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A fundamental feature of the eukaryotic cell membrane is the asymmetric arrangement of lipids in its two leaflets. A cell invests significant energy to maintain this asymmetry and uses it to regulate important biological processes, such as apoptosis and vesiculation. The dynamic coupling of the inner or cytoplasmic and outer or exofacial leaflets is a challenging open question in membrane biology. Here, we combined fluorescence lifetime imaging microscopy (FLIM) with imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) to differentiate the dynamics and organization of the two leaflets of live mammalian cells. We characterized the biophysical properties of fluorescent analogs of phosphatidylcholine, sphingomyelin, and phosphatidylserine in the plasma membrane of two mammalian cell lines (CHO-K1 and RBL-2H3). Because of their specific transverse membrane distribution, these probes allowed leaflet-specific investigation of the plasma membrane. We compared the results of the two methods having different temporal and spatial resolution. Fluorescence lifetimes of fluorescent lipid analogs were in ranges characteristic for the liquid ordered phase in the outer leaflet and for the liquid disordered phase in the inner leaflet. The observation of a more fluid inner leaflet was supported by free diffusion in the inner leaflet, with high average diffusion coefficients. The liquid ordered phase in the outer leaflet was accompanied by slower diffusion and diffusion with intermittent transient trapping. Our results show that the combination of FLIM and ITIR-FCS with specific fluorescent lipid analogs is a powerful tool for investigating lateral and transbilayer characteristics of plasma membrane in live cell lines.

The relevance of mannosyldiinositolphosphorylceramide [M(IP)2C] synthesis, the terminal complex sphingolipid class in the yeast Saccharomyces cerevisiae, for the lateral organization of the plasma membrane, and in particular for sphingolipid-enriched gel-like domains, was investigated by fluorescence spectroscopy and microscopy. We also addressed how changing the complex sphingolipid profile in the plasma membrane could influence the membrane compartments (MC) containing either the arginine/ H+ symporter Can1p (MCC) or the proton ATPase Pma1p (MCP). To achieve these goals, wild-type (wt) and ipt1Δ cells, which are unable to synthesize M(IP)2C accumulating mannosylinositolphosphorylceramide (MIPC), were compared. Living cells, isolated plasma membrane and giant unilamellar vesicles reconstituted from plasma membrane lipids were labelled with various fluorescent membrane probes that report the presence and organization of distinct lipid domains, global order, and dielectric properties. Can1p and Pma1p were tagged with GFP and mRFP, respectively, in both yeast strains, to evaluate their lateral organization using confocal fluorescence intensity and fluorescence lifetime imaging. The results show that IPT1 deletion strongly affects the rigidity of gel-like domains but not their relative abundance, whereas no significant alterations could be perceived in ergosterolenriched domains. Moreover, in these cells lacking M(IP)2C, a clear alteration in Pma1p membrane distribution, but no significant changes in Can1p distribution, were observed. Thus, this work reinforces the notion that sphingolipid-enriched domains distinct from ergosterol-enriched regions are present in the S. cerevisiae plasma membrane and suggests that M(IP)2C is important for a proper hydrophobic chain packing of sphingolipids in the gel-like domains of wt cells. Furthermore, our results strongly support the involvement of sphingolipid domains in the formation and stability of the MCP, possibly being enriched in this compartment.

Circadian clocks are molecular timekeeping mechanisms that allow organisms to anticipate daily changes in their environment. The fundamental cellular basis of these clocks is delayed negative feedback gene regulation with PERIOD and CRYPTOCHROME containing protein complexes as main inhibitory elements. For a correct circadian period, it is essential that such clock protein complexes accumulate in the nucleus in a precisely timed manner, a mechanism that is poorly understood. We performed a systematic RNAi-mediated screen in human cells and identified 15 genes associated with the nucleo-cytoplasmic translocation machinery, whose expression is important for circadian clock dynamics. Among them was Transportin 1 (TNPO1), a non-classical nuclear import carrier, whose knockdown and knockout led to short circadian periods. TNPO1 was found in endogenous clock protein complexes and particularly binds to PER1 regulating its (but not PER2’s) nuclear localization. While PER1 is also transported to the nucleus by the classical, Importin β-mediated pathway, TNPO1 depletion slowed down PER1 nuclear import rate as revealed by fluorescence recovery after photobleaching (FRAP) experiments. In addition, we found that TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization. Together, our RNAi screen knocking down import carriers (but also export carriers) results in short and long circadian periods indicating that the regulatory pathways that control the timing of clock protein subcellular localization are far more complex than previously assumed. TNPO1 is one of the novel players essential for normal circadian periods and potentially for redox regulation of the clock., Author summary Circadian clocks are endogenous timekeeping mechanisms allowing organisms to anticipate daily changes in their environment. In mammals, the fundamental mechanism of these clocks is a delayed negative feedback loop, in which timely auto-repression of clock components is essential. This repression occurs at a transcriptional level and requires clock proteins to enter the nucleus in a precisely timed manner, a regulation that is little understood. We performed a systematic genetic screen for factors modulating subcellular localization in oscillating human cells and identified Transportin 1 (TNPO1) as a non-classical carrier protein required for a normal circadian period. The primary target of TNPO1 within the circadian clockwork is PERIOD1, whose nuclear shuttling is modulated by TNPO1. In addition, TNPO1-mediated nuclear import may constitute a novel input pathway of how cellular redox state signals to the clock, since redox stress increases binding of TNPO1 to PER1 and decreases its nuclear localization.

