Influenza A Virus Infection Alters Lipid Packing and Surface Electrostatic Potential of the Host Plasma Membrane.

The pathogenesis of influenza A viruses (IAVs) is influenced by several factors, including IAV strain origin and reassortment, tissue tropism and host type. While such factors were mostly investigated in the context of virus entry, fusion and replication, little is known about the viral-induced changes to the host lipid membranes which might be relevant in the context of virion assembly. In this work, we applied several biophysical fluorescence microscope techniques (i.e., Förster energy resonance transfer, generalized polarization imaging and scanning fluorescence correlation spectroscopy) to quantify the effect of infection by two IAV strains of different origin on the plasma membrane (PM) of avian and human cell lines. We found that IAV infection affects the membrane charge of the inner leaflet of the PM. Moreover, we showed that IAV infection impacts lipid-lipid interactions by decreasing membrane fluidity and increasing lipid packing. Because of such alterations, diffusive dynamics of membrane-associated proteins are hindered. Taken together, our results indicate that the infection of avian and human cell lines with IAV strains of different origins had similar effects on the biophysical properties of the PM.

Benchmarking of novel green fluorescent proteins for the quantification of protein oligomerization in living cells.

Protein-protein-interactions play an important role in many cellular functions. Quantitative non-invasive techniques are applied in living cells to evaluate such interactions, thereby providing a broader understanding of complex biological processes. Fluorescence fluctuation spectroscopy describes a group of quantitative microscopy approaches for the characterization of molecular interactions at single cell resolution. Through the obtained molecular brightness, it is possible to determine the oligomeric state of proteins. This is usually achieved by fusing fluorescent proteins (FPs) to the protein of interest. Recently, the number of novel green FPs has increased, with consequent improvements to the quality of fluctuation-based measurements. The photophysical behavior of FPs is influenced by multiple factors (including photobleaching, protonation-induced "blinking" and long-lived dark states). Assessing these factors is critical for selecting the appropriate fluorescent tag for live cell imaging applications. In this work, we focus on novel green FPs that are extensively used in live cell imaging. A systematic performance comparison of several green FPs in living cells under different pH conditions using Number & Brightness (N&B) analysis and scanning fluorescence correlation spectroscopy was performed. Our results show that the new FP Gamillus exhibits higher brightness at the cost of lower photostability and fluorescence probability (pf), especially at lower pH. mGreenLantern, on the other hand, thanks to a very high pf, is best suited for multimerization quantification at neutral pH. At lower pH, mEGFP remains apparently the best choice for multimerization investigation. These guidelines provide the information needed to plan quantitative fluorescence microscopy involving these FPs, both for general imaging or for protein-protein-interactions quantification via fluorescence fluctuation-based methods.

Crystal structures of glycoprotein D of equine alphaherpesviruses reveal potential binding sites to the entry receptor MHC-I.

Cell entry of most alphaherpesviruses is mediated by the binding of glycoprotein D (gD) to different cell surface receptors. Equine herpesvirus type 1 (EHV-1) and EHV-4 gDs interact with equine major histocompatibility complex I (MHC-I) to initiate entry into equine cells. We have characterized the gD-MHC-I interaction by solving the crystal structures of EHV-1 and EHV-4 gDs (gD1, gD4), performing protein-protein docking simulations, surface plasmon resonance (SPR) analysis, and biological assays. The structures of gD1 and gD4 revealed the existence of a common V-set immunoglobulin-like (IgV-like) core comparable to those of other gD homologs. Molecular modeling yielded plausible binding hypotheses and identified key residues (F213 and D261) that are important for virus binding. Altering the key residues resulted in impaired virus growth in cells, which highlights the important role of these residues in the gD-MHC-I interaction. Taken together, our results add to our understanding of the initial herpesvirus-cell interactions and will contribute to the targeted design of antiviral drugs and vaccine development.

Thermoplasmonic Vesicle Fusion Reveals Membrane Phase Segregation of Influenza Spike Proteins.

