Membrane Organization Research Articles

Construction and validation of a prognostic model for overall survival time of patients with ovarian cancer by metabolism-related genes.

Ovarian cancer is a female-specific malignancy with high morbidity and mortality. The metabolic reprogramming of tumor cells is closely related to the biological behavior of tumors. The prognostic signature of the metabolism-related gene (MRGs) was established by LASSO-Cox regression analysis. The prognostic signature of MRGs was also prognosticated in each clinical subgroup. These genes were subjected to functional enrichment analysis and tissue expression exploration. Analysis of the MRG prognostic signature in terms of immune cell infiltration and antitumor drug susceptibility was also performed. A MRG prognostic signature including 21 genes was established and validated. Most of the 21 MRGs were expressed at different levels in ovarian cancer than in normal ovarian tissue. The enrichment analysis suggested that MRGs were involved in lipid metabolism, membrane organization, and molecular binding. The MRG prognostic signature demonstrated the predictive value of overall survival time in various clinical subgroups. The monocyte, NKT, Tgd and Tex cell scores showed differences between the groups with high- and low-risk score. The antineoplastic drug analysis we performed provided information on ovarian cancer drug therapy and drug resistance. In vitro experiments verified that PLCH1 in 21 MRGs can regulate the apoptosis and proliferation of ovarian cancer cells. This metabolism-related prognostic signature was a potential prognostic factor in patients with ovarian cancer, demonstrating high stability and accuracy.

THE ORGANIZATION OF THE BASEMENT MEMBRANES IN THE CHOROID PLEXUS VILLI OF THE HUMAN BRAIN

THE ORGANIZATION OF THE BASEMENT MEMBRANES IN THE Choroid PLEXUS VILLI OF THE HUMAN BRAIN Authors O.V. Kirik 1*, O.S. Alekseeva 1,2, I.P. Grigorev 1, E.A. Fedorova 1, A.A. Beketova 1, D.E. Korzhevsky 1 Affiliation 1 Institute of Experimental Medicine, Saint Petersburg, Russian Federation 2 Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, Saint Petersburg, Russian Federation Abstract BACKGROUND: The choroid plexus of the brain is the main component of the blood-cerebrospinal fluid barrier, separating these two biological fluids. Basement membranes of the choroid plexus have a special role in the implementation of barrier functions: they underlie the choroidal epithelium and capillary endothelium, and serve as an additional filter for substances penetrating from the blood into the cerebrospinal fluid. AIM: The purpose of this work was to study the organization of basement membranes in the villi of the human telencephalon choroid plexus using immunohistochemical detection of type IV collagen. METHODS: The study was performed on archival material from the choroid plexus of the human brain (age 29-50 years, n=10) using the immunohistochemical method for detecting type IV collagen. RESULTS: An immunohistochemical reaction using antibodies to type IV collagen showed the distribution of this protein in the subepithelial area and in the stroma of the choroid plexus villi. All immunopositive structures had clear contours; there was no reaction in the cell cytoplasm or a nonspecific background. Contacts of subepithelial and subcapillary basement membranes labeled with antibodies to type IV collagen were not detected. CONCLUSION: The study showed that the basement membranes of the villi of the choroid plexus of the human brain in the subepithelial and perivascular areas have a different organization. In this case, the union of the subepithelial and perivascular components containing type IV collagen, as a rule, does not occur.

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

Structural Basis for the Interaction between the Ezrin FERM-Domain and Human Aquaporins.

The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.

Intrinsically Disordered Membrane Anchors of Rheb, RhoA, and DiRas3 Small GTPases: Molecular Dynamics, Membrane Organization, and Interactions.

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

Functional organization of 3D plant thylakoid membranes as seen by high resolution microscopy

In the field of photosynthesis, only a limited number of approaches of super-resolution fluorescence microscopy can be used, as the functional architecture of the thylakoid membrane in chloroplasts is probed through the natural fluorescence of chlorophyll molecules. In this work, we have used a custom-built fluorescence microscopy method called Single Pixel Reconstruction Imaging (SPiRI) that yields a 1.4 gain in lateral and axial resolution relative to confocal fluorescence microscopy, to obtain 2D images and 3D-reconstucted volumes of isolated chloroplasts, obtained from pea (Pisum sativum), spinach (Spinacia oleracea) and Arabidopsis thaliana. In agreement with previous studies, SPiRI images exhibit larger thylakoid grana diameters when extracted from plants under low-light regimes. The three-dimensional thylakoid architecture, revealing the complete network of the thylakoid membrane in intact, non-chemically-fixed chloroplasts can be visualized from the volume reconstructions obtained at high resolution. From such reconstructions, the stromal connections between each granum can be determined and the fluorescence intensity in the stromal lamellae compared to those of neighboring grana.