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORFall1371encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacteriumAnabaenasp. PCC 7120. Here, ORFall1371was heterologously expressed inEscherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cationsin vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, withkcat = 48.2 s−1at pH 7.5 and 28 °C andKM = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of anAnabaenasp. PCC 7120 mutant strain lackingall1371was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression ofall1371under combined nitrogen deprivation. All1371 might play a substantial role inAnabaenasp. PCC 7120 under these conditions.

When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3-5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ~7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used "model organism" Synechocystis sp. PCC6803 expresses two such genes, nblA16803 and nblA26803, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Forster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N2-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.

Real-time monitoring of acid sphingomyelinase (ASM) activity is crucial for investigating its role in lipid-mediated signaling processes. In this study, we synthesized fluorescent phosphosphingolipids capable of FRET by phosphorodichloridate chemistry. These sphingomyelin analogues are substrates for recombinant human ASM and can be used to monitor ASM activity by fluorescence spectroscopy. Incubation with cell lysates from wild-type and knock-out mice further confirmed probe cleavage to be exclusive to ASM. We also systematically exploited the environmental sensitivity of the fluorophores to achieve significant increases in responsiveness. This concept may be transferred to other lipid probes in the future. The ASM activity in live cells was imaged by two-photon-excitation microscopy.

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells. Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level.

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehythoergosterol (DHE), a well-established marker for cholesterol, by introducing simultaneous imaging of both sterols in model membranes and living cells. BCh2 had a lower affinity than DHE for the biologically relevant liquid-ordered phase in model membranes. Still, DHE and BCh2 trafficked from the plasma membrane to the endocytic recycling compartment (ERC) of BHK cells with identical kinetics. This transport pathway was strongly reduced after energy depletion of cells or expression of the dominant-negative clathrin heavy chain. The partitioning into lipid droplets of BHK and HeLa cells was higher for BCh2 than for DHE. Within droplets, the photodegradation of BCh2 was enhanced and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference of the sterol probe and causes some differential targeting of BCh2 and DHE in cells with high fat content. (c) 2011 Elsevier Ireland Ltd. All rights reserved.

BioMNY, a bacterial high-affinity biotin transporter, is a member of the recently defined class of ECF (energy-coupling factor) transporters. These systems are composed of ABC (ATP-binding-cassette) ATPases (represented by BioM in the case of the biotin transporter), a universally conserved transmembrane protein (BioN) and a core transporter component (BioY), in unknown stoichiometry. The quaternary structure of BioY, which functions as a low-affinity biotin transporter in the absence of BioMN, and of BioMNY was investigated by a FRET (Förster resonance energy transfer) approach using living recombinant Escherichia coli cells. To this end, the donor–acceptor pair, of Cerulean and yellow fluorescent protein respectively, were fused to BioM, BioN and BioY. The fusion proteins were stable and the protein tags did not interfere with transport and ATPase activities. Specific donor–acceptor interactions were characterized by lifetime-based FRET spectroscopy. The results suggest an oligomeric structure for the solitary BioY core transporter and oligomeric forms of BioM and BioY in BioMNY complexes. We surmise that oligomers of BioY are the functional units of the low- and high-affinity biotin transporter in the living cell. Beyond its relevance for clarifying the supramolecular organization of ECF transporters, the results demonstrate the general applicability of lifetime-based FRET studies in living bacteria.

