Phase separation in model lipid membranes investigated with cryogenic electron microscopy.

We describe a method for investigating lateral membrane heterogeneity using cryogenic electron microscopy (cryo-EM) images of liposomes. The method takes advantage of differences in the thickness and molecular density of ordered and disordered phases that are resolvable in phase contrast cryo-EM. Compared to biophysical techniques like FRET or neutron scattering that yield ensemble-averaged information, cryo-EM provides direct visualization of individual vesicles and can therefore reveal variability that would otherwise be obscured by averaging. Moreover, because the contrast mechanism involves inherent properties of the lipid phases themselves, no extrinsic probes are required. We explain and discuss various complementary analyses of spatially resolved thickness and intensity measurements that enable an assessment of the membrane's phase state. The method opens a window to nanodomain structure in synthetic and biological membranes that should lead to an improved understanding of lipid raft phenomena.

Characterization of domain formation in complex membranes.

A widely known property of lipid membranes is their tendency to undergo a separation into disordered (Ld) and ordered (Lo) domains. This impacts the local structure of the membrane relevant for the physical (e.g., enhanced electroporation) and biological (e.g., protein sorting) significance of these regions. The increase in computing power, advancements in simulation software, and more detailed information about the composition of biological membranes shifts the study of these domains into the focus of classical molecular dynamics simulations. In this chapter, we present a versatile yet robust analysis pipeline that can be easily implemented and adapted for a wide range of lipid compositions. It employs Gaussian-based Hidden Markov Models to predict the hidden order states of individual lipids by describing their structure through the area per lipid and the average SCC order parameters per acyl chain. Regions of the membrane with a high correlation between ordered lipids are identified by employing the Getis-Ord local spatial autocorrelation statistic on a Voronoi tessellation of the lipids. As an example, the approach is applied to two distinct systems at a coarse-grained resolution, demonstrating either a strong tendency towards phase separation (1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DIPC), cholesterol) or a weak tendency toward phase separation (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine (PUPC), cholesterol). Explanations of the steps are complemented by coding examples written in Python, providing both a comprehensive understanding and practical guidance for a seamless integration of the workflow into individual projects.

Enhancing extracellular vesicle cargo loading and functional delivery by engineering protein-lipid interactions.

Naturally generated lipid nanoparticles termed extracellular vesicles (EVs) hold significant promise as engineerable therapeutic delivery vehicles. However, active loading of protein cargo into EVs in a manner that is useful for delivery remains a challenge. Here, we demonstrate that by rationally designing proteins to traffic to the plasma membrane and associate with lipid rafts, we can enhance loading of protein cargo into EVs for a set of structurally diverse transmembrane and peripheral membrane proteins. We then demonstrate the capacity of select lipid tags to mediate increased EV loading and functional delivery of an engineered transcription factor to modulate gene expression in target cells. We envision that this technology could be leveraged to develop new EV-based therapeutics that deliver a wide array of macromolecular cargo.

Using lipid binding proteins and advanced microscopy to study lipid domains.

Sphingomyelin is postulated to form clusters with glycosphingolipids, cholesterol and other sphingomyelin molecules in biomembranes through hydrophobic interaction and hydrogen bonds. These clusters form submicron size lipid domains. Proteins that selectively binds sphingomyelin and/or cholesterol are useful to visualize the lipid domains. Due to their small size, visualization of lipid domains requires advanced microscopy techniques in addition to lipid binding proteins. This Chapter describes the method to characterize plasma membrane sphingomyelin-rich and cholesterol-rich lipid domains by quantitative microscopy. This Chapter also compares different permeabilization methods to visualize intracellular lipid domains.

Applications of phase-separating multi-bilayers in protein-membrane domain interactions.

Synthetic model membranes are important tools to elucidate lipid domain and protein interactions due to predefined lipid compositions and characterizable biophysical properties. Here, we introduce a model membrane with multiple lipid bilayers (multi-bilayers) stacked on a mica substrate that is prepared through a spin-coating technique. The spin-coated multi-bilayers are useful in the study of phase separated membranes with a high cholesterol content, mobile lipids, microscopic and reversible phase separation, and easy conjugation with proteins, which make them a good model to study interactions between proteins and membrane domains.

Using the yeast vacuole as a system to test the lipidic drivers of membrane heterogeneity in living cells.

