingomyelin/dioleoylphosphatidylcholine/cholesterol supported bilayers, after dehydration either in the absence or in the presence of several stabilizing substances. Such a study provides information about the effect of extreme environmental conditions on biological membranes and, in particular, on lipidic microdomains. Dehydration stress, indeed, is thought to cause both macroscopical damage and alterations of microdomains in biomembranes, leading to deleterious effects. These phenomena can be avoided if disaccharides are added during dehydration. In this work, we apply AFM imaging to directly visualize damage caused to supported lipid bilayers by water removal. We compare the efficiency of sucrose, trehalose, dextran, dimethyl sulfoxide, and glucose in preserving the structural integrity of domain-exhibiting model membranes. Finally, in addition to confirming previous findings, our results provide further insight into damage and alteration of microdomains in membranes as a consequence of stressful drying conditions.

Disaccharides↗
PubMed2005

Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles.

Much attention has recently been drawn to the hypothesis that cellular membranes organize in functionalized platforms called rafts, enriched in sphingolipids and cholesterol. The notion that glycosylphosphatidylinositol (GPI)-anchored proteins are strongly associated with rafts is based on their insolubility in nonionic detergents. However, detergent-based methodologies for identifying raft association are indirect and potentially prone to artifacts. On the other hand, rafts have proven to be difficult to visualize and investigate in living cells. A number of studies have demonstrated that model membranes provide a valuable tool for elucidating some of the raft properties. Here, we present a model membrane system based on domain-forming giant unilamellar vesicles (GUVs), in which the GPI-anchored protein, human placental alkaline phosphatase (PLAP), has been functionally reconstituted. Raft morphology, protein raft partitioning, and dynamic behavior have been characterized by fluorescence confocal microscopy and fluorescence correlation spectroscopy (FCS). Approximately 20-30% of PLAP associate with sphingomyelin-enriched domains. The affinity of PLAP for the liquid-ordered (l(o)) phase is compared to that of a nonraft protein, bacteriorhodopsin. Next, detergent extraction was carried out on PLAP-containing GUVs as a function of temperature, to relate the lipid and protein organization in distinct phases of the GUVs to the composition of detergent resistant membranes (DRMs). Finally, antibody-mediated cross-linking of PLAP induces a shift of its partition coefficient in favor of the l(o) phase.

Alkaline Phosphatase↗
PubMed2005

Differential lipid packing abilities and dynamics in giant unilamellar vesicles composed of short-chain saturated glycerol-phospholipids, sphingomyelin and cholesterol.

The ability of membrane components to arrange themselves heterogeneously within the bilayer induces the formation of microdomains. Much work has been devoted to mimicking domain-assembly in artificial bilayers and characterizing their physico-chemical properties. Ternary lipid mixtures composed of unsaturated phospholipids, sphingomyelin and cholesterol give rise to large, round domains. Here, we replaced the unsaturated phospholipid in the ternary mixture with sphingomyelin and cholesterol by saturated glycero-phospholipids of different chain length and characterized the critical role of cholesterol in sorting these lipids by confocal imaging and fluorescence correlation spectroscopy (FCS). More cholesterol is needed to obtain phase segregation in ternary mixtures, in which the unsaturated phospholipid is replaced by a saturated one. Finally, lipid dynamics in distinct phases is very low and astonishingly similar, thereby suggesting the poor ability of cholesterol in sorting short-chain saturated glycero-phospholipids and sphingomyelin.

Cholesterol↗
PubMed2004

Characterization of interaction between cationic lipid-oligonucleotide complexes and cellular membrane lipids using confocal imaging and fluorescence correlation spectroscopy.

Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.

Biological Transport↗
PubMed2004

SNAREs prefer liquid-disordered over "raft" (liquid-ordered) domains when reconstituted into giant unilamellar vesicles.

Membrane domains ("rafts") have received great attention as potential platforms for proteins in signaling and trafficking. Because rafts are believed to form by cooperative lipid interactions but are not directly accessible in vivo, artificial phase-separating lipid bilayers are useful model systems. Giant unilamellar vesicles (GUVs) offer large free-standing bilayers, but suitable methods for incorporating proteins are still scarce. Here we report the reconstitution of two water-insoluble SNARE proteins into GUVs without fusogenic additives. Following reconstitution, protein functionality was assayed by confocal imaging and fluorescence auto- and cross-correlation spectroscopy. Incorporation into GUVs containing phase-separating lipids revealed that, in the absence of other cellular factors, both proteins exhibit an intrinsic preference for the liquid-disordered phase. Although the picture from detergent resistance assays on whole cells is ambiguous, reconstitutions of components of the exocytic machinery into GUVs by this new approach should yield insight into the dynamics of protein complex associations with hypothesized liquid-ordered phase microdomains, the correspondence between detergent-resistant membranes and liquid-ordered phase, and the mechanism of SNARE-mediated membrane fusion.

Exocytosis↗
PubMed2004

Lipid domain formation and dynamics in giant unilamellar vesicles explored by fluorescence correlation spectroscopy.