Fusion of influenza virus with the endosomal membrane of the host cell is mediated by the homotrimer-organized glycoprotein hemagglutinin (HA). Its fusion activity is triggered by a low pH-mediated conformational change affecting the structure of the HA1 and HA2 subunits. The HA2 subunits undergo a loop-to-helix transition leading to a coiled-coil structure, a highly conserved motif for many fusion mediating viral proteins. However, experimental studies showed that the HA2 coiled-coil structure is stable at neutral and low pH, implying that there is no direct relationship between low pH and the HA2 loop-to-helix transition. To interpret this observation, we used a computational approach based on the dielectric continuum solvent model to explore the influence of water and pH on the free energy change of the transition. The computations showed that the electrostatic interaction between HA2 fragments and water is the major driving force of the HA2 loop-to-helix transition leading to the coiled-coil structure, as long as the HA1 globular domain covering the HA2 subunits in the nonfusion competent conformation is reorganized and thereby allows water molecules to interact with the whole loop segments of the HA2 subunits. Moreover, we show that the energy released by the loop-to-helix transition may account for those energies required for driving the subsequent steps of membrane fusion. Such a water-driven process may resemble a general mechanism for the formation of the highly conserved coiled-coil motif of enveloped viruses.

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.

A conformational change of the homotrimeric glycoprotein hemagglutinin (HA) of influenza virus mediates fusion between the viral envelope and the endosome membrane. The conformational change of the HA ectodomain is triggered by the acidic pH of the endosome lumen. An essential step of the conformational change is the formation of an extended coiled-coil motif exposing the hydrophobic fusion peptide toward the target membrane. The structures of the neutral-pH, non-fusion active conformation of the HA ectodomain and of a fragment of the ectodomain containing the coiled-coil motif are known. However, it is not known by which mechanism protonation triggers the conformational change of the stable neutral-pH conformation of the ectodomain. Here, recent studies on the stability of the HA ectodomain at neutral pH, the energetics of the conformational change toward the fusion-active state and of the unfolding of the HA ectodomain are summarised. A model for the early steps of the conformational change of the HA ectodomain is presented. The model implicates that protonation leads to a partial dissociation of the distal domains of the HA monomers that is driven by electrostatic repulsion. The opening of the ectodomain enables water to enter the ectodomain. The interaction of water with respective sequences originally shielded from contact with water drives the formation of the coiled-coil structure.

The interaction of a spin-labeled lysophosphatidylcholine analog with intact and bromelain-treated influenza viruses as well as with the bromelain-solubilized hemagglutinin ectodomain has been studied. The inhibition of fusion of influenza viruses with erythrocytes by the lysophosphatidylcholine analog was similar to that observed for non-labeled lysophosphatidylcholine. Only a weak interaction of the lysophosphatidylcholine analog with the hemagglutinin ectodomain was observed even upon triggering the conformational change of the ectodomain at a low pH. A significant interaction of spin-labeled lysophosphatidylcholine with the hemagglutinin ectodomain of intact viruses was observed neither at neutral nor at low pH, whereas a strong interaction of the lipid analog with the viral lipid bilayer was evident. We suggest that the high number of lipid binding sites of the virus bilayer and their affinity compete efficiently with binding sites of the hemagglutinin ectodomain. We conclude that the inhibition of influenza virus fusion by lysolipids is not mediated by binding to the hemagglutinin ectodomain, preventing its interaction with the target membrane. The results unambiguously argue for an inhibition mechanism based on the action of lysolipid inserted into the lipid bilayer.

Treatment of human osteosarcoma cells, expressing CD4 and various chemokine receptors, with the glucosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol (PPMP), blocked target membrane glycosphingolipid (GSL) biosynthesis and reduced the susceptibility of cells to infection and fusion mediated by envelope glycoproteins from a variety of human immunodeficiency virus type 1 (HIV-1) isolates that utilize CXCR4 and/or CCR5. PPMP treatment of the cell lines did not significantly change the cell surface expression of CD4, CXCR4, and/or CCR5, nor did it alter the chemokine receptor association with CD4. PPMP-treated cells exhibited no changes in chemokine-induced Ca2+mobilization and chemotaxis. However, massive envelope glycoprotein conformational changes triggered by CD4 and the appropriate chemokine receptor on the target membrane were inhibited when the target cells were treated with PPMP. Addition of various purified GSLs to PPMP-treated target cells showed that for all isolates tested, globotriaosylceramide (Gb3) was the most potent GSL in restoring the fusion susceptibility of target cells with cells expressing HIV-1 envelope glycoproteins; addition of the monosialoganglioside GM3 yielded a slight enhancement of fusion susceptibility. Our data are consistent with the notion that a limited number of specific GSL species serve as crucial elements in organizing gp120-gp41, CD4, and an appropriate chemokine receptor into a membrane fusion complex.

