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"Lipid raft aging" in nerve cells represents an early event in the development of aging-related neurodegenerative diseases, such as Alzheimer's disease. Lipid rafts are key elements in synaptic plasticity, and their modification with aging alters interactions and distribution of signaling molecules, such as glutamate receptors and ion channels involved in memory formation, eventually leading to cognitive decline. In the present study, we have analyzed, in vivo, the effects of dietary supplementation of n-3 LCPUFA on the lipid structure, membrane microviscosity, domain organization, and partitioning of ionotropic and metabotropic glutamate receptors in hippocampal lipid raffs in female mice. The results revealed several lipid signatures of "lipid rafts aging" in old mice fed control diets, consisting in depletion of n-3 LCPUFA, membrane unsaturation, along with increased levels of saturates, plasmalogens, and sterol esters, as well as altered lipid relevant indexes. These changes were paralleled by increased microviscosity and changes in the raft/non-raft (R/NR) distribution of AMPA-R and mGluR5. Administration of the n-3 LCPUFA diet caused the partial reversion of fatty acid alterations found in aged mice and returned membrane microviscosity to values found in young animals. Paralleling these findings, lipid rafts accumulated mGluR5, NMDA-R, and ASIC2, and increased their R/NR proportions, which collectively indicate changes in synaptic plasticity. Unexpectedly, this diet also modified the lipidome and dimension of lipid rafts, as well as the domain redistribution of glutamate receptors and acid-sensing ion channels involved in hippocampal synaptic plasticity, likely modulating functionality of lipid rafts in memory formation and reluctance to age-associated cognitive decline., (© 2023 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Alzheimer's disease (AD) is associated with a very large burden on global healthcare systems. Thus, it is imperative to find effective treatments of the disease. One feature of AD is the accumulation of neurotoxic β-amyloid peptide (Aβ). Aβ induces multiple pathological processes that are deleterious to nerve cells. Despite the development of medications that target the reduction of Aβ to treat AD, none has proven to be effective to date. Non-pharmacological interventions, such as physical exercise, are also being studied. The benefits of exercise on AD are widely recognized. Experimental and clinical studies have been performed to verify the role that exercise plays in reducing Aβ deposition to alleviate AD. This paper reviewed the various mechanisms involved in the exercise-induced reduction of Aβ, including the regulation of amyloid precursor protein cleaved proteases, the glymphatic system, brain-blood transport proteins, degrading enzymes and autophagy, which is beneficial to promote exercise therapy as a means of prevention and treatment of AD and indicates that exercise may provide new therapeutic targets for the treatment of AD., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

ER lipid raft-associated protein 1 (ERLIN1) and 2 (ERLIN2) are 40 kDa transmembrane glycoproteins belonging to the family of prohibitins, containing a PHB domain. They are generally localized in the endoplasmic reticulum (ER), where ERLIN1 forms a heteroligomeric complex with its closely related ERLIN2. Well-defined functions of ERLINS are promotion of ER-associated protein degradation, mediation of inositol 1,4,5-trisphosphate (IP 3 ) receptors, processing and regulation of lipid metabolism. Until now, ERLINs have been exclusively considered protein markers of ER lipid raft-like microdomains. However, under pathophysiological conditions, they have been described within mitochondria-associated endoplasmic reticulum membranes (MAMs), tethering sites between ER and mitochondria, characterized by the presence of specialized raft-like subdomains enriched in cholesterol and gangliosides, which play a key role in the membrane scrambling and function. In this context, it is emerging that ER lipid raft-like microdomains proteins, i.e., ERLINs, may drive mitochondria-ER crosstalk under both physiological and pathological conditions by association with MAMs, regulating the two main processes underlined, survival and death. In this review, we describe the role of ERLINs in determining cell fate by controlling the "interchange" between apoptosis and autophagy pathways, considering that their alteration has a significant impact on the pathogenesis of several human diseases.

Primary cilia, antenna-like structures of the plasma membrane, detect various extracellular cues and transduce signals into the cell to regulate a wide range of functions. Lipid rafts, plasma membrane microdomains enriched in cholesterol, sphingolipids and specific proteins, are also signalling hubs involved in a myriad of physiological functions. Although impairment of primary cilia and lipid rafts is associated with various diseases, the relationship between primary cilia and lipid rafts is poorly understood. Here, we review a newly discovered interaction between primary cilia and lipid raft dynamics that occurs during Akt signalling in adipogenesis. We also discuss the relationship between primary cilia and lipid raft-mediated Akt signalling in cancer biology. This review provides a novel perspective on primary cilia in the regulation of lipid raft dynamics.

With the discovery of local Ca 2+ signals in the 1990s the concept of 'elementary Ca 2+ signals' and 'fundamental Ca 2+ signals' was developed. While 'elementary Ca 2+ signals' relate to optical signals gained by activity of small clusters of Ca 2+ channels, 'fundamental signals' describe such optical signals that arise from opening of single Ca 2+ channels. In this review, we discuss (i) concepts of local Ca 2+ signals and Ca 2+ microdomains, (ii) molecular mechanisms underlying Ca 2+ microdomains, (iii) functions of Ca 2+ microdomains, and (iv) mathematical modelling of Ca 2+ microdomains. We focus on Ca 2+ microdomains produced by ORAI channels, D-myo-inositol 1,4,5-trisphosphate receptors, or ryanodine receptors. In summary, research on local Ca 2+ signals in different cell models aims to better understand how cells use the Ca 2+ toolkit to produce Ca 2+ microdomains as relevant signals for specific cellular responses, but also how local Ca 2+ signals as building blocks merge into global Ca 2+ signaling., Competing Interests: Declaration of Competing Interest The authors declare that there are no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)

Lateral organization in the plane of the plasma membrane is an important driver of biological processes. The past dozen years have seen increasing experimental support for the notion that lipid organization plays an important role in modulating this heterogeneity. Various biophysical mechanisms rooted in the concept of liquid-liquid phase separation have been proposed to explain diverse experimental observations of heterogeneity in model and cell membranes with distinct but overlapping applicability. In this review, we focus on the evidence for and the consequences of the hypothesis that the plasma membrane is poised near an equilibrium miscibility critical point. Critical phenomena explain certain features of the heterogeneity observed in cells and model systems but also go beyond heterogeneity to predict other interesting phenomena, including responses to perturbations in membrane composition.

