Raman Spectroscopy as a Tool to Study the Pathophysiology of Brain Diseases.

The Raman phenomenon is based on the spontaneous inelastic scattering of light, which depends on the molecular characteristics of the dispersant. Therefore, Raman spectroscopy and imaging allow us to obtain direct information, in a label-free manner, from the chemical composition of the sample. Since it is well established that the development of many brain diseases is associated with biochemical alterations of the affected tissue, Raman spectroscopy and imaging have emerged as promising tools for the diagnosis of ailments. A combination of Raman spectroscopy and/or imaging with tagged molecules could also help in drug delivery and tracing for treatment of brain diseases. In this review, we first describe the basics of the Raman phenomenon and spectroscopy. Then, we delve into the Raman spectroscopy and imaging modes and the Raman-compatible tags. Finally, we center on the application of Raman in the study, diagnosis, and treatment of brain diseases, by focusing on traumatic brain injury and ischemia, neurodegenerative disorders, and brain cancer.

Super-Resolution Microscopy to Study Interorganelle Contact Sites.

Interorganelle membrane contact sites (MCS) are areas of close vicinity between the membranes of two organelles that are maintained by protein tethers. Recently, a significant research effort has been made to study MCS, as they are implicated in a wide range of biological functions, such as organelle biogenesis and division, apoptosis, autophagy, and ion and phospholipid homeostasis. Their composition, characteristics, and dynamics can be studied by different techniques, but in recent years super-resolution fluorescence microscopy (SRFM) has emerged as a powerful tool for studying MCS. In this review, we first explore the main characteristics and biological functions of MCS and summarize the different approaches for studying them. Then, we center on SRFM techniques that have been used to study MCS. For each of the approaches, we summarize their working principle, discuss their advantages and limitations, and explore the main discoveries they have uncovered in the field of MCS.

HIV-1 capture and transmission by dendritic cells: the role of viral glycolipids and the cellular receptor Siglec-1.

Dendritic cells (DCs) are essential in order to combat invading viruses and trigger antiviral responses. Paradoxically, in the case of HIV-1, DCs might contribute to viral pathogenesis through trans-infection, a mechanism that promotes viral capture and transmission to target cells, especially after DC maturation. In this review, we highlight recent evidence identifying sialyllactose-containing gangliosides in the viral membrane and the cellular lectin Siglec-1 as critical determinants for HIV-1 capture and storage by mature DCs and for DC-mediated trans-infection of T cells. In contrast, DC-SIGN, long considered to be the main receptor for DC capture of HIV-1, plays a minor role in mature DC-mediated HIV-1 capture and trans-infection.

Siglec-1 is a novel dendritic cell receptor that mediates HIV-1 trans-infection through recognition of viral membrane gangliosides.

Dendritic cells (DCs) are essential antigen-presenting cells for the induction of immunity against pathogens. However, HIV-1 spread is strongly enhanced in clusters of DCs and CD4(+) T cells. Uninfected DCs capture HIV-1 and mediate viral transfer to bystander CD4(+) T cells through a process termed trans-infection. Initial studies identified the C-type lectin DC-SIGN as the HIV-1 binding factor on DCs, which interacts with the viral envelope glycoproteins. Upon DC maturation, however, DC-SIGN is down-regulated, while HIV-1 capture and trans-infection is strongly enhanced via a glycoprotein-independent capture pathway that recognizes sialyllactose-containing membrane gangliosides. Here we show that the sialic acid-binding Ig-like lectin 1 (Siglec-1, CD169), which is highly expressed on mature DCs, specifically binds HIV-1 and vesicles carrying sialyllactose. Furthermore, Siglec-1 is essential for trans-infection by mature DCs. These findings identify Siglec-1 as a key factor for HIV-1 spread via infectious DC/T-cell synapses, highlighting a novel mechanism that mediates HIV-1 dissemination in activated tissues.

Sialyllactose in viral membrane gangliosides is a novel molecular recognition pattern for mature dendritic cell capture of HIV-1.