Three strains of influenza virus (H1, H2, and H3) exhibited similar characteristics in the ability of their hemagglutinin (HA) to induce membrane fusion, but the HAs differed in their susceptibility to inactivation. The extent of inactivation depended on the pH of preincubation and was lowest for A/Japan (H2 subtype), in agreement with previous studies (A. Puri, F. Booy, R. W. Doms, J. M. White, and R. Blumenthal, J. Virol. 64:3824–3832, 1990). While significant inactivation of X31 (H3 subtype) was observed at 37°C at pH values corresponding to the maximum of fusion (about pH 5.0), no inactivation was seen at preincubation pH values 0.2 to 0.4 pH units higher. Surprisingly, low-pH preincubation under those conditions enhanced the fusion rates and extents of A/Japan as well as those of X31. For A/PR 8/34 (H1 subtype), neither a shift of the pH (to >5.0) nor a decrease of the temperature to 20°C was sufficient to prevent inactivation. We provide evidence that the activated HA is a conformational intermediate distinct from the native structure and from the final structure associated with the conformational change of HA, which is implicated by the high-resolution structure of the soluble trimeric fragment TBHA2 (P. A. Bullough, F. M. Hughson, J. J. Skehel, and D. C. Wiley, Nature 371:37–43, 1994).

The intersection of the HIV and the chemokine fields began with the observation that HIV entry into cells could be blocked by certain chemokines. Subsequent work showed that HIV entry is dependent on the presence of specific chemokine receptors. These observations led us to evaluate a series of compounds, ureido analogs of distamycin previously reported to block HIV entry into cells in vitro, for chemokine antagonist activity. One of the distamycin analogs, 2,2′[4,4′-[[aminocarbonyl]amino]bis[N,4′-di[pyrrole-2-carboxamide-1,1′-dimethyl]]-6,8 napthalenedisulfonic acid] hexasodium salt (NSC 651016), is shown here to inhibit syncytia formation and cell fusion. Mechanistic studies showed that this inhibition was not due to conformational changes in gp120-gp41 induced by target cell CD4 and chemokine co-receptor and was therefore not due to interference with binding of HIV-1. Additional mechanistic studies demonstrated that NSC 651016 inhibited chemokine binding to specific chemokine receptors, induced CXCR4 and CCR5 receptor internalization, and inhibited chemokine-induced chemotaxis by macrophage inflammatory protein (MIP)-1α, MIP-1β, RANTES, and stromal-derived factor-1α but not monocyte chemotactic protein-1. Thus, we describe a novel compound that inhibits in vivo replication of HIV-1 by down-regulation of co-receptors. These data lead us to propose that NSC 651016 may have in vivo anti-inflammatory activity. J. Leukoc. Biol. 64: 6–13; 1998.

Membrane fusion of influenza virus is mediated by a conformational change of the viral membrane protein hemagglutinin (HA) triggered by low pH. By near UV CD spectroscopy, which is sensitive to the arrangement and mobility of aromatic amino acids in proteins, we have monitored continuously with a time resolution of 5 s the kinetics of structural alterations of the ectodomain of HA isolated from different influenza virus strains (H1 (A/PR 8/34), H2 (A/Japan), and H3 (X31)). To establish a functional correlation to structural alterations of the HA ectodomain reflected by the CD, we have measured the kinetics of the virus-erythrocyte fusion and of the inactivation of fusion by low pH preincubation of viruses. At acidic pH we found a multiphasic behavior of the CD signal recorded at 283 nm. Upon lowering the pH we detected first an increase of the CD amplitude, which is associated with the formation of a fusion-competent state of HA. The initial increase was followed by a continuous decline of CD amplitude, which can be ascribed to a transformation into a fusion-inactivated conformation that is in its early phase reversible as found for A/Japan. The half-time of the different phases of the CD signal depended on the virus strain, the temperature, and the acidic pH. The results support recent hypotheses that the fusion-competent conformation is an intermediate of the fusion-inactivated structure of HA.

