Quantitative characterization of membrane-protein reversible association using FCS.

Functionally meaningful reversible protein-membrane interactions mediate many biological events. Fluorescence correlation spectroscopy (FCS) is increasingly used to quantitatively study the non-reversible binding of proteins to membranes using lipid vesicles in solution. However, the lack of a complete description of the phase and statistical equilibria in the case of reversible protein-membrane partitioning has hampered the application of FCS to quantify the partition coefficient (Kx). In this work, we further extend the theory that describes membrane-protein partitioning to account for spontaneous protein-membrane dissociation and reassociation to the same or a different lipid vesicle. We derive the probability distribution of proteins on lipid vesicles for reversible binding and demonstrate that FCS is a suitable technique for accurate Kx quantification of membrane-protein reversible association. We also establish the limits to Kx determination by FCS studying the Cramer-Rao bound on the variance of the retrieved parameters. We validate the mathematical formulation against reaction-diffusion simulations to study phase and statistical equilibria and compare the Kx obtained from a computational FCS titration experiment with the experimental ground truth. Finally, we demonstrate the application of our methodology studying the association of anti-HIV broadly neutralizing antibody (10E8-3R) to the membrane.

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane.

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.

Effects of HIV-1 gp41-Derived Virucidal Peptides on Virus-like Lipid Membranes.

Membrane fusion induced by the envelope glycoprotein enables the intracellular replication of HIV-1; hence, this process constitutes a major target for antiretroviral compounds. It has been proposed that peptides having propensity to interact with membrane interfaces might exert broad antiviral activity against enveloped viruses. To test this hypothesis, in this contribution we have analyzed the antiviral effects of peptides derived from the membrane-proximal external region and the transmembrane domain of the envelope glycoprotein subunit gp41, which display different degrees of interfacial hydrophobicity. Our data support the virucidal activity of a region that combines hydrophobic-at-interface membrane-proximal external region aromatics with hydrophobic residues of the transmembrane domain, and contains the absolutely conserved 679LWYIK/R683 sequence, proposed to embody a "cholesterol recognition/interaction amino acid consensus" motif. We further sought to correlate the antiviral activity of these peptides and their effects on membranes that mimic lipid composition and biophysical properties of the viral envelope. The data revealed that peptides endowed with virucidal activity were membrane active and induced permeabilization and fusion of virus-like lipid vesicles. In addition, they modulated lipid packing and miscibility of laterally segregated liquid domains, two properties that depend on the high cholesterol content of the viral membrane. Thus, the overall experimental evidence is consistent with a pattern of HIV inhibition that involves direct alteration of the physical chemistry of the virus membrane. Furthermore, the sequence-dependent effects observed might guide the development of new virucidal peptides.

End-product diacylglycerol enhances the activity of PI-PLC through changes in membrane domain structure.

Diacylglycerol (DAG)-induced activation of phosphatidylinositol-phospholipase C (PI-PLC) was studied with vesicles containing PI, either pure or in mixtures with dimyristoyl phosphatidylcholine, distearoyl phosphatidylcholine, sphingomyelin, or galactosylceramide, used as substrates. At 22°C, DAG at 33 mol % increased PI-PLC activity in all of the mixtures, but not in pure PI bilayers. DAG also caused an overall decrease in diphenylhexatriene fluorescence polarization (decreased molecular order) in all samples, and increased overall enzyme binding. Confocal fluorescence microscopy of giant unilamellar vesicles of all of the compositions under study, with or without DAG, and quantitative evaluation of the phase behavior using Laurdan generalized polarization, and of enzyme binding to the various domains, indicated that DAG activates PI-PLC whenever it can generate fluid domains to which the enzyme can bind with high affinity. In the specific case of PI/dimyristoyl phosphatidylcholine bilayers at 22°C, DAG induced/increased enzyme binding and activation, but no microscopic domain separation was observed. The presence of DAG-generated nanodomains, or of DAG-induced lipid packing defects, is proposed instead for this system. In PI/galactosylceramide mixtures, DAG may exert its activation role through the generation of small vesicles, which PI-PLC is known to degrade at higher rates. In general, our results indicate that global measurements obtained using fluorescent probes in vesicle suspensions in a cuvette are not sufficient to elucidate DAG effects that take place at the domain level. The above data reinforce the idea that DAG functions as an important physical agent in regulating membrane and cell properties.

