Salvatore Chiantia

Ceramide Triggers Budding of Exosome Vesicles into Multivesicular Endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.

Effect of Line Tension on the Lateral Organization of Lipid Membranes

The principles of organization and functioning of cellular membranes are currently not well understood. The raft hypothesis suggests the existence of domains or rafts in cell membranes, which behave as protein and lipid platforms. They have a functional role in important cellular processes, like protein sorting or cell signaling, among others. Theoretical work suggests that the interfacial energy at the domain edge, also known as line tension, is a key parameter determining the distribution of domain sizes, but there is little evidence of how line tension affects membrane organization. We have investigated the effects of the line tension on the formation and stability of liquid ordered domains in model lipid bilayers with raft-like composition by means of time-lapse confocal microscopy coupled to atomic force microscopy. We varied the hydrophobic mismatch between the two phases, and consequently the line tension, by modifying the thickness of the disordered phase with phosphatidylcholines of different acyl chain length. The temperature of domain formation, the dynamics of domain growth, and the distribution of domain sizes depend strongly on the thickness difference between the domains and the surrounding membrane, which is related to line tension. When considering line tension calculated from a theoretical model, our results revealed a linear increase of the temperature of domain formation and domain growth rate with line tension. Domain budding was also shown to depend on height mismatch. Our experiments contribute significantly to our knowledge of the physical-chemical parameters that control membrane organization. Importantly, the general trends observed can be extended to cellular membranes.

Fluorescence correlation spectroscopy in membrane structure elucidation

This review describes the application of fluorescence correlation spectroscopy (FCS) for the study of biological membranes. Monitoring the fluorescence signal fluctuations, it is possible to obtain diffusion constants and concentrations for several membrane components. Focusing the attention on lipid bilayers, we explain the technical difficulties and the new FCS-based methodologies introduced to overcome them. Finally, we report several examples of studies which apply FCS on both model and biological membranes to obtain interesting insight in the topic of lateral membrane organization.

Accurate Determination of Membrane Dynamics with Line-Scan FCS

Here we present an efficient implementation of line-scan fluorescence correlation spectroscopy (i.e., one-dimensional spatio-temporal image correlation spectroscopy) using a commercial laser scanning microscope, which allows the accurate measurement of diffusion coefficients and concentrations in biological lipid membranes within seconds. Line-scan fluorescence correlation spectroscopy is a calibration-free technique. Therefore, it is insensitive to optical artifacts, saturation, or incorrect positioning of the laser focus. In addition, it is virtually unaffected by photobleaching. Correction schemes for residual inhomogeneities and depletion of fluorophores due to photobleaching extend the applicability of line-scan fluorescence correlation spectroscopy to more demanding systems. This technique enabled us to measure accurate diffusion coefficients and partition coefficients of fluorescent lipids in phase-separating supported bilayers of three commonly used raft-mimicking compositions. Furthermore, we probed the temperature dependence of the diffusion coefficient in several model membranes, and in human embryonic kidney cell membranes not affected by temperature-induced optical aberrations.

Role of ceramide in membrane protein organization investigated by combined AFM and FCS

Ceramide-induced alterations in the lateral organization of membrane proteins can be involved in several biological contexts, ranging from apoptosis to viral infections. In order to investigate such alterations in a simple model, we used a combined approach of atomic force microscopy, scanning fluorescence correlation spectroscopy and confocal fluorescence imaging to study the partitioning of different membrane components in sphingomyelin/dioleoyl-phosphatidylcholine/cholesterol/ceramide supported bilayers. Such model membranes exhibit coexistence of liquid-disordered, liquid-ordered (raft-like) and ceramide-rich lipid phases. Our results show that components with poor affinity toward the liquid-ordered phase, such as several fluorescent lipid analogues or the synaptic protein Synaptobrevin 2, are excluded from ceramide-rich domains. Conversely, we show for the first time that the raft-associated protein placental alkaline phosphatase (GPI-PLAP) and the ganglioside GM1 are enriched in such domains, while exhibiting a strong decrease in lateral diffusion. Analogue modulation of the local concentration and dynamics of membrane proteins/receptors by ceramide can be of crucial importance for the biological functions of cell membranes.

Asymmetric GUVs Prepared by MβCD-Mediated Lipid Exchange: An FCS Study

We report a simple method to obtain stable asymmetric giant unilamellar vesicles (GUVs). Fluorescence correlation spectroscopy was used to quantitatively characterize vesicle properties. After brain sphingomyelin (bSM) was exchanged into dioleoylphosphatidylcholine (DOPC) GUVs, lateral diffusion in the bSM-containing outer leaflet decreased, whereas that in the DOPC-containing inner leaflet was largely unchanged, confirming asymmetry and a lack of coupling between the physical states of the inner and outer leaflets. In contrast, after bSM was exchanged into brain phosphatidylcholine vesicles, lateral diffusion decreased in both leaflets. Thus, asymmetric GUVs should be useful for investigating the molecular mechanisms behind interleaflet coupling.