Muscle disuse and denervation leads to muscle atrophy, but underlying mechanisms can be different. Previously, we have found ceramide (Cer) accumulation and lipid raft disruption after acute hindlimb suspension (HS), a model of muscle disuse. Herein, using biochemical and fluorescent approaches the influence of unilateral denervation itself and in combination with short-term HS on membrane-related parameters of rat soleus muscle was studied. Denervation increased immunoexpression of sphingomyelinase and Cer in plasmalemmal regions, but decreased Cer content in the raft fraction and enhanced lipid raft integrity. Preliminary denervation suppressed (1) HS-induced Cer accumulation in plasmalemmal regions, shown for both nonraft and raft-fractions; (2) HS-mediated decrease in lipid raft integrity. Similar to denervation, inhibition of the sciatic nerve afferents with capsaicin itself increased Cer plasmalemmal immunoexpression, but attenuated the membrane-related effects of HS. Finally, both denervation and capsaicin treatment increased immunoexpression of proapoptotic protein Bax and inhibited HS-driven increase in antiapoptotic protein Bcl-2. Thus, denervation can increase lipid raft formation and attenuate HS-induced alterations probably due to decrease of Cer levels in the raft fraction. The effects of denervation could be at least partially caused by the loss of afferentation. The study points to the importance of motor and afferent inputs in control of Cer distribution and thereby stability of lipid rafts in the junctional and extrajunctional membranes of the muscle.

Astrocytes exhibit spatially-restricted near-membrane microdomain Ca 2+ transients in their fine processes. How these transients are generated and regulate brain function in vivo remains unclear. Here we show that Drosophila astrocytes exhibit spontaneous, activity-independent microdomain Ca 2+ transients in their fine processes. Astrocyte microdomain Ca 2+ transients are mediated by the TRP channel TrpML, stimulated by reactive oxygen species (ROS), and can be enhanced in frequency by the neurotransmitter tyramine via the TyrRII receptor. Interestingly, many astrocyte microdomain Ca 2+ transients are closely associated with tracheal elements, which dynamically extend filopodia throughout the central nervous system (CNS) to deliver O 2 and regulate gas exchange. Many astrocyte microdomain Ca 2+ transients are spatio-temporally correlated with the initiation of tracheal filopodial retraction. Loss of TrpML leads to increased tracheal filopodial numbers, growth, and increased CNS ROS. We propose that local ROS production can activate astrocyte microdomain Ca 2+ transients through TrpML, and that a subset of these microdomain transients promotes tracheal filopodial retraction and in turn modulate CNS gas exchange., Competing Interests: ZM, MF No competing interests declared, (© 2020, Ma and Freeman.)

Cancer represents a severe challenge to healthcare systems and individuals worldwide. The development of multiple drug resistance is a major issue regarding cancer therapy, which can result in the progression of disease. Cholesterol is a major constituent of cell membranes and participates in the regulation of several cellular processes, such as cell growth, proliferation, differentiation, survival and apoptosis. Numerous studies have provided correlative support for a role of cholesterol in cancer development and drug resistance. In the present review, recent insights into the regulation of cholesterol metabolism, the association between cholesterol and the efficacy of antitumor agents in preclinical studies, as well as the possible mechanisms through which cholesterol influences drug resistance, are summarized. Furthermore, the clinical relevance of cholesterol to the development of cancer, as well as strategies targeting cholesterol for therapeutic intervention are detailed. Collectively, studies on various types of cancer have suggested that increased cholesterol levels promote resistance to chemotherapeutic drugs in cancer through a variety of mechanisms, and that the depletion of cholesterol using statins significantly enhances the sensitivity of the therapeutic agents. However, additional studies are required to enhance the current understanding of the involvement of cholesterol in the development of drug‑resistant cancer.

Detergent-resistant membranes (DRMs) microdomains, or "raft lipids", are key components of the plasma membrane (PM), being involved in membrane trafficking, signal transduction, cell wall metabolism or endocytosis. Proteins imbibed in these domains play important roles in these cellular functions, but there are few studies concerning DRMs under abiotic stress. In this work, we determine DRMs from the PM of broccoli roots, the lipid and protein content, the vesicles structure, their water osmotic permeability and a proteomic characterization focused mainly in aquaporin isoforms under salinity (80 mM NaCl). Based on biochemical lipid composition, higher fatty acid saturation and enriched sterol content under stress resulted in membranes, which decreased osmotic water permeability with regard to other PM vesicles, but this permeability was maintained under control and saline conditions; this maintenance may be related to a lower amount of total PIP1 and PIP2. Selective aquaporin isoforms related to the stress response such as PIP1;2 and PIP2;7 were found in DRMs and this protein partitioning may act as a mechanism to regulate aquaporins involved in the response to salt stress. Other proteins related to protein synthesis, metabolism and energy were identified in DRMs independently of the treatment, indicating their preference to organize in DMRs.

There is growing appreciation that the plasma membrane orchestrates a diverse array of functions by segregating different activities into specialized domains that vary in size, stability, and composition. Studies with the budding yeast Saccharomyces cerevisiae have identified a novel type of plasma membrane domain known as the MCC (membrane compartment of Can1)/eisosomes that correspond to stable furrows in the plasma membrane. MCC/eisosomes maintain proteins at the cell surface, such as nutrient transporters like the Can1 arginine symporter, by protecting them from endocytosis and degradation. Recent studies from several fungal species are now revealing new functional roles for MCC/eisosomes that enable cells to respond to a wide range of stressors, including changes in membrane tension, nutrition, cell wall integrity, oxidation, and copper toxicity. The different MCC/eisosome functions are often intertwined through the roles of these domains in lipid homeostasis, which is important for proper plasma membrane architecture and cell signaling. Therefore, this review will emphasize the emerging models that explain how MCC/eisosomes act as hubs to coordinate cellular responses to stress. The importance of MCC/eisosomes is underscored by their roles in virulence for fungal pathogens of plants, animals, and humans, which also highlights the potential of these domains to act as novel therapeutic targets., (Copyright © 2020 American Society for Microbiology.)