Cholesterol-dependent membrane fusion induced by the gp41 membrane-proximal external region-transmembrane domain connection suggests a mechanism for broad HIV-1 neutralization.

UNLABELLED: The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical, and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40 to 50 mol%) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations, and atomic force microscopy observations, we propose that the conserved sequence connecting the MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such a mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody but not by a nonneutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies. IMPORTANCE: Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane-proximal external region (MPER)-transmembrane domain (TMD) junction and block infection. These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combined biochemical assays with molecular dynamics simulations and microscopy observations to characterize the unprecedented fusogenic activity of the MPER-TMD junction. The fact that such activity is dependent on cholesterol and inhibited by the broadly neutralizing 4E10 antibody emphasizes its physiological relevance. Discovery of this functional element adds to our understanding of the mechanisms underlying HIV-1 infection and its blocking by antibodies.

Two-photon Laurdan studies of the ternary lipid mixture DOPC:SM:cholesterol reveal a single liquid phase at sphingomyelin:cholesterol ratios lower than 1.

The ternary lipid mixture DOPC:eggSM:cholesterol in excess water has been studied in the form of giant unilamellar vesicles using two-photon fluorescence microscopy. Previous publications based on single-photon fluorescence microscopy had reported heterogeneous phase behavior (phase coexistence) in the region of the triangular phase diagram corresponding to SM:cholesterol molar ratios <1. We have examined this region by two-photon microscopy of Laurdan-labeled mixtures and have found that, under our conditions, only a single liquid phase exists. We have shown that macroscopic phase separation in the above region can be artifactually induced by one-photon excitation of the fluorescent probes and ensuing photooxidation and is prevented using two-photon excitation. The main effect of increasing the concentration of cholesterol in mixtures containing 30 mol % SM was to increase the rigidity of the disordered domains. Increasing the concentration of SM in mixtures containing 20 mol % cholesterol gradually augmented the rigidity of the ordered domains, while the disordered domains reached minimal order at a SM:cholesterol 2.25:1 molar ratio, which then increased again. Moreover, the detailed measurement of Laurdan generalized polarization across the whole phase diagram allowed the representation, for both the single- and two-phase regions, of the gradual variation of membrane lateral packing along the diagram, which we found to be governed largely by SM:cholesterol interactions.

Acute regulation of PDK1 by a complex interplay of molecular switches.

Phosphoinositide-dependent kinase 1 (PDK1) is the master regulator of at least 23 other AGC kinases whose downstream signalling has often been implicated in various diseases and in particular in cancer. Therefore there has been great interest in determining how PDK1 is controlled and how it regulates its substrates spatially and temporally. The understanding of these mechanisms could offer new possibilities for therapeutic intervention. Over the years, a more comprehensive view of the mechanisms involved in the regulation of PDK1 has emerged and these comprise serine/threonine as well as tyrosine phosphorylation, subcellular localization, regulator binding and conformation status. In the present review, we discuss how various molecular mechanisms are together responsible for the conformational regulation behind the activation of PDK1 in cells.

Multiparametric modulation of magnetic transduction for biomolecular sensing in liquids.

The recent COVID19 pandemic has remarkably boosted the research on in vitro diagnosis assays to detect biomarkers in biological fluids. Specificity and sensitivity are mandatory for diagnostic kits aiming to reach clinical stages. Whilst the modulation of sensitivity can significantly improve the detection of biomarkers in liquids, this has been scarcely explored. Here, we report on the proof of concept and parametrization of a novel biosensing methodology based on the changes of AC magnetic hysteresis areas observed for magnetic nanoparticles following biomolecular recognition in liquids. Several parameters are shown to significantly modulate the transducing capacity of magnetic nanoparticles to detect analytes dispersed in saline buffer at concentrations of clinical relevance. Magnetic nanoparticles were bio-conjugated with an engineered recognition peptide as a receptor. Analytes are engineered tetratricopeptide binding domains fused to the fluorescent protein whose dimerization state allows mono- or divalent variants. Our results unveil that the number of receptors per particle, analyte valency and concentration, nanoparticle composition and concentration, and field conditions play a key role in the formation of assemblies driven by biomolecular recognition. Consequently, all these parameters modulate the nanoparticle transduction capacity. Our study provides essential insights into the potential of AC magnetometry for customizing biomarker detection in liquids.