Role of endosomal pathway in the ciliary transport and the membrane organization of outer segment disc membrane in photoreceptors

A photoreceptor is a specialized neuron that is responsible for the conversion of light into an electrical signal. Photoreceptors are classified into rods and cones, and both photoreceptors possess light-sensing ciliary organelles called outer segments (OSs), anchored in the cells by a microtubule-based axoneme. The OS consists of a stack of disc membranes, which are abundant for the retinal phototransduction proteins such as rhodopsin. Recently, modern protein synchronization techniques using in vivo transfection in rodents revealed that rhodopsin transits through Rab11-positive recycling endosomes, preferentially entering the OS in the dark. Moreover, Peripherin-2 (PRPH2, also called retinal degeneration slow, RDS), a photoreceptor-specific tetraspanin protein essential for the morphogenesis of disc membranes, is delivered to the OS following complementary to that of rhodopsin. Various PRPH2 disease-causing mutations have been found in humans, and most of the mutations in the cytosolic C-terminus of PRPH2 are linked to cone-dominant macular dystrophies. It has been shown that the late endosome is the waystation that sorts newly synthesized PRPH2 into the cilium. The multiple C-terminal motifs of PRPH2 regulate its late endosome and ciliary targeting through ubiquitination and binding to an Endosomal Sorting Complexes Required for Transport (ESCRT) component, Hrs. These findings suggest that the late endosomes play an important role in the biosynthetic pathway of ciliary proteins and can be a new therapeutic target for the diseases caused by ciliary defects.

Positive Allosteric Modulation of NMDARs Prevents the Altered Surface Dynamics Caused by Patients' Antibodies.

A positive allosteric modulator of the NMDAR, SGE-301, has been shown to reverse the alterations caused by the antibodies of patients with anti-NMDAR encephalitis (NMDARe). However, the mechanisms involved beyond receptor modulation are unclear. In this study, we aimed to investigate how this modulator affects NMDAR membrane dynamics. Cultured hippocampal neurons were treated with SGE-301 or vehicle, alongside with immunoglobulins G (IgG) from patients with NMDARe or healthy controls. NMDAR surface dynamics were assessed with single-molecule imaging by photoactivated localization microscopy. NMDAR trajectories from neurons treated with SGE-301 were less confinement, with increased diffusion coefficients. This effect mainly occurred at synapses because extrasynaptic diffusion and confinement were minimally affected by SGE-301. Treatment with patients' IgG reduced NMDAR surface dynamics and increased their confinement. Remarkably, SGE-301 incubation antagonized patients' IgG effects in both synaptic and extrasynaptic membrane compartments, restoring diffusion and confinement values similar to those from neurons exposed to control IgG. We demonstrate that SGE-301 upregulates NMDAR surface diffusion and antagonizes the pathogenic effects of patients' IgG on NMDAR membrane organization. These findings suggest a potential therapeutic strategy for NMDARe.

Distinct Bin/Amphiphysin/Rvs (BAR) family proteins may assemble on the same tubule to regulate membrane organization in vivo

Intracellular membrane tubules play a crucial role in diverse cellular processes, and their regulation is facilitated by Bin-Amphiphysin-Rvs (BAR) domain-containing proteins. This study investigates the roles of Drosophila ICA69 (dICA69) (an N-BAR protein) and Drosophila CIP4 (dCIP4) (an F-BAR protein), focusing on their impact on in vivo membrane tubule organization. In contrast to the prevailing models of BAR-domain protein function, we observed colocalization of endogenous dICA69 with dCIP4-induced tubules, indicating their potential recruitment for tubule formation and maintenance. Moreover, actin-regulatory proteins such as Wasp, SCAR, and Arp2/3 were recruited at the site of CIP4-induced tubule formation. An earlier study indicated that F-BAR proteins spontaneously segregate from the N-BAR domain proteins during membrane tubule formation. In contrast, our observation supports a model in which different BAR-domain family members can associate with the same tubule and cooperate to fine-tune the tubule width, possibly by recruiting actin modulators during the generation of tubules. Our data suggests that cooperative activities of distinct BAR-domain family proteins may determine the length and width of the membrane tubule in vivo.

