Liquid-Ordered Phase Formation by Mammalian and Yeast Sterols: A Common Feature With Organizational Differences.

Here, biophysical properties of membranes enriched in three metabolically related sterols are analyzed both in vitro and in vivo. Unlike cholesterol and ergosterol, the common metabolic precursor zymosterol is unable to induce the formation of a liquid ordered (l o) phase in model lipid membranes and can easily accommodate in a gel phase. As a result, Zym has a marginal ability to modulate the passive membrane permeability of lipid vesicles with different compositions, contrary to cholesterol and ergosterol. Using fluorescence-lifetime imaging microscopy of an aminostyryl dye in living mammalian and yeast cells we established a close parallel between sterol-dependent membrane biophysical properties in vivo and in vitro. This approach unraveled fundamental differences in yeast and mammalian plasma membrane organization. It is often suggested that, in eukaryotes, areas that are sterol-enriched are also rich in sphingolipids, constituting highly ordered membrane regions. Our results support that while cholesterol is able to interact with saturated lipids, ergosterol seems to interact preferentially with monounsaturated phosphatidylcholines. Taken together, we show that different eukaryotic kingdoms developed unique solutions for the formation of a sterol-rich plasma membrane, a common evolutionary trait that accounts for sterol structural diversity.

Full-length cellular ß-secretase has a trimeric subunit stoichiometry, and its sulfur-rich transmembrane interaction site modulates cytosolic copper compartmentalization.

The ß-secretase (BACE1) initiates processing of the amyloid precursor protein (APP) into Aß peptides, which have been implicated as central players in the pathology of Alzheimer disease. BACE1 has been described as a copper-binding protein and its oligomeric state as being monomeric, dimeric, and/or multimeric, but the native cellular stoichiometry has remained elusive. Here, by using single-molecule fluorescence and in vitro cross-linking experiments with photo-activatable unnatural amino acids, we show that full-length BACE1, independently of its subcellular localization, exists as trimers in human cells. We found that trimerization requires the BACE1 transmembrane sequences (TMSs) and cytoplasmic domains, with residues Ala463 and Cys466 buried within the trimer interface of the sulfur-rich core of the TMSs. Our 3D model predicts that the sulfur-rich core of the trimeric BACE1 TMS is accessible to metal ions, but copper ions did not trigger trimerization. The results of functional assays of endogenous BACE1 suggest that it has a role in intracellular copper compartmentalization by transferring cytosolic copper to intracellular compartments, while leaving the overall cellular copper concentration unaltered. Adding to existing physiological models, our results provide novel insight into the atypical interactions between copper and BACE1 and into its non-enzymatic activities. In conclusion, therapeutic Alzheimer disease prevention strategies aimed at decreasing BACE1 protein levels should be regarded with caution, because adverse effects in copper homeostasis may occur.

Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin.

Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and suggest a function of HA palmitoylations in both lateral sorting and anterograde trafficking of the glycoprotein.

The cholesterol-binding motif of the HIV-1 glycoprotein gp41 regulates lateral sorting and oligomerization.

Enveloped viruses often use membrane lipid rafts to assemble and bud, augment infection and spread efficiently. However, the molecular bases and functional consequences of the partitioning of viral glycoproteins into microdomains remain intriguing questions in virus biology. Here, we measured Foerster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) to study the role of distinct membrane proximal regions of the human immunodeficiency virus glycoprotein gp41 for lipid raft partitioning in living Chinese hamster ovary cells (CHO-K1). Gp41 was labelled with a fluorescent protein at the exoplasmic face of the membrane, preventing any interference of the fluorophore with the proposed role of the transmembrane and cytoplasmic domains in lateral organization of gp41. Raft localization was deduced from interaction with an established raft marker, a fluorescently tagged glycophosphatidylinositol anchor and the cholesterol recognition amino acid consensus (CRAC) was identified as the crucial lateral sorting determinant in CHO-K1 cells. Interestingly, the raft association of gp41 indicates a substantial cell-to-cell heterogeneity of the plasma membrane microdomains. In complementary fluorescence polarization microscopy, a distinct CRAC requirement was found for the oligomerization of the gp41 variants. Our data provide further insight into the molecular basis and biological implications of the cholesterol dependent lateral sorting of viral glycoproteins for virus assembly at cellular membranes.

Lipid domain association of influenza virus proteins detected by dynamic fluorescence microscopy techniques.

