Outer Leaflet Of Plasma Membrane Research Articles

Effect of MαCD-mediated phospholipid exchange on plasma membrane ordered lipid domain stability

Using methyl-α-cyclodextrin (MαCD) mediated outer leaflet phospholipid exchange in cells, we recently showed insulin receptor (IR) activity in CHO cells was supported after substitution replacing plasma membrane outer leaflet lipids with sphingomyelin, which has a propensity to form ordered domains, but inhibited upon substitution with unsaturated phosphatidylcholines (PC), which tend to form disordered domains. We also showed IR activity increased as membrane width increased, as seen when plasma membrane outer leaflet lipids were replaced with saturated PCs with different length acyl chains.

Immune-regulating bimetallic metal-organic framework nanoparticles designed for cancer immunotherapy

Immunogenic cell death (ICD) is a promising strategy in cancer immunotherapy to induce high immunogenicity and activate the immune system. However, its efficacy is counteracted by the concurrent exposure of phosphatidylserine (PS), an immunosuppressive signal on the surface of cancer cells. Here we report the synthesis of a bimetallic metal-organic framework (MOF) nanoparticle containing Gd3+ and Zn2+ (Gd-MOF-5) that can be used as an immunomodulator to downregulate the immunosuppressive PS signal and an ICD inducer to upregulate immunostimulatory signals. Gd3+ inhibits PS externalization via inhibiting the activity of scramblase, an enzyme to transfer PS to the outer leaflet of plasma membrane. Moreover, intracellular Zn2+ overload activates endoplasmic reticulum stress for ICD induction. In combination with an immune checkpoint inhibitor (PD-L1 antibody, denoted as aPDL1), Gd-MOF-5 activated potent immune response and effectively inhibited primary and distal tumor growth in a bilateral 4T1 tumor model. This work presents a new strategy using designed MOF materials to modulate the cell signalling and immunosuppressive microenvironment to improve the outcome of cancer immunotherapy.

Intrinsic lipid curvatures of mammalian plasma membrane outer leaflet lipids and ceramides.

We developed a global X-ray data analysis method to determine the intrinsic curvatures of lipids hosted in inverted hexagonal phases. In particular, we combined compositional modelling with molecular shape-based arguments to account for non-linear mixing effects of guest-in-host lipids on intrinsic curvature. The technique was verified by all-atom molecular dynamics simulations and applied to sphingomyelin and a series of phosphatidylcholines and ceramides with differing composition of the hydrocarbon chains. We report positive lipid curvatures for sphingomyelin and all phosphatidylcholines with disaturated and monounsaturated hydrocarbons. Phosphatidylcholines with diunsaturated hydrocarbons in turn yielded intrinsic lipid curvatures with negative values. All ceramides, with chain lengths varying between C2:0 and C24:0, displayed significant negative lipid curvature values. Moreover, we report non-additive mixing for C2:0 ceramide and sphingomyelin. This suggests for sphingolipids that in addition to lipid headgroup and hydrocarbon chain volumes also lipid-specific interactions are important contributors to membrane curvature stress.

Detection of Ganglioside-Specific Toxin Binding with Biomembrane-Based Bioelectronic Sensors.

Gangliosides, glycolipids that are abundant in the plasma membrane outer leaflet, play an integral role in cellular recognition, adhesion, and infection by interacting with different endogenous molecules, viruses, and toxins. Model membrane systems, such as ganglioside-enriched supported lipid bilayers (SLBs), present a useful tool for sensing, characterizing, and quantifying such interactions. In this work, we report the formation of ganglioside GM1-rich SLBs on conducting polymer electrodes using a solvent-assisted lipid bilayer assembly method to investigate changes in membrane electrical properties upon binding of the B subunit of cholera toxin. The sensing capabilities of our platform were investigated by varying both the receptor and the toxin concentrations in the system as well as using a complex sample (milk contaminated with the toxin) and monitoring the changes in the electrical properties of the membrane. Our work highlights the potential of such conducting polymer-supported biomembrane-based platforms for detecting the toxins within a complex environment, studying ganglioside-specific biomolecular interactions with toxins and screening inhibitory molecules to prevent these interactions.

