Transmembrane signaling through single-spanning receptors modulated by phase separation at the cell surface.

A wide variety of transmembrane signals are transduced by cell-surface receptors that activate intracellular signaling molecules. In particular, receptor clustering in the plasma membrane plays a critical role in these processes. Single-spanning or single-pass transmembrane proteins are among the most significant types of membrane receptors, which include adhesion receptors, such as integrins, CD44, cadherins, and receptor tyrosine kinases. Elucidating the molecular mechanisms underlying the regulation of the activity of these receptors is of great significance. Liquid-liquid phase separation (LLPS) is a recently emerging paradigm in cellular physiology for the ubiquitous regulation of the spatiotemporal dynamics of various signaling pathways. This study describes the emerging features of transmembrane signaling through single-spanning receptors from the perspective of phase separation. Possible physicochemical modulations of LLPS-based transmembrane signaling are also discussed.

Spatiotemporal Characteristics Determining the Multifaceted Nature of Reactive Oxygen, Nitrogen, and Sulfur Species in Relation to Proton Homeostasis.

Significance: Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) act as signaling molecules, regulating gene expression, enzyme activity, and physiological responses. However, excessive amounts of these molecular species can lead to deleterious effects, causing cellular damage and death. This dual nature of ROS, RNS, and RSS presents an intriguing conundrum that calls for a new paradigm. Recent Advances: Recent advancements in the study of photosynthesis have offered significant insights at the molecular level and with high temporal resolution into how the photosystem II oxygen-evolving complex manages to prevent harmful ROS production during the water-splitting process. These findings suggest that a dynamic spatiotemporal arrangement of redox reactions, coupled with strict regulation of proton transfer, is crucial for minimizing unnecessary ROS formation. Critical Issues: To better understand the multifaceted nature of these reactive molecular species in biology, it is worth considering a more holistic view that combines ecological and evolutionary perspectives on ROS, RNS, and RSS. By integrating spatiotemporal perspectives into global, cellular, and biochemical events, we discuss local pH or proton availability as a critical determinant associated with the generation and action of ROS, RNS, and RSS in biological systems. Future Directions: The concept of localized proton availability will not only help explain the multifaceted nature of these ubiquitous simple molecules in diverse systems but also provide a basis for new therapeutic strategies to manage and manipulate these reactive species in neural disorders, pathogenic diseases, and antiaging efforts.

The Structural Proteins of Membrane Rafts, Caveolins and Flotillins, in Lung Cancer: More Than Just Scaffold Elements.

Lung cancer is one of the most frequently diagnosed cancers worldwide. Due to its late diagnosis, it remains the leading cause of cancer-related deaths. Despite it is mostly associated to tobacco smoking, recent data suggested that genetic factors are of the highest importance. In this context, different processes meaningful for the development and progression of lung cancer such endocytosis, protein secretion and signal transduction, are controlled by membrane rafts. These highly ordered membrane domains contain proteins such as caveolins and flotillins, which were traditionally considered scaffold proteins but have currently been given a preponderant role in lung cancer. Here, we summarize current knowledge regarding the involvement of caveolins and flotillins in lung cancer from a molecular point of view.

Rafting on the Evidence for Lipid Raft-like Domains as Hubs Triggering Environmental Toxicants' Cellular Effects.

The plasma membrane lipid rafts are cholesterol- and sphingolipid-enriched domains that allow regularly distributed, sub-micro-sized structures englobing proteins to compartmentalize cellular processes. These membrane domains can be highly heterogeneous and dynamic, functioning as signal transduction platforms that amplify the local concentrations and signaling of individual components. Moreover, they participate in cell signaling routes that are known to be important targets of environmental toxicants affecting cell redox status and calcium homeostasis, immune regulation, and hormonal functions. In this work, the evidence that plasma membrane raft-like domains operate as hubs for toxicants' cellular actions is discussed, and suggestions for future research are provided. Several studies address the insertion of pesticides and other organic pollutants into membranes, their accumulation in lipid rafts, or lipid rafts' disruption by polychlorinated biphenyls (PCBs), benzo[a]pyrene (B[a]P), and even metals/metalloids. In hepatocytes, macrophages, or neurons, B[a]P, airborne particulate matter, and other toxicants caused rafts' protein and lipid remodeling, oxidative changes, or amyloidogenesis. Different studies investigated the role of the invaginated lipid rafts present in endothelial cells in mediating the vascular inflammatory effects of PCBs. Furthermore, in vitro and in vivo data strongly implicate raft-localized NADPH oxidases, the aryl hydrocarbon receptor, caveolin-1, and protein kinases in the toxic mechanisms of occupational and environmental chemicals.

