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In nature, the folding and conformation of proteins can control the cell or organelle membrane permeability and regulate the life activities. Here we report the first example of synthetic polypeptide vesicles that regulate their permeability via ordered transition of secondary conformations, in a manner similar to biological systems. The polymersomes undergo a β-sheet to α-helix transition in response to reactive oxygen species (ROS), leading to wall thinning without loss of vesicular integrity. The change of membrane structure increases the vesicular permeability and enables specific transport of payloads with different molecular weights. As a proof-of-concept, the polymersomes encapsulating enzymes could serve as nanoreactors and carries for glucose-stimulated insulin secretion in vivo inspired by human glucokinase, resulting in safe and effective treatment of type 1 diabetes mellitus in mouse models. This study will help understand the biology of biomembranes and facilitate the engineering of nanoplatforms for biomimicry, biosensing, and controlled delivery applications.

Electroporation has been widely used in biology, medicine, and the food industry as a means to transport various molecules through the cell membrane. The phenomenon of electroporation is the result of cell membrane damage caused by the application of an electric field. In order to understand more precisely how cells function, we established a dielectric model of a spherical cell and analyzed its characteristics by the finite element method. The effects of altering different electrical parameters were determined. The results showed that the electric field strength was positively related to the transmembrane voltage (TMV) and pore density. There was a minimum electric field strength necessary to induce a critical TMV for the formation of pores. Pulse width also had to be long enough to charge the cell membrane, compared with the normal membrane charging time constant of about 1 μs. When the pulse width was shorter than the charging time constant, it was necessary to increase pulse frequency to create a high enough TMV. The rise-time of the electric pulse also affected electroporation: a fast rise-time pulse not only allowed penetration of the plasma membrane but also the organelle membrane. With slow rise-time pulse, the organelle was shielded from electroporation. This study defines the response characteristics of electrical parameters on the electric load cell and establishes the specificity of parameters for different purposes.

Family with sequence similarity 134 member B (FAM134B)/RETREG1/JK1 is a novel gene with recently reported roles in various diseases. Understanding the function and mechanism of action of FAM134B is necessary to develop disease therapies. Notably, emerging data are clarifying the molecular mechanisms of FAM134B function in organelle membrane morphogenesis and the regulation of signaling pathways, such as the Wnt and AKT signaling pathways. In addition, transcription factors, RNA N6 -methyladenosine-mediated epigenetic regulation, microRNA, and small molecules are involved in the regulation of FAM134B expression. This review comprehensively considers recent studies on the role of FAM134B and its potential mechanisms in neurodegenerative diseases, obesity, viral diseases, cancer, and other diseases. The functions of FAM134B in maintaining cell homeostasis by regulating Golgi morphology, endoplasmic reticulum autophagy, and mitophagy are also highlighted, which may be the underlying mechanism of FAM134B gene mutation-induced diseases. Moreover, the molecular mechanisms of the FAM134B function during numerous biological processes are discussed. This review provides novel insights into the functions and mechanisms of FAM134B in various diseases, which will inform the development of effective drugs to treat diseases.

The myocardial I/R damage is very complicated. Apoptosis is considered to its a critical mechanism. During the cardiac muscle I/R process, oxygen-free radicals play a pivotal role. Arrhythmias, as well as enlargement of the area of myocardial infarction after cardiac muscle I/R process, are caused by adequate blast generated O2- ion free radicals. During the ischemia-reperfusion process, a large amount of O2- ion free radicals destroyed the cell structure, and it undergoes lipid peroxidation with unsaturated fatty acids that contain a large number of phospholipids in the cell membrane, causing membrane proteins such as ion channels and enzymes on the cell membrane. The activity of cell is reduced, which affects the function of cell membrane and organelle membrane, destroys its integrity and reduces fluidity.We observed the effects of cerium dioxide nanoparticles on glutathione peroxidase as well as superoxide dismutase, also propionate in myocardial tissue of I/R injury in the mouse. Its effects of malondialdehyde and apoptosis were explored to see its protective effect and to provide more preventive measures for ischemia-reperfusion injury.

This article supplements a recent Perspective by Scorrano et al. inNature Communications[10 [ (1)]:1287] in which the properties and functions of inter-organelle membrane contact sites were summarized. It is now clear that inter-organelle membrane contact sites are widespread in eukaryotic cells and that diverse pairs of organelles can be linked via unique protein tethers. An appropriate definition of what constitutes an inter-organelle membrane contact site was proposed in the Perspective. In addition, the various experimental approaches that are frequently used to study these organelle associations, as well as the advantages and disadvantages of each of these methods, were considered. The nature of the tethers that link the pairs of organelles at the contact sites was discussed in detail and some biological functions that have been ascribed to specific membrane contact sites were highlighted. Nevertheless, the functions of most types of organelle contact sites remain unclear. In the current article I have considered some of the points raised in the Perspective but have omitted detailed information on the roles of membrane contact sites in biological functions such as apoptosis, autophagy, calcium homeostasis and mitochondrial fusion. Instead, I have provided some background on the initial discovery of mitochondria-endoplasmic reticulum membrane contact sites, and have focussed on the known roles of membrane contact sites in inter-organelle lipid transport. In addition, potential roles for membrane contact sites in human diseases are briefly discussed.

Lipids play crucial roles as structural elements, signaling molecules and material transporters in cells. However, the functions and dynamics of lipids within cells remain unclear because of a lack of methods to selectively label lipids in specific organelles and trace their movement by live-cell imaging. We describe here a technology for the selective labeling and fluorescence imaging (microscopic or nanoscopic) of phosphatidylcholine in target organelles. This approach involves the metabolic incorporation of azido-choline, followed by a spatially limited bioorthogonal reaction that enables the visualization and quantitative analysis of interorganelle lipid transport in live cells. More importantly, with live-cell imaging, we obtained direct evidence that the autophagosomal membrane originates from the endoplasmic reticulum. This method is simple and robust and is thus powerful for real-time tracing of interorganelle lipid trafficking., 狙った細胞内小器官脂質の可視化に成功 --オートファゴソーム形成機構解明に貢献--. 京都大学プレスリリース. 2020-09-28.

International audience; Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L−1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L−1 and 1.85 mg L−1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.