The biophysical drivers of membrane lateral heterogeneity, often termed lipid rafts, have been largely explored using synthetic liposomes or mammalian plasma membrane-derived giant vesicles. Yeast vacuoles, an organelle comparable to mammalian lysosomes, is the only in vivo system that shows stable micrometer scale phase separation in unperturbed cells. The ease of manipulating lipid metabolism in yeast makes this a powerful system for identifying lipids involved in the onset of vacuole membrane heterogeneity. Vacuole domains are induced by stationary stage growth and nutritional starvation, during which they serve as a docking and internalization site for lipid droplet energy stores. Here we describe methods for characterizing vacuole phase separation, its physiological function, and its lipidic drivers. First, we detail methodologies for robustly inducing vacuole domain formation and quantitatively characterizing during live cell imaging experiments. Second, we detail a new protocol for biochemical isolation of stationary stage vacuoles, which allows for lipidomic dissection of membrane phase separation. Third, we describe biochemical techniques for analyzing lipid droplet internalization in vacuole domains. When combined with genetic or chemical perturbations to lipid metabolism, these methods allow for systematic dissection of lipid composition in the structure and function of ordered membrane domains in living cells.

Pore-spanning membranes as a tool to investigate lateral lipid membrane heterogeneity.

Over the years, it has become more and more obvious that lipid membranes show a very complex behavior. This behavior arises in part from the large number of different kinds of lipids and proteins and how they dynamically interact with each other. In vitro studies using artificial membrane systems have shed light on the heterogeneity based on lipid-lipid interactions in multicomponent bilayer mixtures. Inspired by the raft hypothesis, the coexistence of liquid-disordered (ld) and liquid-ordered (lo) phases has drawn much attention. It was shown that ternary lipid mixtures containing low- and high-melting temperature lipids and cholesterol can phase separate into a lo phase enriched in the high-melting lipids and cholesterol and a ld phase enriched in the low-melting lipids. Depending on the model membrane system under investigation, different domain sizes, shapes, and mobilities have been found. Here, we describe how to generate phase-separated lo/ld phases in model membrane systems termed pore-spanning membranes (PSMs). These PSMs are prepared on porous silicon substrates with pore sizes in the micrometer regime. A proper functionalization of the top surface of the substrates is required to achieve the spreading of giant unilamellar vesicles (GUVs) to obtain PSMs. Starting with lo/ld phase-separated GUVs lead to membrane heterogeneities in the PSMs. Depending on the functionalization strategy of the top surface of the silicon substrate, different membrane heterogeneities are observed in the PSMs employing fluorescence microscopy. A quantitative analysis of the heterogeneity as well as the dynamics of the lipid domains is described.

Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains.

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

Confinement energy landscape classification reveals membrane receptor nano-organization mechanisms.

The cell membrane organization has an essential functional role through the control of membrane receptor confinement in micro- or nanodomains. Several mechanisms have been proposed to account for these properties, although some features have remained controversial, notably the nature, size, and stability of cholesterol- and sphingolipid-rich domains or lipid rafts. Here, we probed the effective energy landscape acting on single-nanoparticle-labeled membrane receptors confined in raft nanodomains- epidermal growth factor receptor (EGFR), Clostridium perfringens ε-toxin receptor (CPεTR), and Clostridium septicum α-toxin receptor (CSαTR)-and compared it with hop-diffusing transferrin receptors. By establishing a new analysis pipeline combining Bayesian inference, decision trees, and clustering approaches, we systematically classified single-protein trajectories according to the type of effective confining energy landscape. This revealed the existence of only two distinct organization modalities: confinement in a quadratic energy landscape for EGFR, CPεTR, and CSαTR (A), and free diffusion in confinement domains resulting from the steric hindrance due to F-actin barriers for transferrin receptor (B). The further characterization of effective confinement energy landscapes by Bayesian inference revealed the role of interactions with the domain environment in cholesterol- and sphingolipid-rich domains with (EGFR) or without (CPεTR and CSαTR) interactions with F-actin to regulate the confinement energy depth. These two distinct mechanisms result in the same organization type (A). We revealed that the apparent domain sizes for these receptor trajectories resulted from Brownian exploration of the energy landscape in a steady-state-like regime at a common effective temperature, independently of the underlying molecular mechanisms. These results highlight that confinement domains may be adequately described as interaction hotspots rather than rafts with abrupt domain boundaries. Altogether, these results support a new model for functional receptor confinement in membrane nanodomains and pave the way to the constitution of an atlas of membrane protein organization.

Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

Partitioning into ER membrane microdomains impacts autophagic protein turnover during cellular aging.

Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.

Normal and Dysregulated Sphingolipid Metabolism: Contributions to Podocyte Injury and Beyond.

Podocyte health is vital for maintaining proper glomerular filtration in the kidney. Interdigitating foot processes from podocytes form slit diaphragms which regulate the filtration of molecules through size and charge selectivity. The abundance of lipid rafts, which are ordered membrane domains rich in cholesterol and sphingolipids, near the slit diaphragm highlights the importance of lipid metabolism in podocyte health. Emerging research shows the importance of sphingolipid metabolism to podocyte health through structural and signaling roles. Dysregulation in sphingolipid metabolism has been shown to cause podocyte injury and drive glomerular disease progression. In this review, we discuss the structure and metabolism of sphingolipids, as well as their role in proper podocyte function and how alterations in sphingolipid metabolism contributes to podocyte injury and drives glomerular disease progression.

Phospholipid scramblase 1: an essential component of the nephrocyte slit diaphragm.

Blood ultrafiltration in nephrons critically depends on specialized intercellular junctions between podocytes, named slit diaphragms (SDs). Here, by studying a homologous structure found in Drosophila nephrocytes, we identify the phospholipid scramblase Scramb1 as an essential component of the SD, uncovering a novel link between membrane dynamics and SD formation. In scramb1 mutants, SDs fail to form. Instead, the SD components Sticks and stones/nephrin, Polychaetoid/ZO-1, and the Src-kinase Src64B/Fyn associate in cortical foci lacking the key SD protein Dumbfounded/NEPH1. Scramb1 interaction with Polychaetoid/ZO-1 and Flotillin2, the presence of essential putative palmitoylation sites and its capacity to oligomerize, suggest a function in promoting SD assembly within lipid raft microdomains. Furthermore, Scramb1 interactors as well as its functional sensitivity to temperature, suggest an active involvement in membrane remodeling processes during SD assembly. Remarkably, putative Ca2+-binding sites in Scramb1 are essential for its activity raising the possibility that Ca2+ signaling may control the assembly of SDs by impacting on Scramb1 activity.

Partitioning to ordered membrane domains regulates the kinetics of secretory traffic.

The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, Retention Using Selective Hooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.

Mechanism of Purinergic Regulation of Neurotransmission in Mouse Neuromuscular Junction: The Role of Redox Signaling and Lipid Rafts.

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic Gi protein-coupled P2Y13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P2Y13 receptors (MRS 2211), Ca2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca2+ with subsequent elevation of ROS levels. The elevation of [Ca2+]in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of Gi protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P2Y13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P2Y13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P2Y13 receptor/Gi protein/Ca2+/protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner.

Exploring the connections between ER-based lipid metabolism and plasma membrane nanodomain signaling.

Recent advancements in our understanding of cell membrane dynamics have shed light on the importance of plasma membrane (PM) nanodomains in plant cell signaling. Nevertheless, many aspects of membrane nanodomains, including their regulatory mechanisms and biological functions, remain enigmatic. To address this knowledge gap, our review article proposes a novel perspective wherein signaling pathways target endoplasmic reticulum (ER)-based lipid metabolism to exert control over the formation and function of membrane nanodomains. Subsequently, these nanodomains reciprocate by influencing the localization and activity of signaling molecules at the PM. We place a specific emphasis on ER-based enzymatic reactions, given the ER's central role in membrane lipid biosynthesis and its capacity to directly impact PM lipid composition, particularly with regard to saturation levels - an essential determinant of nanodomain properties. The interplay among cell signaling, glycerolipid metabolism, and PM nanodomain may create feedforward/feedback loops that fine-tune cellular responses to developmental and environmental cues.

Asymptotic analysis of particle cluster formation in the presence of anchoring sites.