Development of pyrene-based fluorescent ether lipid as inhibitor of SK3 ion channels.

We report the synthesis of three bioactive pyrene-based fluorescent analogues of Ohmline which is the most efficient and selective inhibitor of SK3 ion channel. The interaction of these Ohmline-pyrene (OP1-3) with liposomes of different composition reveals that only OP2 and OP3 are readily integrated into liposomes. Fluorescence measurements indicate that, depending on their concentration, OP2 and OP3 exist either as monomer or as a mixture of monomer and excimers within the liposome bilayer. Among the three Ohmline Pyrene compounds (OP1-3) only OP2 is able to reduce SK3 currents and is the first efficient fluorescent modulator of SK3 channel as revealed by patch clamp measurements (- 71.3 ± 13.3% at 10 µM) and by its inhibition of SK3-dependent cancer cell migration at (-32.5% ± 4.8% at 1 µM). We also report the first fluorescence study on living breast cancer cells (MDA-MB-231) showing that OP2 is rapidly integrated in bio-membranes followed by cell internalization.

Cholesterol displaces palmitoylceramide from its tight packing with palmitoylsphingomyelin in the absence of a liquid-disordered phase.

A set of different biophysical approaches has been used to explore the phase behavior of palmitoylsphingomyelin (pSM)/cholesterol (Chol) model membranes in the presence and absence of palmitoylceramide (pCer). Fluorescence spectroscopy of di-4-ANEPPDHQ-stained pSM/Chol vesicles and atomic force microscopy of supported planar bilayers show gel L(beta)/liquid-ordered (L(o)) phase coexistence within the range X(Chol) = 0-0.25 at 22 degrees C. At the latter compositional point and beyond, a single L(o) pSM/Chol phase is detected. In ternary pSM/Chol/pCer mixtures, differential scanning calorimetry of multilamellar vesicles and confocal fluorescence microscopy of giant unilamellar vesicles concur in showing immiscibility, but no displacement, between L(o) cholesterol-enriched (pSM/Chol) and gel-like ceramide-enriched (pSM/pCer) phases at high pSM/(Chol + pCer) ratios. At higher cholesterol content, pCer is unable to displace cholesterol at any extent, even at X(Chol) < 0.25. It is interesting that an opposite strong cholesterol-mediated pCer displacement from its tight packing with pSM is clearly detected, completely abolishing the pCer ability to generate large microdomains and giving rise instead to a single ternary phase. These observations in model membranes in the absence of the lipids commonly used to form a liquid-disordered phase support the role of cholesterol as the key determinant in controlling its own displacement from L(o) domains by ceramide upon sphingomyelinase activity.

Fluorescence lifetime imaging reveals that the environment of the ATP binding site of myosin in muscle senses force.

Fluorescence lifetime imaging microscopy is used to demonstrate that different loads applied to a muscle fiber change the microenvironment of the nucleotide binding pocket of myosin. Permeabilized skeletal muscle fibers in rigor were labeled with a fluorescent ATP analog, 3'-DEAC-propylenediamine (pda)-ATP (3'-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP), which was hydrolyzed to the diphosphate. Cycles of small-amplitude stretches and releases (<1% of muscle segment length) were synchronized with fluorescence lifetime imaging and force measurements to correlate the effect of force on the lifetime of the ATP analog bound to the actomyosin complex. Analysis of the fluorescence decay resolved two lifetimes, corresponding to the free nucleotide DEAC-pda-ATP (τ(1) = 0.47 ± 0.03 ns; mean ± SD) and nucleotide bound to the actomyosin complex (τ(2) = 2.21 ± 0.06 ns at low strain). Whereas τ(1) did not change with force, τ(2) showed a linear dependence with the force applied to the muscle of 0.43 ± 0.05 ps/kPa. Hence, the molecular environment of the nucleotide binding pocket of myosin is directly affected by a change of length applied at the ends of the fiber segments. These changes may help explain how force modulates the actomyosin ATPase cycle and thus the physiology and energetics of contraction.