This study is to identify the circular RNA (circRNA) expression profile that is functionally related to pancreatic isletβ-cell autophagy and their potential regulation mechanisms in type 2 diabetes mellitus (T2DM). T2DM rat model was constructed by administration of high-fat and high-sugar diet.β-cells were isolated from islets by flow cytometry. CircRNA expression profile inβ-cells was detected by circRNA microarrays, and the differentially expressed circRNAs were identified and validated by qRT-PCR. MicroRNA (miRNA) target prediction software and multiple bioinformatic approaches were used to construct a map of circRNA-miRNA interactions for the differentially expressed circRNAs. A total of 825 differentially expressed circular transcripts were identified in T2DM rats compared with control rats, among which 388 were upregulated and 437 were downregulated. Ten circRNAs were identified to have significant differences by qRT-PCR. GO analysis enriched terms such as organelle membrane and protein binding and the top enriched pathways for the circRNAs included MAPK signaling pathway. The differentially expressed circRNAs might involve in MAPK signaling pathway, apoptosis, and Ras signaling pathway. We speculate that these circRNAs, especially rno_circRNA_008565, can regulate the autophagy of isletβ-cells via interactions with miRNA. Dysregulation of several circRNAs may play a role in T2DM development, and rno_circRNA_008565 may be a potential regulator ofβ-cell autophagy.

The membranes of endosomes, phagosomes and macropinosomes can become damaged by the physical properties of internalized cargo, by active pathogenic invasion or by cellular processes, including endocytic maturation. Loss of membrane integrity is often deleterious and is, therefore, prevented by mitigation and repair mechanisms. However, it can occasionally be beneficial and actively induced by cells. Here, we summarize the mechanisms by which cells, in particular phagocytes, try to prevent membrane damage and how, when this fails, they repair or destroy damaged endocytic organelles. We also detail how one type of phagocyte, the dendritic cell, can deliberately trigger localized damage to endocytic organelles to allow for major histocompatibility complex class I presentation of exogenous antigens and initiation of CD8 + T-cell responses to viruses and tumours. Our review highlights mechanisms for the regulation of endocytic organelle membrane integrity at the intersection of cell biology and immunology that could be co-opted for improving vaccination and intracellular drug delivery.

The macrophage receptor with collagenous structure (MARCO) plays a key role in bacteria-binding innate immune response. According to the transcriptome data MARCO was identified from the spleen (designated as NaMARCO) of Nibea albiflora, its full-length (GenBank accession no. MN243703.1) was 1788 bp, and the open reading frames (ORF) was 1278 bp encoding a polypeptide of 425 amino acids with predicted pI/MW 8.72/43.61 kDa, which had the conserved scavenger receptor cysteine-rich (SRCR) domain similar to its mammalian counterparts. The BLASTp analysis and phylogenetic relationship strongly suggested that NaMARCO shared high similarity with known MARCO, especially at theSRCR domain. The expression of NaMARCO was detected in six tissues (liver, spleen, head kidney, muscle, gill and stomach) and the highest expression appeared in the spleen. After being challenged with Pseudomonas plecoglossicida and poly I:C, NaMARCO expressed with a time-dependent manner in the spleen, and significantly got the peak at 48 h (9.86 times compared with 0 h) for P.plecoglossicida infection, but the peak appeared at 2 h by being challenged with poly I:C (5.18 times compared with 0 h). The results suggested that NaMARCO might be involved in the immune defence of N.albiflora. In addition, subcellular localization of NaMARCO was performed in EPC cells (epithelial cells from fathead minnow), and it was expressed mainly on the cell membrane, the endoplasmic reticulum and the organelle membrane surrounding the nucleus appeared positive signals. These results suggested that NaMARCO, as an important kind of transmembrane scavenger receptors, had a potential role in the immune defense system of N.albiflora against the pathogen.

Objective. Arsenic trioxide (Pishuang, Pishi, arsenolite, As2O3, and CAS 1327-53-3), a naturally occurring and toxic mineral as a drug for more than 2000 years in China, has been found to have a valuable function in hepatocellular carcinoma (HCC) in recent years. However, its exact mechanism remains to be elucidated. Therefore, this study was intended to explore the potential anti-HCC mechanism of arsenic trioxide through network pharmacology. Methods. The potential targets of arsenic trioxide were collected from PubChem and TargetNet. HCC targets were obtained from the GeneCards database. Then, a protein-protein interaction (PPI) network of arsenic trioxide and HCC common targets was established using STRING. GO and KEGG pathway enrichment analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Finally, an arsenic trioxide-target-pathway-HCC network was built by Cytoscape 3.2.1, and network topological analysis was carried out to screen the key candidate targets. Results. A total of 346 corresponding targets of arsenic trioxide and 521 HCC-related targets were collected. After target mapping, a total of 52 common targets were obtained. GO analysis showed that the biological process was mainly involved in the negative regulation of cellular senescence, response to tumor necrosis factor, and cellular response to hypoxia. Molecular functions included NF-kappa B binding, enzyme binding, p53 binding, and transcription factor binding. Cellular components mainly were replication fork, ESC/E(Z) complex, RNA polymerase II transcription factor complex, and organelle membrane. KEGG pathways were mainly enriched in the PI3K-Akt signaling pathway, VEGF signaling pathway, p53 signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, AMPK signaling pathway, NF-kappa B signaling pathway, FoxO signaling pathway, ErbB signaling pathway, and MAPK signaling pathway. In the arsenic trioxide-target-pathway-HCC network, targets such as AKT1, RAF1, RELA, TP53, and PTEN had a higher degree. Conclusions. Our study showed that key targets of arsenic trioxide were mainly involved in multiple biological processes and pathways. It provided a theoretical basis for the screening of drug targets.

