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The interplay between membrane subregions and receptor tyrosine kinases (RTK) will influence signaling in both normal and pathological RTK conditions. In this study, epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor β (PDGFR-β) internalizations were investigated by immunofluorescent microscopy following simultaneous treatment with EGF and PDGF-BB. We found that the two receptors utilize separate routes of internalization, which merges in a common perinuclear endosomal compartment after 45 min of stimulation. This is further strengthened when contrasting the recruitment of either EGFR or PDGFR-β to either clathrin or caveolin-1: PDGFR-β dissociates from caveolin-1 upon stimulation, and engages clathrin, whilst an increased recruitment of EGFR, to both clathrin and caveolin-1, was observed upon EGF stimulation. The association between EGFR and caveolin-1 is supported by the observation that EGFR was localized in lipid raft associated fractions, whereas PDGFR-β was not. We also found that disruption of lipid rafts using MβCD led to an increased EGFR dimerization and phosphorylation in response to ligand, as well as a dramatic decrease in AKT- and a smaller but robust decrease in ERK1/2 phosphorylation. This suggest that lipid rafts may be important to effectively connect the EGFR with downstream proteins to facilitate signaling. Our data implies that cholesterol depletion of the plasma membrane affect the signaling of EGFR and PDGFRβ differently.

The World Health Organization has recognized that testicular function is temperature dependent. Testicular heat exposure caused by occupational factors, lifestyle, and clinical diseases can lead to different degrees of reproductive problems. The aim of this study was to reveal the transcriptional regulatory network and its potential crucial roles in mediating the effects of testicular heat exposure. Testicular tissue was collected from a group of mice subjected to scrotal heat exposure as well as a control group. RNA was isolated from both groups and used for high-throughput sequencing. Using differential transcriptome expression analysis, 172 circRNAs, 279 miRNAs, 465 lncRNAs, and 2721 mRNAs were identified as significantly differentially expressed in mouse testicular tissue after heat exposure compared with the control group. Through Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, differentially expressed lncRNAs and mRNAs were found to have potentially important functions in meiotic cell cycle (GO:0051321), cytoplasm (GO:0005737), membrane raft (GO:0045121), MAPK signaling (mmu04010), purine metabolism (mmu00230), and homologous recombination (mmu03440). Some of the most upregulated and downregulated lncRNAs and circRNAs were predicted to be associated with numerous miRNAs and mRNAs through competing endogenous RNA regulatory network analysis, which were validated with molecular biology experiments. This research provides high-throughput sequencing data of a testicular heat exposure model and lays the foundation for further study on circRNAs, miRNAs, and lncRNAs that are involved in male reproductive diseases related to elevated testicular temperature.

Melanoma is a highly aggressive malignant skin tumor with a high rate of metastasis and mortality. In this study, a comprehensive bioinformatics analysis was used to clarify the hub genes and potential drugs. Download the GSE3189, GSE22301, and GSE35388 microarray datasets from the Gene Expression Omnibus (GEO), which contains a total of 33 normal samples and 67 melanoma samples. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) approach analyze DEGs based on the DAVID. Use STRING to construct protein-protein interaction network, and use MCODE and cytoHubba plug-ins in Cytoscape to perform module analysis and identified hub genes. Use Gene Expression Profile Interactive Analysis (GEPIA) to assess the prognosis of genes in tumors. Finally, use the Drug-Gene Interaction Database (DGIdb) to screen targeted drugs related to hub genes. A total of 140 overlapping DEGs were identified from the three microarray datasets, including 59 up-regulated DEGs and 81 down-regulated DEGs. GO enrichment analysis showed that these DEGs are mainly involved in the biological process such as positive regulation of gene expression, positive regulation of cell proliferation, positive regulation of MAP kinase activity, cell migration, and negative regulation of the apoptotic process. The cellular components are concentrated in the membrane, dendritic spine, the perinuclear region of cytoplasm, extracellular exosome, and membrane raft. Molecular functions include protein homodimerization activity, calmodulin-binding, transcription factor binding, protein binding, and cytoskeletal protein binding. KEGG pathway analysis shows that these DEGs are mainly related to protein digestion and absorption, PPAR signaling pathway, signaling pathways regulating stem cells' pluripotency, and Retinol metabolism. The 23 most closely related DEGs were identified from the PPI network and combined with the GEPIA prognostic analysis, CDH3, ESRP1, FGF2, GBP2, KCNN4, KIT, SEMA4D, and ZEB1 were selected as hub genes, which are considered to be associated with poor prognosis of melanoma closely related. Besides, ten related drugs that may have therapeutic effects on melanoma were also screened. These newly discovered genes and drugs provide new ideas for further research on melanoma.

The local-regional oncological hyperthermia has various electromagnetic methods for energy-transfer. The differences involve conceptual considerations and technical solutions. The most frequently applied energy transfer is capacitive coupling, concentrating the electric field to be the active heating component. The realization of the capacitive coupling set-up is divided into two different categories based on their goals for heating: 1) the homogeneous (conventional) heating, using isothermal conditions for dosing, and 2) the selective heterogeneous heating, using cellularly absorbed energy for dosing. The homogeneous heating utilizes plane-wave matching, absorbing the wave for energy transfer. The heterogenic heating uses impedance matching, selecting the malignant cells by their electromagnetic specialties, like their heterogenic impedance, higher membrane-raft density, and different spatio-temporal (pathologic pattern) arrangements. This article’s objective is to compare and discuss the details of the two kinds of capacitive coupling techniques.

During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1. Electronic supplementary material The online version of this article (10.1007/s00018-020-03663-z) contains supplementary material, which is available to authorized users.

Membrane proteins and lipids have the capacity to associate into lateral domains in cell membranes through mutual or collective interactions. Lipid rafts are functional lateral domains that are formed through collective interactions of certain lipids and which can include or exclude proteins. These domains have been implicated in cell signaling and protein trafficking and seem to be of importance for virus-host interactions. We therefore want to investigate if raft and viral membrane proteins present similar structural features, and how these features are distributed throughout viruses. For this purpose, we performed a bioinformatics analysis of raft and viral membrane proteins from available online databases and compared them to nonraft proteins. In general, transmembrane proteins of rafts and viruses had higher proportions of palmitoyl and phosphoryl residues compared to nonraft proteins. They differed in terms of transmembrane domain length and thickness, with viral proteins being generally shorter and having a smaller accessible surface area per residue. Nontransmembrane raft proteins had increased amounts of palmitoyl, prenyl, and phosphoryl moieties while their viral counterparts were largely myristoylated and phosphorylated. Several of these structural determinants such as phosphorylation are new to the raft field and are extensively discussed in terms of raft functionality and phase separation. Surprisingly, the proportion of palmitoylated viral transmembrane proteins was inversely correlated to the virus size which indicated the implication of palmitoylation in virus membrane curvature and possibly budding. The current results provide new insights into the raft-virus interplay and unveil possible targets for antiviral compounds.

