Membrane nanoclusters-tails of the unexpected.

The existence, nature, and role of highly ordered membrane domains, often referred to as lipid rafts, have been highly debated by cell biologists for many years. In this issue, Raghupathy et al. describe molecular mechanisms leading to the formation of ordered lipid-protein clusters.

The mystery of membrane organization: composition, regulation and roles of lipid rafts.

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors.

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the ß1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

Estrogen receptor activation remodels TEAD1 gene expression to alleviate hepatic steatosis.

Sex-based differences in obesity-related hepatic malignancies suggest the protective roles of estrogen. Using a preclinical model, we dissected estrogen receptor (ER) isoform-driven molecular responses in high-fat diet (HFD)-induced liver diseases of male and female mice treated with or without an estrogen agonist by integrating liver multi-omics data. We found that selective ER activation recovers HFD-induced molecular and physiological liver phenotypes. HFD and systemic ER activation altered core liver pathways, beyond lipid metabolism, that are consistent between mice and primates. By including patient cohort data, we uncovered that ER-regulated enhancers govern central regulatory and metabolic genes with clinical significance in metabolic dysfunction-associated steatotic liver disease (MASLD) patients, including the transcription factor TEAD1. TEAD1 expression increased in MASLD patients, and its downregulation by short interfering RNA reduced intracellular lipid content. Subsequent TEAD small molecule inhibition improved steatosis in primary human hepatocyte spheroids by suppressing lipogenic pathways. Thus, TEAD1 emerged as a new therapeutic candidate whose inhibition ameliorates hepatic steatosis.

Discovery of a novel inhibitor of macropinocytosis with antiviral activity.

Several viruses hijack various forms of endocytosis in order to infect host cells. Here, we report the discovery of a molecule with antiviral properties that we named virapinib, which limits viral entry by macropinocytosis. The identification of virapinib derives from a chemical screen using high-throughput microscopy, where we identified chemical entities capable of preventing infection with a pseudotype virus expressing the spike (S) protein from SARS-CoV-2. Subsequent experiments confirmed the capacity of virapinib to inhibit infection by SARS-CoV-2, as well as by additional viruses, such as mpox virus and TBEV. Mechanistic analyses revealed that the compound inhibited macropinocytosis, limiting this entry route for the viruses. Importantly, virapinib has no significant toxicity to host cells. In summary, we present the discovery of a molecule that inhibits macropinocytosis, thereby limiting the infectivity of viruses that use this entry route such as SARS-CoV2.

Quantification of membrane fluidity in bacteria using TIR-FCS.

Plasma membrane fluidity is an important phenotypic feature that regulates the diffusion, function, and folding of transmembrane and membrane-associated proteins. In bacterial cells, variations in membrane fluidity are known to affect respiration, transport, and antibiotic resistance. Membrane fluidity must therefore be tightly regulated to adapt to environmental variations and stresses such as temperature fluctuations or osmotic shocks. Quantitative investigation of bacterial membrane fluidity has been, however, limited due to the lack of available tools, primarily due to the small size and membrane curvature of bacteria that preclude most conventional analysis methods used in eukaryotes. Here, we develop an assay based on total internal reflection-fluorescence correlation spectroscopy (TIR-FCS) to directly measure membrane fluidity in live bacteria via the diffusivity of fluorescent membrane markers. With simulations validated by experiments, we could determine how the small size, high curvature, and geometry of bacteria affect diffusion measurements and correct subsequent measurements for unbiased diffusion coefficient estimation. We used this assay to quantify the fluidity of the cytoplasmic membranes of the Gram-positive bacteria Bacillus subtilis (rod-shaped) and Staphylococcus aureus (coccus) at high (37°C) and low (20°C) temperatures in a steady state and in response to a cold shock, caused by a shift from high to low temperature. The steady-state fluidity was lower at 20°C than at 37°C, yet differed between B. subtilis and S. aureus at 37°C. Upon cold shock, the membrane fluidity decreased further below the steady-state fluidity at 20°C and recovered within 30 min in both bacterial species. Our minimally invasive assay opens up exciting perspectives for the study of a wide range of phenomena affecting the bacterial membrane, from disruption by chemicals or antibiotics to viral infection or change in nutrient availability.

A Multiparametric and High-Throughput Platform for Host-Virus Binding Screens.

Speed is key during infectious disease outbreaks. It is essential, for example, to identify critical host binding factors to pathogens as fast as possible. The complexity of host plasma membrane is often a limiting factor hindering fast and accurate determination of host binding factors as well as high-throughput screening for neutralizing antimicrobial drug targets. Here, we describe a multiparametric and high-throughput platform tackling this bottleneck and enabling fast screens for host binding factors as well as new antiviral drug targets. The sensitivity and robustness of our platform were validated by blocking SARS-CoV-2 particles with nanobodies and IgGs from human serum samples.