β-Cell keratin 8 maintains islet mechanical integrity, mitochondrial ultrastructure, and β-cell glucose transporter 2 plasma membrane targeting.

Islet β-cell dysfunction is an underlying factor for type I diabetes (T1D) development. Insulin sensing and secretion are tightly regulated in β-cells at multiple subcellular levels. The epithelial intermediate filament (IF) protein keratin (K) 8 is the main β-cell keratin, constituting the filament network with K18. To identify the cell-autonomous functions of K8 in β-cells, mice with targeted deletion of β-cell K8 (K8flox/flox; Ins-Cre) were analyzed for islet morphology, ultrastructure, and integrity, as well as blood glucose regulation and streptozotocin (STZ)-induced diabetes development. Glucose transporter 2 (GLUT2) localization was studied in β-cells in vivo and in MIN6 cells with intact or disrupted K8/K18 filaments. Loss of β-cell K8 leads to a major reduction in K18. Islets without β-cell K8 are more fragile, and these β-cells display disjointed plasma membrane organization with less membranous E-cadherin and smaller mitochondria with diffuse cristae. Lack of β-cell K8 also leads to a reduced glucose-stimulated insulin secretion (GSIS) response in vivo, despite undisturbed systemic blood glucose regulation. K8flox/flox, Ins-Cre mice have a decreased sensitivity to STZ compared with K8 wild-type mice, which is in line with decreased membranous GLUT2 expression observed in vivo, as GLUT2 is required for STZ uptake in β-cells. In vitro, MIN6 cell plasma membrane GLUT2 is rescued in cells overexpressing K8/K18 filaments but mistargeted in cells with disrupted K8/K18 filaments. β-Cell K8 is required for islet and β-cell structural integrity, normal mitochondrial morphology, and GLUT2 plasma membrane targeting, and has implications on STZ sensitivity as well as systemic insulin responses.NEW & NOTEWORTHY Keratin 8 is the main cytoskeletal protein in the cytoplasmic intermediate filament network in β-cells. Here for the first time, we assessed the β-cell autonomous mechanical and nonmechanical roles of keratin 8 in β-cell function. We demonstrated the importance of keratin 8 in islet and β-cell structural integrity, maintaining mitochondrial morphology and GLUT2 plasma membrane targeting.

Membrane lateral organization from potential energy disconnectivity graph

Understanding the thermodynamic and kinetic properties of biomolecules requires elucidation of their complex energy landscape. A disconnectivity graph analysis of the energy landscape provides a framework for mapping the multi-dimensional landscape onto a two-dimensional representation while preserving the key features of the energy landscape. Several studies show that the structure or shape of the disconnectity graph is directly associated with the function of protein and nucleic acid molecules. In this review, we discuss how disconnectivity analysis of the potential energy surface can be extended to lipid molecules to glean important information about membrane organization. The shape of the disconnectivity graphs can be used to predict the lateral organization of multi-component lipid bilayer. We hope that this review encourages the use of disconnectivity graphs routinely by membrane biophysicists to predict the lateral organization of lipids.

Structural Phase Separation of Membranes and Fibers.

Lipid membranes interact with protein filaments on a superstructural level such that they may colocalize or spatially segregate in a living cell, whereas higher-order organization of membranes and fibers is less well explored in artificial systems. Herein, we report on the structural separation of a dispersed, membranous phase and a continuous, fibrous phase in a synthetic system. Systematic characterization of its thermodynamics and kinetics uncovers a physical principle governing phase separation: Interlamellar repulsion, favoring expansion of the membranous phase, is balanced by fibrous network elasticity, preferring the opposite. A direct consequence of this principle is the spatial addressability of the phase separation, preferably localized to soft regions of the fibrous network. Guided by this principle, we design a fibrous network with different spatial heterogeneity to modulate the phase separation, realizing a "memory" effect, patterned separation, and gradient separation. The current spatially addressable phase separation is in great contrast to the conventional ones, in which nucleation is difficult to predict or control. The fact that the membranous and fibrous phases compete for space has implications for the intracellular interactions between endoplasmic reticulum membranes and cytoskeletal filaments.