The influenza virus hemagglutinin (HA) is known to play an essential role in virus infection. It is involved in cell receptor binding and uptake by endocytosis and, after internalization and trafficking to early and late endosomes, induces membrane fusion leading to the release of the viral genome into the cell.This fusion process is mediated by a water-driven “spring- loaded mechanism” which is initiated by protonation of the HA1 globular heads in the acidic environment of the endosome. It has been shown for the HA of influenza virus X-31 that the pH of fusion can be shifted to higher or lower pH values by introducing or deleting salt bridges in the ectodomain of the glycoprotein. In particular, a mutation at the distal end of HA1 leading to the introduction of a salt bridge between two monomers significantly enhanced its stability thereby abolishing membrane fusion. It was suggested that this mutation is also involved in the development of new human pathogenic H5N1 lineages. We hypothesize that salt bridges in the HA1 ectodomain regulate replication efficiency and pathogenesis.As a proof of principle recombinant virus particles containing the wild type or mutant HA of X-31 strain were produced and assayed by fluorescence dequenching. Fusion activity of mutant HA was significantly reduced with increasing pH compared to the wild type, suggesting an influence of the salt bridge on viral fusion. We will apply these results on avian HA and measure virus-endosome fusion by real time imaging inside living cells. Using human and avian cells we will be able to observe possible variations in endosomal pH and in membrane fusion activity.This work was supported by the Marie Curie Actions 7th framework program of Initial Training Networks.

Octadecylrhodamine (R18) has often been used to measure membrane fusion of enveloped viruses by fluorescence dequenching. In order to see whether non-specific R18 exchange between non-fused membranes occurs we have measured fusion of influenza virus with erythrocyte membranes by utilizing dequenching of the non-exchangeable lipid analogue N-(lissamine-rhodamine B-sulfonyl)diacylphosphatidylethanolamine (N-Rh-PE). Rather low concentration of N-Rh-PE (< 0.1 mol%) were required to assess fusion since self-quenching in the influenza virus membrane was more efficient in comparison to R18. For both markers we observed the same kinetics as well as the same extent of fluorescence dequenching upon triggering low pH-induced fusion. Non-specific marker transfer was not observed. Haemolysis was not affected by either type of fluorophore. Our results confirm that R18 is a valuable tool to investigate membrane fusion of enveloped viruses in a quantitative manner. Differences in the efficiency of self-quenching of both markers are discussed.

The hydropathy profile of hemagglutinin (HA) subunits HA1 and HA2 of influenza virus X31 and A/PR 8/34 is analyzed at different pH. At neutral pH (7.4) pronounced hydrophobic sequences of HA correspond to the N-terminus and the transmembrane spanning sequence of HA2. At pH 5.0 where influenza virus is known to fuse with biological membranes several hydrophobic sequences in the ectodomain exist which are comparable in both the hydrophobicity and length of the N-terminus of HA2. It is suggested that these hydrophobic stretches are important for the fusion complex, in addition to the N-terminal site of HA2.

It has been supposed that the HA (haemagglutinin) of influenza virus must be recruited to membrane rafts to perform its function in membrane fusion and virus budding. In the present study, we aimed at substantiating this association in living cells by biophysical methods. To this end, we fused the cyan fluorescent protein Cer (Cerulean) to the cytoplasmic tail of HA. Upon expression in CHO (Chinese-hamster ovary) cells HA–Cer was glycosylated and transported to the plasma membrane in a similar manner to authentic HA. We measured FLIM-FRET (Förster resonance energy transfer by fluorescence lifetime imaging microscopy) and showed strong association of HA–Cer with Myr-Pal–YFP (myristoylated and palmitoylated peptide fused to yellow fluorescent protein), an established marker for rafts of the inner leaflet of the plasma membrane. Clustering was significantly reduced when rafts were disintegrated by cholesterol extraction and when the known raft-targeting signals of HA, the palmitoylation sites and amino acids in its transmembrane region, were removed. FRAP (fluorescence recovery after photobleaching) showed that removal of raft-targeting signals moderately increased the mobility of HA in the plasma membrane, indicating that the signals influence access of HA to slowly diffusing rafts. However, Myr-Pal–YFP exhibited a much faster mobility compared with HA–Cer, demonstrating that HA and the raft marker do not diffuse together in a stable raft complex for long periods of time.