Development of an Effective Functional Lipid Anchor for Membranes (FLAME) for the Bioorthogonal Modification of the Lipid Bilayer of Mesenchymal Stromal Cells.

The glycosylation of cellular membranes is crucial for the survival and communication of cells. As our target is the engineering of the glycocalyx, we designed a functionalized lipid anchor for the introduction into cellular membranes called Functional Lipid Anchor for MEmbranes (FLAME). Since cholesterol incorporates very effectively into membranes, we developed a twice cholesterol-substituted anchor in a total synthesis by applying protecting group chemistry. We labeled the compound with a fluorescent dye, which allows cell visualization. FLAME was successfully incorporated in the membranes of living human mesenchymal stromal cells (hMSC), acting as a temporary, nontoxic marker. The availability of an azido function─a bioorthogonal reacting group within the compound─enables the convenient coupling of alkyne-functionalized molecules, such as fluorophores or saccharides. After the incorporation of FLAME into the plasma membrane of living hMSC, we were able to successfully couple our molecule with an alkyne-tagged fluorophore via click reaction. This suggests that FLAME is useful for the modification of the membrane surface. Coupling FLAME with a galactosamine derivative yielded FLAME-GalNAc, which was incorporated into U2OS cells as well as in giant unilamellar vesicles (GUVs) and cell-derived giant plasma membrane vesicles (GPMVs). With this, we have shown that FLAME-GalNAc is a useful tool for studying the partitioning in the liquid-ordered (Lo) and the liquid-disordered (Ld) phases. The molecular tool can also be used to analyze the diffusion behavior in the model and the cell membranes by fluorescence correlation spectroscopy (FCS).

Influence of the extracellular domain size on the dynamic behavior of membrane proteins.

The dynamic behavior of plasma membrane proteins mediates various cellular processes such as cellular motility, communication, and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interactions of the intracellular domain with cytosolic components such as cortical actin. Although initiation of different cellular signaling events at the plasma membrane has been attributed to the extracellular domain (ECD) properties recently, the impact of ECDs on the dynamic behavior of membrane proteins is rather unexplored. Here, we investigate how ECD properties influence protein dynamics in the lipid bilayer by reconstituting ECDs of different sizes or glycosylation in model membrane systems and analyzing ECD-driven protein sorting in lipid domains as well as protein mobility. Our data show that increasing the ECD mass or glycosylation leads to a decrease in ordered domain partitioning and diffusivity. Our data reconcile different mechanisms proposed for the initiation of cellular signaling by linking the ECD size of membrane proteins with their localization and diffusion dynamics in the plasma membrane.

Influenza A viruses use multivalent sialic acid clusters for cell binding and receptor activation.

Influenza A virus (IAV) binds its host cell using the major viral surface protein hemagglutinin (HA). HA recognizes sialic acid, a plasma membrane glycan that functions as the specific primary attachment factor (AF). Since sialic acid alone cannot fulfill a signaling function, the virus needs to activate downstream factors to trigger endocytic uptake. Recently, the epidermal growth factor receptor (EGFR), a member of the receptor-tyrosine kinase family, was shown to be activated by IAV and transmit cell entry signals. However, how IAV's binding to sialic acid leads to engagement and activation of EGFR remains largely unclear. We used multicolor super-resolution microscopy to study the lateral organization of both IAV's AFs and its functional receptor EGFR at the scale of the IAV particle. Intriguingly, quantitative cluster analysis revealed that AFs and EGFR are organized in partially overlapping submicrometer clusters in the plasma membrane of A549 cells. Within AF domains, the local AF concentration reaches on average 10-fold the background concentration and tends to increase towards the cluster center, thereby representing a multivalent virus-binding platform. Using our experimentally measured cluster characteristics, we simulated virus diffusion on a flat membrane. The results predict that the local AF concentration strongly influences the distinct mobility pattern of IAVs, in a manner consistent with live-cell single-virus tracking data. In contrast to AFs, EGFR resides in smaller clusters. Virus binding activates EGFR, but interestingly, this process occurs without a major lateral EGFR redistribution, indicating the activation of pre-formed clusters, which we show are long-lived. Taken together, our results provide a quantitative understanding of the initial steps of influenza virus infection. Co-clustering of AF and EGFR permit a cooperative effect of binding and signaling at specific platforms, thus linking their spatial organization to their functional role during virus-cell binding and receptor activation.