Multidimensional Spectral Phasors of LAURDAN's Excitation-Emission Matrices: The Ultimate Sensor for Lipid Phases?

The impact of lipid diversity on the lateral organization of biological membranes remains a topic of debate. While the existence of domains in lamellar membranes is well-established, the nonlamellar phases occurring in biological systems are less explored due to technical constraints. Here, we present the measurement of the excitation-emission matrices (EEM) of LAURDAN in several lipid structures. LAURDAN is a fluorescence probe widely used for characterizing lipid assemblies. The EEMs were analyzed by multidimensional spectral phasors (MdSP), an approach that seizes information from both the excitation and emission spectra. We developed a computer algorithm to construct EEM data based on a model for LAURDAN's photophysics. The MdSP calculated from the simulated EEMs reveals that all feasible possibilities lie inside a universal triangle in the phasor's plot. We use this triangle to propose a ternary representation for the phasors, allowing a better assessment of LAURDAN's surroundings in terms of hydration, water mobility, and local electronic environment. Building upon this foundation, we constructed a theoretical "phase map" that can assess both lamellar and nonlamellar membranes. We thoroughly validated this theory using well-known lipid mixtures under different phase-state conditions and enzymatically generated systems. Our results confirm that the use of MdSP is a powerful tool for obtaining quantitative information on both lamellar and nonlamellar structures. This study not only advances our understanding of the impact of lipid diversity on membrane organization but also provides a robust and general framework for the assessment of fluorescence properties that can be further extended to other probes.

Synthetic control of actin polymerization and symmetry breaking in active protocells.

Nonlinear biomolecular interactions on membranes drive membrane remodeling crucial for biological processes including chemotaxis, cytokinesis, and endocytosis. The complexity of biomolecular interactions, their redundancy, and the importance of spatiotemporal context in membrane organization impede understanding of the physical principles governing membrane mechanics. Developing a minimal in vitro system that mimics molecular signaling and membrane remodeling while maintaining physiological fidelity poses a major challenge. Inspired by chemotaxis, we reconstructed chemically regulated actin polymerization inside vesicles, guiding membrane self-organization. An external, undirected chemical input induced directed actin polymerization and membrane deformation uncorrelated with upstream biochemical cues, suggesting symmetry breaking. A biophysical model incorporating actin dynamics and membrane mechanics proposes that uneven actin distributions cause nonlinear membrane deformations, consistent with experimental findings. This protocellular system illuminates the interplay between actin dynamics and membrane shape during symmetry breaking, offering insights into chemotaxis and other cell biological processes.

Ankyrin-B is required for the establishment and maintenance of lens cytoarchitecture, mechanics, and clarity.

This study illustrates a vital role for ankyrin-B in lens architecture, growth and function through its involvement in membrane protein and spectrin-actin cytoskeletal organization and stability The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis, but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, channel proteins, cell-cell adhesion, shape change, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired ball-and-socket membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane protein and cytoskeletal organization.

Glomerular Endothelial Cell Receptor Adhesion G-Protein-Coupled Receptor F5 (ADGRF5) and the Integrity of the Glomerular Filtration Barrier.