HIV-1 is internalized into mature dendritic cells (mDCs) via an as yet undefined mechanism with subsequent transfer of stored, infectious virus to CD4+ T lymphocytes. Thus, HIV-1 subverts a DC antigen capture mechanism to promote viral spread. Here, we show that gangliosides in the HIV-1 membrane are the key molecules for mDC uptake. HIV-1 virus-like particles and liposomes mimicking the HIV-1 lipid composition were shown to use a common internalization pathway and the same trafficking route within mDCs. Hence, these results demonstrate that gangliosides can act as viral attachment factors, in addition to their well known function as cellular receptors for certain viruses. Furthermore, the sialyllactose molecule present in specific gangliosides was identified as the determinant moiety for mDC HIV-1 uptake. Thus, sialyllactose represents a novel molecular recognition pattern for mDC capture, and may be crucial both for antigen presentation leading to immunity against pathogens and for succumbing to subversion by HIV-1.

Comparative lipidomics analysis of HIV-1 particles and their producer cell membrane in different cell lines.

Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that obtains its lipid envelope by budding through the plasma membrane of infected host cells. Various studies indicated that the HIV-1 membrane differs from the producer cell plasma membrane suggesting virus budding from pre-existing subdomains or virus-mediated induction of a specialized budding membrane. To perform a comparative lipidomics analysis by quantitative mass spectrometry, we first evaluated two independent methods to isolate the cellular plasma membrane. Subsequent lipid analysis of plasma membranes and HIV-1 purified from two different cell lines revealed a significantly different lipid composition of the viral membrane compared with the host cell plasma membrane, independent of the cell type investigated. Virus particles were significantly enriched in phosphatidylserine, sphingomyelin, hexosylceramide and saturated phosphatidylcholine species when compared with the host cell plasma membrane of the producer cells; they showed reduced levels of unsaturated phosphatidylcholine species, phosphatidylethanolamine and phosphatidylinositol. Cell type-specific differences in the lipid composition of HIV-1 and donor plasmamembranes were observed for plasmalogen-phosphatidylethanolamine and phosphatidylglycerol, which were strongly enriched only in HIV-1 derived from MT-4 cells. MT-4 cell-derived HIV-1 also contained dihydrosphingomyelin as reported previously, but this lipid class was also enriched in the host cell membrane. Taken together, these data strongly support the hypothesis that HIV-1 selects a specific lipid environment for its morphogenesis.

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells.

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DCs) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single-particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment.

Shedding light on membrane rafts structure and dynamics in living cells.

Cellular membranes are fundamental building blocks regulating an extensive repertoire of biological functions. These structures contain lipids and membrane proteins that are known to laterally self-aggregate in the plane of the membrane, forming defined membrane nanoscale domains essential for protein activity. Membrane rafts are described as heterogeneous, dynamic, and short-lived cholesterol- and sphingolipid-enriched membrane nanodomains (10-200 nm) induced by lipid-protein and lipid-lipid interactions. Those membrane nanodomains have been extensively characterized using model membranes and in silico methods. However, despite the development of advanced fluorescence microscopy techniques, undoubted nanoscale visualization by imaging techniques of membrane rafts in the membrane of unperturbed living cells is still uncompleted, increasing the skepticism about their existence. Here, we broadly review recent biochemical and microscopy techniques used to investigate membrane rafts in living cells and we enumerate persistent open questions to answer before unlocking the mystery of membrane rafts in living cells.

Role of Protein-Lipid Interactions in Viral Entry.

The viral entry consists of several sequential events that ensure the attachment of the virus to the host cell and the introduction of its genetic material for the continuation of the replication cycle. Both cellular and viral lipids have gained a wider focus in recent years in the field of viral entry, as they are found to play key roles in different steps of the process. The specific role is summarized that lipids and lipid membrane nanostructures play in viral attachment, fusion, and immune evasion and how they can be targeted with antiviral therapies. Finally, some of the limitations of techniques commonly used for protein-lipid interactions studies are discussed, and new emerging tools are reviewed that can be applied to this field.

Identification of a New Cholesterol-Binding Site within the IFN-γ Receptor that is Required for Signal Transduction.

The cytokine interferon-gamma (IFN-γ) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN-γ exerts it signaling action by binding to a specific cell surface receptor, the IFN-γ receptor (IFN-γR), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN-γR signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN-γR2 chains into plasma membrane lipid nanodomains, orchestrating IFN-γR oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol-binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN-γR transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol-IFNγR2 interaction may represent a potential therapeutic strategy for various IFN-γ-dependent diseases.