Background Age-related macular degeneration (AMD) represents the leading cause of visual impairment in the aging population. The goal of this study was to identify aberrantly-methylated, differentially-expressed genes (MDEGs) in AMD and explore the involved pathways via integrated bioinformatics analysis. Methods Data from expression profile GSE29801 and methylation profile GSE102952 were obtained from the Gene Expression Omnibus database. We analyzed differentially-methylated genes and differentially-expressed genes using R software. Functional enrichment and protein–protein interaction (PPI) network analysis were performed using the R package and Search Tool for the Retrieval of Interacting Genes online database. Hub genes were identified using Cytoscape. Results In total, 827 and 592 genes showed high and low expression, respectively, in GSE29801; 4117 hyper-methylated genes and 511 hypo-methylated genes were detected in GSE102952. Based on overlap, we categorized 153 genes as hyper-methylated, low-expression genes (Hyper-LGs) and 24 genes as hypo-methylated, high-expression genes (Hypo-HGs). Four Hyper-LGs (CKB, PPP3CA, TGFB2, SOCS2) overlapped with AMD risk genes in the Public Health Genomics and Precision Health Knowledge Base. KEGG pathway enrichment analysis indicated that Hypo-HGs were enriched in the calcium signaling pathway, whereas Hyper-LGs were enriched in sphingolipid metabolism. In GO analysis, Hypo-HGs were enriched in fibroblast migration, membrane raft, and coenzyme binding, among others. Hyper-LGs were enriched in mRNA transport, nuclear speck, and DNA binding, among others. In PPI network analysis, 23 nodes and two edges were established from Hypo-HGs, and 151 nodes and 73 edges were established from Hyper-LGs. Hub genes (DHX9, MAPT, PAX6) showed the greatest overlap. Conclusion This study revealed potentially aberrantly MDEGs and pathways in AMD, which might improve the understanding of this disease.

An optimized support vector machine model was used to construct a lung cancer diagnosis model based on serological indicators, and a molecular regulation model of Wogonin, a component of Scutellaria baicalensis, was established. Serological indexes of patients were collected, the grid search method was used to identify the optimal penalty coefficient C and parameter g of the support vector machine model, and the benign and malignant auxiliary diagnosis model of isolated pulmonary nodules based on serological indicators was established. The regulatory network and key targets of Wogonin in lung cancer were analyzed by network pharmacology, and key targets were detected by western blot. The relationship between serological susceptibility genes and key targets of Wogonin was established, and the signaling pathway of Wogonin regulating lung cancer was constructed. After support vector machine parameter optimization (C = 90.597, g = 32), the accuracy of the model was 90.8333%, with nine false positives and two false negative cases. Ontology functional analysis of 67 common genes between Wogonin targets and lung cancer–related genes showed that the targets were associated with biological processes involved in peptidye-serine modification and regulation of protein kinase B signaling; cell components in the membrane raft and chromosomal region; and molecular function in protein serine/threonine kinase activity and heme binding. Kyoto Encyclopedia of Genes and Genomes analysis showed that the regulation pathways involved the PI3K-Akt signaling pathway, ERBB signaling pathway, and EGFR tyrosine kinase inhibitor resistance. In vitro analyses using lung cancer cells showed that Wogonin led to significantly increased levels of cleaved caspase-3 and Bad and significantly decreased Bcl-2 expression in a concentration-dependent manner. ErbB4 expression also significantly decreased in lung cancer cells after treatment with Wogonin. A regulatory network of Wogonin regulating lung cancer cell apoptosis was constructed, including the participation of serological susceptibility genes. There is a certain regulatory effect between the serological indexes that can be used in the diagnosis of lung cancer and the key targets of Chinese herbal medicine treatment of lung cancer, which provides a new idea for the diagnosis, treatment and prognosis of clinical lung cancer.

Membrane rafts are heterogeneous and dynamic domains that are characterized by tight packing of lipids. They are enriched in cholesterol, sphingolipids, and certain types of proteins. Among these are various cell signaling proteins, which indicate that rafts play an important role in cell signal transduction pathways, including some involved in cancer development, progression, and invasiveness. Due to their increased cholesterol content, raft domains exhibit lower fluidity than the surrounding membrane. The cell membranes of some solid tumors, such as breast and prostate cancer, contain higher levels of cholesterol, which means larger raft domain can form in those membranes. This may stimulate signaling pathways to promote tumor growth and progression. This review focuses on the known raft-dependent regulatory mechanisms that promote prostate cancer progression.Impact statementProstate cancer remains the most common malignancy and second most frequent cause of cancer-related death in men. Cholesterol levels are usually higher in prostate cancer cells. This affects the cell membrane composition, with cholesterol and sphingolipid-containing raft membrane domains becoming a greater component. In addition to polar lipids, these domains recruit and regulate certain types of protein, including various cell signaling proteins that are critical to cancer cell survival and invasiveness. This suggests that membrane rafts have a regulatory role in tumor progression, making them a potential target in prostate cancer treatment.

Background The roles played by cholesterol in cancer development and progression represent a popular field in the cancer community. High cholesterol levels are positively correlated with the risk of various types of cancer. APOA-I binding protein (AIBP) promotes the reverse cholesterol transport pathway (RCT) in cooperation with Apolipoprotein A-I (APOA-I) or high-density lipoprotein cholesterol. However, the combined effect of AIBP and APOA-I on intestinal tumor cells is still unclear. Methods Immunohistochemistry, western blot and qPCR were performed to investigate the expression of AIBP and APOA-I in intestinal tumor tissues and cell lines. The anti-tumor activity of AIBP and APOA-I was evaluated by overexpression or recombinant protein treatment. Cholesterol efflux and localization of lipid raft-related proteins were analyzed by a cholesterol efflux assay and lipid raft fraction assay, respectively. Results Here, we reported that both AIBP expression and APOA-I expression were associated with the degree of malignancy in intestinal tumors. Co-overexpression of AIBP and APOA-I more potently inhibited colon cancer cell-mediated tumor growth and metastasis compared to overexpression of each protein individually. Additionally, the recombinant fusion proteins of AIBP and APOA-I exhibited a significant therapeutic effect on tumor growth in Apcmin/+ mice as an inherited intestinal tumor model. The synergistic effect of the two proteins inhibited colon cancer cell migration, invasion and tumor-induced angiogenesis by promoting cholesterol efflux, reducing the membrane raft content, and eventually disrupting the proper localization of migration- and invasion-related proteins on the membrane raft. Moreover, cyclosporine A, a cholesterol efflux inhibitor, rescued the inhibitory effect induced by the combination of AIBP and APOA-I. Conclusions These results indicate that the combination of APOA-I and AIBP has an obvious anticancer effect on colorectal cancer by promoting cholesterol efflux. Electronic supplementary material The online version of this article (10.1186/s12967-019-1910-7) contains supplementary material, which is available to authorized users.

NEW FINDINGS What is the central question of this study? Is the membrane raft redox signalling pathway involved in blood pressure increase, endothelial dysfunction and vascular remodelling in an angiotensin II-induced hypertensive animal model? What is the main finding and its importance? The membrane raft redox signalling pathway was involved in endothelial dysfunction and medial remodelling in angiotensin II-induced hypertension. ABSTRACT The membrane raft (MR) redox pathway is characterized by NADPH oxidase activation via the clustering of its subunits through lysosome fusion and the activation of acid sphingomyelinase (ASMase). Our previous study shows that the MR redox signalling pathway is associated with angiontensin II (AngII)-induced production of reactive oxygen species (ROS) and endothelial dysfunction in rat mesenteric arteries. In the present study, we hypothesized that this signalling pathway is involved in blood pressure increase, endothelial dysfunction and vascular remodelling in an AngII-induced hypertensive animal model. Sixteen-week-old male Sprague-Dawley rats were subjected to AngII infusion for 2 weeks with or without treatment with the lysosome fusion inhibitor bafilomycin A1 and ASMase inhibitor amitriptyline. After treatments, aortas were harvested for further study. The results showed that the MR redox signalling pathway was activated as indicated by the increase of MR formation, ASMase activity and ROS production in aorta from AngII-infused rats compared with that from control rats. MR formation and ROS production were significantly inhibited in thoracic aorta from AngII-induced rats treated with bafilomycin A1 and amitriptyline. Both treatments significantly attenuated blood pressure increase, endothelial dysfunction and vascular remodelling including medial hypertrophy, and increased collagen and fibronectin deposition in thoracic aortas from AngII-infused rats. Finally, both treatments significantly prevented the increase of inflammatory factors including monocyte chemotactic protein 1, intercellular adhesion molecule 1 and tumour necrosis factor α in thoracic aorta from AngII-infused rats. In conclusion, the present study demonstrates that the MR redox signalling pathway was involved in endothelial dysfunction and medial remodelling in AngII-induced hypertension.