Tetraspanins comprise a family of proteins embedded in the membrane through four transmembrane domains. One of the most distinctive features of tetraspanins is their ability to interact with other proteins in the membrane using their extracellular, transmembrane and cytoplasmic domains, allowing them to incorporate several proteins into clusters called tetraspanin-enriched microdomains. The spatial proximity of signaling proteins and their regulators enables a rapid functional cross-talk between these proteins, which is required for a rapid translation of extracellular signals into intracellular signaling cascades. In this article, we highlight a few examples that illustrate how tetraspanin-mediated interactions between cell surface proteins allow their functional cross-talk to regulate intracellular signaling.

Objective: Endothelial Cav-1 (caveolin-1) expression plays a relevant role during atherogenesis by controlling NO production, vascular inflammation, LDL (low-density lipoprotein) transcytosis, and extracellular matrix remodeling. Additional studies have identified cholesterol-rich membrane domains as important regulators of autophagy by recruiting ATGs (autophagy-related proteins) to the plasma membrane. Here, we investigate how the expression of Cav-1 in the aortic endothelium influences autophagy and whether enhanced autophagy contributes to the atheroprotective phenotype observed in Cav-1-deficient mice. Approach and Results: To analyze the impact of Cav-1 deficiency on regulation of autophagy in the aortic endothelium during the progression of atherosclerosis, we fed Ldlr -/- and Cav-1 -/- Ldlr -/- mice a Western diet and assessed autophagy in the vasculature. We observe that the absence of Cav-1 promotes autophagy activation in athero-prone areas of the aortic endothelium by enhancing autophagic flux. Mechanistically, we found that Cav-1 interacts with the ATG5-ATG12 complex and influences the cellular localization of autophagosome components in lipid rafts, which controls the autophagosome formation and autophagic flux. Pharmacological inhibition of autophagy attenuates the atheroprotection observed in Cav-1 -/- mice by increasing endothelial inflammation and macrophage recruitment, identifying a novel molecular mechanism by which Cav-1 deficiency protects against the progression of atherosclerosis., Conclusions: These results identify Cav-1 as a relevant regulator of autophagy in the aortic endothelium and demonstrate that pharmacological suppression of autophagic flux in Cav-1-deficient mice attenuates the atheroprotection observed in Cav-1 -/- mice. Additionally, these findings suggest that activation of endothelial autophagy by blocking Cav-1 might provide a potential therapeutic strategy for cardiovascular diseases including atherosclerosis.

Drug repurposing is potentially the fastest available option in the race to identify safe and efficacious drugs that can be used to prevent and/or treat COVID-19. By describing the life cycle of the newly emergent coronavirus, SARS-CoV-2, in light of emerging data on the therapeutic efficacy of various repurposed antimicrobials undergoing testing against the virus, we highlight in this review a possible mechanistic convergence between some of these tested compounds. Specifically, we propose that the lysosomotropic effects of hydroxychloroquine and several other drugs undergoing testing may be responsible for their demonstrated in vitro antiviral activities against COVID-19. Moreover, we propose that Niemann-Pick disease type C (NPC), a lysosomal storage disorder, may provide new insights into potential future therapeutic targets for SARS-CoV-2, by highlighting key established features of the disorder that together result in an "unfavorable" host cellular environment that may interfere with viral propagation. Our reasoning evolves from previous biochemical and cell biology findings related to NPC, coupled with the rapidly evolving data on COVID-19. Our overall aim is to suggest that pharmacological interventions targeting lysosomal function in general, and those particularly capable of reversibly inducing transient NPC-like cellular and biochemical phenotypes, constitute plausible mechanisms that could be used to therapeutically target COVID-19., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Antarctic shallow coastal marine communities were long thought to be isolated from their nearest neighbours by hundreds of kilometres of deep ocean and the Antarctic Circumpolar Current. The discovery of non-native kelp washed up on Antarctic beaches led us to question the permeability of these barriers to species dispersal. According to the literature, over 70 million kelp rafts are afloat in the Southern Ocean at any one time. These living, floating islands can play host to a range of passenger species from both their original coastal location and those picked in the open ocean. Driven by winds, currents and storms towards the coast of the continent, these rafts are often cited as theoretical vectors for the introduction of new species into Antarctica and the sub-Antarctic islands. We found non-native kelps, with a wide range of "hitchhiking" passenger organisms, on an Antarctic beach inside the flooded caldera of an active volcanic island. This is the first evidence of non-native species reaching the Antarctic continent alive on kelp rafts. One passenger species, the bryozoan Membranipora membranacea, is found to be an invasive and ecologically harmful species in some cold-water regions, and this is its first record from Antarctica. The caldera of Deception Island provides considerably milder conditions than the frigid surrounding waters and it could be an ideal location for newly introduced species to become established. These findings may help to explain many of the biogeographic patterns and connections we currently see in the Southern Ocean. However, with the impacts of climate change in the region we may see an increase in the range and number of organisms capable of surviving both the long journey and becoming successfully established.

Whether respiratory epithelial cells regulate the final transit of extravasated neutrophils into the inflamed airspace or are a passive barrier is poorly understood. Alveolar epithelial type 1 (AT1) cells, best known for solute transport and gas exchange, have few established immune roles. Epithelial membrane protein 2 (EMP2), a tetraspan protein that promotes recruitment of integrins to lipid rafts, is highly expressed in AT1 cells but has no known function in lung biology. Here, we show that Emp2-/- mice exhibit reduced neutrophil influx into the airspace after a wide range of inhaled exposures. During bacterial pneumonia, Emp2-/- mice had attenuated neutrophilic lung injury and improved survival. Bone marrow chimeras, intravital neutrophil labeling, and in vitro assays suggested that defective transepithelial migration of neutrophils into the alveolar lumen occurs in Emp2-/- lungs. Emp2-/- AT1 cells had dysregulated surface display of multiple adhesion molecules, associated with reduced raft abundance. Epithelial raft abundance was dependent upon putative cholesterol-binding motifs in EMP2, whereas EMP2 supported adhesion molecule display and neutrophil transmigration through suppression of caveolins. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular neutrophil traffic into the alveolus.

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.

Plasma membranes are heterogeneous and laterally compartmentalized into distinct microdomains. These membrane microdomains consist of special lipids and proteins and are thought to act as signaling platforms. In plants, membrane microdomains have been detected by super-resolution microscopy, and there is evidence that they play roles in several biological processes. Here, we review current knowledge about the lipid and protein components of membrane microdomains. Furthermore, we summarize the dynamics of membrane microdomains in response to different stimuli. We also explore the biological functions associated with membrane microdomains as signal integration hubs. Finally, we outline challenges and questions for further studies.

