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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 the 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 shows that increasing the ECD mass or glycosylation leads to a decrease in ordered domain partitioning and diffusivity. Our data reconciles 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.SIGNIFICANCE STATEMENTWe studied how the size and glycosylation of the proteins influences their dynamic behavior in a lipid bilayer by reconstituting the ECDs of different sizes or glycosylation in model membrane systems and analyzing their sorting into lipid domains as well as their mobility. We observe that increasing the ECD apparent mass leads to a decrease in membrane ordered domain partitioning and diffusivity. Our data reconciles multiple mechanisms proposed for the initiation of cellular signaling by linking the ECD properties of membrane proteins with their localization and diffusion dynamics in the plasma membrane.

T cells rely for their development and function on the correct folding and turnover of proteins generated in response to a broad range of molecular cues. In the absence of the eukaryotic type II chaperonin complex, CCT, T cell activation induced changes in the proteome are compromised including the formation of nuclear actin filaments and the formation of a normal cell stress response. Consequently, thymocyte maturation and selection, and T cell homeostatic maintenance and receptor-mediated activation are severely impaired. In the absence of CCT-controlled protein folding, Th2 polarization diverges from normal differentiation with paradoxical continued IFN-γ expression. As a result, CCT-deficient T cells fail to generate an efficient immune protection against helminths as they are unable to sustain a coordinated recruitment of the innate and adaptive immune systems. These findings thus demonstrate that normal T cell biology is critically dependent on CCT-controlled proteostasis and that its absence is incompatible with protective immunity., Communications Biology, 4 (1), ISSN:2399-3642

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four.Graphical abstractHighlightsBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles.BSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV.tSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs.

Multiple myeloma (MM) is a fatal hematological malignancy, where the majority of patients are diagnosed with, or develop, destructive and debilitating osteolytic bone lesions. Current treatments for MM bone disease such as the bisphosphonate zoledronic acid can result in deleterious side effects at high doses. In this study, eliglustat, an FDA approved glycosphingolipid inhibitor, was shown to reduce MM bone disease in preclinical models of MM. Mechanistically, eliglustat alters the lipid composition and plasma membrane fluidity and acts as an autophagy flux inhibitor in bone-resorbing osteoclasts (OC). Autophagic degradation of the signaling molecule TRAF3 is key step in OC differentiation; this was prevented by eliglustat in OC precursors. In addition, eliglustat works depend on TRAF3 in vivo. Furthermore, the combination of eliglustat and zoledronic acid was found to have an additive effect to reduce MM bone disease, suggesting the potential for combination therapies that would allow for drug dose reductions. Taken together, this project identifies a novel mechanism in which glycosphingolipid inhibition reduces osteoclastogenesis via autophagy and highlights the translational potential of eliglustat for the treatment of bone loss disorders such as MM.One Sentence SummaryTranslational use of eliglustat as an autophagy inhibitor to limit bone lesions in multiple myeloma.

Wnt/&beta, catenin signaling controls many biological processes for the generation and sustainability of proper tissue size, organization and function during development and homeostasis. Consequently, mutations in the Wnt pathway components and modulators cause diseases, including genetic disorders and cancers. Targeted treatment of pathway-associated diseases entails detailed understanding of the regulatory mechanisms that fine-tune Wnt signaling. Here, we identify the neurotrophin receptor-associated death domain (Nradd), a homolog of p75 neurotrophin receptor (p75NTR), as a negative regulator of Wnt/&beta, catenin signaling in zebrafish embryos and in mammalian cells. Nradd significantly suppresses Wnt8-mediated patterning of the mesoderm and neuroectoderm during zebrafish gastrulation. Nradd is localized at the plasma membrane, physically interacts with the Wnt receptor complex and enhances apoptosis in cooperation with Wnt/&beta, catenin signaling. Our functional analyses indicate that the N-glycosylated N-terminus and the death domain-containing C-terminus regions are necessary for both the inhibition of Wnt signaling and apoptosis. Finally, Nradd can induce apoptosis in mammalian cells. Thus, Nradd regulates cell death as a modifier of Wnt/&beta, catenin signaling during development.

A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4+ T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.

Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways. However, the impact of intracellular metabolites on Treg fate has not been well explored. Here we show that the α-ketoglutarate (αKG) tricarboxylic acid (TCA) cycle metabolite increases oxidative phosphorylation (OXPHOS) in naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating Foxp3+ Treg differentiation and increasing inflammatory cytokine expression. Adoptive transfer of these T cells into tumor-bearing mice results in enhanced tumor infiltration and decreased Foxp3 expression. Mechanistically, αKG-induced OXPHOS is associated with lipidome remodelling, characterized by augmented mitochondrial lipids and triacylglyceride (TAG) stores. Inhibiting succinate dehydrogenase, the bridging enzyme between the TCA cycle and the ETC, enforces Treg differentiation. Strikingly, TAG storage directly promotes an inflammatory phenotype –– Treg differentiation is restored by inhibiting DGAT2-mediated TAG synthesis. Thus, we identify a novel crosstalk between αKG, mitochondrial metabolism and DGAT2-mediated TAG synthesis in controlling Treg fate.

Dendritic cells (DCs) are professional antigen-presenting cells of the immune system. Upon sensing pathogenic material in their environment, DCs start to mature, which includes cellular processes, such as antigen uptake, processing and presentation, as well as upregulation of costimulatory molecules and cytokine secretion. During maturation, DCs detach from peripheral tissues, migrate to the nearest lymph node, and find their way into the correct position in the net of the lymph node microenvironment to meet and interact with the respective T cells. We hypothesize that the maturation of DCs is well prepared and optimized leading to processes that alter various cellular characteristics from mechanics and metabolism to membrane properties. Here, we investigated the mechanical properties of monocyte-derived dendritic cells (moDCs) using real-time deformability cytometry to measure cytoskeletal changes and found that mature moDCs were stiffer compared to immature moDCs. These cellular changes likely play an important role in the processes of cell migration and T cell activation. As lipids constitute the building blocks of the plasma membrane, which, during maturation, need to adapt to the environment for migration and DC-T cell interaction, we performed an unbiased high-throughput lipidomics screening to identify the lipidome of moDCs. These analyses revealed that the overall lipid composition was significantly changed during moDC maturation, even implying an increase of storage lipids and differences of the relative abundance of membrane lipids upon maturation. Further, metadata analyses demonstrated that lipid changes were associated with the serum low-density lipoprotein (LDL) and cholesterol levels in the blood of the donors. Finally, using lipid packing imaging we found that the membrane of mature moDCs revealed a higher fluidity compared to immature moDCs. This comprehensive and quantitative characterization of maturation associated changes in moDCs sets the stage for improving their use in clinical application.

International audience; Imaging of biological membranes by environment-sensitive solvatochromic probes, such as Laurdan, provides information about organization of lipids, their ordering and uneven distribution. To address a key drawback of Laurdan linked to its rapid internalization and subsequent labelling 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 that redistributes within both plasma membrane leaflets and intracellular membranes. Finally, its operating range in the blue spectral region ensures absence of cross-talk 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.

The differentiation of CD4 T cells to a specific effector fate is metabolically regulated, integrating glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) with transcriptional and epigenetic changes. OXPHOS is tightly coordinated with the tricarboxylic acid (TCA) cycle but the precise role of TCA intermediates in CD4 T cell differentiation remain unclear. Here we demonstrate that α-ketoglutarate (αKG) inhibited regulatory T cell (Treg) generation while conversely, increasing Th1 polarization. In accord with these data, αKG promoted the effector profile of Treg-polarized chimeric antigen receptor-engineered T cells against the ErbB2 tumor antigen. Mechanistically, αKG significantly altered transcripts of genes involved in lipid-related processes, inducing a robust lipidome-wide remodelling and decreased membrane fluidity. A massive increase in storage and mitochondria lipids was associated with expression of mitochondrial genes and a significantly augmented OXPHOS. Notably, inhibition of succinate dehydrogenase activity, the bridge between the TCA cycle and the electron transport chain, enforced Treg generation. Thus, our study identifies novel connections between αKG, lipidome remodelling and OXPHOS in CD4 T cell fate decisions.

Super-resolution STED microcopy provides optical resolution beyond the diffraction limit. The resolution can be increased laterally (xy/2D) or axially (z/3D). 2D 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 approximately 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.