Background The mammalian epididymis is responsible for the provision of a highly specialized environment in which spermatozoa acquire functional maturity and are subsequently stored in preparation for ejaculation. Making important contributions to both processes are epididymosomes, small extracellular vesicles released from the epididymal soma via an apocrine secretory pathway. While considerable effort has been focused on defining the cargo transferred between epididymosomes and spermatozoa, comparatively less is known about the mechanistic basis of these interactions. To investigate this phenomenon, we have utilized an in vitro co-culture system to track the transfer of biotinylated protein cargo between mouse epididymosomes and recipient spermatozoa isolated from the caput epididymis; an epididymal segment that is of critical importance for promoting sperm maturation. Results Our data indicate that epididymosome-sperm interactions are initiated via tethering of the epididymosome to receptors restricted to the post-acrosomal domain of the sperm head. Thereafter, epididymosomes mediate the transfer of protein cargo to spermatozoa via a process that is dependent on dynamin, a family of mechanoenzymes that direct intercellular vesicle trafficking. Notably, upon co-culture of sperm with epididymosomes, dynamin 1 undergoes a pronounced relocation between the peri- and post-acrosomal domains of the sperm head. This repositioning of dynamin 1 is potentially mediated via its association with membrane rafts and ideally locates the enzyme to facilitate the uptake of epididymosome-borne proteins. Accordingly, disruption of membrane raft integrity or pharmacological inhibition of dynamin both potently suppress the transfer of biotinylated epididymosome proteins to spermatozoa. Conclusion Together, these data provide new mechanistic insight into epididymosome-sperm interactions with potential implications extending to the manipulation of sperm maturation for the purpose of fertility regulation. Electronic supplementary material The online version of this article (10.1186/s12915-019-0653-5) contains supplementary material, which is available to authorized users.

The proliferation epidermal growth factor (EGF) is known to acquire contradictory apoptotic activities upon conjugation with gold nanoparticles (GNPs) through hitherto unknown mechanisms. Here, we identified an essential role of membrane rafts in the drastic activity switching of EGF-GNPs through the following intracellular signaling. (1) In contrast to the rapid diffusion of activated EGF receptor after the soluble EGF stimulation, the receptor is confined within membrane rafts upon binding to the EGF-GNPs. (2) This initial receptor confinements switch its endocytosis process from normal clathrin-mediated endocytosis to caveolin-mediated one, changing the phosphorylation dynamics of essential downstream kinases, i.e., extracellular signal-regulated kinase and AKT. Importantly, the destruction of membrane rafts by β-cyclodextrin reversed this trafficking and signaling, restoring EGF-GNPs to lost anti-apoptotic property. These results reveal the importance of GNP-mediated signal condensation at membrane rafts in conferring the unique apoptotic activity on EGF-nanoparticle conjugates. STATEMENT OF SIGNIFICANCE: Epidermal growth factor (EGF) is a small secretory protein that induces cell proliferation upon binding to its receptor existed on cellular plasma membranes. One interesting feature of the protein in the nanobiology field is, its acquisition of apoptosis-inducing (cellular suicide) activity rather than proliferative one upon conjugation to gold nanoparticles through hitherto unknown mechanisms. Here, we identified the involvement of membrane rafts, plasma membrane nanodomains enriched with cholesterol, in the apoptosis processes by changing the receptor trafficking and downstream signal transduction pathways. Moreover, the destruction of lipid rafts restored the EGF-nanoparticle conjugates with lost anti-apoptotic activity. These finding highlight potential applications of EGF-nanoparticle conjugates to cancer therapy, as the EGF receptor are highly expressed in cancer cells.

Transmembrane protein 30A (Tmem30a) is the β-subunit of P4-ATPases which function as flippase that transports aminophospholipids such as phosphatidylserine from the outer to the inner leaflets of the plasma membrane to maintain asymmetric distribution of phospholipids. It has been documented that deficiency of Tmem30a led to exposure of phosphatidylserine. However, the role of Tmem30a in vivo remains largely unknown. Here we found that Vav-Cre-driven conditional deletion of Tmem30a in hematopoietic cells led to embryonic lethality due to severe anemia by embryonic day 16.5. The numbers of erythroid colonies and erythroid cells were decreased in the Tmem30a deficient fetal liver. This was accompanied by increased apoptosis of erythroid cells. Confocal microscopy analysis revealed an increase of localization of erythropoietin receptor to areas of membrane raft microdomains in response to erythropoietin stimulation in Ter119-erythroid progenitors, which was impaired in Tmem30a deficient cells. Moreover, erythropoietin receptor (EPOR)-mediated activation of the STAT5 pathway was significantly reduced in Tmem30a deficient fetal liver cells. Consistently, knockdown of TMEM30A in human CD34+ cells also impaired erythropoiesis. Our findings demonstrate that Tmem30a plays a critical role in erythropoiesis by regulating the EPOR signaling pathway through the formation of membrane rafts in erythroid cells.

This work is devoted to the phenomenon of liquid-liquid phase separation (LLPS), which has come to be recognized as fundamental organizing principle of living cells. We distinguish separation processes with different dimensions. Well-known 3D-condensation occurs in aqueous solution and leads to membraneless organelle (MLOs) formation. 2D-films may be formed near membrane surfaces and lateral phase separation (membrane rafts) occurs within the membranes themselves. LLPS may also occur on 1D structures like DNA and the cyto- and nucleoskeleton. Phase separation provides efficient transport and sorting of proteins and metabolites, accelerates the assembly of metabolic and signaling complexes, and mediates stress responses. In this work, we propose a model in which the processes of polymerization (1D structures), phase separation in membranes (2D structures), and LLPS in the volume (3D structures) influence each other. Disordered proteins and whole condensates may provide membrane raft separation or polymerization of specific proteins. On the other hand, 1D and 2D structures with special composition or embedded IDRs can nucleate condensates. We hypothesized that environmental change may trigger a LLPS which can propagate within the cell interior moving along the cytoskeleton or as an autowave. New phase propagation quickly and using a low amount of energy adjusts cell signaling and metabolic systems to new demands. Cumulatively, the interconnected phase separation phenomena in different dimensions represent a previously unexplored system of intracellular communication and regulation which cannot be ignored when considering both physiological and pathological cell processes.