Diatoms are unicellular algae, characterized by silica cell walls. The silica elements are formed intracellularly in a membrane-bound silica deposition vesicle (SDV), and are exocytosed after completion. How diatoms maintain membrane homeostasis during the exocytosis of these large and rigid silica elements is a long-standing enigma. We studied membrane dynamics during cell wall formation and exocytosis in two model diatom species, using live-cell confocal microscopy, transmission electron microscopy and cryo-electron tomography. Our results show that during the formation of the mineral phase it is in tight association with the SDV membranes, which are forming a precise mold of the delicate geometrical patterns. During exocytosis, the distal SDV membrane and the plasma membrane gradually detach from the mineral and disintegrate in the extracellular space, without any noticeable endocytic retrieval or extracellular repurposing. Within the cell, there is no evidence for the formation of a new plasma membrane, thus the proximal SDV membrane becomes the new barrier between the cell and its environment, and assumes the role of a new plasma membrane. These results provide direct structural observations of diatom silica exocytosis, and point to an extraordinary mechanism in which membrane homeostasis is maintained by discarding, rather than recycling, significant membrane patches.Significance StatementExocytosis is a fundamental process for cell metabolism, communication, and growth. During exocytosis, an intracellular vesicle fuses with the plasma membrane to release its contents. In classical exocytosis, where the exocytosed vesicles are much smaller than the cell, membrane homeostasis is maintained by recycling excess membranes back into the cell. However, an extreme case of exocytosis is the extrusion of large and rigid cell wall elements by unicellular marine algae. During this process, the cell needs to deal with a potential doubling of its plasma membrane. This study reports on a unique exocytosis mechanism used by these organisms, in which the cells cope with the geometrical and physical challenges of exocytosis by releasing a significant amount of membranes to the extracellular space.

Background Whole-proteome distributions of protein isoelectric point (pI) values in different organisms are bi- or trimodal with some variations. It was suggested that the observed multimodality of the proteome-wide pI distributions is associated with subcellular localization-specific differences in the local pI distributions. However, the factors responsible for variation of the intracellular localization-specific pI profiles have not been investigated in detail. Results In this work, we explored proteome-wide pI distributions of 32,138 human proteins predicted to reside in 10 subcellular compartments, as well as the pI distributions of experimentally observed lysosomal and Golgi proteins. The distributions were found to differ significantly, although all of them adhered to the major recurrent bimodal pattern. Grossly, acid-biased and alkaline-biased patterns with various minor statistical features were observed at different subcellular locations. Bioinformatics analysis revealed the existence of strong statistically significant correlations between protein pI and subcellular localization. Most markedly, protein pI was found to correlate positively with nuclear and mitochondrial locations and negatively with cytoskeletal, cytoplasmic, lysosomal and peroxisomal environment. Further analysis demonstrated that subcellular compartment-specific pI distributions are greatly influenced by local pH and organelle membrane charge. Multiple nonlinear regression analysis identified a polynomial function of the two variables that best fitted the mean pI values of the localization-specific pI distributions. A high coefficient of determination calculated for this regression (R2 = 0.98) suggests that local pH and organelle membrane charge are the major factors responsible for variation of the intracellular localization-specific pI profiles. Conclusions Our study demonstrates that strong correlations exist between protein pI and subcellular localization. The specific pI distributions at different subcellular locations are defined by local environment. Predominantly, it is the local pH and membrane charge that shape the organelle-specific protein pI patterns. These findings expand our understanding of spatial organization of the human proteome. Electronic supplementary material The online version of this article (10.1186/s12860-019-0221-4) contains supplementary material, which is available to authorized users.

This paper considers the electroporation characteristics of a cell under high-frequency nanosecond pulse bursts. A typical two-dimensional cell system, including an organelle membrane, was discretized into nodes using MATLAB, and the mesh transport network method model was established after the bidirectional coupling properties of electrical transport and pore transport were assigned to the nodes within this model. The dynamic process in single-cell electroporation under the application of 10 unipolar high-frequency nanosecond pulse bursts at 1 Hz to the target system was simulated and analyzed. The electroporation characteristics of a single pulse burst and multiple pulse bursts were evaluated. Particularly, the effect of intra-burst frequency on the average pore radius was examined. For the plasma membrane, when multiple pulses were applied, the pore number remained unchanged while the pore radius at the transition region exhibited a more obvious cumulative effect at higher intra-burst frequencies. For the organelle membrane, the number of pores significantly increased when the intra-burst frequency was high (1 MHz). Repetitive pulse bursts expanded the pore radius but do not change the pore number. The results of the present study further enrich our understanding of the electroporation mechanism of a pulsed electric field acting on biological cells.

Lysosomes, the cell’s degradation center, are filled with acidic hydrolases. Lysosomes generate nutrient-sensitive signals to regulate the import of H(+), hydrolases, and endocytic and autophagic cargos, and the export of the degradation products (catabolites). In response to environmental and cellular signals, lysosomes change their positioning, number, morphology, size, composition, and activity within minutes to hours to meet the changing cellular needs. Ion channels in the lysosome are essential transducers that mediate signal-initiated Ca(2+)/Fe(2+)/Zn(2+) release and H(+)/Na(+)/K(+)-dependent changes of membrane potential across the perimeter membrane. Dysregulation of lysosomal ion flux impairs lysosome movement, membrane trafficking, nutrient-sensing, membrane repair, organelle membrane contact, and lysosome biogenesis and adaptation. Hence, activation and inhibition of lysosomal channels by synthetic modulators may tune lysosome function to maintain cellular health and promote cellular clearance in lysosome storage disorders.

Despite having a membrane that is impermeable to all but the smallest of metabolites, peroxisomes acquire their newly synthesized (cytosolic) matrix proteins in an already folded conformation. In some cases, even oligomeric proteins have been reported to translocate the organelle membrane. The protein sorting machinery that accomplishes this feat must be rather flexible and, unsurprisingly, several of its key components have large intrinsically disordered domains. Here, we provide an overview on these domains and their interactions trying to infer their functional roles in this protein sorting pathway.

Membrane trafficking is essential for processing and transport of proteins and lipids and to establish cell compartmentation and tissue organization. Cells respond to their needs and control the quantity and quality of protein secretion accordingly. In this review, we focus on a particular membrane trafficking route from the trans-Golgi network (TGN) to the cell surface: protein kinase D (PKD)-dependent pathway for constitutive secretion mediated by carriers of the TGN to the cell surface (CARTS). Recent findings highlight the importance of lipid signaling by organelle membrane contact sites (MCSs) in this pathway. Finally, we discuss our current understanding of multiple signaling pathways for membrane trafficking regulation mediated by PKD, G protein-coupled receptors (GPCRs), growth factors, metabolites, and mechanosensors.

