Quantifying membrane binding and diffusion with fluorescence correlation spectroscopy diffusion laws.

Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g., single/multiple particle tracking, fluorescence recovery after photobleaching, and fluorescence correlation spectroscopy (FCS), enable non-invasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non-Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KD, the diffusion coefficient on the membrane D2D, and the diffusion coefficient in the cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells, and we quantified KD and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KD and D2D) for peripheral membrane molecules.

[Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy]. / Apport de la microscopie de fluorescence super-résolue : dans l'intimité des virus !

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy.

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

Mapping spatio-temporal dynamics of single biomolecules in living cells.

We present a Bayesian framework for inferring spatio-temporal maps of diffusivity and potential fields from recorded trajectories of single molecules inside living cells. The framework naturally lets us regularise the high-dimensional inference problem using prior distributions in order to obtain robust results. To overcome the computational complexity of inferring thousands of map parameters from large single particle tracking datasets, we developed a stochastic optimisation method based on local mini-batches and parsimonious gradient calculation. We quantified the gain in convergence speed on numerical simulations, and we demonstrated for the first time temporal regularisation and aligned values of the inferred potential fields across multiple time segments. As a proof-of-concept, we mapped the dynamics of HIV-1 Gag proteins involved in the formation of virus-like particles (VLPs) on the plasma membrane of live T cells at high spatial and temporal resolutions. We focused on transient aggregation events lasting only on tenth of the time required for full VLP formation. The framework and optimisation methods are implemented in the TRamWAy open-source software platform for analysing single biomolecule dynamics.

Coxiella burnetii effector CvpB modulates phosphoinositide metabolism for optimal vacuole development.

The Q fever bacterium Coxiella burnetii replicates inside host cells within a large Coxiella-containing vacuole (CCV) whose biogenesis relies on the Dot/Icm-dependent secretion of bacterial effectors. Several membrane trafficking pathways contribute membranes, proteins, and lipids for CCV biogenesis. These include the endocytic and autophagy pathways, which are characterized by phosphatidylinositol 3-phosphate [PI(3)P]-positive membranes. Here we show that the C. burnetii secreted effector Coxiella vacuolar protein B (CvpB) binds PI(3)P and phosphatidylserine (PS) on CCVs and early endosomal compartments and perturbs the activity of the phosphatidylinositol 5-kinase PIKfyve to manipulate PI(3)P metabolism. CvpB association to early endosome triggers vacuolation and clustering, leading to the channeling of large PI(3)P-positive membranes to CCVs for vacuole expansion. At CCVs, CvpB binding to early endosome- and autophagy-derived PI(3)P and the concomitant inhibition of PIKfyve favor the association of the autophagosomal machinery to CCVs for optimal homotypic fusion of the Coxiella-containing compartments. The importance of manipulating PI(3)P metabolism is highlighted by mutations in cvpB resulting in a multivacuolar phenotype, rescuable by gene complementation, indicative of a defect in CCV biogenesis. Using the insect model Galleria mellonella, we demonstrate the in vivo relevance of defective CCV biogenesis by highlighting an attenuated virulence phenotype associated with cvpB mutations.

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.

UNLABELLED: During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCE: During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.

Influence of Adsorption on Proteins and Amyloid Detection by Silicon Nitride Nanopore.

For the past 2 decades, emerging single-nanopore technologies have opened the route to multiple sensing applications. Besides DNA sensing, the identification of proteins and amyloids is a promising field for early diagnosis. However, the influence of the interactions between the nanopore surface and proteins should be taken into account. In this work, we have selected three proteins (avidin, lysozyme, and IgG) that exhibit different affinities with the SiNx surface, and we have also examined lysozyme amyloid. Our results show that the piranha treatment of SiNx significantly decreases protein adsorption. Moreover, we have successfully detected all proteins (pore diameter 17 nm) and shown the possibility of discriminating between denatured lysozyme and its amyloid. For all proteins, the capture rates are lower than expected, and we evidence that they are correlated with the affinity of proteins to the surface. Our result confirms that proteins interacting only with the nanopore surface wall stay long enough to be detected. For lysozyme amyloid, we show that the use of the nanopore is suitable for determining the number of monomer units even if only the proteins interacting with the nanopore are detected.