Ordered lipid domains (rafts) are generally considered to be features of eukaryotic cells, but ordered lipid domains formed by cholesterol lipids have been identified in bacteria from the genus Borrelia, and similar cholesterol lipids exist in the bacterium Helicobacter pylori. To determine whether H. pylori lipids could form ordered membrane domains, we investigated domain formation in aqueous dispersions of H. pylori whole lipid extracts, individual H. pylori lipids, or defined mixtures of H. pylori lipids and other membrane-forming lipids. DPH (1,6-diphenyl-1,3,5-hexatriene) anisotropy measurements were used to assay membrane order and FRET (Förster resonance energy transfer) was used to detect the presence of co-existing ordered and disordered domains. We found that H. pylori membrane lipid extracts spontaneously formed lipid domains. Domain formation was more stable when lipids were extracted from H. pylori cells grown in the presence of cholesterol. Certain isolated H. pylori lipids (by themselves or when mixed with other lipids) also had the ability to form ordered domains. To be specific, H. pylori cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside (CAG) and cholesterol-6-O-phosphatidyl-α-D-glucopyranoside (CPG) had the ability to form and/or stabilize ordered domain formation, while H. pylori phosphatidylethanolamine did not, behaving similarly to unsaturated phosphatidylethanolamines. We conclude that specific H. pylori cholesterol lipids have a marked ability to form ordered lipid domains., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Flotillin-1 (Flot1) is an evolutionary conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired because of a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. In this study, we analyzed morphology and migration parameters of murine wild-type and Flot1 -/- CD8 + T cells using in vitro assays and intravital two-photon microscopy of lymphoid and nonlymphoid tissues. Flot1 -/- CD8 + T cells displayed significant alterations in cell shape and motility parameters in vivo but showed comparable homing to lymphoid organs and intact in vitro migration to chemokines. Furthermore, their clonal expansion and infiltration into nonlymphoid tissues during primary and secondary antiviral immune responses was comparable to wild-type CD8 + T cells. Taken together, Flot1 plays a detectable but unexpectedly minor role for CD8 + T cell behavior under physiological conditions., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Objective: Clinical and laboratory studies have demonstrated that platelets become hyperactive and prothrombotic in conditions of inflammation. We have previously shown that the proinflammatory cytokine interleukin (IL)-6 forms a complex with soluble IL-6 receptor α (sIL-6Rα) to prime platelets for activation by subthreshold concentrations of collagen. Upon being stimulated with collagen, the transcription factor signal transducer and activator of transcription (STAT) 3 in platelets is phosphorylated and dimerized to act as a protein scaffold to facilitate the catalytic action between the kinase Syk and the substrate phospholipase Cγ2 (PLCγ2) in collagen-induced signaling. However, it remains unknown how collagen induces phosphorylation and dimerization of STAT3., Methods and Results: We conducted complementary in vitro experiments to show that the IL-6 receptor subunit glycoprotein 130 (GP130) was in physical proximity to the collagen receptor glycoprotein VI (GPVI in membrane lipid rafts of platelets. This proximity allows collagen to induce STAT3 activation and dimerization, and the IL-6-sIL-6Rα complex to activate the kinase Syk and the substrate PLCγ2 in the GPVI signal pathway, resulting in an enhanced platelet response to collagen. Disrupting lipid rafts or blocking GP130-Janus tyrosine kinase (JAK)-STAT3 signaling abolished the cross-activation and reduced platelet reactivity to collagen., Conclusion: These results demonstrate cross-talk between collagen and IL-6 signal pathways. This cross-talk could potentially provide a novel mechanism for inflammation-induced platelet hyperactivity, so the IL-6-GP130-JAK-STAT3 pathway has been identified as a potential target to block this hyperactivity., (© 2019 International Society on Thrombosis and Haemostasis.)

Despite knowledge that glucose metabolism is essential for the regulation of signaling cascades in the sperm that are pre-assembled into specific areas and function at multistage for fertilization, the physiological roles of glucose in avian sperm are poorly understood. Accumulated results of studies conducted in our laboratory and others indicate that sperm possess membrane microdomains, or membrane rafts, which play important roles in several processes, including the induction of acrosome reaction (AR). When characterizing proteomes associated with chicken sperm rafts, we observed marked enrichment of glucose transporter 3 (GLUT3). Here we show that glucose uptake is mediated by membrane rafts and stimulates AR induction by activating AMP-activated protein kinase (AMPK). Using a specific antibody, we observed that GLUT3 is localized to the entire flagellum and acrosome region and highly associated with membrane rafts. The addition of glucose stimulated AR in a dose-dependent manner without affecting sperm motility. AR and glucose uptake assays were performed using both inhibitors and activators, and demonstrated that glucose-dependent AR results from the activity of a glucose transporter located in membrane rafts and associated with AMPK. To better understand the mechanism of AMPK activation by glucose, we evaluated localization and phosphorylation status of AMPKα, showing that glucose uptake stimulates AMPKα phosphorylation, leading to its complete activation. Together, these results lead us to propose a novel mechanism by which glucose uptake stimulates the AMPK signaling pathway via membrane rafts, resulting in maximal acrosomal responsiveness in avian sperm as migrating upward to a fertilization site., (© The Author(s) 2019. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Temperature plays an important role in the immune response. Acclimatization occurs when there are changes in ambient temperature over a long period. In this study, we used the human leukemic Jurkat T cell line to study the effect of temperature on the immune system using concanavalin A (ConA), a plant-derived immunostimulant, as a trigger for T-cell activation. Previously, we have reported endocytic intracellular cluster formation during T-cell activation by ConA with the aid of rafts and polymerization of the cytoskeleton (actin and microtubules). Here, we investigated the effect of temperature on cluster formation (with the aid of three-dimensional images of the cells) and on the stability of rafts, actin, and microtubules. When the temperature was changed between 23°C and 37°C (physiological temperature), clusters could be observed throughout this temperature range. Raft structure was stabilized at lower temperatures but destabilized at higher temperatures. Actin was stable when the temperature was higher than 27°C. When actin was depolymerized, clustering was not observed at 37°C but could be observed at 23°C. There were no changes in microtubules within this temperature range. Thus, raft clustering may be associated with raft stability at lower temperatures (<27°C) and with actin at higher temperatures (≥27°C). Hence, we provided insight into the associations between temperature, rafts, actin, and microtubules in the immune response., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