A vast majority of cellular processes take root at the surface of biological membranes. By providing a two-dimensional platform with limited diffusion, membranes are, by nature, perfect devices to concentrate signaling and metabolic components. As such, membranes often act as “key processors” of cellular information. Biological membranes are highly dynamic and deformable and can be shaped into curved, tubular, or flat conformations, resulting in differentiated biophysical properties. At membrane contact sites, membranes from adjacent organelles come together into a unique 3D configuration, forming functionally distinct microdomains, which facilitate spatially regulated functions, such as organelle communication. Here, we describe the diversity of geometries of contact site-forming membranes in different eukaryotic organisms and explore the emerging notion that their shape, 3D architecture, and remodeling jointly define their cellular activity. The review also provides selected examples highlighting changes in membrane contact site architecture acting as rapid and local responses to cellular perturbations, and summarizes our current understanding of how those structural changes confer functional specificity to those cellular territories.

Phosphatidylethanolamine is an abundant component of most cellular membranes whose physical and chemical properties modulate multiple aspects of organelle membrane dynamics. An evolutionarily ancient mechanism for producing phosphatidylethanolamine is to decarboxylate phosphatidylserine and the enzyme catalyzing this reaction, phosphatidylserine decarboxylase, localizes to the inner membrane of the mitochondrion. We characterize a second form of phosphatidylserine decarboxylase, termed PISD-LD, that is generated by alternative splicing of PISD pre-mRNA and localizes to lipid droplets and to mitochondria. Sub-cellular targeting is controlled by a common segment of PISD-LD that is distinct from the catalytic domain and is regulated by nutritional state. Growth conditions that promote neutral lipid storage in lipid droplets favors targeting to lipid droplets, while targeting to mitochondria is favored by conditions that promote consumption of lipid droplets. Depletion of both forms of phosphatidylserine decarboxylase impairs triacylglycerol synthesis when cells are challenged with free fatty acid, indicating a crucial role phosphatidylserine decarboxylase in neutral lipid storage. The results reveal a previously unappreciated role for phosphatidylserine decarboxylase in lipid droplet biogenesis., Summary: A form of mitochondrial phosphatidylserine decarboxylase that is generated by alternative pre-mRNA splicing is shown to localize to the surface of lipid droplets.

Significance Lysosomes degrade and recycle cell components and integrate environmental and intracellular cues to regulate cell growth, metabolism, and autophagy. The lysosomal transporter PQLC2 exports cationic amino acids from lysosomes, and under amino acid starvation, it recruits to lysosomes a signaling complex implicated in neurological diseases. In this study, we show that PQLC2 transport activity is uncoupled from the lysosomal pH gradient and other ion gradients and that it is selectively modulated by arginine through a trans-inhibition mechanism. Kinetic modeling suggests that arginine accelerates the closing of its cytosolic gate. We propose a signaling model in which PQLC2 transduces the nutrient status to its cognate complex through opposing effects of lysosomal membrane potential and cytosolic arginine on its conformational state., Lysosomes degrade excess or damaged cellular components and recycle their building blocks through membrane transporters. They also act as nutrient-sensing signaling hubs to coordinate cell responses. The membrane protein PQ-loop repeat-containing protein 2 (PQLC2; “picklock two”) is implicated in both functions, as it exports cationic amino acids from lysosomes and serves as a receptor and amino acid sensor to recruit the C9orf72/SMCR8/WDR41 complex to lysosomes upon nutrient starvation. Its transport activity is essential for drug treatment of the rare disease cystinosis. Here, we quantitatively studied PQLC2 transport activity using electrophysiological and biochemical methods. Charge/substrate ratio, intracellular pH, and reversal potential measurements showed that it operates in a uniporter mode. Thus, PQLC2 is uncoupled from the steep lysosomal proton gradient, unlike many lysosomal transporters, enabling bidirectional cationic amino acid transport across the organelle membrane. Surprisingly, the specific presence of arginine, but not other substrates (lysine, histidine), in the discharge (“trans”) compartment impaired PQLC2 transport. Kinetic modeling of the uniport cycle recapitulated the paradoxical substrate-yet-inhibitor behavior of arginine, assuming that bound arginine facilitates closing of the transporter’s cytosolic gate. Arginine binding may thus tune PQLC2 gating to control its conformation, suggesting a potential mechanism for nutrient signaling by PQLC2 to its interaction partners.

Ferroptosis is an iron-dependent regulated necrosis characterized by the peroxidation damage of lipid molecular containing unsaturated fatty acid long chain on the cell membrane or organelle membrane after cellular deactivation restitution system, resulting in the cell membrane rupture. Ferroptosis is biochemically and morphologically distinct and disparate from other forms of regulated cell death. Recently, mounting studies have investigated the mechanism of ferroptosis, and numerous proteins play vital roles in regulating ferroptosis. With detailed studies, emerging evidence indicates that ferroptosis is found in multiple lung diseases, demonstrating that ferroptosis appears to be particularly important for lung diseases. The mounting interest in ferroptosis drugs specifically targeting the ferroptosis mechanism holds substantial therapeutic promise in lung diseases. The present review emphatically summarizes the functions and integrated molecular mechanisms of ferroptosis in various lung diseases, proposing that multiangle regulation of ferroptosis might be a promising strategy for the clinical treatment of lung diseases.

Membrane contact sites (MCS) play crucial roles in cell physiology with dysfunction in several MCS proteins being linked with neurological and optic nerve diseases. Although there have been significant advances in imaging these interactions over the past two decades with advanced electron microscopy techniques, super-resolution imaging and proximity-dependent fluorescent reporters, a technique to observe and quantify MCS in mammalian optic nerve tissues has not yet been reported. We demonstrate for the first time that proximity ligation assay (PLA), a technique already used in mammalian cell lines, can be used as an efficient method of quantifying inter-organelle contact sites, namely mitochondria-endoplasmic reticulum (ER) and mitochondria-late-endosomes, in mammalian optic nerve tissues treated with adeno-associated virus (AAV) gene therapy with wild-type or phosphomimetic (active) protrudin. PLA utilises complementary single-stranded DNA oligomers bound to secondary antibodies that hybridise and complete a circular piece of DNA when the primary antibodies of interest interact. These interactions can be detected by amplifying the circular DNA and adding fluorescent probes. We show that PLA is a useful method that can be used to quantify MCS in optic nerve tissues. We have found that upregulation of protrudin with gene therapy significantly increases the number of mitochondria-ER and mitochondria-Rab7-late endosomes contact sites in optic nerves.