Influenza virus binds via its envelope protein hemagglutinin (HA) sialic acid (SA) residues of glycoproteins on the plasma membrane of the host cell. The affinity of a single HA-SA pair is low (103 M−1) compared to the overall affinity between virus and cell surface caused by multiple simultaneous interactions. For example, the affinity between virus and erythrocytes - serving as a model for a host cell - is about 1013 M−1. Multivalency is ubiquitous in biology and can dramatically enhance affinities. We use synthetic polymers with multiple sialic acid moieties to investigate their inhibitory effect on virus binding and fusion. The aim of this study is to gain insights into the molecular mechanism and the effect principles of multivalent inhibitors. Furthermore we aim to measure the accurate affinity between a virus particle and the target erythrocyte membrane by using a new developed optical tweezers system. With this approach we are able to explore the action of multivalent inhibitors on the single molecule level. Furthermore, we study binding of labelled virus particles to human erythrocytes using fluorescence activated cell sorting (FACS). The fusion activity is examined by detection of fluorescence de-quenching of R 18 labeled viruses attached to human erythrocyte ghosts.

Hemagglutinin (HA) of influenza virus undergoes an irreversible conformational change at acidic pH, mediating viral fusion with the host endosomal membrane. To unravel the molecular basis of the pH-dependent stability of HA, we demonstrate by mutagenesis of the prototype HA of virus strain X31 (H3 subtype) that salt bridges, especially a tetrad salt bridge within the monomers, are crucial for folding and stability of the trimeric ectodomain. This complex (tetrad) salt bridge is highly conserved among influenza virus subtypes. Introducing additional sites of electrostatic attraction between monomers in the distal region enhanced the stability of ectodomain at low pH mimicking the natural variant H2 subtype. We propose that distinct salt bridges in the distal domain may contribute to the enhanced stability of HA of natural virus variants. This hypothesis may provide clues to understanding adaptations of virus strains (for example, avian influenza viruses) in order to preserve stability of the protein in the host-specific environment.

The homotrimeric spike glycoprotein hemagglutinin (HA) of influenza virus undergoes a low pH-mediated conformational change which mediates the fusion of the viral envelope with the target membrane. Previous approaches predict that the interplay of electrostatic interactions between and within HA subunits, HA 1 and HA2, are essential for the metastability of the HA ectodomain. Here, we show that suspension media of low ionic concentration promote fusion of fluorescent labelled influenza virus X31 with erythrocyte ghosts and with ganglioside containing liposomes. By measuring the low pH mediated inactivation of the fusion competence of HA and the Proteinase K sensitivity of low pH incubated HA we show that the conformational change is promoted by low ionic concentration. We surmise that electrostatic attraction within the HA ectodomain is weakened by lowering the ionic concentration facilitating the conformational change at low pH.

In mammals, circadian rhythms are generated by delayed negative feedback, in which period (PER1-PER3) and cryptochrome (CRY1, CRY2) proteins gradually accumulate in the nucleus to suppress the transcription of their own genes. Although the importance of nuclear import and export signals for the subcellular localization of clock proteins is well established, little is known about the dynamics of these processes as well as their importance for the generation of circadian rhythms. We show by pharmacological perturbations of oscillating cells that nuclear import and export are of crucial importance for the circadian period. Live-cell fluorescence microscopy revealed that nuclear import of the key circadian protein PER2 is fast and further accelerated by CRY1. Moreover, PER2 nuclear import is crucially dependent on a specific nuclear-receptor-binding motif in PER2 that also mediates nuclear immobility. Nuclear export, however, is relatively slow, supporting a model of PER2 nuclear accumulation by rapid import, slow export and substantial nuclear degradation.

We have continuously measured CD4-induced conformational changes of cell surface-expressed human immunodeficiency virus type-1 envelope glycoprotein gp120-gp41 in situ using 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid, a fluorescent probe that binds to hydrophobic groups. CD4-expressing human T cell lines induced significant and rapid conformational changes (

Binding of the fluorophore 1,1′-bis(4-anili-no) naphthalene-5,5′-disulfonic acid (bis-ANS) to influenza virus A/PR 8/34 is strongly enhanced at low pH. Binding is accompanied by a significant increase in fluorescence intensity. The binding and the fluorescence increase are associated with the low-pH induced conformational change of the viral spike protein, hemagglutinin, exposing hydrophobic binding sites. The data indicate that in addition to the hydrophobic N-terminus of HA2 other hydrophobic sequences of the HA ectodomain become accessible to bis-ANS at low pH. It is shown that the time course of the fluorescence increase of bis-ANS at low pH is determined by the conformational change of HA. The application of this assay for continuously monitoring the kinetics of the structural alteration in HA is discussed and its relevance for elucidating the temporal relationship between the conformational change of HA and virus-membrane fusion is outlined.