Background Posterior fossa ependymoma (EPN-PF) can be classified into Group A posterior fossa ependymoma (EPN-PFA) and Group B posterior fossa ependymoma (EPN-PFB) according to DNA CpG island methylation profile status and gene expression. EPN-PFA usually occurs in children younger than 5 years and has a poor prognosis. Methods Using epigenome and transcriptome microarray data, a multi-component weighted gene co-expression network analysis (WGCNA) was used to systematically identify the hub genes of EPN-PF. We downloaded two microarray datasets (GSE66354 and GSE114523) from the Gene Expression Omnibus (GEO) database. The Limma R package was used to identify differentially expressed genes (DEGs), and ChAMP R was used to analyze the differential methylation genes (DMGs) between EPN-PFA and EPN-PFB. GO and KEGG enrichment analyses were performed using the Metascape database. Results GO analysis showed that enriched genes were significantly enriched in the extracellular matrix organization, adaptive immune response, membrane raft, focal adhesion, NF-kappa B pathway, and axon guidance, as suggested by KEGG analysis. Through WGCNA, we found that MEblue had a significant correlation with EPN-PF (R = 0.69, P = 1 × 10–08) and selected the 180 hub genes in the blue module. By comparing the DEGs, DMGs, and hub genes in the co-expression network, we identified five hypermethylated, lower expressed genes in EPN-PFA (ATP4B, CCDC151, DMKN, SCN4B, and TUBA4B), and three of them were confirmed by IHC. Conclusion ssGSEA and GSVA analysis indicated that these five hub genes could lead to poor prognosis by inducing hypoxia, PI3K-Akt-mTOR, and TNFα-NFKB pathways. Further study of these dysmethylated hub genes in EPN-PF and the pathways they participate in may provides new ideas for EPN-PF treatment.

Background Caveolae proteins play diverse roles in cancer development and progression. In prostate cancer, non‐caveolar caveolin‐1 (CAV1) promotes metastasis, while CAVIN1 attenuates CAV1‐induced metastasis. Here, we unveil a novel mechanism linking CAV1 to selective loading of exosomes with metastasis‐promoting microRNAs. Results We identify hnRNPK as a CAV1‐regulated microRNA binding protein. In the absence of CAVIN1, non‐caveolar CAV1 drives localisation of hnRPNK to multi‐vesicular bodies (MVBs), recruiting AsUGnA motif‐containing miRNAs and causing their release within exosomes. This process is dependent on the lipid environment of membranes as shown by cholesterol depletion using methyl‐β‐cyclodextrin or by treatment with n‐3 polyunsaturated fatty acids. Consistent with a role in bone metastasis, knockdown of hnRNPK in prostate cancer PC3 cells abolished the ability of PC3 extracellular vesicles (EV) to induce osteoclastogenesis, and biofluid EV hnRNPK is elevated in metastatic prostate and colorectal cancer. Conclusions Taken together, these results support a novel pan‐cancer mechanism for CAV1‐driven exosomal release of hnRNPK and associated miRNA in metastasis, which is modulated by the membrane lipid environment., Highlights 1. Non‐caveolar caveolin‐1 drives non‐canonical localization of hnRNPK to multivesicular bodies (MVB) via membrane raft‐dependent mechanism. 2. MVB localized hnRNPK mediates EV loading of AsUGnA motif‐containing miRNA, which is required for EV‐induced in vitro osteoclastogenesiss. 3. Elevated EV hnRNPK in biofluids from metastatic cancer patients suggest a role as a biomarker.

Background. Parkinson’s disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. Liuwei Dihuang (LWDH) pills have a good effect on PD, but its mechanism remains unclear. Network pharmacology is the result of integrating basic theories and research methods of medicine, biology, computer science, bioinformatics, and other disciplines, which can systematically and comprehensively reflect the mechanism of drug intervention in disease networks. Methods. The main components and targets of herbs in LWDH pills were obtained through Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP). Its active components were screened based on absorption, distribution, metabolism, and excretion (ADME); the PD-related targets were obtained from the Genecards, OMIM, TTD, and DRUGBANK databases. We used R to take the intersection of LWDH- and PD-related targets and Cytoscape software to construct the drug-component-target network. Moreover, STRING and Cytoscape software was used to analyze protein–protein interactions (PPI), construct a PPI network, and explore potential protein functional modules in the network. The Metascape platform was used to perform KEGG pathway and GO function enrichment analyses. Finally, molecular docking was performed to verify whether the compound and target have good binding activity. Results. After screening and deduplication, 210 effective active ingredients, 204 drug targets, 4333 disease targets, and 162 drug-disease targets were obtained. We consequently constructed a drug-component-targets network and a PPI-drug-disease-targets network. The results showed that the hub components of LWDH pills were quercetin, stigmasterol, kaempferol, and beta-sitosterol; the hub targets were AKT1, VEGFA, and IL6. GO and KEGG enrichment analyses showed that these targets are involved in neuronal death, G protein-coupled amine receptor activity, reactive oxygen species metabolic processes, membrane rafts, MAPK signaling pathways, cellular senescence, and other biological processes. Molecular docking showed that the hub components were in good agreement with the hub targets. Conclusion. LWDH pills have implications for the treatment of PD since they contain several active components, target multiple ligands, and activate various pathways. The hub components possibly include quercetin, stigmasterol, kaempferol, and beta-sitosterol and act through pairing with hub targets, such as AKT1, VEGFA, and IL6, to regulate neuronal death, G protein-coupled amine receptor activity, reactive oxygen species metabolic process, membrane raft, MAPK signaling pathway, and cellular senescence for the treatment of PD.

PurposeChanges in the retina and the choroid blood vessels are regularly observed in myopia. The aim of this study is to test if the retinal glial cells, which directly contact blood vessels, play a role in mammalian myopia.MethodWe adapted the common form-deprivation myopia mouse model and used the retina slice and whole-mount immunofluorescence technique to evaluate changes in the morphology, and distribution of retinal glial cells. We then searched the Gene Expression Omnibus database for series on myopia and retinal glial cells (astrocytes and Müller cells). Using review articles and the National Center for Biotechnology Information gene database, we obtained clusters of myopia-related gene lists. By searching SwissTargetPrediction, we collected information on atropine target proteins. We then used online tools to find the Gene Ontology analysis enriched clusters, pathways, and proteins that provide correlative evidence.ResultGlial fibrillary acidic protein fluorescence was observed in mice eyes that were covered and deprived of light for five days compared to uncovered eyes, and the cell morphology became more star-like. From in silico experiments, we identified several pathways and proteins that were common to both myopia and retinal glial cells in hypoxic conditions. The common pathways in human astrocytes represented response to mechanical stimuli, peptide secretion, skeletal system development, and monosaccharide binding. In mouse Müller cells, the pathways represented membrane raft and extracellular structure organisation. The proteins common to myopia and hypoxic glial cells were highly relevant to atropine target proteins.ConclusionRetinal astrocytes and Müller cells under hypoxic conditions contribute to the development of myopia, and may be a valid target for atropine.

Processing of the amyloid precursor protein (APP) via the amyloidogenic pathway is associated with the etiology of Alzheimer’s disease. The cleavage of APP by β-secretase to generate the transmembrane 99-residue C-terminal fragment (C99) and subsequent processing of C99 by γ-secretase to yield amyloid-β (Aβ) peptides are essential steps in this pathway. Biochemical evidence suggests amyloidogenic processing of C99 occurs in cholesterol- and sphingolipid-enriched liquid ordered phase membrane raft domains. However, direct evidence that C99 preferentially associates with rafts has remained elusive. Here, we test this idea by quantifying the affinity of C99-GFP for raft domains in cell-derived giant plasma membrane vesicles. We find that C99 is essentially excluded from ordered domains in HeLa cells, SH-SY5Y cells and neurons, instead exhibiting a strong (roughly 90%) affinity for disordered domains. The strong association of C99 with disordered domains occurs independently of its cholesterol binding activity, homodimerization, or the familial Alzheimer disease Arctic mutation. Finally, we confirm previous studies suggesting that C99 is processed in the plasma membrane by α-secretase, in addition to the well-known γ-secretase. These findings suggest that C99 itself lacks an intrinsic affinity for raft domains, implying either that amyloidogenic processing of the protein occurs in disordered regions of the membrane, that processing involves a marginal sub-population of C99 found in rafts, or that as-yet-unidentified protein-protein interactions involving C99 in living cells drive it into rafts to promote its cleavage therein.