Within the cell, proteins are segregated into different organelles depending on their function and activation status. In response to stimulus, posttranslational modifications or loss of organelle membrane integrity lead to the movement of proteins from one compartment to another. This movement of proteins or protein translocation, exerts a significant effect on protein function. This is clearly demonstrated in the context of apoptosis wherein the cytoplasmic translocation of the mitochondrial resident protein, cytochrome C, initiates the activation of the intrinsic arm of the apoptotic pathway. Experimentally, protein translocation can be demonstrated by subcellular fractionation and subsequent western blot analysis of the isolated fractions. This chapter describes the step-by-step procedure in obtaining mitochondrial and cytoplasmic fractions from cell pellets and determining their purity and integrity.

Phosphatidylethanolamine is an abundant component of most cellular membranes whose physical and chemical properties modulate multiple aspects of organelle membrane dynamics. An evolutionarily ancient mechanism for producing phosphatidylethanolamine is to decarboxylate phosphatidylserine and the enzyme catalyzing this reaction, phosphatidylserine decarboxylase, localizes to the inner membrane of the mitochondrion. We characterize a second form of phosphatidylserine decarboxylase, termed PISD-LD, that is generated by alternative splicing of PISD pre-mRNA and localizes to lipid droplets and to mitochondria. Sub-cellular targeting is controlled by a common segment of PISD-LD that is distinct from the catalytic domain and is regulated by nutritional state. Growth conditions that promote neutral lipid storage in lipid droplets favors targeting to lipid droplets, while targeting to mitochondria is favored by conditions that promote consumption of lipid droplets. Depletion of both forms of phosphatidylserine decarboxylase impairs triacylglycerol synthesis when cells are challenged with free fatty acid, indicating a crucial role phosphatidylserine decarboxylase in neutral lipid storage. The results reveal a previously unappreciated role for phosphatidylserine decarboxylase in lipid droplet biogenesis.

The interactions between different intra-cellular organelles, including the endoplasmic reticulum, have recently been in focus thanks to the tremendous progress in imaging them using cryogenic transmission electron microscopy. However, they are still difficult to study in cellulo, and reconstituting these systems has been a standing challenge. Here we achieve this task using a giant unilamellar vesicle (GUV) and supported lipid bilayer (SLB) system. The tethers, which may reside in the cytosol when unbound, are mimicked by single (or double) stranded DNA sequences of two different lengths with ends that are self-sticky, and with terminal cholesterol moieties which insert into GUV or SLB membranes. The DNA-tethers, bound by their sticky-end, can exist in two possible states - either with both cholesterols in the same membrane or each cholesterol in a different membrane, the latter conformation leading to adhesion. Exchange of tether-molecules between the membranes occurs through the aqueous phase. By developing theoretical arguments that are supported in our experiments, we show that this possibility of exchange and the relative difference in the projected area between the two states drives the adhesion due to collective entropic considerations, rather than the usually considered enthalpy of binding. The establishment of this fundamentally different interaction between two membranes suggests that in physiological conditions, the regulation of contact formation inside cells may be very different from the case of the much studied ligand-receptor pairing on the external cell membrane.

Mitochondrial dysfunction and failing mitochondrial quality control (MQC) are major determinants of aging. Far from being standalone organelles, mitochondria are intricately related with cellular other compartments, including lysosomes. The intimate relationship between mitochondria and lysosomes is reflected by the fact that lysosomal degradation of dysfunctional mitochondria is the final step of mitophagy. Inter-organelle membrane contact sites also allow bidirectional communication between mitochondria and lysosomes as part of nondegradative pathways. This interaction establishes a functional unit that regulates metabolic signaling, mitochondrial dynamics, and, hence, MQC. Contacts of mitochondria with the endoplasmic reticulum (ER) have also been described. ER-mitochondrial interactions are relevant to Ca2+ homeostasis, transfer of phospholipid precursors to mitochondria, and integration of apoptotic signaling. Many proteins involved in mitochondrial contact sites with other organelles also participate to degradative MQC pathways. Hence, a comprehensive assessment of mitochondrial dysfunction during aging requires a thorough evaluation of degradative and nondegradative inter-organelle pathways. Here, we present a geroscience overview on (1) degradative MQC pathways, (2) nondegradative processes involving inter-organelle tethering, (3) age-related changes in inter-organelle degradative and nondegradative pathways, and (4) relevance of MQC failure to inflammaging and age-related conditions, with a focus on Parkinson’s disease as a prototypical geroscience condition.

Inter-organelle membrane contact sites (MCSs) are classically defined as areas of close proximity between heterologous membranes and established by specific proteins (termed tethers). The interest on MCSs has rapidly increased in the last years, since MCSs play a crucial role in the transfer of cellular components between different organelles and have been involved in important cellular functions such as apoptosis, organelle division and biogenesis, and cell growth. Recently, an unprecedented depth and breadth in insights into the details of MCSs have been uncovered. On one hand, extensive MCSs (organelles interactome) are revealed by comprehensive analysis of organelle network with high temporal-spatial resolution at the system level. On the other hand, more and more tethers involving in MCSs are identified and further works are focusing on addressing the role of these tethers in regulating the function of MCSs at the molecular level. These enormous progresses largely depend on the powerful approaches, including several different types of microscopies and various biochemical techniques. These approaches have greatly accelerated recent advances in MCSs at the system and molecular level. In this review, we summarize the current and emerging approaches for studying MCSs, such as various microscopies, proximity-driven fluorescent signal generation and proximity-dependent biotinylation. In addition, we highlight the advantages and disadvantages of the techniques to provide a general guidance for the study of MCSs.

Gene coexpression analysis is widely used to infer gene modules associated with diseases and other clinical traits. However, a systematic view and comparison of gene coexpression networks and modules across a cohort of tissues are more or less ignored. In this study, we first construct gene coexpression networks and modules of 52 GTEx tissues and cell lines. The network modules are enriched in many tissue-common functions like organelle membrane and tissue-specific functions. We then study the correlation of tissues from the network point of view. As a result, the network modules of most tissues are significantly correlated, indicating a general similar network pattern across tissues. However, the level of similarity among the tissues is different. The tissues closing in a physical location seem to be more similar in their coexpression networks. For example, the two adjacent tissues fallopian tube and bladder have the highest Fisher’s exact test p value 8.54E-291 among all tissue pairs. It is known that immune-associated modules are frequently identified in coexperssion modules. In this study, we found immune modules in many tissues like liver, kidney cortex, lung, uterus, adipose subcutaneous, and adipose visceral omentum. However, not all tissues have immune-associated modules, for example, brain cerebellum. Finally, by the clique analysis, we identify the largest clique of modules, in which the genes in each module are significantly overlapped with those in other modules. As a result, we are able to find a clique of size 40 (out of 52 tissues), indicating a strong correlation of modules across tissues. It is not surprising that the 40 modules are most commonly enriched in immune-related functions.