We study the crucial role of membrane fluctuations in maintaining a narrow gap between a fluid membrane tube and an enclosed solid particle. Solvent flows can occur in this gap, hence giving rise to a finite particle mobility along the tube. While our study has relevance for how cells are able to transport large organelles or other cargo along connecting membrane tubes, known as tunneling nanotubes, our calculations are also framed so that they can be tested by a specific in vitro experiment: A tubular membrane tether can be pulled from a membrane reservoir, such as an aspirated Giant Unilamellar Vesicle (GUV), e.g. using a conjugated bead that binds to the membrane and is held in a laser trap. We compute the subsequent mobility of colloidal particles trapped in the tube, focusing on the case when the particle is large compared to the equilibrium tube radius. We predict that the particle mobility should scale as ∼ σ-2/3, with σ the membrane tension., Competing Interests: The author has declared that no competing interests exist.

Ganglioside GM3 is strongly related with human tumors, such as lung, brain cancers and melanomas, and more and more evidences have revealed that GM3 possesses powerful effects on cancer development and progression. GM3 is over expressed on several types of cancers, and can be as a tumor-associated carbohydrate antigen, used for immunotherapy of cancers. GM3 can also inhibit tumor cells growth by anti-angiogenesis or motility and so on. Especially, GM3 has effects on the EGFR tyrosine kinase signaling, uPAR-related signaling and glycolipid-enriched microdomains, which are essential for cancer signaling conduction. It is obvious that GM3 will be a promising target for cancer treatment., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Both transcriptionally and translationally inactive sperm need preassembled pathways into specific cellular compartments to function. Although initiation of the acrosome reaction (AR) involves several signaling pathways including protein kinase A (PKA) activation, how these are regulated remains poorly understood in avian sperm. Membrane rafts are specific membrane regions enriched in sterols and functional proteins and play important roles in diverse cellular processes, including signal transduction. Our recent studies on chicken sperm demonstrated that membrane rafts exist and play a role in multistage fertilization. These, combined with the functional importance of membrane rafts in mammalian sperm AR, prompted us to investigate the roles of membrane rafts in signaling pathways leading to AR in chicken sperm. Using 2-hydroxypropyl-β-cyclodextrin (2-OHCD), we found that the disruption of membrane rafts inhibits PKA activity and AR without affecting protein tyrosine phosphorylation; however, these inhibitions were abolished in the presence of a cyclic 3,5-adenosine monophosphate (cAMP) analog. In addition, biochemical experiments showed a decrease in cAMP content in 2-OHCD-treated sperm, suggesting the involvement of soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclase (tmAC). Pharmacological experiments, combined with transcriptome analysis, showed that sAC and tmAC are present and involved in AR induction in chicken sperm. Furthermore, stimulation of both isoforms reversed the inhibition of PKA activity and AR in 2-OHCD-treated sperm. In conclusion, our results demonstrated that membrane rafts play an important role in AR induction by regulating the cAMP-dependent pathway and that they provide a mechanistic insight into membrane regulation of AR and sperm function in birds.

Docosahexaenoic acid (DHA, 22:6) is an n-3 polyunsaturated fatty acid (n-3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state 2 H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The plasma membrane is of central importance for defining the closed volume of cells in contradistinction to the extracellular environment. The plasma membrane not only serves as a boundary, but it also mediates the exchange of physical and chemical information between the cell and its environment in order to maintain intra- and intercellular functions. Artificial lipid- and cell-derived membrane vesicles have been used as closed-volume containers, representing the simplest cell model systems to study transmembrane processes and intracellular biochemistry. Classical examples are studies of membrane translocation processes in plasma membrane vesicles and proteoliposomes mediated by transport proteins and ion channels. Liposomes and native membrane vesicles are widely used as model membranes for investigating the binding and bilayer insertion of proteins, the structure and function of membrane proteins, the intramembrane composition and distribution of lipids and proteins, and the intermembrane interactions during exo- and endocytosis. In addition, natural cell-released microvesicles have gained importance for early detection of diseases and for their use as nanoreactors and minimal protocells. Yet, in most studies, ensembles of vesicles have been employed. More recently, new micro- and nanotechnological tools as well as novel developments in both optical and electron microscopy have allowed the isolation and investigation of individual (sub)micrometer-sized vesicles. Such single-vesicle experiments have revealed large heterogeneities in the structure and function of membrane components of single vesicles, which were hidden in ensemble studies. These results have opened enormous possibilities for bioanalysis and biotechnological applications involving unprecedented miniaturization at the nanometer and attoliter range. This review will cover important developments toward single-vesicle analysis and the central discoveries made in this exciting field of research.

Sphingolipids are found in abundance at synapses and have been implicated in regulation of synapse structure, function, and degeneration. Their precise role in these processes, however, remains obscure. Serine Palmitoyl-transferase (SPT) is the first enzymatic step for synthesis of sphingolipids. Analysis of the Drosophila larval neuromuscular junction (NMJ) revealed mutations in the SPT enzyme subunit, lace/SPTLC2 resulted in deficits in synaptic structure and function. Although NMJ length is normal in lace mutants, the number of boutons per NMJ is reduced to ∼50% of the wild type number. Synaptic boutons in lace mutants are much larger but show little perturbation to the general ultrastructure. Electrophysiological analysis of lace mutant synapses revealed strong synaptic transmission coupled with predominance of depression over facilitation. The structural and functional phenotypes of lace mirrored aspects of Basigin (Bsg), a small Ig-domain adhesion molecule also known to regulate synaptic structure and function. Mutant combinations of lace and Bsg generated large synaptic boutons, while lace mutants showed abnormal accumulation of Bsg at synapses, suggesting that Bsg requires sphingolipid to regulate structure of the synapse. In support of this, we found Bsg to be enriched in lipid rafts. Our data points to a role for sphingolipids in the regulation and fine-tuning of synaptic structure and function while sphingolipid regulation of synaptic structure may be mediated via the activity of Bsg., (© 2018 The Authors. The Journal of Comparative Neurology published by Wiley Periodicals, Inc.)