Lipid peroxidation drives liquid-liquid phase separation and disrupts raft protein partitioning in biological membranes.

The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes the structure, conformation and physicochemical properties of lipids, leading to major membrane alterations including bilayer thinning, altered fluidity, and increased permeability. Whether and how lipid peroxidation impacts the lateral organization of proteins and lipids in biological membranes, however, remains poorly understood. Here, we employ cell-derived giant plasma membrane vesicles (GPMVs) as a model to investigate the impact of lipid peroxidation on ordered membrane domains, often termed membrane rafts. We show that lipid peroxidation induced by the Fenton reaction dramatically enhances phase separation propensity of GPMVs into co-existing liquid ordered (raft) and liquid disordered (non-raft) domains and increases the relative abundance of the disordered, non-raft phase. Peroxidation also leads to preferential accumulation of peroxidized lipids and 4-hydroxynonenal (4-HNE) adducts in the disordered phase, decreased lipid packing in both raft and non-raft domains, and translocation of multiple classes of proteins out of rafts. These findings indicate that peroxidation of plasma membrane lipids disturbs many aspects of membrane rafts, including their stability, abundance, packing, and protein and lipid composition. We propose that these disruptions contribute to the pathological consequences of lipid peroxidation during aging and disease, and thus serve as potential targets for therapeutic intervention.

Composition of raft-like cell membrane microdomains resistant to styrene-maleic acid copolymer (SMA) solubilization.

An advantageous alternative to the use of detergents in biochemical studies on membrane proteins are the recently developed styrene-maleic acid (SMA) amphipathic copolymers. In our recent study [1] we demonstrated that using this approach, most T cell membrane proteins were fully solubilized (presumably in small nanodiscs), while two types of raft proteins, GPI-anchored proteins and Src family kinases, were mostly present in much larger (>250 nm) membrane fragments markedly enriched in typical raft lipids, cholesterol and lipids containing saturated fatty acid residues. In the present study we demonstrate that disintegration of membranes of several other cell types by means of SMA copolymer follows a similar pattern and we provide a detailed proteomic and lipidomic characterization of these SMA-resistant membrane fragments (SRMs).

HER2 expression defines unique requirements for flotillin and c-Src in EGFR signaling.

The epidermal growth factor receptor (EGFR) controls many cellular functions. Upon binding its ligand, the receptor undergoes dimerization, phosphorylation and activation of signals including the phosphoinositide-3-kinase (PI3K)-Akt pathway. Although some studies have indicated that EGFR signaling may be controlled by signal enrichment within various membrane rafts, such as flotillin nanodomains, others have found a limited effect of disruption of these nanodomains on EGFR signaling, suggesting that specific factors may define context-specific control of EGFR signaling. Ligand-bound EGFR can homodimerize or instead undergo heterodimerization with the related receptor HER2 (also known as ERBB2) when the latter is expressed. We examined how EGFR signaling in the presence of HER2 distinctly requires flotillin nanodomains. Induction of HER2 expression altered EGFR signaling duration, which is consistent with EGFR-HER2 heterodimer formation. EGFR and c-Src (also known as SRC) localized within plasma membrane structures demarked by flotillin-1 more prominently in HER2-expressing cells. Consistently, HER2-expressing cells, but not cells lacking HER2, were dependent on flotillin-1 and c-Src for EGFR signaling leading to Akt activation and cell proliferation. Hence, HER2 expression establishes a requirement for flotillin membrane rafts and c-Src in EGFR signaling.

Membrane-Mediated Regulation of Sperm Fertilization Potential in Poultry.

Fertilization requires successful completion of molecular events taking place at different spatiotemporal scales. Transcriptionally and translationally inactive sperm need to rely on pre-assembled pathways modulated by extracellular signals that traverse the plasma membranes. However, species differences in how sperm respond to them delay the progress toward a comprehensive understanding of how activation of the signaling cascades is coordinated in poultry sperm. In chickens, recent studies have found that membrane rafts are present on the sperm surface and play important roles in regulating multistage fertilization. In this review, we focus on three steps in which membrane alteration plays a key role. The first is post-testicular maturation, in which bird sperm acquire fertilization functions through biochemical changes. The second part of this review concerns membrane regulation of sperm-egg binding and the acrosome reaction. Finally, we extend our discussion to the translation of membrane raft theory into a technical principle for the commercial production and genetic preservation of poultry.