Dispatching plasma membrane cholesterol and Sonic Hedgehog dispatch: two sides of the same coin?

Vertebrate and invertebrate Hedgehog (Hh) morphogens signal over short and long distances to direct cell fate decisions during development and to maintain tissue homeostasis after birth. One of the most important questions in Hh biology is how such Hh signaling to distant target cells is achieved, because all Hh proteins are secreted as dually lipidated proteins that firmly tether to the outer plasma membrane leaflet of their producing cells. There, Hhs multimerize into light microscopically visible storage platforms that recruit factors required for their regulated release. One such recruited release factor is the soluble glycoprotein Scube2 (Signal sequence, cubulin domain, epidermal-growth-factor-like protein 2), and maximal Scube2 function requires concomitant activity of the resistance-nodulation-division (RND) transporter Dispatched (Disp) at the plasma membrane of Hh-producing cells. Although recently published cryo-electron microscopy-derived structures suggest possible direct modes of Scube2/Disp-regulated Hh release, the mechanism of Disp-mediated Hh deployment is still not fully understood. In this review, we discuss suggested direct modes of Disp-dependent Hh deployment and relate them to the structural similarities between Disp and the related RND transporters Patched (Ptc) and Niemann-Pick type C protein 1. We then discuss open questions and perspectives that derive from these structural similarities, with particular focus on new findings that suggest shared small molecule transporter functions of Disp to deplete the plasma membrane of cholesterol and to modulate Hh release in an indirect manner.

Citral derivative activates cell cycle arrest and apoptosis signaling pathways in Candida albicans by generating oxidative stress

For combating life-threatening infections caused by Candida albicans there is an urgent requirement of new antifungal agents with a targeted activity and low host cytotoxicity. Manipulating the mechanistic basis of cell death decision in yeast may provide an alternative approach for future antifungal therapeutics. Herein, the effect of an active citral derivative (Cd1) over the physiology of cell death in C. albicans was assessed. The viability of C. albicans SC5314 cells was determined by broth microdilution assay. The crucial morphological changes and apoptotic markers in Cd1-exposed yeast cells were analyzed. Subsequently the results confirmed that Cd1 arrested growth and caused death in yeast cells. Furthermore, this molecule inhibited antioxidant enzymes that resulted in production of reactive oxygen species. DNA fragmentation and condensation, phosphatidylserine exposure at the outer leaflet of cell membrane, mitochondrial disintegration as well as accumulation of cells at G2/M phase of the cell cycle were recorded. Altogether, this derivative induced apoptotic-type cell death in C. albicans SC5314.

Phospholipid exchange shows insulin receptor activity is supported by both the propensity to form wide bilayers and ordered raft domains

Insulin receptor (IR) is a membrane tyrosine kinase that mediates the response of cells to insulin. IR activity has been shown to be modulated by changes in plasma membrane lipid composition, but the properties and structural determinants of lipids mediating IR activity are poorly understood. Here, using efficient methyl-alpha-cyclodextrin mediated lipid exchange, we studied the effect of altering plasma membrane outer leaflet phospholipid composition upon the activity of IR in mammalian cells. After substitution of endogenous lipids with lipids having an ability to form liquid ordered (Lo) domains (sphingomyelins) or liquid disordered (Ld) domains (unsaturated phosphatidylcholines (PCs)), we found that the propensity of lipids to form ordered domains is required for high IR activity. Additional substitution experiments using a series of saturated PCs showed that IR activity increased substantially with increasing acyl chain length, which increases both bilayer width and the propensity to form ordered domains. Incorporating purified IR into alkyl maltoside micelles with increasing hydrocarbon lengths also increased IR activity, but more modestly than by increasing lipid acyl chain length in cells. These results suggest that the ability to form Lo domains as well as wide bilayer width contributes to increased IR activity. Inhibition of phosphatases showed that some of the lipid dependence of IR activity upon lipid structure reflected protection from phosphatases by lipids that support Lo domain formation. These results are consistent with a model in which a combination of bilayer width and ordered domain formation modulates IR activity via IR conformation and accessibility to phosphatases.