Zika virus (ZIKV) is a major public health concern in the Americas. We report that ZIKV infection and RNA extracted from ZIKV infected cells potently activated the induction of type I interferons (IFNs). This effect was fully dependent on the mitochondrial antiviral signaling protein (MAVS), implicating RIG‐I‐like receptors (RLRs) as upstream sensors of viral RNA. Indeed, RIG‐I and the related RNA sensor MDA5 contributed to type I IFN induction in response to RNA from infected cells. We found that ZIKV NS5 from a recent Brazilian isolate blocked type I IFN induction downstream of RLRs and also inhibited type I IFN receptor (IFNAR) signaling. We defined the ZIKV NS5 nuclear localization signal and report that NS5 nuclear localization was not required for inhibition of signaling downstream of IFNAR. Mechanistically, NS5 blocked IFNAR signaling by both leading to reduced levels of STAT2 and by blocking phosphorylation of STAT1, two transcription factors activated by type I IFNs. Taken together, our observations suggest that ZIKV infection induces a type I IFN response via RLRs and that ZIKV interferes with this response by blocking signaling downstream of RLRs and IFNAR.

Cell-free studies have demonstrated how collective action of actin-associated proteins can organize actin filaments into dynamic patterns, such as vortices, asters and stars. Using complementary microscopic techniques, we here show evidence of such self-organization of the actin cortex in living HeLa cells. During cell adhesion, an active multistage process naturally leads to pattern transitions from actin vortices over stars into asters. This process is primarily driven by Arp2/3 complex nucleation, but not by myosin motors, which is in contrast to what has been theoretically predicted and observed in vitro. Concomitant measurements of mechanics and plasma membrane fluidity demonstrate that changes in actin patterning alter membrane architecture but occur functionally independent of macroscopic cortex elasticity. Consequently, tuning the activity of the Arp2/3 complex to alter filament assembly may thus be a mechanism allowing cells to adjust their membrane architecture without affecting their macroscopic mechanical properties., Wellcome Trust (WT103883)

Plasma membranes dynamically respond to external cues and changing environment. Quantitative measurements of these adaptations can elucidate the mechanism that cells exploit to survive, adapt and function. However, cell-based assays are affected by active processes while measurements on synthetic models suffer from compositional limitations. Here, as a model system we employ giant plasma membrane vesicles (GPMVs), which largely preserve the plasma membrane lipidome and proteome. From analysis of fluorescence emission and lifetime of environment-sensitive dyes, and membrane shape fluctuations, we investigate how plasma membrane order, viscosity and bending rigidity are affected by different stimuli such as cell seeding density in three different cell models. Our studies reveal that bending rigidity of plasma membranes vary with lipid order and microviscosity in a highly correlated fashion. Thus, readouts from polarity- and viscosity-sensitive probes represent a promising indicator of membrane mechanical properties. Quantitative analysis of the data allows for comparison to synthetic lipid membranes as plasma membrane mimetics.

Cholesterol constitutes approximately 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 analog, whereas the other one was significantly faster. When a cholesterol analog 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.

[Image: see text] 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.

For many years, the high-affinity receptor for immunoglobulin E (IgE) FceRI, which is expressed by mast cells and basophils, has been widely held to be the exemplar of cross-linking (that is, aggregation dependent) signaling receptors. We found, however, that FceRI signaling could occur in the presence or absence of receptor cross-linking. Using both cell and cell-free systems, we showed that FceRI signaling was stimulated by surface-associated monovalent ligands through the passive, size-dependent exclusion of the receptor-type tyrosine phosphatase CD45 from plasma membrane regions of FceRI-ligand engagement. Similarly to the T cell receptor, FceRI signaling could also be initiated in a ligand-independent manner. These data suggest that a simple mechanism of CD45 exclusion-based receptor triggering could function together with cross-linking-based FceRI signaling, broadening mast cell and basophil reactivity by enabling these cells to respond to both multivalent and surface-presented monovalent antigens. These findings also strengthen the case that a size-dependent, phosphatase exclusion-based receptor triggering mechanism might serve generally to facilitate signaling by noncatalytic immune receptors.

Although liquid–liquid phase separation of cytoplasmic or nuclear components in cells has been a major focus in cell biology, it is only recently that the principle of phase separation has been a long-standing concept and extensively studied in biomembranes. Membrane phase separation has been reconstituted in simplified model systems, and its detailed physicochemical principles, including essential phase diagrams, have been extensively explored. These model membrane systems have proven very useful to study the heterogeneity in cellular membranes, however, concerns have been raised about how reliably they can represent native membranes. In this review, we will discuss how phase-separated membrane systems can mimic cellular membranes and where they fail to reflect the native cell membrane heterogeneity. We also include a few humble suggestions on which phase-separated systems should be used for certain applications, and which interpretations should be avoided to prevent unreliable conclusions.