Coarse-grained simulations of the HIV-1 matrix protein anchoring: revisiting its assembly on membrane domains.

In the accepted model for human immunodeficiency virus preassembly in infected host cells, the anchoring to the intracellular leaflet of the membrane of the matrix domain (MA) that lies at the N-terminus of the viral Gag protein precursor appears to be one of the crucial steps for particle assembly. In this study, we simulated the membrane anchoring of human immunodeficiency virus-1 myristoylated MA protein using a coarse-grained representation of both the protein and the membrane. Our calculations first suggest that the myristoyl group could spontaneously release from its initial hydrophobic pocket before MA protein interacts with the lipid membrane. All-atom simulations confirmed this possibility with a related energy cost estimated to be ~5 kcal.mol(-1). The phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) head binds preferentially to the MA highly basic region as described in available NMR data, but interestingly without flipping of its 2' acyl chain into the MA protein. Moreover, MA was able to confine PI(4,5)P2 lipids all around its molecular surface after having found a stable orientation at the membrane surface. Our results suggest that this orientation is dependent on Myr anchoring and that this confinement induces a lateral segregation of PI(4,5)P2 in domains. This is consistent with a PI(4,5)P2 enrichment of the virus envelope as compared to the host cell membrane.

Centrosomal targeting of Syk kinase is controlled by its catalytic activity and depends on microtubules and the dynein motor.

The nonreceptor Syk kinase is detected in epithelial cells, where it acts as a tumor suppressor, in addition to its well-established role in immunoreceptor-based signal transduction in hematopoietic cells. Thus, several carcinomas and melanomas have subnormal concentrations of Syk. Although Syk is mainly localized at the plasma membrane, it is also present in centrosomes, where it is involved in the control of cell division. The mechanisms responsible for its centrosomal localization and action are unknown. We used wild-type and mutant fluorescent Syk fusion proteins in live-cell imaging (fluorescence recovery after photobleaching, total internal reflection fluorescence, and photoactivation) combined with mathematical modeling to demonstrate that Syk is actively transported to the centrosomes via the microtubules and that this transport depends on the dynein/dynactin molecular motor. Syk can only target the centrosomes if its kinase activity is intact and it is catalytically active at the centrosomes. We showed that the autophosphorylated Y130 Syk residue helps to uncouple Syk from the plasma membrane and to promote its translocation to the centrosome, suggesting that the subcellular location of Syk depends on its autophosphorylation on specific tyrosine residues. We have thus established the details of how Syk is trafficked intracellularly and found evidence that its targeting to the centrosomes is controlled by autophosphorylation.

Evidence for electro-induced membrane defects assessed by lateral mobility measurement of a GPi anchored protein.

Electrotransfer is a method by which molecules can be introduced into living cells via plasma membrane electropermeabilization. Here, we show that electropermeabilization affects the lateral mobility of Rae-1, a GPi anchored protein. Our results suggest that 10-20 % of the membrane surface is occupied by defects or pores and that these structures propagate rapidly (<1 min) over the cell surface. Electrotransfer of plasmid DNA (pDNA) also affects the lateral mobility of Rae-1. Furthermore, we clearly show that, once inserted into the plasma membrane, pDNA is completely immobile and excludes Rae-1; this indicates that the pDNA molecules are tightly packed together to form aggregates occupying at least the outer leaflet of the plasma membrane.

Visualizing HIV-1 Assembly at the T-Cell Plasma Membrane Using Single-Molecule Localization Microscopy.

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis.

HIV-1 diverts cortical actin for particle assembly and release.