A new paradigm in molecular recognition? Specific antibody binding to membrane-inserted HIV-1 epitopes.

The conserved membrane proximal external region (MPER), adjacent to the transmembrane domain (TMD) of human immunodeficiency virus type-1 (HIV-1) gp41 glycoprotein subunit, is accessible to the broadly neutralizing 4E10 and 2F5 monoclonal antibodies (mAbs) and, therefore, constitutes a potential target for vaccine design. This gp41 domain is postulated to be functional during the Env glycoprotein-mediated fusion reaction by destabilizing the highly rigid viral envelope. To perform this task, the aromatic-rich MPER is believed to insert into the interfacial region of the viral membrane external monolayer, thereby inducing the restructuring of the lipid bilayer required for fusion-pore opening. This model predicts that: (i) 2F5 and 4E10 mAbs are capable of binding epitopes inserted into the membrane interface; (ii) in-membrane binding will result in effective blocking of MPER membrane activity; and (iii) both processes, in-membrane recognition and blocking of membrane activity, can be modulated by altering both the lipid composition and the MPER amino acid sequence. We review here recently reported experimental data consistent with those predictions, and further speculate on their relevance for prospective anti-HIV vaccine development.

Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV-1 Env Clustering.

HIV-1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751-854 in the cytoplasmic tail (CT751-854). Super-resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751-854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.

Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells.

Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N3 ) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.

Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission.

Membrane fusion and fission underlie two limiting steps of enveloped virus replication cycle: access to the interior of the host-cell (entry) and dissemination of viral progeny after replication (budding), respectively. These dynamic processes proceed mediated by specialized proteins that disrupt and bend the lipid bilayer organization transiently and locally. We introduced Wimley-White membrane-water partitioning free energies of the amino acids as an algorithm for predicting functional domains that may transmit protein conformational energy into membranes. It was found that many viral products possess unusually extended, aromatic-rich pre-transmembrane stretches predicted to stably reside at the membrane interface. Here, we review structure-function studies, as well as data reported on the interaction of representative peptides with model membranes, all of which sustain a functional role for these domains in viral fusion and fission. Since pre-transmembrane sequences also constitute antigenic determinants in a membrane-bound state, we also describe some recent results on their recognition and blocking at membrane interface by neutralizing antibodies.

Poliovirus 2b insertion into lipid monolayers and pore formation in vesicles modulated by anionic phospholipids.

Enterovirus 2B viroporin has been involved in membrane permeabilization processes occurring late during cell infection. Even though 2B lacks an obvious signal sequence for translocation, the presence of a Lys-based amphipathic domain suggests that this product bears the intrinsic capacity for partitioning into negatively charged cytofacial membrane surfaces. Pore formation by poliovirus 2B attached to a maltose-binding protein (MBP) has been indeed demonstrated in pure lipid vesicles, a fact supporting spontaneous insertion into and direct permeabilization of membranes. Here, biochemical evidence is presented indicating that both processes are modulated by phosphatidylinositol and phosphatidylserine, the main anionic phospholipids existing in membranes of target organelles. Insertion into lipid monolayers and partitioning into phospholipid bilayers were sustained by both phospholipids. However, MBP-2B inserted into phosphatidylserine bilayers did not promote membrane permeabilization and addition of this lipid inhibited the leakage observed in phosphatidylinositol vesicles. Mathematical modelling of pore formation in membranes containing increasing phosphatidylserine percentages was consistent with its inhibitory effect arising from a higher reversibility of MBP-2B surface aggregation. These results support that 2B insertion and pore-opening are mechanistically distinguishable events modulated by the target membrane anionic phospholipids.

The broadly neutralizing anti-human immunodeficiency virus type 1 4E10 monoclonal antibody is better adapted to membrane-bound epitope recognition and blocking than 2F5.