GSL synthesis and metabolism are carried out via endosomal organelle of endoplasmic reticulum (ER) and Golgi systemic network, in the secretory organelle membrane system [16]. As a starting lipid structure to generate GSLs, the Cer is used. As described earlier, the aminoalcohol sphingosine with long chain is a key constituent of Cer [1]. Sphingosine is synthesized from serine and palmitoyl-CoA with fatty acid-amide linkage because the GSL biosynthesis is initiated by the serine palmitoyltransferase, an amino acid-fatty acid ligase. The serine palmitoyltransferase catalyzes the synthesis of 3-keto-sphinganine from palmitoyl-CoA and serine. The keto group is further reduced to the hydroxyl group to synthesize the sphinganine named dihydrosphingosine and the C-4 position of the formed dihydrosphingosine is further hydroxylated to make the phytosphingosine (4-hydroxysphinganine). Dihydrosphingosine and phytosphingosine are then converted to dihydroceramide or phytoceramide, respectively, by each substrate-specific acyl transferase. Ceramide is converted from dihydroceramide by a desaturase, which is the enzyme to form a double bond at position C-4 of a precursor dihydroceramide [55].

© The Author(s) 2020., Cell biologists generally consider that microtubules and actin play complementary roles in long- and short-distance transport in animal cells. On the contrary, using melanosomes of melanocytes as a model, we recently discovered that the motor protein myosin-Va works with dynamic actin tracks to drive long-range organelle dispersion in opposition to microtubules. This suggests that in animals, as in yeast and plants, myosin/actin can drive long-range transport. Here, we show that the SPIRE-type actin nucleators (predominantly SPIRE1) are Rab27a effectors that co-operate with formin-1 to generate actin tracks required for myosin-Va-dependent transport in melanocytes. Thus, in addition to melanophilin/myosin-Va, Rab27a can recruit SPIREs to melanosomes, thereby integrating motor and track assembly activity at the organelle membrane. Based on this, we suggest a model in which organelles and force generators (motors and track assemblers) are linked, forming an organelle-based, cell-wide network that allows their collective activity to rapidly disperse the population of organelles long-distance throughout the cytoplasm., This work was supported by a Medical Research Council New Investigator Award to A.N.H. (grant reference G1100063), Wellcome Trust Grant Awards (grant reference 108429/Z/15/Z to E.V.S. and 204843/Z/16/Z to A.N.H.), a Biotechnology and Biological Sciences Research Council (grant reference BB/F016956/1) and University of Nottingham funded PhD studentship awarded to C.L.R. D.A.B. was funded by Biochemical Society Vacation Studentship. L.M. is funded through the Spanish Ministry of Economy. Industry and Competitiveness (MINECO) [BIO2015-70978-R]. E.K. and T.W. are funded by DFG SPP1464, KE 447/10-2” and DFG KE 447/18-1. F.S. is funded by DFG Research Training Group, GRK 2174 Award. N.A. is supported by a PhD studentship from Taibah University, Medina, Kingdom of Saudi Arabia. W.W.K. and E.W.T. are funded by The Royal Society (Newton International Fellowship grant NF161582 to W.W.K.), European Commission (Marie Sklodowska Curie Individual Fellowship grant 752165 to W.W.K.), and Cancer Research UK (C29637/A20183 to E.W.T.). A.K.M. acknowledges financial support of the John and Lucille van Geest Foundation.

Stroke is one of the leading causes of death worldwide and therapies are limited. According to the mechanisms underlying stroke, direct cell demise, destruction of organelles, blood-brain barrier disruption, neuroinflammation are potential therapeutic targets. Gasdermin (GSDM) family is a series of recently discovered proteins, which acts as the executor of pyroptosis and protagonist of membrane pore formation. It can be cleaved by inflammatory caspases that are activated by multi-protein complexes, named as inflammasomes, and divided into two domains. The N-terminal domain interacts with lipid localized in the plasma membrane as well as organelle membrane and perforates them. After the formation of pores, the dysfunctional structures suffer lytic death and their contents are released. In this review, we discuss the biological characteristic of GSDM family and their possible roles in stroke-induced brain injury, and based on the review, we hypothesize the feasibility of using GSDMs as a potential target for stroke treatment.

Summary Nature has evolved elaborate, dynamic organelle morphologies for optimal organelle functions. Among them, cisternae stacks are the universal structure for most organelles. However, compared with the well-studied spherical cell/organelle membrane mimic, the fabrication of the ubiquitously present cisternal organelle-like membrane structures for organelle mimic remains a challenging task. Herein, rough endoplasmic reticulum (RER)-like helicoidal cisternae stacks were assembled to mimic the enzyme crowded environment in spatially confined RER cisternae. RER-like single helicoid, multiple helicoids, and secondary helix are all observed. Membrane electrostatics drives their formation and controls the percentages, which indicates the possible role of membrane electrostatics in RER shaping. The organelle-like cisternae stacks can reversibly expand and compress, which provides modulated crowded or de-crowded enzyme environment for biochemical reactions. This work provides advanced membrane models, and novel mechanisms for organelle shaping and helicoids formation, and holds great potential in biomimetics, cell biology, and advanced materials design., Graphical Abstract, Highlights • Rough endoplasmic reticulum-like helicoids were formed from phospholipids • Membrane electrostatics controls helicoids formation and probably organelle shaping • The helicoids reversibly expand and compress to control loaded molecule concentration • A “breathing” modulated proto-organelle model is proposed based on the helicoids, Biomaterials; Materials Science; Molecular Self-Assembly

Membrane contact sites, where two organelles are in close proximity, are critical regulators of cellular membrane homeostasis, with roles in signaling, lipid metabolism, and ion dynamics. A growing catalog of specialized lipid transfer proteins carry out lipid exchange at these sites. Currently characterized eukaryotic lipid transport proteins are shuttles that typically extract a single lipid from the membrane of the donor organelle, solubilize it during transport through the cytosol, and deposit it in the acceptor organelle membrane. Here, we highlight the recently identified chorein_N family of lipid transporters, including the Vps13 proteins and the autophagy protein Atg2. These are elongated proteins that, distinct from previously characterized transport proteins, bind tens of lipids at once. They feature an extended channel, most likely lined with hydrophobic residues. We discuss the possibility that they are not shuttles but instead are bridges between membranes, with lipids traversing the cytosol via the hydrophobic channel.