Enveloped viruses assemble and bud from the host cell membranes. Any role of cortical actin in these processes have often been a source of debate. Here, we assessed if cortical actin was involved in HIV-1 assembly in infected CD4 T lymphocytes. Our results show that preventing actin branching not only increases HIV-1 particle release but also the number of individual HIV-1 Gag assembly clusters at the T cell plasma membrane. Indeed, in infected T lymphocytes and in in vitro quantitative model systems, we show that HIV-1 Gag protein prefers areas deficient in F-actin for assembling. Finally, we found that the host factor Arpin, an inhibitor of Arp2/3 branched actin, is recruited at the membrane of infected T cells and it can associate with the viral Gag protein. Altogether, our data show that, for virus assembly and particle release, HIV-1 prefers low density of cortical actin and may favor local actin debranching by subverting Arpin.

Virus Detection and Identification in Minutes Using Single-Particle Imaging and Deep Learning.

The increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive diagnostic methods. Here, we present a methodology for virus detection and identification that uses a convolutional neural network to distinguish between microscopy images of fluorescently labeled intact particles of different viruses. Our assay achieves labeling, imaging, and virus identification in less than 5 min and does not require any lysis, purification, or amplification steps. The trained neural network was able to differentiate SARS-CoV-2 from negative clinical samples, as well as from other common respiratory pathogens such as influenza and seasonal human coronaviruses. We were also able to differentiate closely related strains of influenza, as well as SARS-CoV-2 variants. Additional and novel pathogens can easily be incorporated into the test through software updates, offering the potential to rapidly utilize the technology in future infectious disease outbreaks or pandemics. Single-particle imaging combined with deep learning therefore offers a promising alternative to traditional viral diagnostic and genomic sequencing methods and has the potential for significant impact.

Spatially Addressable Multiplex Biodetection by Calibrated Micro/Nanostructured Surfaces.

A challenge of any biosensing technology is the detection of very low concentrations of analytes. The fluorescence interference contrast (FLIC) technique improves the fluorescence-based sensitivity by selectively amplifying, or suppressing, the emission of a fluorophore-labeled biomolecule immobilized on a transparent layer placed on top of a mirror basal surface. The standing wave of the reflected emission light means that the height of the transparent layer operates as a surface-embedded optical filter for the fluorescence signal. FLIC extreme sensitivity to wavelength is also its main problem: small, e.g., 10 nm range, variations of the vertical position of the fluorophore can translate in unwanted suppression of the detection signal. Herein, we introduce the concept of quasi-circular lenticular microstructured domes operating as continuous-mode optical filters, generating fluorescent concentric rings, with diameters determined by the wavelengths of the fluorescence light, in turn modulated by FLIC. The critical component of the lenticular structures was the shallow sloping side wall, which allowed the simultaneous separation of fluorescent patterns for virtually any fluorophore wavelength. Purposefully designed microstructures with either stepwise or continuous-slope dome geometries were fabricated to modulate the intensity and the lateral position of a fluorescence signal. The simulation of FLIC effects induced by the lenticular microstructures was confirmed by the measurement of the fluorescence profile for three fluorescent dyes, as well as high-resolution fluorescence scanning using stimulated emission depletion (STED) microscopy. The high sensitivity of the spatially addressable FLIC technology was further validated on a diagnostically important target, i.e., the receptor-binding domain (RBD) of the SARS-Cov2 via the detection of RBD:anti-S1-antibody.

Electromediated formation of DNA complexes with cell membranes and its consequences for gene delivery.

Electroporation is a physical method to induce the uptake of therapeutic drugs and DNA, by eukaryotic cells and tissues. The phenomena behind electro-mediated membrane permeabilization to plasmid DNA have been shown to be significantly more complex than those for small molecules. Small molecules cross the permeabilized membrane by diffusion whereas plasmid DNA first interacts with the electropermeabilized part of the cell surface, forming localized aggregates. The dynamics of this process is still poorly understood because direct observations have been limited to scales of the order of seconds. Here, cells are electropermeabilized in the presence of plasmid DNA and monitored with a temporal resolution of 2 ms. This allows us to show that during the first pulse application, plasmid complexes, or aggregates, start to form at distinct sites on the cell membrane. FRAP measurements show that the positions of these sites are remarkably immobile during the application of further pluses. A theoretical model is proposed to explain the appearance of distinct interaction sites, the quantitative increase in DNA and also their immobility leading to a tentative explanation for the success of electro-mediated gene delivery.