Platelets protect the vascular system during damage or inflammation, but platelet activation can result in pathological thrombosis. Activated platelets release a variety of extracellular vesicles (EVs). EVs shed from the plasma membrane often expose phosphatidylserine (PS). These EVs are pro-thrombotic and increased in number in many cardiovascular and metabolic diseases. The mechanisms by which PS-exposing EVs are shed from activated platelets are not well characterised. Cholesterol-rich lipid rafts provide a platform for coordinating signalling through receptors and Ca 2+ channels in platelets. We show that cholesterol depletion with methyl-β-cyclodextrin or sequestration with filipin prevented the Ca 2+ -triggered release of PS-exposing EVs. Although calpain activity was required for release of PS-exposing, calpain-dependent cleavage of talin was not affected by cholesterol depletion. P2Y 12 and TPα, receptors for ADP and thromboxane A 2 , respectively, have been reported to be in platelet lipid rafts. However, the P2Y 12 antagonist, AR-C69931MX, or the cyclooxygenase inhibitor, aspirin, had no effect on A23187-induced release of PS-exposing EVs. Together, these data show that lipid rafts are required for release of PS-exposing EVs from platelets.

Lipid rafts are highly dynamic membrane domains featured by distinctive biochemical composition and physicochemical properties compared with the surrounding plasma membrane. These microstructures are associated not only with cellular signaling and communication in normal nerve cells but also with pathological processing of amyloid precursor protein in Alzheimer's disease. Using lipid rafts isolated from human frontal cortex in nondemented subjects aging 24 to 85 years, we demonstrate here that lipid structure of lipid rafts undergo significant alterations of specific lipid classes and phospholipid-bound fatty acids as brain cortex correlating with aging. Main changes affect levels of plasmalogens, polyunsaturated fatty acids (especially docosahexaenoic acid and arachidonic acid), total polar lipids (mainly phosphatidylinositol, sphingomyelin, sulfatides, and cerebrosides), and total neutral lipids (particularly cholesterol and sterol esters). Besides, relevant relationships between main fatty acids and/or lipid classes were altered in an age-related manner. This "lipid raft aging" exhibits clear gender differences and appear to be more pronounced in women than in men, especially in older (postmenopausal) women. The outcomes led us to conclude that human cortical lipid rafts are modified by aging in a gender-dependent fashion. Given the central role of bilayer lipid matrix in lipid rafts functionality and neuronal signaling, we hypothesize that these findings might underlie the higher prevalence of cognitive decline evolving toward Alzheimer's disease in postmenopausal women., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Hepatocyte proliferation during liver regeneration is a well-coordinated process regulated by the activation of several growth factor receptors, including the insulin receptor (IR). The IR can be localized in part to cholesterol-enriched membrane microdomains, but the role of such domains in insulin-mediated events in hepatocytes is not known. We investigated whether partitioning of IRs into cholesterol-enriched membrane rafts is important for the mitogenic effects of insulin in the hepatic cells. IR and lipid rafts were labeled in HepG2 cells and primary rat hepatocytes. Membrane cholesterol was depleted in vitro with metyl-β-cyclodextrin (MβCD) and in vivo with lovastatin. Insulin-induced calcium (Ca 2+ ) signals studies were examined in HepG2 cells and in freshly isolated rat hepatocytes as well as in whole liver in vivo by intravital confocal imaging. Liver regeneration was studied by 70% partial hepatectomy (PH), and hepatocyte proliferation was assessed by PCNA staining. A subpopulation of IR was found in membrane microdomains enriched in cholesterol. Depletion of cholesterol from plasma membrane resulted in redistribution of the IR along the cells, which was associated with impaired insulin-induced nuclear Ca 2+ signals, a signaling event that regulates hepatocyte proliferation. Cholesterol depletion also led to ERK1/2 hyper-phosphorylation. Lovastatin administration to rats decreased hepatic cholesterol content, disrupted lipid rafts and decreased insulin-induced Ca 2+ signaling in hepatocytes, and delayed liver regeneration after PH. Therefore, membrane cholesterol content and lipid rafts integrity showed to be important for the proliferative effects of insulin in hepatic cells. NEW & NOTEWORTHY One of insulin's actions is to stimulate liver regeneration. Here we show that a subpopulation of insulin receptors is in a specialized cholesterol-enriched region of the cell membrane and this subfraction is important for insulin's proliferative effects.

The plasma membrane is a ∼4 nm thick phospholipid bilayer that defines the boundary of a cell, segregating internal content from the external environment. Its hydrophobic interior presents a barrier to the exchange of ions and polar solutes between the inside and outside of the cell, as well as to the spontaneous reorientation of lipids between the two leaflets of the bilayer. Specific transport systems, e.g. ion channels and lipid flippases, are needed to enable the passage of these molecules across the plasma membrane at physiologically relevant rates. Although the influential fluid mosaic membrane model of 1972 depicted the membrane as an archipelago of protein islands within a uniform sea of lipids, its micrometer-scale lateral heterogeneity was recognized relatively quickly, evolving into the current picture of structural granularity at the nanoscale., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

During metastatic dissemination, cancer cells experience shear stresses in narrow confinements of in vivo vasculature. Such stresses are currently known to influence a gamut of cellular processes. While a host of cells emanating from a primary tumor perish in circulation due to shear, some cells manage to migrate to distant niches and form secondary tumors. Current research focuses on how cancer cells avert such mechanical stresses and adapt themselves in order to survive. This study deals with the autophagic response of cervical cancer cells HeLa and its subline HeLa 229, exposed to physiological shear stresses in vitro, and evaluates its role as a pro-survival mechanism. It also delineates the probable mechanotransduction pathway that is involved in eliciting the stress-adaptive response in cervical cancer cells. Our results show that shear stress of physiological regime elicits protective autophagy in cervical cancer cells as an immediate response and inhibiting the same, leads to early onset of apoptosis. An effort to study the underlying mechanotransduction revealed that autophagy induction by shear stress requires intact lipid rafts which serve as signalling platforms to trigger phosphorylation of p38 mitogen activated protein kinases, leading to autophagy. This study thus gives novel insights into the mechanobiology of cervical cancer and hints at promising therapeutic targets in metastasis, the major cause of cancer mortality.