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

10-gingerol is a major phenolic lipid found in the rhizomes of ginger (Zingiber officinale). Being amphiphilic in nature, phenolic lipids have the ability to incorporate into cell membranes and modulate membrane properties. The purpose of the present study was to evaluate the effects of 10-gingerol on lipid raft/membrane raft modulation in radio-resistant triple negative breast cancer (MDA-MB-231/IR) cells. The effects of 10-gingerol on MDA-MB-231/IR cells&rsquo, proliferation, clonogenic growth, migration, and invasion were assayed using MTT, colony formation, cell migration, and invasion assays, respectively. Sucrose density gradient centrifugation was used to extract lipid rafts. Western blotting and immunofluorescence were employed to assess the effects of 10-gingerol on lipid raft/membrane raft modulation and lipid rafts-associated PI3K/Akt signaling. Cholesterol measurements were carried out using a commercially available kit. 10-gingerol suppressed the proliferation, migration, invasion, and induced apoptosis through targeting the PI3K/Akt signaling pathway in MDA-MB-231/IR cells. Moreover, 10-gingerol was found to modulate the lipid rafts of MDA-MB-231/IR cells and attenuate the key PI3K/Akt signaling components in lipid rafts. The cholesterol content of the lipid rafts and rafts-resident Akt signaling were also affected by exposure to 10-gingerol. The results of the present study highlight rafts-associated PI3K/Akt signaling as a new target of 10-gingerol in MDA-MB-231/IR cells, thus rationalizing a new rafts-mediated treatment approach for radio-resistant triple negative breast cancer cells.

Background The underlying mechanism involved in ovarian cancer stemness and chemoresistance remains largely unknown. Here, we explored whether the regulation of c-Kit and plasma membrane prohibitin (PHB) affects ovarian cancer stemness and chemotherapy resistance. Methods Mass spectrum analysis and an in vitro kinase assay were conducted to examine the phosphorylation of PHB at tyrosine 259 by c-Kit. The in vitro effects of c-Kit on membrane raft-PHB in ovarian cancer were determined using tissue microarray (TMA)-based immunofluorescence, western blotting, immunoprecipitation, colony and spheroid formation, cell migration and cell viability assays. In vivo tumor initiation and carboplatin treatment were conducted in nude mice. Results We found that c-Kit and PHB colocalized in the raft domain and were positively correlated in human ovarian serous carcinoma. c-Kit interacted with PHB and facilitated the phosphorylation of PHB at tyrosine 259 (phospho-PHBY259) in the membrane raft to enhance ovarian cancer cell motility. The generation of SKOV3GL-G4, a metastatic phenotype of SKOV3 green fluorescent protein and luciferase (GL) ovarian cancer cells, in xenograft murine ascites showed a correlation between metastatic potential and stem cell characteristics, as indicated by the expression of c-Kit, Notch3, Oct4, Nanog and SOX2. Further study revealed that after activation by c-Kit, raft-phospho-PHBY259 interacted with Notch3 to stabilize Notch3 and increase the downstream target PBX1. Downregulation of raft-phospho-PHBY259 increased the protein degradation of Notch3 through a lysosomal pathway and inhibited the β-catenin—ABCG2 signaling pathway. Moreover, raft-phospho-PHBY259 played an important role in ovarian cancer stemness and tumorigenicity as well as resistance to platinum drug treatment in vitro and in vivo. Conclusions These findings thus reveal a hitherto unreported interrelationship between c-Kit and PHB as well as the effects of raft-phospho-PHBY259 on ovarian cancer stemness and tumorigenicity mediated by the Notch3 and β-catenin signaling pathways. Targeting the c-Kit/raft-phospho-PHBY259 axis may provide a new therapeutic strategy for treating patients with ovarian cancer.

Cells 9(3), 607 (2020). doi:10.3390/cells9030607, Published by MDPI, Basel

Several G protein-coupled receptors are present in lipid rafts. We have shown that most of the P2Y2 receptor (P2Y2R) protein is fractionated into lipid rafts in COS 7 cells. In the same cells, about 25–30% of the bradykinin B2 receptor (B2R) protein is also fractionated into lipid rafts. When both P2Y2R and B2R are co-expressed, the distribution of P2Y2R remained unchanged, but more B2R shifted into the raft fraction. This indicates that the interaction between both receptors recruited B2R into the lipid rafts. After 15 min of UTP stimulation, both receptors almost completely disappeared from the cell surface by endocytosis as observed with a confocal fluorescence microscope. Furthermore, with bradykinin stimulation for 15 min, portions of both receptors disappeared from the cell surface and were endocytosed. As we reported previously with both CHO-K1 cells and HEK 293 cells, continuous stimulation of COS7 cells with GT1b and CSC resulted in the disappearance of both P2Y2R and B2R from the cell membrane surface. Thus, both P2Y2R and B2R migrate into membrane rafts and are endocytosed in parallel with signal crosstalk, clearly indicating that both closely interact on membrane rafts. The P2Y2R N-glycosylation deficient mutant does not migrate to the cell surface. It remains predominantly in the endoplasmic reticulum and is fractionated into raft fractions. In the presence of this glycosylation mutant, most of B2R remains in the endoplasmic reticulum, and is fractionated into the raft fraction. These findings demonstrate that in the membrane rafts of the endoplasmic reticulum, both receptors are already closely associated, and B2R shifts into the rafts by affinity with P2Y2R.

Hongyi Liao,1,* Xianbiao Xie,2,* Yuanyuan Xu,3 Gang Huang2 1Department of Orthopedic Surgery, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen, People’s Republic of China; 2Department of Orthopedic Oncology, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China; 3Department of Pediatrics, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of China *These authors contributed equally to this work Background: The aim of this study was to identify the driver genes associated with chemotherapy resistance of Ewing’s sarcoma and potential targets for Ewing’s sarcoma treatment.Methods: Two mRNA microarray datasets, GSE12102 and GSE17679, were downloaded from the Gene Expression Omnibus database, which contain 94 human Ewing’s sarcoma samples, including 65 from those who experienced a relapse and 29 from those with no evidence of disease. The differen­tially expressed genes (DEGs) were identified using LIMMA package R. Subsequently, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed for DEGs using Database for Annotation, Visualization and Integrated Analysis. The protein–protein interaction network was constructed using Cytoscape software, and module analysis was performed using Molecular Complex Detection.Results: A total of 206 upregulated DEGs and 141 downregulated DEGs were identified. Upregulated DEGs were primarily enriched in DNA replication, nucleoplasm and protein kinase binding for biological processes, cellular component and molecular functions, respectively. Downregulated DEGs were predominantly involved in receptor clustering, membrane raft, and ligand-dependent nuclear receptor binding. The protein–protein interaction network of DEGs consisted of 150 nodes and 304 interactions. Thirteen hub genes were identified, and biological analysis revealed that these genes were primarily enriched in cell division, cell cycle, and mitosis. Furthermore, based on closeness centrality, betweenness centrality, and degree centrality, the three most significant genes were identified as GAPDH, AURKA, and EHMT2. Furthermore, the significant network module was composed of nine genes. These genes were primarily enriched in mitotic nuclear division, mitotic chromosome condensation, and nucleoplasm.Conclusion: These hub genes, especially GAPDH, AURKA, and EHMT2, may be closely associated with the progression of Ewing’s sarcoma chemotherapy resistance, and further experiments are needed for confirmation. Keywords: Ewing’s sarcoma, chemotherapy resistance, differentially expressed gene, enrichment analysis, network

The membrane raft has been a focus of intensive research for the past two decades. Liquid-ordered domains form in artificial liposomes containing sterol and saturated lipids, but their presence in living cell membranes has been controversial. The yeast vacuole is exceptional in that micron-sized raft-like domains form in the stationary phase and under several other conditions. The sterol content of the vacuole in the log phase is much lower than that of liposomes showing liquid-ordered domains, suggesting that sterols may need to be supplied to the vacuole for the raft-like domain formation. We will discuss how lipids and lipid domains are organized in the vacuolar membrane and examine whether evidence is strong enough to conclude that the observed micron-sized domains are rafts., ファイル公開：2019/10/19

Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC.