A Complex Interplay of Anionic Phospholipid Binding Regulates 3'-Phosphoinositide-Dependent-Kinase-1 Homodimer Activation.

3'-Phosphoinositide-dependent-Kinase-1 (PDK1) is a master regulator whereby its PI3-kinase-dependent dysregulation in human pathologies is well documented. Understanding the direct role for PtdIns(3,4,5)P3 and other anionic phospholipids in the regulation of PDK1 conformational dynamics and its downstream activation remains incomplete. Using advanced quantitative-time-resolved imaging (Fluorescence Lifetime Imaging and Fluorescence Correlation Spectroscopy) and molecular modelling, we show an interplay of antagonistic binding effects of PtdIns(3,4,5)P3 and other anionic phospholipids, regulating activated PDK1 homodimers. We demonstrate that phosphatidylserine maintains PDK1 in an inactive conformation. The dysregulation of the PI3K pathway affects the spatio-temporal and conformational dynamics of PDK1 and the activation of its downstream substrates. We have established a new anionic-phospholipid-dependent model for PDK1 regulation, depicting the conformational dynamics of multiple homodimer states. We show that the dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 homodimer conformations. Our findings provide a role for the PtdSer binding site and its previously unrewarding role in PDK1 downregulation, suggesting a possible therapeutic strategy where the constitutively active dimer conformer of PDK1 may be rendered inactive by small molecules that drive it to its PtdSer-bound conformer.

Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET.

Quantification of the intracellular equilibrium dissociation constant of the interaction, Kd , is challenging due to the variability of the relative concentrations of the interacting proteins in the cell. Fluorescence lifetime imaging microscopy (FLIM) of the donor provides an accurate measurement of the molecular fraction of donor involved in FRET, but the fraction of bound acceptor is also needed to reliably estimate Kd . We present a method that exploits the spectroscopic properties of the widely used eGFP - mCherry FRET pair to rigorously determine the intracellular Kd based on imaging the fluorescence lifetime of only the donor (single-channel FLIM). We have assessed the effect of incomplete labelling and determined its range of application for different Kd using Monte Carlo simulations. We have demonstrated this method estimating the intracellular Kd for the homodimerisaton of the oncogenic protein 3-phosphoinositide-dependent kinase 1 (PDK1) in different cell lines and conditions, revealing a competitive mechanism for its regulation. The measured intracellular Kd was validated against in-vitro data. This method provides an accurate and generic tool to quantify protein interactions in situ.

Rapid hyperspectral fluorescence lifetime imaging.

We report a rapid hyperspectral fluorescence lifetime imaging (FLIM) instrument that exploits high-speed FLIM technology in a line-scanning microscope. We demonstrate the acquisition of whole-field optically sectioned hyperspectral fluorescence lifetime image stacks (with 32 spectral bins) in less than 40 s and illustrate its application to unstained biological tissue.

Functional organization of the HIV lipid envelope.

The chemical composition of the human immunodeficiency virus type 1 (HIV-1) membrane is critical for fusion and entry into target cells, suggesting that preservation of a functional lipid bilayer organization may be required for efficient infection. HIV-1 acquires its envelope from the host cell plasma membrane at sites enriched in raft-type lipids. Furthermore, infectious particles display aminophospholipids on their surface, indicative of dissipation of the inter-leaflet lipid asymmetry metabolically generated at cellular membranes. By combining two-photon excited Laurdan fluorescence imaging and atomic force microscopy, we have obtained unprecedented insights into the phase state of membranes reconstituted from viral lipids (i.e., extracted from infectious HIV-1 particles), established the role played by the different specimens in the mixtures, and characterized the effects of membrane-active virucidal agents on membrane organization. In determining the molecular basis underlying lipid packing and lateral heterogeneity of the HIV-1 membrane, our results may help develop compounds with antiviral activity acting by perturbing the functional organization of the lipid envelope.

Time-resolved fluorescence imaging of solvent interactions in microfluidic devices.