Understanding immune signaling using advanced imaging techniques.

Advanced imaging is key for visualizing the spatiotemporal regulation of immune signaling which is a complex process involving multiple players tightly regulated in space and time. Imaging techniques vary in their spatial resolution, spanning from nanometers to micrometers, and in their temporal resolution, ranging from microseconds to hours. In this review, we summarize state-of-the-art imaging methodologies and provide recent examples on how they helped to unravel the mysteries of immune signaling. Finally, we discuss the limitations of current technologies and share our insights on how to overcome these limitations to visualize immune signaling with unprecedented fidelity.

z-STED Imaging and Spectroscopy to Investigate Nanoscale Membrane Structure and Dynamics.

Super-resolution stimulated emission depletion (STED) microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy) or axially (z). Two-dimensional STED has been extensively used to elucidate the nanoscale membrane structure and dynamics via imaging or combined with spectroscopy techniques such as fluorescence correlation spectroscopy (FCS) and spectral imaging. On the contrary, z-STED has not been used in this context. Here, we show that a combination of z-STED with FCS or spectral imaging enables us to see previously unobservable aspects of cellular membranes. We show that thanks to an axial resolution of ∼100 nm, z-STED can be used to distinguish axially close-by membranes, early endocytic vesicles, or tubular membrane structures. Combination of z-STED with FCS and spectral imaging showed diffusion dynamics and lipid organization in these structures, respectively.

Lipoprotein Particles Interact with Membranes and Transfer Their Cargo without Receptors.

Lipid transfer from lipoprotein particles to cells is essential for lipid homeostasis. High-density lipoprotein (HDL) particles are mainly captured by cell membrane-associated scavenger receptor class B type 1 (SR-B1) from the bloodstream, while low-density and very-low-density lipoprotein (LDL and VLDL, respectively) particles are mostly taken up by receptor-mediated endocytosis. However, the role of the target lipid membrane itself in the transfer process has been largely neglected so far. Here, we study how lipoprotein particles (HDL, LDL, and VLDL) interact with synthetic lipid bilayers and cell-derived membranes and transfer their cargo subsequently. Employing cryo-electron microscopy, spectral imaging, and fluorescence (cross) correlation spectroscopy allowed us to observe integration of all major types of lipoprotein particles into the membrane and delivery of their cargo in a receptor-independent manner. Importantly, the biophysical properties of the target cell membranes change upon delivery of cargo. The concept of receptor-independent interaction of lipoprotein particles with membranes helps us to better understand lipoprotein particle biology and can be exploited for novel treatments of dyslipidemia diseases.

Nanoscale dynamics of cholesterol in the cell membrane.

Cholesterol constitutes ∼30-40% of the mammalian plasma membrane, a larger fraction than of any other single component. It is a major player in numerous signaling processes as well as in shaping molecular membrane architecture. However, our knowledge of the dynamics of cholesterol in the plasma membrane is limited, restricting our understanding of the mechanisms regulating its involvement in cell signaling. Here, we applied advanced fluorescence imaging and spectroscopy approaches on in vitro (model membranes) and in vivo (live cells and embryos) membranes as well as in silico analysis to systematically study the nanoscale dynamics of cholesterol in biological membranes. Our results indicate that cholesterol diffuses faster than phospholipids in live membranes, but not in model membranes. Interestingly, a detailed statistical diffusion analysis suggested two-component diffusion for cholesterol in the plasma membrane of live cells. One of these components was similar to a freely diffusing phospholipid analogue, whereas the other one was significantly faster. When a cholesterol analogue was localized to the outer leaflet only, the fast diffusion of cholesterol disappeared, and it diffused similarly to phospholipids. Overall, our results suggest that cholesterol diffusion in the cell membrane is heterogeneous and that this diffusional heterogeneity is due to cholesterol's nanoscale interactions and localization in the membrane.

Reconstitution of immune cell interactions in free-standing membranes.

The spatiotemporal regulation of signalling proteins at the contacts formed between immune cells and their targets determines how and when immune responses begin and end. Therapeutic control of immune responses therefore relies on thorough elucidation of the molecular processes occurring at these interfaces. However, the detailed investigation of each component's contribution to the formation and regulation of the contact is hampered by the complexities of cell composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially in the use of advanced imaging technology. One approach that circumvents these problems is to establish in vitro systems that faithfully mimic immune cell interactions, but allow complexity to be 'dialled-in' as needed. Here, we present an in vitro system that makes use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we began to explore the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and how this changes during the initiation of signalling. The GUV/cell system presented here is expected to be widely applicable.