Background: Matrix vesicles (MVs) are released from hypertrophic chondrocytes and from mature osteoblasts, the cells responsible for endochondral and membranous ossification. Under pathological conditions, they can also be released from cells of non-skeletal tissues such as vascular smooth muscle cells. MVs are extracellular vesicles of approximately 100-300nm diameter harboring the biochemical machinery needed to induce mineralization., Scope of the Review: The review comprehensively delineates our current knowledge of MV biology and highlights open questions aiming to stimulate further research. The review is constructed as a series of questions addressing issues of MVs ranging from their biogenesis and functions, to biomimetic models. It critically evaluates experimental data including their isolation and characterization methods, like lipidomics, proteomics, transmission electron microscopy, atomic force microscopy and proteoliposome models mimicking MVs., Major Conclusions: MVs have a relatively well-defined function as initiators of mineralization. They bind to collagen and their composition reflects the composition of lipid rafts. We call attention to the as yet unclear mechanisms leading to the biogenesis of MVs, and how minerals form and when they are formed. We discuss the prospects of employing upcoming experimental models to deepen our understanding of MV-mediated mineralization and mineralization disorders such as the use of reconstituted lipid vesicles, proteoliposomes and, native sample preparations and high-resolution technologies., General Significance: MVs have been extensively investigated owing to their roles in skeletal and ectopic mineralization. MVs serve as a model system for lipid raft structures, and for the mechanisms of genesis and release of extracellular vesicles., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The plasma membrane of living cells is compartmentalized at multiple spatial scales ranging from the nano- to the mesoscale. This nonrandom organization is crucial for a large number of cellular functions. At the nanoscale, cell membranes organize into dynamic nanoassemblies enriched by cholesterol, sphingolipids, and certain types of proteins. Investigating these nanoassemblies known as lipid rafts is of paramount interest in fundamental cell biology. However, this goal requires simultaneous nanometer spatial precision and microsecond temporal resolution, which is beyond the reach of common microscopes. Optical antennas based on metallic nanostructures efficiently enhance and confine light into nanometer dimensions, breaching the diffraction limit of light. In this Perspective, we discuss recent progress combining optical antennas with fluorescence correlation spectroscopy (FCS) to monitor microsecond dynamics at nanoscale spatial dimensions. These new developments offer numerous opportunities to investigate lipid and protein dynamics in both mimetic and native biological membranes.

The field of tissue engineering has not yet provided knowledge on which a consensus for the complex physiological relevance (CPR) of neuronal cultures could be established. The CPR of 3D neuronal cultures can have a profound impact on the drug discovery process through the validation of in vitro models for the study of neuropsychiatric and degenerative diseases, as well as screening for neurotoxicity during drug development. Herein, we assemble evidence in support of the potential of [Ca 2+ ] i oscillation frequency as a CPR outcome that can demonstrate the in vivo-like behavior of 3D cultures and differentiate them from 2D monolayers. We demonstrate that [Ca 2+ ] i oscillation frequencies in 2D cultures are significantly higher than those found in 3D cultures, and provide a possible molecular explanation., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: Adhesion of cells to the extracellular matrix is facilitated by integrin receptors. We recently found that a nanoscale organization of plasma membrane located integrins containing the β1 subunit is responsible for an enhanced radio-resistance in 3D cultured cells over cells grown in 2D. While ionizing radiation is known to have broad effects on the lipid composition of the plasma membrane and their organization in lipid-rafts, it is not clear whether the effects of ionizing radiation on the nanoscale clustering of integrins is lipid-raft dependent., Results: Using single molecule microscopy we can show that β1 integrins colocalize with cholesterol in lipid-rafts. Ionizing radiation, as an extrinsic stressor, causes the separation of β1 integrins from cholesterol lipid raft suggesting that the effects of ionizing radiation on the clustering of β1 integrins are lipid-raft independent.

Infectious diseases pose major socioeconomic and health-related threats to millions of people across the globe. Strategies to combat infectious diseases derive from our understanding of the complex interactions between the host and specific bacterial, viral, and fungal pathogens. Lipid rafts are membrane microdomains that play important role in life cycle of microbes. Interaction of microbial pathogens with host membrane rafts influences not only their initial colonization but also their spread and the induction of inflammation. Therefore, intervention strategies aimed at modulating the assembly of membrane rafts and/or regulating raft-directed signaling pathways are attractive approaches for the. management of infectious diseases. The current review discusses the latest advances in terms of techniques used to study the role of membrane microdomains in various pathological conditions and provides updated information regarding the role of membrane rafts during bacterial, viral and fungal infections.

Autophagy plays important roles in maintaining cellular homeostasis. It uses double- or multiple-membrane vesicles termed autophagosomes to remove protein aggregates and damaged organelles from the cytoplasm for recycling. Hepatitis C virus (HCV) has been shown to induce autophagy to enhance its own replication. Here we describe a procedure that combines membrane flotation and affinity chromatography for the purification of autophagosomes from cells that harbor an HCV subgenomic RNA replicon. The purified autophagosomes had double- or multiple-membrane structures with a diameter ranging from 200 nm to 600 nm. The analysis of proteins associated with HCV-induced autophagosomes by proteomics led to the identification of HCV nonstructural proteins as well as proteins involved in membrane trafficking. Notably, caveolin-1, caveolin-2, and annexin A2, which are proteins associated with lipid rafts, were also identified. The association of lipid rafts with HCV-induced autophagosomes was confirmed by Western blotting, immunofluorescence microscopy, and immunoelectron microscopy. Their association with autophagosomes was also confirmed in HCV-infected cells. The association of lipid rafts with autophagosomes was specific to HCV, as it was not detected in autophagosomes induced by nutrient starvation. Further analysis indicated that the autophagosomes purified from HCV replicon cells could mediate HCV RNA replication in a lipid raft-dependent manner, as the depletion of cholesterol, a major component of lipid rafts, from autophagosomes abolished HCV RNA replication. Our studies thus demonstrated that HCV could specifically induce the association of lipid rafts with autophagosomes for its RNA replication. IMPORTANCE HCV can cause severe liver diseases, including cirrhosis and hepatocellular carcinoma, and is one of the most important human pathogens. Infection with HCV can lead to the reorganization of membrane structures in its host cells, including the induction of autophagosomes. In this study, we developed a procedure to purify HCV-induced autophagosomes and demonstrated that HCV could induce the localization of lipid rafts to autophagosomes to mediate its RNA replication. This finding provided important information for further understanding the life cycle of HCV and its interaction with the host cells., (Copyright © 2017 American Society for Microbiology.)