We present the application of wide-field time-resolved fluorescence imaging methods for the study of solvent interactions and mixing in microfluidic devices. Time-resolved imaging of fluorescence polarization anisotropy allows us to image the local viscosity of fluorescence in three dimensions in order to directly monitor solvent mixing within a microfluidic channel. This provides a viscosity image acquisition time of the order of minutes, and has been applied to a steady-state laminar flow configuration. To image dynamic fluid mixing in real-time, we demonstrate high-speed fluorescence lifetime imaging at 12.3 Hz applied to DASPI, which directly exhibits a solvent viscosity-dependant fluorescence lifetime. These two methods facilitate a high degree of quantification of microfluidic flow in 3-D and/or at high speed, providing a tool for studying fluid dynamics and for developing enhanced microfluidic assays.

Fluorescence nanoscopy. Methods and applications.

Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffraction-limited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

Fluorescent labeling of Acanthamoeba assessed in situ from corneal sectioned microscopy.

Acanthamoeba keratitis is a serious pathogenic corneal disease, with challenging diagnosis. Standard diagnostic methods include corneal biopsy (involving cell culture) and in vivo reflection corneal microscopy (in which the visualization of the pathogen is challenged by the presence of multiple reflectance corneal structures). We present a new imaging method based on fluorescence sectioned microscopy for visualization of Acanthamoeba. A fluorescent marker (MT-11-BDP), composed by a fluorescent group (BODIPY) inserted in miltefosine (a therapeutic agent against Acanthamoeba), was developed. A custom-developed fluorescent structured illumination sectioned corneal microscope (excitation wavelength: 488 nm; axial/lateral resolution: 2.6 µm/0.4-0.6 µm) was used to image intact enucleated rabbit eyes, injected with a solution of stained Acanthamoeba in the stroma. Fluorescent sectioned microscopic images of intact enucleated rabbit eyes revealed stained Acanthamoeba trophozoites within the stroma, easily identified by the contrasted fluorescent emission, size and shape. Control experiments show that the fluorescent maker is not internalized by corneal cells, making the developed marker specific to the pathogen. Fluorescent sectioned microscopy shows potential for specific diagnosis of Acanthamoeba keratitis. Corneal confocal microscopy, provided with a fluorescent channel, could be largely improved in specificity and sensitivity in combination with specific fluorescent marking.

Wide-field fluorescence lifetime imaging of cancer.

Optical imaging of tissue autofluorescence has the potential to provide rapid label-free screening and detection of surface tumors for clinical applications, including when combined with endoscopy. Quantitative imaging of intensity-based contrast is notoriously difficult and spectrally resolved imaging does not always provide sufficient contrast. We demonstrate that fluorescence lifetime imaging (FLIM) applied to intrinsic tissue autofluorescence can directly contrast a range of surface tissue tumors, including in gastrointestinal tissues, using compact, clinically deployable instrumentation achieving wide-field fluorescence lifetime images of unprecedented clarity. Statistically significant contrast is observed between cancerous and healthy colon tissue for FLIM with excitation at 355 nm. To illustrate the clinical potential, wide-field fluorescence lifetime images of unstained ex vivo tissue have been acquired at near video rate, which is an important step towards real-time FLIM for diagnostic and interoperative imaging, including for screening and image-guided biopsy applications.

Fluorescence lifetime imaging to detect actomyosin states in mammalian muscle sarcomeres.

We investigated the use of fluorescence lifetime imaging microscopy (FLIM) of a fluorescently labeled ATP analog (3'-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP) to probe in permeabilized muscle fibers the changes in the environment of the nucleotide binding pocket caused by interaction with actin. Spatial averaging of FLIM data of muscle sarcomeres reduces photon noise, permitting detailed analysis of the fluorescence decay profiles. FLIM reveals that the lifetime of the nucleotide, in its ADP form because of the low concentration of nucleotide present, changes depending on whether the nucleotide is free in solution or bound to myosin, and on whether the myosin is bound to actin in an actomyosin complex. Characterization of the fluorescence decays by a multiexponential function allowed us to resolve the lifetimes and amplitudes of each of these populations, namely, the fluorophore bound to myosin, bound to actin, in an actomyosin complex, and free in the filament lattice. This novel application of FLIM to muscle fibers shows that with spatial averaging, detailed information about the nature of nucleotide complexes can be derived.