The emerging diverse roles of ether (phospho)lipids in nervous system development and function in health and disease are currently attracting growing interest. Plasmalogens, a subgroup of ether lipids, are important membrane components involved in vesicle fusion and membrane raft composition. They store polyunsaturated fatty acids and may serve as antioxidants. Ether lipid metabolites act as precursors for the formation of glycosyl-phosphatidyl-inositol anchors; others, like platelet-activating factor, are implicated in signaling functions. Consolidating the available information, we attempt to provide molecular explanations for the dramatic neurological phenotype in ether lipid-deficient human patients and mice by linking individual functional properties of ether lipids with pathological features. Furthermore, recent publications have identified altered ether lipid levels in the context of many acquired neurological disorders including Alzheimer’s disease (AD) and autism. Finally, current efforts to restore ether lipids in peroxisomal disorders as well as AD are critically reviewed.

In neurons, intracellular membrane rafts are essential for specific actions of brain-derived neurotrophic factor (BDNF), which include the regulation of axon outgrowth, growth cone turning and synaptic transmission. Virtually, all the actions of BDNF are mediated by binding to its receptor, TrkB. The association of TrkB with the tyrosine kinase, Fyn, is critical for its localization to intracellular membrane rafts. Here, we show that synapsins, a family of highly amphipathic neuronal phosphoproteins, regulate membrane raft lipid composition and consequently, the ability of BDNF to regulate axon/neurite development and potentiate synaptic transmission. In the brains of mice lacking all synapsins, the expression of both BDNF and TrkB were increased, suggesting that BDNF/TrkB-mediated signaling is impaired. Consistent with this finding, synapsin-depleted neurons exhibit altered raft lipid composition, deficient targeting of Fyn to rafts, attenuated TrkB activation, and abrogation of BDNF-stimulated axon outgrowth and synaptic potentiation. Conversely, overexpression of synapsins in neuroblastoma cells results in corresponding reciprocal changes in raft lipid composition, increased localization of Fyn to rafts and promotion of BDNF-stimulated neurite formation. In the presence of synapsins, the ratio of cholesterol to estimated total phospholipids converged to 1, suggesting that synapsins act by regulating the ratio of lipids in intracellular membranes, thereby promoting lipid raft formation. These studies reveal a mechanistic link between BDNF and synapsins, impacting early development and synaptic transmission.

Aim: Heating by nanoparticles, which are located in the tissue to be treated, is a well-recognized method in hyperthermic oncology. Our objective is to investigate selective, nanoscopic heating without concentrating extra artificial nanoparticles. We have in silico calculation to study the heating of the transmembrane protein clusters (rafts) on cell-membrane. The transmembrane protein domains have significantly higher dielectric constant than their lipid neighborhood in the membrane. This difference causes a local gradient in the Specific Absorption Rate (SAR), which could be a factor of heating of the membranes locally, as well as exciting the receptors for various signal transduction in the cells. We suppose that this process determines the observed cellular effects of modulated electro-hyperthermia (mEHT, trade-name: oncothermia). Materials and Methods: In silico models with highly specialized software (Computer Simulation Technology (CST), Darmstadt, Germany) were performed visualizing the selectivity for the membrane domains. Local raft models were created to simulate the electromagnetic (EM) effect of a 13.56 MHz excitation between two perfect electrical conductor plates, simulating the equipotential conditions of the sides of the membrane in the vicinity of the raft. The simulations were performed with near-field (EQS) solver of CST. The electric field, current density, and electric loss density were monitored by the simulations. The applied material properties and parameters refer to the recent literature. Results: In silico models show ten times higher energy-absorption of the transmembrane domains than that of its lipid-membrane surrounding, and intra- and extracellular neighborhood. Depending on the size, orientation, and location of the membrane rafts, the value of SAR varies, but we use only two simplified models to see the absorption properties. Taking into account the characteristics of the EM field effects we showed that the selective energy-absorption increased further by the cell-cell interactions. The model-calculation could confirm the opportunity of the local membrane heating. Conclusion: Our results indicate the heating in nanoscopic range with energy-absorption by the transmembrane proteins. The heated protein-clusters (membrane rafts) are used the same way as the artificial nanoparticles, while these absorbers are natural parts of the biological system.

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

Vi capsular polysaccharide (Vi) is a major virulence factor of human typhoid-causing pathogen Salmonella enterica serovar Typhi (S. Typhi). It distinguishes S. Typhi from closely related non-typhoidal Salmonella serovars such as S. Typhimurium which do not normally cause systemic infection in humans. Vi not only forms a capsule around S. Typhi but it is also readily released from this pathogen. We have previously reported that Vi targets prohibitin to inhibit cellular responses activated through immune receptors. Here, we show that engagement of membrane prohibitin with Vi prevents Salmonella-induced activation of small Rho-family GTPases, Rac1, and Cdc42, and suppresses actin cytoskeletal rearrangements resulting in reduced invasion and highly subdued inflammatory responses. Cells infected with S. Typhimurium in the presence of Vi show poor activation of NF-kB and MAP-kinase pathways of intracellular signaling. Treatment with Vi brings about redistribution of Rac-1, prohibitin, and ganglioside GM1 in membrane raft domains. Vi-mediated interference with activation of Rho-family GTPases represents a previously unrecognized mechanism by which S. Typhi can limit its invasion and alarming of the host.

Biological membranes are organized in various microdomains, one of the best known being called membrane rafts. The major function of these is thought to organize signaling partners into functional complexes. An important protein found in membrane raft microdomains of erythroid and other blood cells is MPP1 (membrane palmitoylated protein 1)/p55. MPP1 (p55) belongs to the MAGUK (membrane-associated guanylate kinase homolog) family and it is a major target of palmitoylation in the red blood cells (RBCs) membrane. The well-known function of this protein is to participate in formation of the junctional complex of the erythrocyte mem-brane skeleton. However, its function as a "raft organizer" is not well understood. In this review we focus on recent reports concerning MPP1 participation in membrane rafts organization in erythroid cells, including its role in signal transduction. Currently it is not known whether MPP1 could have a similar role in cell types other than erythroid lineage. We present also preliminary data regarding the expression level of MPP1 gene in several non-erythroid cell lines.

Globally, diabetes mellitus (DM) is one of the most occurred metabolic diseases, involving an increased level of blood glucose in the body. In general, DM patients grow many comorbidities including coronary heart disease, overweight gain, and stroke. Myocardial ischemia (MI) is a type of coronary heart disease, it occurs when heart muscles lost the ability to pump blood accurately. Though DM and MI are not associated but many biological functions of both disease match with each other. Our objectives were to uncover the association between the DM and MI using a bioinformatics approach with bio-molecular signature. We compare both datasets with the Venn diagram, the outcomes showed that 144 genes were overlapped in both disease. The GO analysis revealed that the majority amount of genes were connected with the cytokine-mediated signaling pathway, cytokine activity, and membrane raft. Pathway analysis explored the common 144 genes that were enriched with the AGE-RAGE signaling pathway in diabetic complications, malaria, and pathways in cancer. A protein-protein interaction (PPI) network was constructed using the Cytoscape. From where we identified 10 significant genes (INS, ALB, IL6, TNF, VEGFA, IL10, CCL2, IL1B, CXCL8, and ICAM1) according to their connectivity range using cytoHubba tool. Drug target analysis reveals that the dexamethasone CTD 00005779 target is the most associated with all the hub genes. Our analysis showed the genetic and biological functional associations between DM and MI. The outcomes of the study will help in future medications development of DM and MI.