Differential impact of lipid raft depletion on platelet-derived growth factor (PDGF)-induced ERK1/2 MAP-kinase, SRC and AKT signaling.

It has become clear that lipid rafts functions as signaling hotspots connecting cell surface receptors to intracellular signaling pathways. However, the exact involvement of lipid rafts in receptor tyrosine kinase signaling is still poorly understood. In this study, we have analyzed platelet-derived growth factor (PDGF) receptor ß (PDGFR-ß) signaling in two different cell lines depleted of cholesterol, and as a consequence, disruption of lipid rafts. Cholesterol depletion of BJ-hTERT fibroblasts using methyl-ß-cyclodextrin (MßCD) did not affect PDGFR-ß activation as measured by its tyrosine phosphorylation. However, we did observe a small reduction in AKT phosphorylation and a more robust decrease of ERK1/2 activation. In contrast, in the osteosarcoma cell line U2OS, we noticed a deficient receptor activation. Interestingly, in U2OS cells, the ERK1/2 pathway was unaffected, but instead AKT and SRC signaling was reduced. These results suggest that cell type specific wiring of signaling pathways can lead to differential sensitivity to cholesterol depletion. Furthermore, MßCD treatment had a much more pronounced morphological effect on U2OS compared to BJ-hTERT cells. This is consistent with a previous report claiming that cancer cells are more sensitive to cholesterol depletion than normal cells. Our data supports the possibility that cholesterol lowering drugs may impede tumor growth.

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

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

High-Level Expression of Palmitoylated MPP1 Recombinant Protein in Mammalian Cells.

Our recent studies have pointed to an important role of the MAGUK family member, MPP1, as a crucial molecule interacting with flotillins and involved in the lateral organization of the erythroid plasma membrane. The palmitoylation of MPP1 seems to be an important element in this process; however, studies on the direct effect of palmitoylation on protein-protein or protein-membrane interactions in vitro are still challenging due to the difficulties in obtaining functional post-translationally modified recombinant proteins and the lack of comprehensive protocols for the purification of palmitoylated proteins. In this work, we present an optimized approach for the high-yield overexpression and purification of palmitoylated recombinant MPP1 protein in mammalian HEK-293F cells. The presented approach facilitates further studies on the molecular mechanism of lateral membrane organization and the functional impact of the palmitoylation of MPP1, which could also be carried out for other palmitoylated proteins.

Cholera Toxin as a Probe for Membrane Biology.

Cholera toxin B-subunit (CTxB) has emerged as one of the most widely utilized tools in membrane biology and biophysics. CTxB is a homopentameric stable protein that binds tightly to up to five GM1 glycosphingolipids. This provides a robust and tractable model for exploring membrane structure and its dynamics including vesicular trafficking and nanodomain assembly. Here, we review important advances in these fields enabled by use of CTxB and its lipid receptor GM1.

Dynamic spatial reorganization of BSK1 complexes in the plasma membrane underpins signal-specific activation for growth and immunity.

Growth and immunity are opposing processes that compete for cellular resources, and proper resource allocation is crucial for plant survival. BSK1 plays a key role in the regulation of both growth and immunity by associating with BRI1 and FLS2, respectively. However, it remains unclear how two antagonistic signals co-opt BSK1 to induce signal-specific activation. Here we show that the dynamic spatial reorganization of BSK1 within the plasma membrane underlies the mechanism of signal-specific activation for growth or immunity. Resting BSK1 localizes to membrane rafts as complexes. Unlike BSK1-associated FLS2 and BRI1, flg22 or exogenous brassinosteroid (BR) treatment did not decrease BSK1 levels at the plasma membrane (PM) but rather induced BSK1 multimerization and dissociation from FLS2/BSK1 or BRI1/BSK1, respectively. Moreover, flg22-activated BSK1 translocated from membrane rafts to non-membrane-raft regions, whereas BR-activated BSK1 remained in membrane rafts. When applied together with flg22, BR suppressed various flg22-induced BSK1 activities such as BSK1 dissociation from FLS2/BSK1, BSK1 interaction with MAPKKK5, and BSK translocation together with MAPKKK5. Taken together, this study provides a unique insight into how the precise control of BSK1 spatiotemporal organization regulates the signaling specificity to balance plant growth and immunity.