Influenza virus is thought to assemble in raft domains of the plasma membrane, but many of the conclusions were based on (controversial) Triton extraction experiments. Here we review how sophisticated methods of fluorescence microscopy, such as FPALM, FRET and FRAP, contributed to our understanding of lipid domain association of the viral proteins HA and M2. The results are summarized in light of the current model for virus assembly and lipid domain organization. Finally, it is described how the signals that govern domain association in transfected cells affect replication of influenza virus.

Benzothiazinones: a novel class of adenosine receptor antagonists structurally unrelated to xanthine and adenine derivatives.

2-(Acyl)amino-4H-3,1-benzothiazin-4-ones and related thienothiazinones were identified as structurally novel antagonists at adenosine receptors (ARs). 6-Methyl-2-benzoylamino-4H-3,1-benzothiazin-4-one (10d) was found to be a balanced AR antagonist with affinity for all human (h) subtypes (K(i) hA(1) 65.6 nM; hA(2A) 120 nM; hA(2B) 360 nM; hA(3) 30.4 nM), while in rat (r), 10d was a highly potent A(1)-selective antagonist (rA(1) 7.7 nM; rA(2A) 546 nM; rA(2B) 679 nM, rA(3) >10000 nM). 2-(4-Methylbenzoylamino)-4H-3,1-benzothiazin-4-one (10g) was found to be a potent antagonist at human A(2A) (68.8 nM) and A(3) ARs (23.0 nM) with high selectivity versus the other human AR subtypes. In contrast to A(1) and A(3) ARs, A(2A) and A(2B) ARs tolerated bulky 2-acyl substituents. tert-Butyl (4-oxo-4H-3,1-benzothiazin-2-ylcarbamoyl)benzylcarbamate (15g, K(i) hA(2B) 186 nM; hA(2A) 603 nM) and 4-(4-benzylpiperazine-1-carbonyl)-N-(4-oxo-4H-3,1-benzothiazin-2-yl)benzamide (15k, hA(2A) 69.5 nM; hA(2B) 178 nM) were highly selective versus the other AR subtypes. 2-Acylamino-3,1-benzothiazin-4-ones represent novel scaffolds suitable for the development of potent and selective AR antagonists for each of the four receptor subtypes.

Applications of fluorescence lifetime spectroscopy and imaging to lipid domains in vivo.

Lipid domains are part of the current description of cell membranes and their involvement in many fundamental cellular processes is currently acknowledged. However, their study in living cells is still a challenge. Fluorescence lifetimes have and will continue to play an important role in unraveling the properties and function of lipid domains, and their use in vivo is expected to increase in the near future, since their extreme sensitivity to the physical properties of the membrane and the possibility of optical imaging are particularly suited to deal with the hurdles that are met by researchers. In this review, a practical guide on the use of fluorescence lifetimes for the study of this subject is given. A section is devoted to studies in vitro, particularly membrane model systems, and how they are used to better design and correctly interpret results obtained in living cells. Criteria are presented for selecting suitable probes to solve each problem, drawing attention to factors sometimes overlooked and which may affect the fluorescence lifetime such as subcellular distribution and concentration of the probe. The principal groups of lifetime probes for lipid domains: (i) fluorescent lipid analogues, (ii) other lipophilic probes, and (iii) fluorescent proteins, and respective applications are briefly described and lab tips about the labeling of living cells are provided. The advantages and complementarities of spectroscopy (cuvette) work and fluorescence lifetime imaging microscopy are presented and illustrated with three selected case studies: (i) the finding of a new type of lipid rafts in yeast cells; (ii) the detection of liquid ordered type heterogeneity in animal cells below optical resolution; and (iii) establish a role for the transmembrane domain of influenza virus hemagglutinin with cholesterol-enriched domains in mammalian cells.

Unraveling interactions of cell cycle-regulating proteins Sic1 and B-type cyclins in living yeast cells: a FLIM-FRET approach.