Dipyrromethene difluoride-cholesterol (TopFluor-Cholesterol, TF-Chol) is a widely used cholesterol analogue due to its excellent fluorescence properties and considerable similarity with natural cholesterol in terms of membrane partitioning. However, the suitability of TF-Chol for detecting lysosomal cholesterol deposition has recently been questioned. Here, we highlight the fact that the method of lipid delivery and the analysis of time-point both affect the membrane distribution and labeling pattern of TF-Chol, similarly as with radiolabeled cholesterol. Lysosomal sterol accumulation characteristic to a lysosomal storage disease is most readily detected when the probe is introduced via the physiological route, i.e. as a sterol fatty acid ester in low-density lipoprotein particles. When administered to cells from solvent, lysosomal sterol sequestration becomes evident after an overnight equilibration between membranes.

Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes.

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. It is important, therefore, to be able to elucidate 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 complexity of cellular composition and architecture. Moreover, the transient nature of these interactions creates additional challenges, especially for using advanced imaging technology. One approach to circumventing these problems is to establishin vitrosystems that faithfully mimic immune cell interactions, incorporating complexity that can be ‘dialled-in’ as needed. Here, we present anin vitrosystem making use of synthetic vesicles that mimic important aspects of immune cell surfaces. Using this system, we begin to investigate the spatial distribution of signalling molecules (receptors, kinases and phosphatases) and the intracellular rearrangements that accompany the initiation of signalling in T cells. The model system presented here is expected to be widely applicable.Summary StatementImmune cell-cell interactions are reconstituted in free-standing vesicles wherein spatiotemporal aspects of immune synapse formation can be investigated.

Heterogeneous diffusion dynamics of molecules play an important role in many cellular signaling events, such as of lipids in plasma membrane bioactivity. However, these dynamics can often only be visualized by single-molecule and super-resolution optical microscopy techniques. Using fluorescence lifetime correlation spectroscopy (FLCS, an extension of fluorescence correlation spectroscopy, FCS) on a super-resolution stimulated emission depletion (STED) microscope, we here extend previous observations of nanoscale lipid dynamics in the plasma membrane of living mammalian cells. STED-FLCS allows an improved determination of spatiotemporal heterogeneity in molecular diffusion and interaction dynamics via a novel gated detection scheme, as demonstrated by a comparison between STED-FLCS and previous conventional STED-FCS recordings on fluorescent phosphoglycerolipid and sphingolipid analogues in the plasma membrane of live mammalian cells. The STED-FLCS data indicate that biophysical and biochemical parameters such as the affinity for molecular complexes strongly change over space and time within a few seconds. Drug treatment for cholesterol depletion or actin cytoskeleton depolymerization not only results in the already previously observed decreased affinity for molecular interactions but also in a slight reduction of the spatiotemporal heterogeneity. STED-FLCS specifically demonstrates a significant improvement over previous gated STED-FCS experiments and with its improved spatial and temporal resolution is a novel tool for investigating how heterogeneities of the cellular plasma membrane may regulate biofunctionality.

Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic. To unravel the molecular basis of CTxB binding to GM1 and bdGM1, we explored the partitioning and the headgroup presentation of these gangliosides in the Lo and Ld phases using atomistic molecular dynamics simulations complemented by CTxB binding experiments. The conformation of both GM1 and bdGM1 was shown to be largely similar in the Lo and Ld phases. However, bdGM1 showed reduction in receptor availability when reconstituted into synthetic bilayer mixtures, highlighting that membrane phase partitioning of the gangliosides plays a considerable role in CTxB binding. Our results suggest that the CTxB binding is predominately modulated by the partitioning of the receptor to an appropriate membrane phase. Further, given that the Lo and Ld partitioning of bdGM1 differs from those of GM1, usage of bdGM1 for studying GM1 behavior in cells can lead to invalid interpretation of experimental data. publishedVersion

Lipid packing is a crucial feature of cellular membranes. Quantitative analysis of membrane lipid packing can be achieved using polarity sensitive probes whose emission spectrum depends on the lipid packing. However, detailed insights into the exact mechanisms that cause the changes in the spectra are necessary to interpret experimental fluorescence emission data correctly. Here, we analysed frequently used polarity sensitive probes, Laurdan and di-4-ANEPPDHQ, to test whether the underlying physical mechanisms of their spectral changes are the same and, thus, whether they report on the same physico-chemical properties of the cell membrane. Steady-state spectra as well as time-resolved emission spectra of the probes in solvents and model membranes revealed that they probe different properties of the lipid membrane. Our findings are important for the application of these dyes in cell biology.