Not Just Another Scaffolding Protein Family: The Multifaceted MPPs.

Membrane palmitoylated proteins (MPPs) are a subfamily of a larger group of multidomain proteins, namely, membrane-associated guanylate kinases (MAGUKs). The ubiquitous expression and multidomain structure of MPPs provide the ability to form diverse protein complexes at the cell membranes, which are involved in a wide range of cellular processes, including establishing the proper cell structure, polarity and cell adhesion. The formation of MPP-dependent complexes in various cell types seems to be based on similar principles, but involves members of different protein groups, such as 4.1-ezrin-radixin-moesin (FERM) domain-containing proteins, polarity proteins or other MAGUKs, showing their multifaceted nature. In this review, we discuss the function of the MPP family in the formation of multiple protein complexes. Notably, we depict their significant role for cell physiology, as the loss of interactions between proteins involved in the complex has a variety of negative consequences. Moreover, based on recent studies concerning the mechanism of membrane raft formation, we shed new light on a possible role played by MPPs in lateral membrane organization.

Use of Streptolysin O (SLO) to Study the Function of Lipid Rafts.

Group A Streptococcus (GAS) produces the pore-forming toxin, streptolysin O (SLO). SLO sequesters cholesterol and induces a plasma membrane repair process that removes the pores via a lipid raft-mediated endocytosis. The impact SLO has on membranes makes it an effective toxin for investigating the function of lipid rafts in cellular processes. Lipid rafts are essential for B-cell activation. Indeed, antigen-stimulated B-cell receptors (BCRs) require localization with lipid rafts for efficient signaling and internalization. SLO treatment impairs BCR activation by competing for lipid rafts. Here, disrupting lipid rafts using SLO and assessing the effects on BCR activation by fluorescence microscopy and flow cytometry are described.

Membrane Rearrangements in the Maturation of Circulating Human Reticulocytes.

Red blood cells (RBCs) begin their circulatory life as reticulocytes (Retics) after their egress from the bone marrow where, as R1 Retics, they undergo significant rearrangements in their membrane and intracellular components, via autophagic, proteolytic, and vesicle-based mechanisms. Circulating, R2 Retics must complete this maturational process, which involves additional loss of significant amounts of membrane and selected membrane proteins. Little is known about the mechanism(s) at the basis of this terminal differentiation in the circulation, which culminates with the production of a stable biconcave discocyte. The membrane of R1 Retics undergoes a selective remodeling through the release of exosomes that are enriched in transferrin receptor and membrane raft proteins and lipids, but are devoid of Band 3, glycophorin A, and membrane skeletal proteins. We wondered whether a similar selective remodeling occurred also in the maturation of R2 Retics. Peripheral blood R2 Retics, isolated by an immunomagnetic method, were compared with mature circulating RBCs from the same donor and their membrane protein and lipid content was analyzed. Results show that both Band 3 and spectrin decrease from R2 Retics to RBCs on a "per cell" basis. Looking at membrane proteins that are considered as markers of membrane rafts, flotillin-2 appears to decrease in a disproportionate manner with respect to Band 3. Stomatin also decreases but in a more proportionate manner with respect to Band 3, hinting at a heterogeneous nature of membrane rafts. High resolution lipidomics analysis, on the contrary, revealed that those lipids that are typically representative of the membrane raft phase, sphingomyelin and cholesterol, are enriched in mature RBCs with respct to Retics, relative to total cell lipids, strongly arguing in favor of the selective retention of at least certain subclasses of membrane rafts in RBCs as they mature from Retics. Our hypothesis that rafts serve as additional anchoring sites for the lipid bilayer to the underlying membrane-skeleton is corroborated by the present results. It is becoming ever more clear that a proper lipid composition of the reticulocyte is necessary for the production of a normal mature RBC.

Fluorinated Galactoses Inhibit Galactose-1-Phosphate Uridyltransferase and Metabolically Induce Galactosemia-like Phenotypes in HEK-293 Cells.