Many cellular processes involve the lateral organization of integral and peripheral membrane proteins into nanoscale domains. Despite the biological significance, the mechanisms that facilitate membrane protein clustering into nanoscale lipid domains remain enigmatic. In cells, the analysis of membrane protein phase affinity is complicated by the size and temporal nature of ordered and disordered lipid domains. To overcome these limitations, we developed a method for delivering membrane proteins from transfected cells into phase-separated model membranes that combines optical trapping with thermoplasmonic-mediated membrane fusion and confocal imaging. Using this approach, we observed clear phase partitioning into the liquid disordered phase following the transfer of GFP-tagged influenza hemagglutinin and neuraminidase from transfected cell membranes to giant unilamellar vesicles. The generic platform presented here allows investigation of the phase affinity of any plasma membrane protein which can be labeled or tagged with a fluorescent marker.

Advances in fluorescence microscopy for orthohantavirus research.

Orthohantaviruses are important zoonotic pathogens responsible for a considerable disease burden globally. Partly due to our incomplete understanding of orthohantavirus replication, there is currently no effective antiviral treatment available. Recently, novel microscopy techniques and cutting-edge, automated image analysis algorithms have emerged, enabling to study cellular, subcellular and even molecular processes in unprecedented detail and depth. To date, fluorescence light microscopy allows us to visualize viral and cellular components and macromolecular complexes in live cells, which in turn enables the study of specific steps of the viral replication cycle such as particle entry or protein trafficking at high temporal and spatial resolution. In this review, we highlight how fluorescence microscopy has provided new insights and improved our understanding of orthohantavirus biology. We discuss technical challenges such as studying live infected cells, give alternatives with recombinant protein expression and highlight future opportunities, for example, the application of super-resolution microscopy techniques, which has shown great potential in studies of different cellular processes and viral pathogens.

Biomimetic asymmetric bacterial membranes incorporating lipopolysaccharides.

Gram-negative bacteria are equipped with a cell wall that contains a complex matrix of lipids, proteins, and glycans, which form a rigid layer protecting bacteria from the environment. Major components of this outer membrane are the high-molecular weight and amphiphilic lipopolysaccharides (LPSs). They form the extracellular part of a heterobilayer with phospholipids. Understanding LPS properties within the outer membrane is therefore important to develop new antimicrobial strategies. Model systems, such as giant unilamellar vesicles (GUVs), provide a suitable platform for exploring membrane properties and interactions. However, LPS molecules contain large polysaccharide parts that confer high water solubility, which makes LPS incorporation in artificial membranes difficult; this hindrance is exacerbated for LPS with long polysaccharide chains, i.e., the smooth LPS. Here, a novel emulsification step of the inverted emulsion method is introduced to incorporate LPS in the outer or the inner leaflet of GUVs, exclusively. We developed an approach to determine the LPS content on individual GUVs and quantify membrane asymmetry. The asymmetric membranes with outer leaflet LPS show incorporations of 1-16 mol % smooth LPS (corresponding to 16-79 wt %), while vesicles with inner leaflet LPS reach coverages of 2-7 mol % smooth LPS (28-60 wt %). Diffusion coefficient measurements in the obtained GUVs showed that increasing LPS concentrations in the membranes resulted in decreased diffusivity.

Characterization of Hantavirus N Protein Intracellular Dynamics and Localization.

Hantaviruses are enveloped viruses that possess a tri-segmented, negative-sense RNA genome. The viral S-segment encodes the multifunctional nucleocapsid protein (N), which is involved in genome packaging, intracellular protein transport, immunoregulation, and several other crucial processes during hantavirus infection. In this study, we generated fluorescently tagged N protein constructs derived from Puumalavirus (PUUV), the dominant hantavirus species in Central, Northern, and Eastern Europe. We comprehensively characterized this protein in the rodent cell line CHO-K1, monitoring the dynamics of N protein complex formation and investigating co-localization with host proteins as well as the viral glycoproteins Gc and Gn. We observed formation of large, fibrillar PUUV N protein aggregates, rapidly coalescing from early punctate and spike-like assemblies. Moreover, we found significant spatial correlation of N with vimentin, actin, and P-bodies but not with microtubules. N constructs also co-localized with Gn and Gc albeit not as strongly as the glycoproteins associated with each other. Finally, we assessed oligomerization of N constructs, observing efficient and concentration-dependent multimerization, with complexes comprising more than 10 individual proteins.

Mandipropamid as a chemical inducer of proximity for in vivo applications.

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins.

Influenza A virus hemagglutinin prevents extensive membrane damage upon dehydration.