Proving Lipid Rafts Exist: Membrane Domains in the Prokaryote Borrelia burgdorferi Have the Same Properties as Eukaryotic Lipid Rafts

Lipid rafts in eukaryotic cells are sphingolipid and cholesterol-rich, ordered membrane regions that have been postulated to play roles in many membrane functions, including infection. We previously demonstrated the existence of cholesterol-lipid-rich domains in membranes of the prokaryote, B. burgdorferi, the causative agent of Lyme disease [LaRocca et al. (2010) Cell Host & Microbe 8, 331–342]. Here, we show that these prokaryote membrane domains have the hallmarks of eukaryotic lipid rafts, despite lacking sphingolipids. Substitution experiments replacing cholesterol lipids with a set of sterols, ranging from strongly raft-promoting to raft-inhibiting when mixed with eukaryotic sphingolipids, showed that sterols that can support ordered domain formation are both necessary and sufficient for formation of B. burgdorferi membrane domains that can be detected by transmission electron microscopy or in living organisms by Förster resonance energy transfer (FRET). Raft-supporting sterols were also necessary and sufficient for formation of high amounts of detergent resistant membranes from B. burgdorferi. Furthermore, having saturated acyl chains was required for a biotinylated lipid to associate with the cholesterol-lipid-rich domains in B. burgdorferi, another characteristic identical to that of eukaryotic lipid rafts. Sterols supporting ordered domain formation were also necessary and sufficient to maintain B. burgdorferi membrane integrity, and thus critical to the life of the organism. These findings provide compelling evidence for the existence of lipid rafts and show that the same principles of lipid raft formation apply to prokaryotes and eukaryotes despite marked differences in their lipid compositions.

Analysis of Prototype Foamy Virus particle-host cell interaction with autofluorescent retroviral particles

BackgroundThe foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive.ResultsIn order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction.ConclusionsWe have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV - host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines resistant to Prototype Foamy Virus Env-mediated gene transfer. Interestingly, both cell lines still displayed FV Env-dependent attachment of fluorescent retroviral particles, implying a post-binding block potentially due to lack of putative FV entry cofactors. These cell lines might ultimately lead to the identification of the currently unknown ubiquitous cellular entry receptor(s) of FVs.

Acyl Chain Length and Saturation Modulate Interleaflet Coupling in Asymmetric Bilayers: Effects on Dynamics and Structural Order

A long-standing question about membrane structure and function is the degree to which the physical properties of the inner and outer leaflets of a bilayer are coupled to one another. Using our recently developed methods to prepare asymmetric vesicles, coupling was investigated for vesicles containing phosphatidylcholine (PC) in the inner leaflet and sphingomyelin (SM) in the outer leaflet. The coupling of both lateral diffusion and membrane order was monitored as a function of PC and SM acyl chain structure. The presence in the outer leaflet of brain SM, which decreased outer-leaflet lateral diffusion, had little effect upon lateral diffusion in inner leaflets composed of dioleoyl PC (i.e., diffusion was only weakly coupled in the two leaflets) but did greatly reduce lateral diffusion in inner leaflets composed of PC with one saturated and one oleoyl acyl chain (i.e., diffusion was strongly coupled in these cases). In addition, reduced outer-leaflet diffusion upon introduction of outer-leaflet milk SM or a synthetic C24:0 SM, both of which have long interdigitating acyl chains, also greatly reduce diffusion of inner leaflets composed of dioleoyl PC, indicative of strong coupling. Strikingly, several assays showed that the ordering of the outer leaflet induced by the presence of SM was not reflected in increased lipid order in the inner leaflet, i.e., there was no detectable coupling between inner and outer leaflet membrane order. We propose a model for how lateral diffusion can be coupled in opposite leaflets and discuss how this might impact membrane function.

pH-Controlled two-step uncoating of influenza virus.

Upon endocytosis in its cellular host, influenza A virus transits via early to late endosomes. To efficiently release its genome, the composite viral shell must undergo significant structural rearrangement, but the exact sequence of events leading to viral uncoating remains largely speculative. In addition, no change in viral structure has ever been identified at the level of early endosomes, raising a question about their role. We performed AFM indentation on single viruses in conjunction with cellular assays under conditions that mimicked gradual acidification from early to late endosomes. We found that the release of the influenza genome requires sequential exposure to the pH of both early and late endosomes, with each step corresponding to changes in the virus mechanical response. Step 1 (pH 7.5-6) involves a modification of both hemagglutinin and the viral lumen and is reversible, whereas Step 2 (pH <6.0) involves M1 dissociation and major hemagglutinin conformational changes and is irreversible. Bypassing the early-endosomal pH step or blocking the envelope proton channel M2 precludes proper genome release and efficient infection, illustrating the importance of viral lumen acidification during the early endosomal residence for influenza virus infection.