Deciphering the Assembly of Enveloped Viruses Using Model Lipid Membranes.

The cell plasma membrane is mainly composed of phospholipids, cholesterol and embedded proteins, presenting a complex interface with the environment. It maintains a barrier to control matter fluxes between the cell cytosol and its outer environment. Enveloped viruses are also surrounded by a lipidic membrane derived from the host-cell membrane and acquired while exiting the host cell during the assembly and budding steps of their viral cycle. Thus, model membranes composed of selected lipid mixtures mimicking plasma membrane properties are the tools of choice and were used to decipher the first step in the assembly of enveloped viruses. Amongst these viruses, we choose to report the three most frequently studied viruses responsible for lethal human diseases, i.e., Human Immunodeficiency Type 1 (HIV-1), Influenza A Virus (IAV) and Ebola Virus (EBOV), which assemble at the host-cell plasma membrane. Here, we review how model membranes such as Langmuir monolayers, bicelles, large and small unilamellar vesicles (LUVs and SUVs), supported lipid bilayers (SLBs), tethered-bilayer lipid membranes (tBLM) and giant unilamellar vesicles (GUVs) contribute to the understanding of viral assembly mechanisms and dynamics using biophysical approaches.

Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles.

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

The control of transcriptional memory by stable mitotic bookmarking.

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is mediated, in part, by transcription factors (TF) mitotic bookmarking. However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that GAGA pioneer factor (GAF) acts as a stable mitotic bookmarker during zygotic genome activation. We show that, during mitosis, GAF remains associated to a large fraction of its interphase targets, including at cis-regulatory sequences of key developmental genes with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we report that GAF binding establishes competence for rapid activation upon mitotic exit.

The FDA-approved drug Auranofin has a dual inhibitory effect on SARS-CoV-2 entry and NF-κB signaling.

Patients with severe COVID-19 show an altered immune response that fails to control the viral spread and suffer from exacerbated inflammatory response, which eventually can lead to death. A major challenge is to develop an effective treatment for COVID-19. NF-κB is a major player in innate immunity and inflammatory process. By a high-throughput screening approach, we identified FDA-approved compounds that inhibit the NF-κB pathway and thus dampen inflammation. Among these, we show that Auranofin prevents post-translational modifications of NF-κB effectors and their recruitment into activating complexes in response to SARS-CoV-2 infection or cytokine stimulation. In addition, we demonstrate that Auranofin counteracts several steps of SARS-CoV-2 infection. First, it inhibits a raft-dependent endocytic pathway involved in SARS-CoV-2 entry into host cells; Second, Auranofin alters the ACE2 mobility at the plasma membrane. Overall, Auranofin should prevent SARS-CoV-2 infection and inflammatory damages, offering new opportunities as a repurposable drug candidate to treat COVID-19.

FCS diffusion laws in two-phase lipid membranes: determination of domain mean size by experiments and Monte Carlo simulations.

Many efforts have been undertaken over the last few decades to characterize the diffusion process in model and cellular lipid membranes. One of the techniques developed for this purpose, fluorescence correlation spectroscopy (FCS), has proved to be a very efficient approach, especially if the analysis is extended to measurements on different spatial scales (referred to as FCS diffusion laws). In this work, we examine the relevance of FCS diffusion laws for probing the behavior of a pure lipid and a lipid mixture at temperatures below, within and above the phase transitions, both experimentally and numerically. The accuracy of the microscopic description of the lipid mixtures found here extends previous work to a more complex model in which the geometry is unknown and the molecular motion is driven only by the thermodynamic parameters of the system itself. For multilamellar vesicles of both pure lipid and lipid mixtures, the FCS diffusion laws recorded at different temperatures exhibit large deviations from pure Brownian motion and reveal the existence of nanodomains. The variation of the mean size of these domains with temperature is in perfect correlation with the enthalpy fluctuation. This study highlights the advantages of using FCS diffusion laws in complex lipid systems to describe their temporal and spatial structure.