Nanotubes (NTs) are thin, long membranous structures forming novel, yet poorly known communication pathways between various cell types. Key mechanisms controlling their growth still remained poorly understood. Since NT-forming capacity of immature and mature B cells was found largely different, we investigated how lipid composition and molecular order of the membrane affect NT-formation. Screening B cell lines with various differentiation stages revealed that NT-growth linearly correlates with membrane ganglioside levels, while it shows maximum as a function of cholesterol level. NT-growth of B lymphocytes is promoted by raftophilic phosphatidylcholine and sphingomyelin species, various glycosphingolipids, and docosahexaenoic acid-containing inner leaflet lipids, through supporting membrane curvature, as demonstrated by comparative lipidomic analysis of mature versus immature B cell membranes. Targeted modification of membrane cholesterol and sphingolipid levels altered NT-forming capacity confirming these findings, and also highlighted that the actual lipid raft number may control NT-growth via defining the number of membrane-F-actin coupling sites. Atomic force microscopic mechano-manipulation experiments further proved that mechanical properties (elasticity or bending stiffness) of B cell NTs also depend on the actual membrane lipid composition. Data presented here highlight importance of the lipid side in controlling intercellular, nanotubular, regulatory communications in the immune system., (Copyright © 2017. Published by Elsevier B.V.)

Endogenous electric fields (EFs) are involved in developmental regulation and wound healing. Although the phenomenon is known for more than a century, it is not clear how cells perceive the external EF. Membrane proteins, responding to electrophoretic and electroosmotic forces, have long been proposed as the sensing molecules. However, specific charge modification of surface proteins did not change cell migration motility nor directionality in EFs. Moreover, symmetric alternating current (AC) EF directs cell migration in a frequency-dependent manner. Due to their charge and ability to coalesce, glycolipids are therefore the likely primary EF sensor driving polarization of membrane proteins and intracellular signaling. We demonstrate that detergent-resistant membrane nanodomains, also known as lipid rafts, are the primary response element in EF sensing. The clustering and activation of caveolin and signaling proteins further stabilize raft structure and feed-forward downstream signaling events, such as rho and PI3K activation. Theoretical modeling supports the experimental results and predicts AC frequency-dependent cell and raft migration. Our results establish a fundamental mechanism for cell electrosensing and provide a role in lipid raft mechanotransduction., Competing Interests: The authors declare no conflict of interest.

In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or "lipid rafts." Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered ( l o ) domains in the freestanding parts of the PSMs: ( i ) immobile domains that were attached to the pore rims and ( ii ) mobile, round-shaped l o domains within the center of the PSMs. Analysis of the diffusion of the mobile l o domains by video microscopy and particle tracking showed that the domains' mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms., Competing Interests: The authors declare no conflict of interest.

Phosphatidylinositol-3-phosphate (PI3P) is a key player in membrane dynamics and trafficking regulation. Most PI3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI3P synthesis, VPS34 and proteins such as Beclin1 and ATG14L that regulate PI3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI3P-binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI3P during autophagy, notably during the autophagosome biogenesis sequence., (© 2016 Federation of European Biochemical Societies.)

The observation of acetylcholinesterase (AChE) type H (AChE H ), which is the predominant AChE variant in visceral organs and immune cells, in lipid rafts of muscle supports functional reasons for the raft targeting of glypiated AChE H The search for these reasons revealed that liver AChE activity is mostly confined to rafts and that the liver is able to make N-extended AChE variants and target them to rafts. These results prompted us to test whether AChE and muscarinic receptors existed in the same raft. Isolation of flotillin-2-rich raft fractions by their buoyancy in sucrose gradients, followed by immunoadsorption and matrix-assisted laser desorption ionization-time of flight-mass spectrometry application, gave the following results: 1) most hepatic AChE activity emanates from AChE-H mRNA, and its product, glypiated AChE H , accumulates in rafts; 2) N-extended N-AChE readthrough variant, nonglypiated N-AChE H , and N-AChE tailed variant were all identified in liver rafts; and 3) M3 AChRs were observed in rafts, and coprecipitation of raft-confined N-AChE and M3 receptors by using anti-M3 antibodies showed that enzyme and receptor reside in the same raft unit. A raft domain that harbors tightly packed muscarinic receptor and AChE may represent a molecular device that, by means of which, the intensity and duration of cholinergic inputs are regulated.-Montenegro, M. F., Cabezas-Herrera, J., Campoy, F. J., Muñoz-Delgado, E., Vidal, C. J. Lipid rafts of mouse liver contain nonextended and extended acetylcholinesterase variants along with M3 muscarinic receptors., (© FASEB.)

Objective/background: Mycobacterium lepraemurium (MLM), the etiologic agent of murine leprosy, is an intracellular parasite of macrophages; the mechanism used by this bacterium to enter macrophages is not known. The fate of the MLM phagosome inside macrophages is also unknown. This study was conducted to investigate how MLM enters macrophages and to define the maturation process of MLM phagosome inside macrophages., Materials and Methods: Peritoneal macrophages were incubated in the presence of mannan-bovine serum albumin (BSA), and antibodies to known macrophage receptors, including, anti-FcγRIII/RII (anti-CD16/32), anti-CD35 (anti-CR1), anti-TLR2, anti-TLR4, anti-TLR6, anti-CD14, and anti-dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). Then, macrophages were challenged with Iris Fuchsia-stained MLM, at a multiplicity of infection of 50:1. The blocking effect of the antibodies (and mannan-BSA) used was analyzed using direct microscopy and flow cytometry. The maturation process of MLM phagosomes was visualized by their interaction with antibodies to Rab5, Rab7, proton ATPase, and cathepsin D, by confocal microscopy., Results: Only mannan-BSA and anti-TLR6 antibody significantly blocked the entry of MLM into macrophages. None of the other antibodies, including that for DC-SIGN, meaningfully inhibited the endocytic process. We also found that MLM is a fusiogenic mycobacterium. This was deduced from the orderly association of MLM phagosomes with Rab5, Rab7, Proton ATPase, and lysosomes (cathepsin D)., Conclusion: Fusion of MLM phagosomes with lysosomes seems to be a necessary event for the intracellular multiplication of MLM; similar to Mycobacterium leprae, this microorganism hardly grows on artificial, synthetic, bacteriologic media.