Dextran sulfate suppresses the low pH-induced fusion of influenza virus A/Brazil 11/78 with erythrocyte membranes, as shown by fluorescence dequenching assay, using the fluorophore octadecylrhodamine B chloride (R18). Inhibition of fusion was maximal at pH 5.0, while at higher pH values (> 5.6) fusion was not affected. Hemolysis of intact red blood cells by influenza A virus at low pH values is also prevented by dextran sulfate. The inhibiting effect of the polymer is mainly ascribed to repression of virus attachment. Evidence is given that the conformational change of the virus envelope protein hemagglutinin (HA) responsible for triggering fusion is not affected by the polymer.

To elucidate the role of phospholipid asymmetry in calcium-phosphate-induced fusion of human erythrocytes, we examined the interaction of erythrocyte membranes with asymmetric and symmetric bilayer distributions of phospholipids. Fusion of human erythrocytes was monitored by light microscopy as well as spectrophotometrically by the octadecylrhodamine dequenching assay. Phospholipid translocation and distribution between the inner and the outer leaflet of intact red blood cells were determined with spin-labeled phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Significant fusion of lipid-asymmetric red blood cells where PS and PE are predominantly oriented to the inner leaflet was only observed at Ca2+ concentrations greater than or equal to 10 mM (in the presence of 10 mM phosphate buffer) while fusion of lipid-symmetric erythrocyte membranes was established at greater than or equal to 1.5 mM Ca2+. The Ca2+ threshold of fusion of lipid-asymmetric red blood cells was significantly reduced (i) after exposure of PS to the outer layer but not after redistribution of PE alone, and (ii) upon incorporation of spin-labeled PS into the outer leaflet of red blood cells. Spin-labeled PE or PC did not affect fusion, suggesting that the serine headgroup is an important factor in calcium-phosphate-induced fusion.

To elucidate the role of the fusion peptide in influenza hemagglutinin (HA)-mediated fusion, we compared pH-dependent conformational changes and fusion mediated by wild-type and a mutant HA in which Glu residues at positions 11 and 15 of the fusion peptide are substituted for valine. The pH dependence of conformational changes and kinetics of fusion with erythrocytes was the same for both forms of HA. The time for commitment and the temperature dependence of HA-mediated fusion were also the same. However, striking differences were observed between wild-type and mutant fusion peptides in their interactions with lipid membranes at neutral and acidic pH. Since elimination of the negatively charged residues allows the exposed fusion peptide to penetrate the bilayer at pH values closer to neutral, but does not affect conformational changes and fusion activity in intact HA, we conclude that conformational changes are tightly coupled to fusion peptide insertion in the overall HA-mediated fusion cascade.

The influenza virus hemagglutinin (HA) is known to play an essential role in virus infection. It is not only involved in cell receptor binding and uptake by endocytosis, but moreover, after internalization and trafficking to early and late endosomes, induces membrane fusion which leads to the release of the viral genome into the cell. It has been shown for several influenza strains that the pH of fusion of HA can be shifted by modulating its stability which results in altered infectivity of the virus. However, the underlying molecular mechanisms that lead to an altered pH dependence of membrane fusion are still unknown.Using experimental approaches and Molecular Dynamics studies we demonstrate that the introduction of charges in the vicinity of certain His residues can change its protonation state, thereby influencing the pH dependant conformational change of the HA protein. Histidines at key structural locations were identified and neighboring residues were mutated in the HA of highly (HP) or low pathogenic (LP) H5N1 and X31. Fusion activity and stability of recombinant proteins and resulting virus particles were assessed by fluorescence dequenching.First results showed that charges, especially at position 216, in the vicinity of His184 have a significant effect on the stability of the HA1 ectodomain in both H3 and H5 HA. Interestingly, Arginine at position 216 is naturally present in the H5 HP whereas the H5 LP has a Glutamate at this position. We could show that the exchange of these charges in both variants rescues the pH dependence of fusion of HP and LP, respectively. We suggest that influenza viruses adjust their pH of activation by the exchange of charged residues in the vicinity of conserved Histidines for optimized spread and stability.