Sic1, cyclin-dependent kinase inhibitor of budding yeast, is synthesized in anaphase and largely degraded at the S-phase onset to regulate timing of DNA synthesis. Sic1 interacts with phase-specific B-type cyclin (Clb)-kinase (Cdk1) complexes, central regulators in cell cycle control. Its appearance is timed to mediate reduction in kinase activities at appropriate stages. Clbs are unstable proteins with extremely short half-lives. Interactions of Sic1 with Clbs have been detected both in vitro and in vivo by high-throughput genome-wide screenings. Furthermore, we have recently shown that Sic1 regulates waves of Clbs, acting as a timer in their appearance, thus controlling Cdk1-Clbs activation. The molecular mechanism is not yet fully understood but is hypothesized to occur via stoichiometric binding of Sic1 to Cdk1-Clb complexes. Using Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy (FLIM), we showed association of Sic1 to Clb cyclins in living yeast cells. This finding is consistent with the notion that inhibition of kinase activity can occur over the whole cell cycle progression despite variable Sic1 levels. Specifically, Sic1/Clb3 interaction was observed for the first time, and Sic1/Clb2 and Sic1/Clb5 pairs were confirmed, but no Sic1/Clb4 interaction was found, which suggests that, despite high functional homology between Clbs, only some of them can target Sic1 function in vivo.

Lipid domain specific recruitment of lipophilic nucleic acids: a key for switchable functionalization of membranes.

Lipid domains in mammalian plasma membranes serve as platforms for specific recruitment or separation of proteins involved in various functions. Here, we have applied this natural strategy of lateral separation to functionalize lipid membranes at micrometer scale in a switchable and reversible manner. Membrane-anchored peptide nucleic acid and DNA, differing in their lipophilic moieties, partition into different lipid domains in model and biological membranes. Separation was visualized by hybridization with the respective complementary fluorescently labeled DNA strands. Upon heating, domains vanished, and both lipophilic nucleic acid structures intermixed with each other. Reformation of the lipid domains by cooling led again to separation of membrane-anchored nucleic acids. By linking appropriate structures/functions to complementary strands, this approach offers a reversible tool for triggering interactions among the structures and for the arrangement of reactions and signaling cascades on biomimetic surfaces.

Interaction of mammalian seminal plasma protein PDC-109 with cholesterol: implications for a putative CRAC domain.

Seminal plasma proteins of the fibronectin type II (Fn2) family modulate mammalian spermatogenesis by triggering the release of the lipids phosphatidylcholine and cholesterol from sperm cells. Whereas the specific interaction of these proteins with phosphatidylcholine is well-understood, their selectivity for cholesterol is unknown. To characterize the interaction between the bovine Fn2 protein PDC-109 and cholesterol, we have investigated the effect of PDC-109 on the dynamics of fluorescent cholesterol analogues in lipid vesicles by time-resolved fluorescence anisotropy. The data show that PDC-109 decreases the rotational mobility of cholesterol within the membrane and that the extent of this impact depends on the cholesterol structure, indicating a specific influence of PDC-109 on cholesterol. We propose that the cholesterol recognition/interaction amino acid consensus (CRAC) regions of PDC-109 are involved in the interaction with cholesterol.

Subunit composition of an energy-coupling-factor-type biotin transporter analysed in living bacteria.

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.

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.

FLIM-FRET and FRAP reveal association of influenza virus haemagglutinin with membrane rafts.

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.

Lateral distribution of the transmembrane domain of influenza virus hemagglutinin revealed by time-resolved fluorescence imaging.

Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577-589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.

Evaluation of anticonvulsant and analgesic effects of benzyl- and benzhydryl ureides.

The anticonvulsant effects of benzyl- and benzhydryl ureides in mice models of seizures (maximal electroshock seizure test, pentylenetetrazol test, picrotoxin-induced seizure test) and the influence on spontaneous locomotor activity has been assessed. Furthermore, the analgesic effect of ureide derivatives was studied in the hot-plate test in mice. Selected compounds were investigated for their in vitro interaction with adenosine receptors as well as the benzodiazepine binding site of GABA(A) receptors. This study demonstrated the strong anticonvulsant activity of several ureides in electrically or chemically induced seizure models, and structure-activity relationships were discussed. 1-Benzyl-3-butyrylurea (9) was found to be equipotent to ethosuximide in the pentylenetetrazol test with regard to the number of attacks as well as the time of the onset of seizures. The ureide 9 also revealed the highest protective activity against seizures in the other models, maximal electroshock seizure and picrotoxin test. Moreover, 1-benzyl-3-butyrylurea was not neurotoxic at doses up to 200 mg/kg. Benzylureides 8-10 showed affinity to the adenosine A1 receptors at low micromolar concentrations. However, the apparent anticonvulsant activity in different seizure models does not appear to result from direct activation of adenosine A1 receptors or GABA(A) receptors, respectively. In the hot-plate test, the majority of investigated compounds exhibited analgesic activity. Again, compound 9 was superior to the other substances investigated, suggesting a potential therapeutic value of that ureide derivative.