While the molecular mechanisms of virus infectivity are rather well known, the detailed consequences of environmental factors on virus biophysical properties are poorly understood. Seasonal influenza outbreaks are usually connected to the low winter temperature, but also to the low relative air humidity. Indeed, transmission rates increase in cold regions during winter. While low temperature must slow degradation processes, the role of low humidity is not clear. We studied the effect of relative humidity on a model of Influenza A H1N1 virus envelope, a supported lipid bilayer containing the surface glycoprotein hemagglutinin (HA), which is present in the viral envelope in very high density. For complete cycles of hydration, dehydration and rehydration, we evaluate the membrane properties in terms of structure and dynamics, which we assess by combining confocal fluorescence microscopy, raster image correlation spectroscopy, line-scan fluorescence correlation spectroscopy and atomic force microscopy. Our findings indicate that the presence of HA prevents macroscopic membrane damage after dehydration. Without HA, fast membrane disruption is followed by irreversible loss of lipid and protein mobility. Although our model is principally limited by the membrane composition, the macroscopic effects of HA under dehydration stress reveal new insights on the stability of the virus at low relative humidity.

Influenza A M2 recruits M1 to the plasma membrane: A fluorescence fluctuation microscopy study.

Influenza A virus (IAV) is a respiratory pathogen that causes seasonal epidemics with significant mortality. One of the most abundant proteins in IAV particles is the matrix protein 1 (M1), which is essential for the virus structural stability. M1 organizes virion assembly and budding at the plasma membrane (PM), where it interacts with other viral components. The recruitment of M1 to the PM as well as its interaction with the other viral envelope proteins (hemagglutinin [HA], neuraminidase, matrix protein 2 [M2]) is controversially discussed in previous studies. Therefore, we used fluorescence fluctuation microscopy techniques (i.e., scanning fluorescence cross-correlation spectroscopy and number and brightness) to quantify the oligomeric state of M1 and its interactions with other viral proteins in co-transfected as well as infected cells. Our results indicate that M1 is recruited to the PM by M2, as a consequence of the strong interaction between the two proteins. In contrast, only a weak interaction between M1 and HA was observed. M1-HA interaction occurred only in the event that M1 was already bound to the PM. We therefore conclude that M2 initiates the assembly of IAV by recruiting M1 to the PM, possibly allowing its further interaction with other viral proteins.

Multicolor fluorescence fluctuation spectroscopy in living cells via spectral detection.

Signaling pathways in biological systems rely on specific interactions between multiple biomolecules. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Cross-correlation analysis of spectrally separated fluctuations provides information about intermolecular interactions but is usually limited to two fluorophore species. Here, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), a versatile approach that can be implemented on commercial confocal microscopes, allowing the investigation of interactions between multiple protein species at the plasma membrane. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus.

Pantoea stewartii WceF is a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like fold.

Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties of a biofilm and result in a highly viscous environment. Bacterial communities regulate this biofilm state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation of this glycan matrix, however, requires the action of extracellular glycosidases. So far, these were mainly described for bacteriophages that must degrade biofilms for gaining access to host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes the protein WceF within its EPS synthesis cluster. WceF has homologs in various biofilm forming plant pathogens of the Erwinia family. In this work, we show that WceF is a glycosidase active on stewartan, the main P. stewartii EPS biofilm component. WceF has remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed a native trimer of right-handed parallel ß-helices. Despite its similar fold, WceF lacks the high stability found in bacteriophage TSPs. WceF is a stewartan hydrolase and produces oligosaccharides, corresponding to single stewartan repeat units. However, compared with a stewartan-specific glycan hydrolase of bacteriophage origin, WceF showed lectin-like autoagglutination with stewartan, resulting in notably slower EPS cleavage velocities. This emphasizes that the bacterial enzyme WceF has a role in P. stewartii biofilm glycan matrix reorganization clearly different from that of a bacteriophage exopolysaccharide depolymerase.

Differentially-Charged Liposomes Interact with Alphaherpesviruses and Interfere with Virus Entry.

Exposure of phosphatidylserine (PS) in the outer leaflet of the plasma membrane is induced by infection with several members of the Alphaherpesvirinae subfamily. There is evidence that PS is used by the equine herpesvirus type 1 (EHV-1) during entry, but the exact role of PS and other phospholipids in the entry process remains unknown. Here, we investigated the interaction of differently charged phospholipids with virus particles and determined their influence on infection. Our data show that liposomes containing negatively charged PS or positively charged DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium) inhibited EHV-1 infection, while neutral phosphatidylcholine (PC) had no effect. Inhibition of infection with PS was transient, decreased with time, and was dose dependent. Our findings indicate that both cationic and anionic phospholipids can interact with the virus and reduce infectivity, while, presumably, acting through different mechanisms. Charged phospholipids were found to have antiviral effects and may be used to inhibit EHV-1 infection.