Acquired hearing loss is the predominant neurodegenerative condition associated with aging in humans. Although mutations on several genes are known to cause congenital deafness in newborns, few genes have been implicated in age-related hearing loss (ARHL), perhaps because its cause is likely polygenic. Here, we generated mice lacking lysosomal calcium channel mucolipins 3 and 1 and discovered that both male and female mice suffered a polygenic form of hearing loss. Whereas mucolipin 1 is ubiquitously expressed in all cells, mucolipin 3 is expressed in a small subset of cochlear cells, hair cells (HCs) and marginal cells of the stria vascularis, and very few other cell types. Mice lacking both mucolipins 3 and 1, but not either one alone, experienced hearing loss as early as at 1 month of age. The severity of hearing impairment progressed from high to low frequencies and increased with age. Early onset of ARHL in these mice was accompanied by outer HC (OHC) loss. Adult mice conditionally lacking mucolipins in HCs exhibited comparable auditory phenotypes, thereby revealing that the reason for OHC loss is mucolipin codeficiency in the HCs and not in the stria vascularis. Furthermore, we observed that OHCs lacking mucolipins contained abnormally enlarged lysosomes aggregated at the apical region of the cell, whereas other organelles appeared normal. We also demonstrated that these aberrant lysosomes in OHCs lost their membrane integrity through lysosomal membrane permeabilization, a known cause of cellular toxicity that explains why and how OHCs die, leading to premature ARHL.SIGNIFICANCE STATEMENTPresbycusis, or age-related hearing loss (ARHL), is a common characteristic of aging in mammals. Although many genes have been identified to cause deafness from birth in both humans and mice, only a few are known to associate with progressive ARHL, the most prevalent form of deafness. We have found that mice lacking two lysosomal channels, mucolipins 3 and 1, suffer accelerated ARHL due to auditory outer hair cell degeneration, the most common cause of hearing loss and neurodegenerative condition in humans. Lysosomes lacking mucolipins undergo organelle membrane permeabilization and promote cytotoxicity with age, revealing a novel mechanism of outer hair cell degeneration and ARHL. These results underscore the importance of lysosomes in hair cell survival and the maintenance of hearing.

Microalgae based biofuel has been extensively researched with huge investment over more than a past decade. However, integration of lipid accumulation and recovery needs to be optimized for economical production of biofuel. Microalgae are promising feedstocks for carbon-neutral biofuels and their commercialization depends on strain improvement by targeted inhibition of desired gene to increase lipid content. The current review describes the triacylglycerol, phospholipid, fatty acid, starch biosynthetic pathway and target gene for metabolic engineering. Further, it focuses on genome editing techniques viz., Clustered Regularly Interspaced Short Palindromic Repeats, RNA Interference, Transcription Activator-Like Effector Nucleases, Mutation and chemical inhibitor to enhance triacylglycerol content together with cultivation and biomass harvesting for fuel precursor extraction. It also highlights the latest development, challenges for integrating single cell oil accumulation and extraction methods like, biomass pretreatment, solvent based cell lysis and solvent free green technologies for fuel conversion. Future efforts on microalgal extracellular matrix, organelle membrane biology, continuous production of oil and non-destructive oil recovery from growth media need to be addressed for large scale commercial production of biofuel.

Stroke is a major cause of death and disability worldwide that triggers a variety of neuropathological conditions, leading to the initiation of several pro-inflammatory mediators and neuronal damage. Neuroinflammation has been considered the potential therapeutic target and contributes to the pathology of ischemia and reperfusion. Pyroptosis is an inflammatory form of programmed cell death that plays an important role in immune protection against stroke. Gasdermin D (GSDMD) is the final executor of pyroptosis upon cleavage by caspases-1/4/5/11, followed by canonical and noncanonical inflammasome activation, leading to a series of inflammatory responses. GSDMD N-terminal domain assembles plasma membrane as well as organelle membrane pores to induce cytolysis, thereby triggering cytokine release and inflammatory-related cell death. In our review, we concisely summarized and highlighted the potential role of GSDMD-regulated pyroptosis and the biological characteristic of GSDMD as a therapeutic target in ischemic stroke. A better understanding of the roles of GSDMD may provide a theoretical basis for the design of novel therapeutic interventions for the treatment of ischemic stroke.

In this study, we examined the role of autophagy in the initiation of lipid increases in renal epithelial HK2 cells. We found that trivalent chromium [Cr(III)] induced autophagy by activating sphingomyelin phosphodiesterase 2 (SMPD2). SMPD2 increases levels of ceramide and other lipids. Confocal immunofluorescence microscopy showed that signals of ceramide overlapped with LC3, suggesting that ceramide might play an important role in the formation of autophagosome. In conclusion, our data indicate that Cr(III) induces autophagy via structural aberration of organelle membrane, in particular by the increase of lipid compositions in addition to autophagy-associated proteins. This article is protected by copyright. All rights reserved

Diglycerol monolaurate (DGL) has been manufactured as a novel type of food emulsifier and is being considered for further application as a food preservative. DGL lethality was thus examined against Saccharomyces cerevisiae as a model of a yeast that causes food spoilage. In spite of its molecular structure as a nonionic surfactant, DGL could exhibit lethality at a concentration lower than that which caused disruptive damage to the yeast plasma membrane. DGL lethality was rather accompanied by a dynamic intracellular event such as a marked vacuolar membrane fragmentation. In DGL-treated cells, the tiny dots or particles of fragmented vacuolar membranes failed to fuse into the original large rounded architecture after its removal from medium, which were distinguished from those generated as a result of vacuolar fission normally accelerated under hyperosmotic conditions. Such an irreversible structural damage of the organelle membrane was considered a cause of DGL lethality.