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signalling. These have been suggested to be strongly associated with the actin cytoskeleton. Here, we utilise super-resolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access the sub-diffraction diffusion regime of different fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the cellular plasma membrane, and compare it to the diffusion regime of these molecules in cell-derived actin-free giant plasma membrane vesicles (GPMVs). We show that phospholipids and sphingomyelin, which undergo hindered diffusion in the live cell membrane, diffuse freely in the GPMVs. In contrast to sphingomyelin, which is transiently trapped on molecular-scale complexes in intact cells, diffusion of the ganglioside lipid GM1 suggests transient incorporation into nanodomains, which is less influenced by the actin cortex. Finally, our data on GPI-APs indicate two molecular pools in living cells, one pool showing high mobility with trapped and compartmentalized diffusion, and the other forming immobile clusters both of which disappear in GPMVs. Our data underlines the crucial role of the actin cortex in maintaining hindered diffusion modes of most but not all membrane molecules.

Nature of the cellular membrane heterogeneity has been a long-standing question in cell biology. One crucial measure for the membrane heterogeneity is the lipid packing. Measuring changes of lipid packing in the plasma membrane during cellular events has been an important part of elucidating the biological function of membrane physical chemistry. Here, we apply conventional and spectral imaging to figure out the nature of membrane heterogeneity and its role in immune signalling events.We observe bio-membranes sampling different lipid packing states that form coexisting domains (i.e., relatively ordered and disordered domains) with a variety of distinct compositions and functional characteristics. We first investigate the connection between membrane composition and lipid packing in synthetic liposomes and show that both the relatively ordered and disordered phase, and the difference between them, are tuned by lipid composition. We then give an example of live cells regulating the lipid packing phenotypes of their plasma membranes, accessing a variety of both ordered and disordered domains as a function of cellular activity. Finally, we find that in both synthetic and natural membranes, the tunable interdomain lipid packing disparity (i.e. the difference between coexisting domains) regulates component partitioning between domains and the functionality of membrane embedded lipidic receptors. By applying advanced image processing with spectral imaging, we show that even the marginal modulation of membrane heterogeneity is crucial for immune signalling events such as FCR and B-cell signalling.Membrane heterogeneity is an organizing principle for the membrane bio-activity by either concentrating certain molecules in transient domains or by physically adjusting the conformity of the molecules to favour or hinder their interactions in the membrane.

Cholesterol is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of cholesterol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently-labeled cholesterol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction and trafficking in cells, hence it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase separated giant unilamellar vesicles (GUVs) and giant plasma membrane vesicles (GPMVs); 2) cellular trafficking, specifically subcellular localization in Niemann-Pick C (NPC) disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS) and super-resolution STED-FCS based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent cholesterol analogs in visualizing cellular cholesterol dynamics.

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.

Liquid-ordered phases are one of two biochemically active membrane states, which until now were thought to be a unique consequence of the interactions between eukaryotic membrane lipids. The formation of a liquid-ordered phase depends crucially on the ordering properties of sterols. However, it is not known whether this capacity exists in organisms that lack sterols, such as bacteria. We show that diplopterol, the simplest bacterial hopanoid, has similar properties and that hopanoids are bacterial “sterol surrogates” with the ability to order saturated lipids and to form a liquid-ordered phase in model membranes. These observations suggest that the evolution of an ordered biochemically active liquid membrane could have evolved before the oxygenation of Earth’s surface and the emergence of sterols.

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., Author summary The plasma membrane is the major interface between a cell and its environment. This complex and dynamic organelle needs to protect, as a barrier, but also transmit subtle signals into and out of the cell. For the enveloped virus IAV, the plasma membrane represents both a major obstacle to overcome during infection, and the site for the assembly of progeny virus particles. However, the organisation of the plasma membrane–a key to understanding how viral entry works—at the scale of an infecting particle (length scales < 100 nm) remains largely unknown. Sialylated glycans serve as IAV attachment factors but are not able to transmit signals across the plasma membrane. Receptor tyrosine kinases were identified to be activated upon virus binding and serve as functional receptors. How IAV engages and activates its functional receptors while initially binding glycans still remains speculative. Here, we use super resolution microscopy to study the lateral organization of plasma membrane-bound molecules involved in IAV infection, as well as their functional relationship. We find that molecules are organized in submicrometer nanodomains and, in combination with virus diffusion simulations, present a mechanistic model for how IAV first engages with AFs in the plasma membrane to subsequently engage and trigger entry-associated membrane receptors.