Effect of Erufosine on Membrane Lipid Order in Breast Cancer Cell Models.

Alkylphospholipids are a novel class of antineoplastic drugs showing remarkable therapeutic potential. Among them, erufosine (EPC3) is a promising drug for the treatment of several types of tumors. While EPC3 is supposed to exert its function by interacting with lipid membranes, the exact molecular mechanisms involved are not known yet. In this work, we applied a combination of several fluorescence microscopy and analytical chemistry approaches (i.e., scanning fluorescence correlation spectroscopy, line-scan fluorescence correlation spectroscopy, generalized polarization imaging, as well as thin layer and gas chromatography) to quantify the effect of EPC3 in biophysical models of the plasma membrane, as well as in cancer cell lines. Our results indicate that EPC3 affects lipid-lipid interactions in cellular membranes by decreasing lipid packing and increasing membrane disorder and fluidity. As a consequence of these alterations in the lateral organization of lipid bilayers, the diffusive dynamics of membrane proteins are also significantly increased. Taken together, these findings suggest that the mechanism of action of EPC3 could be linked to its effects on fundamental biophysical properties of lipid membranes, as well as on lipid metabolism in cancer cells.

Fluorescence microscopy methods for the study of protein oligomerization.

Protein-protein interactions (PPIs) are of fundamental importance in several cellular processes. While "classical" biochemical methods are commonly used to monitor protein multimerization in biological samples, fluorescence microscopy offers the possibility to investigate PPIs directly in living cells, even distinguishing among different cellular compartments. In this chapter, we shortly describe the most common procedures used to label proteins with fluorescent probes. Furthermore, we discuss a variety of fluorescence microscopy techniques that can be used to obtain quantitative information about protein multimerization. Special emphasis is given to fluorescence fluctuation techniques and their applications in the context of, e.g., receptor multimerization and virus assembly.

Purely Polysaccharide-Based Biofilm Matrix Provides Size-Selective Diffusion Barriers for Nanoparticles and Bacteriophages.

Biofilms are complex mixtures of proteins, DNA, and polysaccharides surrounding bacterial communities as protective barriers that can be biochemically modified during the bacterial life cycle. However, their compositional heterogeneity impedes a precise analysis of the contributions of individual matrix components to the biofilm structural organization. To investigate the structural properties of glycan-based biofilms, we analyzed the diffusion dynamics of nanometer-sized objects in matrices of the megadalton-sized anionic polysaccharide, stewartan, the major biofilm component of the plant pathogen, Pantoea stewartii. Fluorescence correlation spectroscopy and single-particle tracking of nanobeads and bacteriophages indicated notable subdiffusive dynamics dependent on probe size and stewartan concentration, in contrast to free diffusion of small molecules. Stewartan enzymatic depolymerization by bacteriophage tailspike proteins rapidly restored unhindered diffusion. We, thus, hypothesize that the glycan polymer stewartan determines the major physicochemical properties of the biofilm, which acts as a selective diffusion barrier for nanometer-sized objects and can be controlled by enzymes.

Influenza A matrix protein M1 induces lipid membrane deformation via protein multimerization.

The matrix protein M1 of the Influenza A virus (IAV) is supposed to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle toward the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In the present study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed able to cause membrane curvature in lipid bilayers containing negatively charged lipids, in the absence of other viral components. Furthermore, we prove that protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1-M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context, M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains).

Self-association and subcellular localization of Puumala hantavirus envelope proteins.

Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement. In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

Optimal fluorescent protein tags for quantifying protein oligomerization in living cells.

Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive analysis of molecular interactions in living cells. The quantification of protein oligomerization in the native cellular environment is highly relevant for a detailed understanding of complex biological processes. An important parameter in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, inducing non-fluorescent states. Here, we show how these processes strongly affect molecular brightness measurements. We perform a systematic characterization of non-fluorescent states for commonly used FPs and provide a simple guideline for accurate, unbiased oligomerization measurements in living cells. Further, we focus on novel red FPs and demonstrate that mCherry2, an mCherry variant, possesses superior properties with regards to precise quantification of oligomerization.