Lipids in eukaryotic cell membranes have been shown to cluster in "rafts" with different lipid/protein compositions and molecular packing. Model membranes such as giant unilamellar vesicles (GUVs) provide a key system to elucidate the physical mechanisms of raft assembly. Despite the large amount of work devoted to the detection and characterization of rafts, one of the most important pieces of information still missing in the picture of the cell membrane is dynamics: how lipids organize and move in rafts and how they modulate membrane fluidity. This missing element is of crucial importance for the trafficking at and from the periphery of the cell regulated by endo- and exocytosis and, in general, for the constant turnover which redistributes membrane components. Here, we review studies of combined confocal fluorescence microscopy and fluorescence correlation spectroscopy on lipid dynamics and organization in rafts assembled in GUVs prepared from various lipid mixtures which are relevant to the problem of raft formation.

Biological Transport↗
PubMed2004

Fluorescence correlation spectroscopy relates rafts in model and native membranes.

The lipid raft model has evoked a new perspective on membrane biology. Understanding the structure and dynamics of lipid domains could be a key to many crucial membrane-associated processes in cells. However, one shortcoming in the field is the lack of routinely applicable techniques to measure raft association without perturbation by detergents. We show that both in cell and in domain-exhibiting model membranes, fluorescence correlation spectroscopy (FCS) can easily distinguish a raft marker (cholera toxin B subunit bound to ganglioside (GM1) and a nonraft marker (dialkylcarbocyanine dye diI)) by their decidedly different diffusional mobilities. In contrast, these markers exhibit only slightly different mobilities in a homogeneous artificial membrane. Performing cholesterol depletion with methyl-beta-cyclodextrin, which disrupts raft organization, we find an analogous effect of reduced mobility for the nonraft marker in domain-exhibiting artificial membranes and in cell membranes. In contrast, cholesterol depletion has differential effects on the raft marker, cholera toxin B subunit-GM1, rendering it more mobile in artificial domain-exhibiting membranes but leaving it immobile in cell membranes, where cytoskeleton disruption is required to achieve higher mobility. Thus, fluorescence correlation spectroscopy promises to be a valuable tool to elucidate lipid raft associations in native cells and to gain deeper insight into the correspondence between model and natural membranes.

Animals↗
PubMed2003

Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy.

Confocal fluorescence microscopy and fluorescence correlation spectroscopy (FCS) have been employed to investigate the lipid spatial and dynamic organization in giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol. For a certain range of cholesterol concentration, formation of domains with raft-like properties was observed. Strikingly, the lipophilic probe 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-C18) was excluded from sphingomyelin-enriched regions, where the raft marker ganglioside GM1 was localized. Cholesterol was shown to promote lipid segregation in dioleoyl-phosphatidylcholine-enriched, liquid-disordered, and sphingomyelin-enriched, liquid-ordered phases. Most importantly, the lipid mobility in sphingomyelin-enriched regions significantly increased by increasing the cholesterol concentration. These results pinpoint the key role, played by cholesterol in tuning lipid dynamics in membranes. At cholesterol concentrations >50 mol%, domains vanished and the lipid diffusion slowed down upon further addition of cholesterol. By taking the molecular diffusion coefficients as a fingerprint of membrane phase compositions, FCS is proven to evaluate domain lipid compositions. Moreover, FCS data from ternary and binary mixtures have been used to build a ternary phase diagram, which shows areas of phase coexistence, transition points, and, importantly, how lipid dynamics varies between and within phase regions.

Carbocyanines↗
PubMed2003

Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol.

In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2002

Spatial organization of bacteriorhodopsin in model membranes. Light-induced mobility changes.

Bacteriorhodopsin is a proton-transporting membrane protein in Halophilic archaea, and it is considered a prototype of membrane transporters and a model for G-protein-coupled receptors. Oligomerization of the protein has been reported, but it is unknown whether this feature is correlated with, for instance, light activation. Here, we have addressed this issue by reconstituting bacteriorhodopsin into giant unilamellar vesicles. The dynamics of the fully active protein was investigated using fluorescence correlation spectroscopy and freeze fracture electron microscopy. At low protein-to-lipid ratios (<1:10 w/w), a decrease in mobility was observed upon protein photoactivation. This process occurred on a second time scale and was fully reversible, i.e. when the dark-adapted state was reestablished the lateral diffusion rate of the protein was returned to that prior to activation. A similar decrease in lateral mobility as observed upon photoactivation was obtained when bacteriorhodopsin was reconstituted at high protein-to-lipid ratios (>1:10 w/w). We interpret the shifts in mobility during light adaptation as being caused by transient photoinduced oligomerization of bacteriorhodopsin. These observations are fully supported by freeze-fracture electron microscopy, and the size of the clusters during photoactivation was estimated to consist of two or three trimers.

Bacteriorhodopsins↗
PubMedDate not supplied

Fluorescence correlation studies of lipid domains in model membranes.

Advances in optical microscopy techniques and single-molecule detection have paved the way to exploring new approaches for investigating membrane dynamics and organization, thereby revealing details on the processing of signals, complex association/dissociation, chemical reactions and transport at and around the membrane. These events rely on a tight regulation of lipid-protein and protein-protein interactions in space and time. Fluorescence Correlation Spectroscopy (FCS) provides exquisite sensitivity in measuring local concentrations, association/dissociation constants, chemical rate constants and, in general, in probing the chemical environment of the species of interest and its interactions with potential partners. Here, we review some applications of FCS to lipid and protein organization in biomimetic membranes with lateral heterogeneities, which share some physico-chemical properties with cellular rafts. What we learn from investigations of lipid-lipid and lipid-protein interactions in simple model membranes can be regarded as an essential basic lecture for studies in more complex cellular membranes.

Animals↗