// Jayshree L. Hirpara 1,2 , Thomas Loh 3 , Siok Bian Ng 4 , Wee Joo Chng 5 and Shazib Pervaiz 1,6,7,8 1 Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 2 Experimental Therapeutics Program, Cancer Science Institute, National University Healthcare System, Singapore 3 Department of Otolaryngology, National University Healthcare System, Singapore 4 Department of Pathology, National University Healthcare System, Singapore 5 Cancer Science Institute, National University Healthcare System, Singapore 6 NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore 7 National University Cancer Institute, National University Healthcare System, 8 School of Biomedical Sciences, Curtin University, Perth, Australia Correspondence to: Shazib Pervaiz, email: // Keywords : DLBCL, apoptosome, Apaf-1, lipid rafts, ROS, Autophagy Received : April 15, 2016 Accepted : November 08, 2016 Published : November 14, 2016 Abstract Resistance to chemotherapy remains a challenge in the clinical management of diffuse B cell lymphomas despite aggressive chemotherapy such as CHOP and monoclonal CD20. Here we provide evidence that the apoptosome adaptor protein, Apaf-1, is mislocalized in primary cells derived from patients with diffuse large B cell lymphomas (DLBCL). Whereas, the total expression of Apaf-1 did not change, its sub-cellular localization was significantly different in DLBCL, compared to T cell lymphomas as well as cells derived from reactive lymphadenopathy biopsies. As expected, Apaf-1 was detected in the cytosolic fractions of non-B cell lymphomas and non-cancerous tissues; however, in B cell derived lymphomas the protein was detected in membrane raft sub-domains rather than the cytosol. Disruption of lipid raft structures resulted in the redistribution of Apaf-1 to the cytosol and restored apoptosis sensitivity of DLBCL. Furthermore, we identified novel small molecule compounds that target DLBCL by promoting Apaf-1 release form lipid rafts via mechanisms that involve an increase in intracellular reactive oxygen species production. Taken together, our results implicate Apaf-1 mislocalization as a potential diagnostic and prognostic marker for DLBCL, and provide a novel therapeutic strategy for circumventing the drug refractory nature of this sub-class of B cell lymphoma.

Lipids are the fundamental structural components of biological membranes. For a long time considered as simple barriers segregating aqueous compartments, membranes are now viewed as dynamic interfaces providing a molecular environment favorable to the activity of membrane-associated proteins. Interestingly, variations in membrane lipid composition, whether quantitative or qualitative, play a crucial role in regulation of membrane protein functionalities. Indeed, a variety of alterations in brain lipid composition have been associated with the processes of normal and pathological aging. Although not establishing a direct cause-and-effect relationship between these complex modifications in cerebral membranes and the process of cognitive decline, evidence shows that alterations in membrane lipid composition affect important physicochemical properties notably impacting the lateral organization of membranes, and thus microdomains. It has been suggested that preservation of microdomain functionality may represent an effective strategy for preventing or decelerating neuronal dysfunction and cerebral vulnerability, processes that are both aggravated by aging. The working hypothesis developed in this review proposes that preservation of membrane organization, for example, through nutritional supplementation of docosahexaenoic acid, could prevent disturbances in and preserve effective cerebral function.

Interleukin-1 (IL-1) binding to its receptor triggers signaling events at the plasma membrane that are essential but not sufficient for the induction of the IL-1-dependent gene expression. In addition, the ligand-induced endocytosis of the IL-1 receptor and signaling events that are initiated after the internalization of the IL-1 receptor presumably involving signaling endosomes are critical for the IL-1-induced gene expression. In this study, we investigate the role of membrane domains, commonly denoted as lipid rafts, in the IL-1-induced signal transduction. We demonstrate that the internalization of the IL-1 receptor depends on the integrity of lipid rafts and that the disruption of lipid rafts strongly reduces the IL-1-induced gene expression. Interestingly, the IL-1-dependent signaling events activated at the plasma membrane are not influenced by the disruption of lipid rafts suggesting that IL-1 signaling is initiated in a non-raft domain of the plasma membrane. Subsequently, the IL-1 receptor is translocated to lipid rafts where receptor endocytosis occurs to enable the internalization-dependent IL-1 signaling to activate the IL-1-induced gene expression.

Paracoccidioides brasiliensis is one of the etiological agents of paracoccidioidomycosis, a human systemic mycosis, highly prevalent in Latin America. In the present work, we demonstrate that P. brasiliensis yeasts promote IL-6 and IL-8 secretion by the human lung epithelial cell line A549 in an integrin-dependent manner. In fact, small interfering RNA directed to α3 and α5 integrins decreased IL-6 and IL-8 levels in P. brasiliensis-infected A549 cell cultures. This fungus also led to an increase in the expression of α3 and α5 integrins in this epithelial cell line. In addition, P. brasiliensis yeasts promoted α3 and α5 integrins clustering into A549 cell membrane rafts. Furthermore, epithelial cell membrane raft disruption with nystatin decreased IL-6 and IL-8 levels in P. brasiliensis-A549 cell cultures. Therefore, by increasing host α3 and α5 integrins levels and clustering these receptors into membrane rafts, P. brasiliensis yeasts may modulate host inflammation.

The present study explored the molecular mechanisms by which instant cell membrane resealing (CMR) controls the activation of NOD-like receptor pyrin domain containing 3 (Nlrp3) inflammasomes. Using wavelength-switching fluorescent microscopy with PI and fura-2 as indicators, we monitored instant CMR simultaneously with (Ca(2+))i in mouse microvascular endothelial cell (MVECs). LCWE or saponin wad found to produce membrane injury, which was resealed in a Ca(2+)-dependent manner, but abolished by FasL, a membrane raft (MR) clustering stimulator. Even in the presence of Ca(2+), FasL prolonged the CMR time as shown by an earlier onset of PI influx (48±12 sec vs. 17±3 min. of control). These effects of FasL were substantially blocked by an MR disruptor, methyl-beta-cyclodextrin (MCD). The failure of CMR upon FasL activated Nlrp3 inflammasomes, which was blocked by MCD, a membrane resealing compound, VA64 or siRNA of an MR-facilitating enzyme, acid sphingomyelinase. This inflammasome activation was due to increased lysosomal permeability and cathepsin B release. It is concluded that an MR-associated CMR protects ECs from Nlrp3 inflammasome activation induced by membrane injury.