Background: Glomerular endothelial cells are recognized to be important for maintaining the glomerular filtration barrier. ADGRF5, an adhesion G protein-coupled receptor, has been suggested to be involved in endothelial cell function. However, the role of ADGRF5 in the glomerular filtration barrier integrity remains elusive. Methods: Cellular expression of ADGRF5 in mouse glomerulus was determined by histological analyses. The impact of ADGRF5 deletion on the glomerular morphology, kidney function, and glomerular endothelial gene/protein expression was then analyzed using ADGRF5 knockout (Adgrf5 −/−) mice and human primary glomerular endothelial cells. Results: ADGRF5 was specifically expressed in the capillary endothelial cells within the glomerulus. Adgrf5 −/− mice developed albuminuria and impaired kidney function with morphological defects in the glomeruli, namely glomerular hypertrophy, glomerular basement membrane splitting and thickening, diaphragmed fenestration and detachment of the glomerular endothelial cells, and mesangial interposition. These defects were accompanied by the altered expression of genes responsible for glomerular basement membrane organization (type IV collagens and laminins) and Krüppel-like factor 2 (Klf2) in glomerular endothelial cells. Moreover, ADGRF5 knockdown decreased COL4A3 and COL4A4 expression and increased KLF2 expression in human primary glomerular endothelial cells. Conclusions: The loss of ADGRF5 resulted in altered gene expression in glomerular endothelial cells, and perturbed the structure and permselectivity of the glomerular filtration barrier.

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.

Editorial: Emergence of dynamic membranes – role of terpenoids in the evolution of membrane organization and regulation

Editorial: Emergence of dynamic membranes – role of terpenoids in the evolution of membrane organization and regulation

UV-B Radiation Disrupts Membrane Lipid Organization and Suppresses Protein Mobility of GmNARK in Arabidopsis.

While it is well known that plants interpret UV-B as an environmental cue and a potential stressor influencing their growth and development, the specific effects of UV-B-induced oxidative stress on the dynamics of membrane lipids and proteins remain underexplored. Here, we demonstrate that UV-B exposure notably increases the formation of ordered lipid domains on the plasma membrane (PM) and significantly alters the behavior of the Glycine max nodule autoregulation receptor kinase (GmNARK) protein in Arabidopsis leaves. The GmNARK protein was located on the PM and accumulated as small particles in the cytoplasm. We found that UV-B irradiation interrupted the lateral diffusion of GmNARK proteins on the PM. Furthermore, UV-B light decreases the efficiency of surface molecule internalization by clathrin-mediated endocytosis (CME). In brief, UV-B irradiation increased the proportion of the ordered lipid phase and disrupted clathrin-dependent endocytosis; thus, the endocytic trafficking and lateral mobility of GmNARK protein on the plasma membrane are crucial for nodule formation tuning. Our results revealed a novel role of low-intensity UV-B stress in altering the organization of the plasma membrane and the dynamics of membrane-associated proteins.

PMP22 duplication dysregulates lipid homeostasis and plasma membrane organization in developing human Schwann cells.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy caused by a 1.5 Mb tandem duplication of chromosome 17 harbouring the PMP22 gene. This dose-dependent overexpression of PMP22 results in disrupted Schwann cell myelination of peripheral nerves. To obtain better insights into the underlying pathogenic mechanisms in CMT1A, we investigated the role of PMP22 duplication in cellular homeostasis in CMT1A mouse models and in patient-derived induced pluripotent stem cells differentiated into Schwann cell precursors (iPSC-SCPs). We performed lipidomic profiling and bulk RNA sequencing (RNA-seq) on sciatic nerves of two developing CMT1A mouse models and on CMT1A patient-derived iPSC-SCPs. For the sciatic nerves of the CMT1A mice, cholesterol and lipid metabolism was downregulated in a dose-dependent manner throughout development. For the CMT1A iPSC-SCPs, transcriptional analysis unveiled a strong suppression of genes related to autophagy and lipid metabolism. Gene ontology enrichment analysis identified disturbances in pathways related to plasma membrane components and cell receptor signalling. Lipidomic analysis confirmed the severe dysregulation in plasma membrane lipids, particularly sphingolipids, in CMT1A iPSC-SCPs. Furthermore, we identified reduced lipid raft dynamics, disturbed plasma membrane fluidity and impaired cholesterol incorporation and storage, all of which could result from altered lipid storage homeostasis in the patient-derived CMT1A iPSC-SCPs. Importantly, this phenotype could be rescued by stimulating autophagy and lipolysis. We conclude that PMP22 duplication disturbs intracellular lipid storage and leads to a more disordered plasma membrane owing to an alteration in the lipid composition, which might ultimately lead to impaired axo-glial interactions. Moreover, targeting lipid handling and metabolism could hold promise for the treatment of patients with CMT1A.