Ultrahigh pressure extraction (UPE) technique displays great advantages in natural products extraction. According to literature, compared to conventional methods, UPE shows the significant advantages of less time consuming, higher extraction yields, less solvent consumption, higher purity of product. Moreover, UPE minimizes the heat generation which contributes to thermal degradation on bioactive components. So UPE has been considered to be a promising technique on extracting bioactive ingredients from biological material. Recent research declared that the high extraction efficiency of UPE should be attributed to disruption of cellular organelle, which causes enhancement of diffusion and mass transfer. The aim of this work is to explore the micro-mechanism of cell disruption and mass transfer across cell membrane when UPE works. Attempting finite element method, it showed that strain and stress concentration occurred on the organelle membrane under high hydrostatic pressure. The results might reveal the deformation of cellular structure, formation and development of cell failure. It helps establish mechanism model of cell disruption and mass transfer. In addition, it was found that cell with irregular shape performed higher sensitivity to pressure in simulation analysis. This result might provide new ideas in technique improvement.

Background The function of the prion protein, involved in the so-called prion diseases, remains a subject of intense debate and the possibility that it works as a pleiotropic protein through the interaction with multiple membrane proteins is somehow supported by recent reports. Therefore, the use of proteomic and bioinformatics combined to uncover cellular processes occurring together with changes in the expression of the prion protein may provide further insight into the putative pleiotropic role of the prion protein. Results This study assessed the membrane-enriched proteome changes accompanying alterations in the expression of the prion protein. A 2D-DIGE approach was applied to two cell lines after prefractionation towards the membrane protein subset: an embryonic stem cell line and the PK1 subline of neuroblastoma cells which efficiently propagates prion infection. Several proteins were differentially abundant with the increased expression of the prion protein during neural differentiation of embryonic stem cells and with the knockdown of the prion protein in PK1 cells. The identity of around 20% of the differentially abundant proteins was obtained by tandem MS. The catalytic subunit A of succinate dehydrogenase, a key enzyme for the aerobic energy metabolism and redox homeostasis, showed a similar abundance trend as the prion protein in both proteomic experiments. A gene ontology analysis revealed “myelin sheath”, “organelle membrane” and “focal adhesion” associated proteins as the main cellular components, and “protein folding” and “ATPase activity” as the biological processes enriched in the first set of differentially abundant proteins. The known interactome of these differentially abundant proteins was customized to reveal four interactors with the prion protein, including two heat shock proteins and a protein disulfide isomerase. Conclusions Overall, our study shows that expression of the prion protein occurs concomitantly with changes in chaperone activity and cell-redox homeostasis, emphasizing the functional link between these cellular processes and the prion protein. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3694-6) contains supplementary material, which is available to authorized users.

In contrast to many protein translocases that use ATP or GTP hydrolysis as the driving force to transport proteins across biological membranes, the peroxisomal matrix protein import machinery relies on a regulated self-assembly mechanism for this purpose and uses ATP hydrolysis only to reset its components. The ATP-dependent protein complex in charge of resetting this machinery—the Receptor Export Module (REM)—comprises two members of the “ATPases Associated with diverse cellular Activities” (AAA+) family, PEX1 and PEX6, and a membrane protein that anchors the ATPases to the organelle membrane. In recent years, a large amount of data on the structure/function of the REM complex has become available. Here, we discuss the main findings and their mechanistic implications. This work was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior in the framework of the project PTDC/BEX-BCM/2311/2014 (POCI-01-0145-FEDER-016613) and the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274). This work is a result of the project NORTE-01-0145-FEDER-000008—Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). A.B.-B., A.G.P., M.J.F., T.F. and T.A.R. are supported by Fundação para a Ciência e Tecnologia, Programa Operacional Potencial Humano do QREN, and Fundo Social Europeu.

Though the toxicity of strobilurins on non-target aquatic organisms has been characterized, the associated toxic mechanisms have not been fully explored. The present study showed that the larval stage was the most sensitive developmental stage in zebrafish, and pyraclostrobin (PY) had the highest acute toxicity to embryos, larvae, juvenile and adult with 96 h-LC50 at 0.048 mg/L, 0.029 mg/L, 0.039 mg/L, 0.031 mg/L respectively, when compared with the toxicity of trifloxystrobin (TR), kresoxim-methyl (KM) and azoxystrobin (AZ) at corresponding developmental stage. Then we investigated the transcriptomics and developmental toxicity of TR, KM, AZ and PY on zebrafish embryos after 72 h exposure. RNA-seq revealed that the pathways related to cell apoptosis and cancer, and cellular components organelle membrane and mitochondrion, were markedly affected after TR, KM, AZ and PY exposure during zebrafish early life stages. The results were further confirmed by the induction of antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities, the elevation of H2O2, malondialdehyde (MDA) and reactive oxygen species (ROS) level, as well as the reduction of intracellular calcium ions (Ca2+) and mitochondrial membrane potential (MMP), which indicated that strobilurins could cause mitochondrial dysfunction and cell apoptosis. The present study was performed a systematic analysis of strobilurins to zebrafish at multi-levels, which provided suggestions for further investigation of molecular mechanisms underlying the toxicity induced by strobilurins on aquatic organisms.

Postsynaptic density protein-93 (PSD-93) is enriched in the postsynaptic density and is involved in N-methyl-d-aspartate receptor (NMDAR) triggered neurotoxicity through PSD-93/NMDAR/nNOS signaling pathway. In the present study, we found that PSD-93 deficiency reduced infarcted volume and neurological deficits induced by transient middle cerebral artery occlusion (tMCAO) in the mice. To identify novel targets of PSD-93 related neurotoxicity, we applied isobaric tags for relative and absolute quantitative (iTRAQ) labeling and combined this labeling with on-line two-dimensional LC/MS/MS technology to elucidate the changes in protein expression in PSD-93 knockout mice following tMCAO. The proteomic data set consisted of 1892 proteins. Compared to control group, differences in expression levels in ischemic group >1.5-fold and

Apoptosis is accompanied by distinct morphological changes at the plasma and organelle membrane level. Involvement of certain lipids in apoptosis has been established; however, we have limited understanding of the specific lipid structures that participate in this process. We used untargeted comparative lipidomics to study the changes in lipid composition during staurosporine-induced apoptosis in HCT-116. Our results revealed that ceramides, dihydroceramides, and sphingomyelins, with defined acyl chains, constitute the majority of changes in the lipidome. Expression levels and activities of enzymes responsible for the biosynthesis of lipids that change suggest that de novo synthesis causes these specific changes. Further analysis of the lipidome during apoptosis in other cancer and non-cancer cell lines suggested that accumulation of ceramides and dihydroceramides is specific to cancer cells. Taken together, our data propose that these molecules are regulated at the lipid-specific level during apoptosis and that this regulation differs between cancer and non-cancer cells.