Study hypothesis Given the importance of the chaperone Heat Shock Protein A2 (HSPA2) in the regulation of male fertility, this study aimed to identify and characterize additional proteins that may rely on the activity of this chaperone in human spermatozoa. Study finding In view of the findings in this study we propose that angiotensin converting enzyme (ACE) and protein disulfide isomerase A6 (PDIA6) are novel interacting proteins of HSPA2 and that this multimeric complex may participate in key elements of the fertilization cascade. What is known already The molecular chaperone HSPA2 plays a pivotal role in the remodelling of the sperm surface during capacitation. Indeed, human spermatozoa that are deficient in HSPA2 protein expression lack the ability to recognize human oocytes, resulting in repeated IVF failure in a clinical setting. Moreover, our recent work has shown that defective HSPA2 function induced by oxidative stress leads to the aberrant surface expression of one of its interacting proteins, arylsulfatase A, and thus contributes to a loss of sperm-zona pellucida adhesion. Study design, samples/materials, methods Human spermatozoa were collected from fertile donors, capacitated and prepared for Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) analysis. Protein complexes resolved via BN-PAGE were excised and their constituents were identified using mass spectrometry. The interactions between ACE, PDIA6 and HSPA2 were then confirmed using immunoprecipitation and proximity ligation assays and the localization of these proteins was assessed in isolated spermatozoa and commercially available human testis tissue sections. Finally, pharmacological inhibition of ACE was performed to assess the role of ACE in human sperm capacitation. Main results and the role of chance Herein we have identified ACE and PDIA6 as potential HSPA2-interacting proteins and shown that this assemblage resides in membrane raft microdomains located in the peri-acrosomal region of the sperm head. Additionally, the surface expression of PDIA6, but not ACE, was shown to be dynamically regulated during sperm capacitation and, like that of previously characterized HSPA2-interacting proteins, this surface expression proved vulnerable to oxidative stress. In terms of the functional significance of this protein complex, pharmacological inhibition of ACE significantly reduced the ability of human spermatozoa to undergo an agonist induced acrosome reaction (P Limitations, reasons for caution While these results provide a descriptive analysis of the PDIA6/ACE/HSPA2 complex, this study provides the impetus for further investigation into the role of PDIA6 and ACE in human sperm function. Wider implications of the findings As our research group, and others, have shown that HSPA2 is compromised in the spermatozoa of men with oocyte recognition defects, the characterization of these HSPA2-interacting proteins provides important insight into the complexity of the cellular pathways that may be affected in the spermatozoa of infertile individuals. Large scale data Large scale proteomics data can be accessed through the Proteomics Identifications Database (PRIDE). Study funding/competing interests This work was supported by the National Health and Medical Research Council. Grant # APP1046346. The authors have no competing interests to declare.

© 2020 Elsevier B.V. The antioxidant vitamin E is a commonly used vitamin supplement. Although the multi-billion dollar vitamin and nutritional supplement industry encourages the use of vitamin E, there is very little evidence supporting its actual health benefits. Moreover, vitamin E is now marketed as a lipid raft destabilizing anti-cancer agent, in addition to its antioxidant behaviour. Here, we studied the influence of vitamin E and some of its vitamers on membrane raft stability using phase separating unilamellar lipid vesicles in conjunction with small-angle scattering techniques and fluorescence microscopy. We find that lipid phase behaviour remains unperturbed well beyond physiological concentrations of vitamin E (up to a mole fraction of 0.10). Our results are consistent with a proposed line active role of vitamin E at the domain boundary. We discuss the implications of these findings as they pertain to lipid raft modification in native membranes, and propose a new hypothesis for the antioxidant mechanism of vitamin E.

Platycodin D (PTD) is an oleanane-type terpenoid saponin, isolated from the plant Platycodon grandiflorus. PTD displays multiple pharmacological effects, notably significant anticancer activities in vitro and in vivo. Recently, PTD was shown to trigger the extracellular release of the immunologic checkpoint glycoprotein PD-L1. The reduction of PD-L1 expression at the surface of cancer cells leads to interleukin-2 secretion and T cells activation. In the present review, we have analyzed the potential origin of this atypical PTD-induced PD-L1 release to propose a mechanistic explanation. For that, we considered all published scientific information, as well as the physicochemical characteristics of the natural product (a modeling analysis of PTD and the related saponin β -escin is provided). On this basis, we raise the hypothesis that the capacity of PTD to induce PD-L1 extracellular release derives from two main mechanisms: (i) a drug-promoted shedding of membrane PD-L1 by metalloproteases or more likely, (ii) a cholesterol binding-related effect, that would lead to perturbation of membrane raft domains, limiting the recruitment of proteins like TLR4. The drug-induced membrane effects (frequently observed with saponin drugs), associated with a production of interferon-γ,can favor the release of proteins like PD-L1 into membrane vesicles. Our analysis supports the hypothesis that PTD is a cholesterol-dependent lipid raft-modulating agent able to promote the formation of PD-L1 containing extracellular vesicles. The anticancer potential of PTD and its capacity to modulate the functioning of the PD-1/PD-L1 checkpoint should be further considered.

Plants depend on both preformed and inducible defence responses to defend themselves against biotic stresses stemming from pathogen attacks. In this regard, plants perceive pathogenic threats from the environment through pattern recognition receptors (PRRs) that recognise microbe-associated molecular patterns (MAMPs), and so induce plant defence responses against invading pathogens. Close to thirty PRR proteins have been identified in plants, however, the molecular mechanisms underlying MAMP perception by these receptors/receptor complexes are not fully understood. As such, knockout (KO) of genes that code for PRRs and co-receptors/defence-associated proteins is a valuable tool to study plant immunity. The loss of gene activity often causes changes in the phenotype of the model plant, allowing in vivo studies of gene function and associated biological mechanisms. Here, we review the functions of selected PRRs, brassinosteroid insensitive 1 (BRI1) associated receptor kinase 1 (BAK1) and other associated defence proteins that have been identified in plants, and also outline KO lines generated by T-DNA insertional mutagenesis as well as the effect on MAMP perception—and triggered immunity (MTI). In addition, we further review the role of membrane raft domains in flg22-induced MTI in Arabidopsis, due to the vital role in the activation of several proteins that are part of the membrane raft domain theory in this regard.

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

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 membrane raft hypothesis, proposed in 1997 by Simons and Ikonen, has played a paradoxical role in the history of biomembrane research. While it has generated a large amount of investigations, thus helping to increase our understanding of membranes, the object that gives name to the hypothesis, i.e. the raft itself, has been and still is an object of controversy, in which its very reality is often questioned. In this contribution I review the history of the hypothesis and its reception by membrane biologists, and summarize some of the valuable physico-chemical results that have been obtained while testing the raft hypothesis. To save a useful concept from its many misuses I propose that the expression "(transient) nanodomains" be employed instead of "rafts".

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement.

Helium ion microscopy (HIM) scans samples with a fine ion beam exploiting the very short de Broglie wavelength of helium ions. Because the radiation induces only a small sample region to emit secondary electrons (SEs), very high resolution is expected. In order to explore the applications of SE-HIM in biology, COS7 kidney fibroblast cells and C2C12 myoblast cells cultured on a silicon (Si) nitride (SiN)/Si bilayer were dried and directly observed in high vacuum, without coating or staining. High contrast, high depth-of-field images were obtained revealing the nucleus, endoplasmic reticulum, cytoskeleton and putative mitochondria above a bright background from the support. Gold-tagged antibodies were employed to aid organelle identification. Signals from the gold tags were most clearly distinguishable by secondary electron (SE)-HIM when cells were grown on thin SiN film, and the minimum gap measured between gold particles showed the resolution to be 2 nm. Wheat germ agglutinin-gold labeling revealed clusters of gold particles ~50-200 nm in diameter on COS7 cells, which might represent assemblies of glycosylated proteins, suggesting the formation of membrane raft structures that include membrane proteins. SE-HIM also delivered high contrast images of unstained, uncoated, thin sections of Epon‑embedded mouse kidney tissues mounted on a SiN/Si bilayer, revealing the details of sub-tissues and cell organelles. A charge-coupled mechanism explaining the observed SE-HIM contrast is proposed. Ionoluminescence-HIM was also performed targeting zinc oxide particles on cells. In conclusion, the high depth-of-field, high-resolution imaging achieved using HIM may have applications in various fields, including soft materials.