The broadly neutralizing 2F5 and 4E10 monoclonal antibodies (MAbs) recognize epitopes within the membrane-proximal external region (MPER) that connects the human immunodeficiency virus type 1 (HIV-1) envelope gp41 ectodomain with the transmembrane anchor. By adopting different conformations that stably insert into the virion external membrane interface, such as helical structures, a conserved aromatic-rich sequence within the MPER is thought to participate in HIV-1-cell fusion. Recent experimental evidence suggests that the neutralizing activity of 2F5 and 4E10 might correlate with the MAbs' capacity to recognize epitopes inserted into the viral membrane, thereby impairing MPER fusogenic activity. To gain new insights into the molecular mechanism underlying viral neutralization by these antibodies, we have compared the capacities of 2F5 and 4E10 to block the membrane-disorganizing activity of MPER peptides inserted into the surface bilayer of solution-diffusing unilamellar vesicles. Both MAbs inhibited leakage of vesicular aqueous contents (membrane permeabilization) and intervesicular lipid mixing (membrane fusion) promoted by MPER-derived peptides. Thus, our data support the idea that antibody binding to a membrane-inserted epitope may interfere with the function of the MPER during gp41-induced fusion. Antibody insertion into a cholesterol-containing, uncharged virion-like membrane is mediated by specific epitope recognition, and moreover, partitioning-coupled folding into a helix reduces the efficiency of 2F5 MAb binding to its epitope in the membrane. We conclude that the capacity to interfere with the membrane activity of conserved MPER sequences is best correlated with the broad neutralization of the 4E10 MAb.

PI(4,5)P2 degradation promotes the formation of cytoskeleton-free model membrane systems.

Giant plasma membrane vesicles (GPMVs) are cell-derived model membrane systems that undergo large-scale lipid phase separation when cooled below room temperature. Because of their presumably more physiological lipid composition, they are increasingly used as alternatives to synthetic model membranes. However, the exact mechanism of GPMV formation, and thus, effects of this process on the physiological integrity of the membrane are still unclear. Herein, we identify the key steps of GPMV formation and characterize their differences with respect to the plasma membrane of intact cells. Addition of GPMV-inducing reagents triggers a steady Ca2+ influx that is accompanied by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] degradation and phosphatidyl serine (PS) externalization before detachment of the cytoskeleton and the onset of vesicle formation. When comparing GPMVs to other cell-derived model systems, PI(4,5)P2 is not detectable in phase-separating plasma membrane spheres (PMSs) either, but is present in non-phase-separating blebs. GPMVs differ from the physiological state of the plasma membrane in the presence of specific lipids, which limits their use as model systems. Furthermore, we propose that PI(4,5)P2 influences the phase-separation behavior.

Lipid modulation of membrane-bound epitope recognition and blocking by HIV-1 neutralizing antibodies.

The conserved, aromatic-rich membrane-proximal external region (MPER) of gp41 is functional in human immunodeficiency virus (HIV)-cell fusion by perturbing membrane integrity. Broadly-neutralizing 2F5 and 4E10 monoclonal antibodies (MAb-s) recognize amino- and carboxy-terminal epitope sequences within this domain, respectively. An MPER peptide overlapping 2F5 and 4E10 epitope sequences was capable of breaching the permeability barrier of lipid vesicles. Cholesterol and sphingomyelin raft-lipids, present at high quantities in the HIV-1 envelope, promoted exposure or occlusion of 4E10 epitope, respectively. Conversely, 2F5 epitope accessibility was affected to a lesser extent by these envelope lipids. These observations support the idea that MPER epitopes on membranes are segmented in terms of how they are affected by envelope lipids, which may have implications for MPER-based vaccine development.

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure.

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development.

Membrane-transferring sequences of the HIV-1 Gp41 ectodomain assemble into an immunogenic complex.

The membrane-proximal stem region of gp41 has been postulated to host the two conserved membrane-transferring domains that promote HIV-1 fusion: the amino-terminal fusion peptide (FP) and the highly aromatic pre-transmembrane sequence. Our results confirm that the hydrophobic FP and membrane-proximal sequences come into contact and form structurally defined complexes. These complexes are immunogenic and evoke responses in rabbits that compete with the recognition of native functional gp41 by the 2F5 monoclonal antibody. We conclude that co-assembly of the FP and the pre-transmembrane sequences might exert a constraint that helps maintain a gp41 stem region pre-fusion structure.