Redesigning Solvatochromic Probe Laurdan for Imaging Lipid Order Selectively in Cell Plasma Membranes.

Imaging of biological membranes by environmentally sensitive solvatochromic probes, such as Laurdan, provides information about the organization of lipids, their ordering, and their uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labeling of internal membranes, we redesigned it by introducing a membrane anchor group based on negatively charged sulfonate and dodecyl chain. The obtained probe, Pro12A, stains exclusively the outer leaflet of lipid bilayers of liposomes, as evidenced by leaflet-specific fluorescence quenching with a viologen derivative, and shows higher fluorescence brightness than Laurdan. Pro12A also exhibits stronger spectral change between liquid-ordered and liquid-disordered phases in model membranes and distinguishes better lipid domains in giant plasma membrane vesicles (GPMVs) than Laurdan. In live cells, it stains exclusively the cell plasma membranes, in contrast to Laurdan and its carboxylate analogue C-Laurdan. Owing to its outer leaflet binding, Pro12A is much more sensitive to cholesterol extraction than Laurdan, which is redistributed within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures the absence of crosstalk with a number of orange/red fluorescent proteins and dyes. Thus, Pro12A will enable accurate multicolor imaging of lipid organization of cell plasma membranes in the presence of fluorescently tagged proteins of interest, which will open new opportunities in biomembrane research.

High photon count rates improve the quality of super-resolution fluorescence fluctuation spectroscopy.

Probing the diffusion of molecules has become a routine measurement across the life sciences, chemistry and physics. It provides valuable insights into reaction dynamics, oligomerisation, molecular (re-)organisation or cellular heterogeneities. Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. This technique relies on the temporal autocorrelation of intensity fluctuations but recording these fluctuations has thus far been limited by the detection electronics, which could not efficiently and accurately time-tag photons at high count rates. This has until now restricted the range of measurable dye concentrations, as well as the data quality of the FCS recordings, especially in combination with super-resolution stimulated emission depletion (STED) nanoscopy. Here, we investigate the applicability and reliability of (STED-)FCS at high photon count rates (average intensities of more than 1 MHz) using novel detection equipment, namely hybrid detectors and real-time gigahertz sampling of the photon streams implemented on a commercial microscope. By measuring the diffusion of fluorophores in solution and cytoplasm of live cells, as well as in model and cellular membranes, we show that accurate diffusion and concentration measurements are possible in these previously inaccessible high photon count regimes. Specifically, it offers much greater flexibility of experiments with biological samples with highly variable intensity, e.g. due to a wide range of expression levels of fluorescent proteins. In this context, we highlight the independence of diffusion properties of cytosolic GFP in a concentration range of approx. 0.01-1 µm. We further show that higher photon count rates also allow for much shorter acquisition times, and improved data quality. Finally, this approach also pronouncedly increases the robustness of challenging live cell STED-FCS measurements of nanoscale diffusion dynamics, which we testify by confirming a free diffusion pattern for a fluorescent lipid analogue on the apical membrane of adherent cells.

Receptor-Independent Transfer of Low Density Lipoprotein Cargo to Biomembranes.

The fundamental task of lipoprotein particles is extracellular transport of cholesterol, lipids, and fatty acids. Besides, cholesterol-rich apoB-containing lipoprotein particles (i.e., low density lipoprotein LDL) are key players in progression of atherosclerotic cardiovascular disease and are associated with familial hypercholesterolemia (FH). So far, lipoprotein particle binding to the cell membrane and subsequent cargo transfer is directly linked to the lipoprotein receptors on the target cell surface. However, our observations showed that lipoprotein particle cargo transport takes place even in the absence of the receptor. This finding suggests that an alternative mechanism for lipoprotein-particle/membrane interaction, besides the receptor-mediated one, exists. Here, we combined several complementary biophysical techniques to obtain a comprehensive view on the nonreceptor mediated LDL-particle/membrane. We applied a combination of atomic force and single-molecule-sensitive fluorescence microscopy (AFM and SMFM) to investigate the LDL particle interaction with membranes of increasing complexity. We observed direct transfer of fluorescently labeled amphiphilic lipid molecules from LDL particles into the pure lipid bilayer. We further confirmed cargo transfer by fluorescence cross-correlation spectroscopy (FCCS) and spectral imaging of environment-sensitive probes. Moreover, the integration of the LDL particle into the membranes was directly visualized by high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-EM). Overall, our data show that lipoprotein particles are able to incorporate into lipid membranes upon contact to transfer their cargo in the absence of specific receptors.