The aggregation or clustering of proteins and other macromolecules plays an important role in the formation of large-scale molecular assemblies within cell membranes. Examples of such assemblies include lipid rafts, and postsynaptic domains (PSDs) at excitatory and inhibitory synapses in neurons. PSDs are rich in scaffolding proteins that can transiently trap transmembrane neurotransmitter receptors, thus localizing them at specific spatial positions. Hence, PSDs play a key role in determining the strength of synaptic connections and their regulation during learning and memory. Recently, a two-dimensional (2D) diffusion-mediated aggregation model of PSD formation has been developed in which the spatial locations of the clusters are determined by a set of fixed anchoring sites. The system is kept out of equilibrium by the recycling of particles between the cell membrane and interior. This results in a stationary distribution consisting of multiple clusters, whose average size can be determined using an effective mean-field description of the particle concentration around each anchored cluster. In this paper, we derive corrections to the mean-field approximation by applying the theory of diffusion in singularly perturbed domains. The latter is a powerful analytical method for solving two-dimensional (2D) and three-dimensional (3D) diffusion problems in domains where small holes or perforations have been removed from the interior. Applications range from modeling intracellular diffusion, where interior holes could represent subcellular structures such as organelles or biological condensates, to tracking the spread of chemical pollutants or heat from localized sources. In this paper, we take the bounded domain to be the cell membrane and the holes to represent anchored clusters. The analysis proceeds by partitioning the membrane into a set of inner regions around each cluster, and an outer region where mean-field interactions occur. Asymptotically matching the inner and outer stationary solutions generates an asymptotic expansion of the particle concentration, which includes higher-order corrections to mean-field theory that depend on the positions of the clusters and the boundary of the domain. Motivated by a recent study of light-activated protein oligomerization in cells, we also develop the analogous theory for cluster formation in a three-dimensional (3D) domain. The details of the asymptotic analysis differ from the 2D case due to the contrasting singularity structure of 2D and 3D Green's functions.

Measuring the Transmembrane Registration of Lipid Domains in Droplet Interface Bilayers through Tensiometry.

Diverse collections of lipids self-assemble into domains within biological membranes, and these domains are typically organized in both the transverse and lateral directions of the membrane. The ability of the membrane to link these domains across the membrane's interior grants cells control over features on the external cellular surface. Numerous hypothesized factors drive the cross-membrane (or transverse) coupling of lipid domains. In this work we seek to isolate these transverse lipid-lipid influences in a simple model system using droplet interface bilayers (DIBs) to better understand the associated mechanics. DIBs enable symmetric and asymmetric combinations of domain-forming lipid mixtures within a model bilayer, and the evolving energetics of the membrane may be tracked using drop-shape analysis. We find that symmetric distributions of domain-forming lipids produce long-lasting, gradual shifts in the DIB membrane energetics that are not observed in asymmetric distributions of the lipids where the domain-forming lipids are only within one leaflet. The approach selected for this work provides experimental measurement of the mismatch penalty associated with antiregistered lipid domains as well as measurements of the influence of rafts on DIB behaviors with suggestions for their future use as a model platform.

Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

Anti-Nociceptive Effects of Sphingomyelinase and Methyl-Beta-Cyclodextrin in the Icilin-Induced Mouse Pain Model.

The thermo- and pain-sensitive Transient Receptor Potential Melastatin 3 and 8 (TRPM3 and TRPM8) ion channels are functionally associated in the lipid rafts of the plasma membrane. We have already described that cholesterol and sphingomyelin depletion, or inhibition of sphingolipid biosynthesis decreased the TRPM8 but not the TRPM3 channel opening on cultured sensory neurons. We aimed to test the effects of lipid raft disruptors on channel activation on TRPM3- and TRPM8-expressing HEK293T cells in vitro, as well as their potential analgesic actions in TRPM3 and TRPM8 channel activation involving acute pain models in mice. CHO cell viability was examined after lipid raft disruptor treatments and their effects on channel activation on channel expressing HEK293T cells by measurement of cytoplasmic Ca2+ concentration were monitored. The effects of treatments were investigated in Pregnenolone-Sulphate-CIM-0216-evoked and icilin-induced acute nocifensive pain models in mice. Cholesterol depletion decreased CHO cell viability. Sphingomyelinase and methyl-beta-cyclodextrin reduced the duration of icilin-evoked nocifensive behavior, while lipid raft disruptors did not inhibit the activity of recombinant TRPM3 and TRPM8. We conclude that depletion of sphingomyelin or cholesterol from rafts can modulate the function of native TRPM8 receptors. Furthermore, sphingolipid cleavage provided superiority over cholesterol depletion, and this method can open novel possibilities in the management of different pain conditions.