Genetic defects of human galactose-1-phosphate uridyltransferase (hGALT) and the partial loss of enzyme function result in an altered galactose metabolism with serious long-term developmental impairment of organs in classic galactosemia patients. In search for cellular pathomechanisms induced by the stressor galactose, we looked for ways to induce metabolically a galactosemia-like phenotype by hGALT inhibition in HEK293 cells. In kinetic studies, we provide evidence for 2-fluorinated galactose-1-phosphate (F-Gal-1-P) to competitively inhibit recombinant hGALT with a KI of 0.9 mM. Contrasting with hepatic cells, no alterations of N-glycoprofiles in MIG (metabolic induction of galactosemia)-HEK293 cells were revealed for an inducible secretory netrin-1 probe by MALDI-MS. Differential fluorescence-activated cell sorting demonstrated reduced surface expression of N-glycosylated CD109, EGFR, DPP4, and rhMUC1. Membrane raft proteomes exhibited dramatic alterations pointing to an affection of the unfolded protein response, and of targeted protein traffick. Most prominent, a negative regulation of oxidative stress was revealed presumably as a response to a NADPH pool depletion during reduction of Gal/F-Gal. Cellular perturbations induced by fluorinated galactoses in normal epithelial cells resemble proteomic changes revealed for galactosemic fibroblasts. In conclusion, the metabolic induction of galactosemia-like phenotypes in healthy epithelial/neuronal cells could support studies on the molecular pathomechanisms in classic galactosemia, in particular under conditions of low galactose stress and residual GALT activity.

A Model for Primary Cilium Biogenesis by Polarized Epithelial Cells: Role of the Midbody Remnant and Associated Specialized Membranes.

Primary cilia are solitary, microtubule-based protrusions surrounded by a ciliary membrane equipped with selected receptors that orchestrate important signaling pathways that control cell growth, differentiation, development and homeostasis. Depending on the cell type, primary cilium assembly takes place intracellularly or at the cell surface. The intracellular route has been the focus of research on primary cilium biogenesis, whereas the route that occurs at the cell surface, which we call the "alternative" route, has been much less thoroughly characterized. In this review, based on recent experimental evidence, we present a model of primary ciliogenesis by the alternative route in which the remnant of the midbody generated upon cytokinesis acquires compact membranes, that are involved in compartmentalization of biological membranes. The midbody remnant delivers part of those membranes to the centrosome in order to assemble the ciliary membrane, thereby licensing primary cilium formation. The midbody remnant's involvement in primary cilium formation, the regulation of its inheritance by the ESCRT machinery, and the assembly of the ciliary membrane from the membranes originally associated with the remnant are discussed in the context of the literature concerning the ciliary membrane, the emerging roles of the midbody remnant, the regulation of cytokinesis, and the role of membrane compartmentalization. We also present a model of cilium emergence during evolution, and summarize the directions for future research.

Effects of vanadium(IV) compounds on plasma membrane lipids lead to G protein-coupled receptor signal transduction.

Luteinizing hormone receptors (LHR), expressed at physiological numbers <30,000 receptors per cell, translocate to and signal within membrane rafts following binding of human chorionic gonadotropin (hCG). Similarly LHR signal in cells when treated with bis(maltolato)oxovanadium(IV) (BMOV), bis(ethylmaltolato)oxovanadium(IV) (BEOV) or VOSO4, which decrease membrane lipid packing. Overexpressed LHR (>85,000 receptors per cell) are found in larger clusters in polarized homo-transfer fluorescence resonance energy transfer (homo-FRET) studies that were not affected by either hCG or vanadium compounds. Intracellular cyclic adenylate monophosphate (cAMP) levels indicate that only clustered LHR are active and produce the intracellular second messenger, cAMP. When LHR are over-expressed, cell signaling is unaffected by binding of hCG or vanadium compounds. To confirm the existence of intact complex, the EPR spectra of vanadium compounds in cell media were obtained using 1 mM BMOV, BEOV or VOSO4. These data were used to determine intact complex in a 10 µM solution and verified by speciation calculations. Effects of BMOV and BEOV samples were about two-fold greater than those of aqueous vanadium(IV) making it likely that intact vanadium complex are responsible for effects of LHR function. This represents a new mechanism for activation of a G protein-coupled receptor; perturbations in the lipid bilayer by vanadium compounds lead to aggregation and accumulation of physiological numbers of LHR in membrane raft domains where they initiate signal transduction and production of cAMP, a second messenger involved in signaling.