Altered cellular localisation and expression, together with unconventional protein trafficking, of prion protein, PrPC, in type 1 diabetes.

Normal cellular prion protein (PrPC) is a conserved mammalian glycoprotein found on the outer plasma membrane leaflet through a glycophosphatidylinositol anchor. Although PrPC is expressed by a wide range of tissues throughout the body, the complete repertoire of its functions has not been fully determined. The misfolded pathogenic isoform PrPSc (the scrapie form of PrP) is a causative agent of neurodegenerative prion diseases. The aim of this study is to evaluate PrPC localisation, expression and trafficking in pancreases from organ donors with and without type 1 diabetes and to infer PrPC function through studies on interacting protein partners. In order to evaluate localisation and trafficking of PrPC in the human pancreas, 12 non-diabetic, 12 type 1 diabetic and 12 autoantibody-positive organ donor tissue samples were analysed using immunofluorescence analysis. Furthermore, total RNA was isolated from 29 non-diabetic, 29 type 1 diabetic and 24 autoantibody-positive donors to estimate PrPC expression in the human pancreas. Additionally, we performed PrPC-specific immunoblot analysis on total pancreatic protein from non-diabetic and type 1 diabetic organ donors to test whether changes in PrPC mRNA levels leads to a concomitant increase in PrPC protein levels in human pancreases. In non-diabetic and type 1 diabetic pancreases (the latter displaying both insulin-positive [INS(+)] and -negative [INS(-)] islets), we found PrPC in islets co-registering with beta cells in all INS(+) islets and, strikingly, unexpected activation of PrPC in alpha cells within diabetic INS(-) islets. We found PrPC localised to the plasma membrane and endoplasmic reticulum (ER) but not the Golgi, defining two cellular pools and an unconventional protein trafficking mechanism bypassing the Golgi. We demonstrate PrPC co-registration with established protein partners, neural cell adhesion molecule 1 (NCAM1) and stress-inducible phosphoprotein 1 (STI1; encoded by STIP1) on the plasma membrane and ER, respectively, linking PrPC function with cyto-protection, signalling, differentiation and morphogenesis. We demonstrate that both PRNP (encoding PrPC) and STIP1 gene expression are dramatically altered in type 1 diabetic and autoantibody-positive pancreases. As the first study to address PrPC expression in non-diabetic and type 1 diabetic human pancreas, we provide new insights for PrPC in the pathogenesis of type 1 diabetes. We evaluated the cell-type specific expression of PrPC in the human pancreas and discovered possible connections with potential interacting proteins that we speculate might address mechanisms relevant to the role of PrPC in the human pancreas.

Biosynthesis and Functions of Very-Long-Chain Fatty Acids in the Responses of Plants to Abiotic and Biotic Stresses.

Very-long-chain fatty acids (i.e., fatty acids with more than 18 carbon atoms; VLCFA) are important molecules that play crucial physiological and structural roles in plants. VLCFA are specifically present in several membrane lipids and essential for membrane homeostasis. Their specific accumulation in the sphingolipids of the plasma membrane outer leaflet is of primordial importance for its correct functioning in intercellular communication. VLCFA are found in phospholipids, notably in phosphatidylserine and phosphatidylethanolamine, where they could play a role in membrane domain organization and interleaflet coupling. In epidermal cells, VLCFA are precursors of the cuticular waxes of the plant cuticle, which are of primary importance for many interactions of the plant with its surrounding environment. VLCFA are also major components of the root suberin barrier, which has been shown to be fundamental for nutrient homeostasis and plant adaptation to adverse conditions. Finally, some plants store VLCFA in the triacylglycerols of their seeds so that they later play a pivotal role in seed germination. In this review, taking advantage of the many studies conducted using Arabidopsis thaliana as a model, we present our current knowledge on the biosynthesis and regulation of VLCFA in plants, and on the various functions that VLCFA and their derivatives play in the interactions of plants with their abiotic and biotic environment.

Cell surface glycosaminoglycans exacerbate plasma membrane perturbation induced by the islet amyloid polypeptide

Glycosaminoglycans (GAGs) are long and unbranched anionic heteropolysaccharides that have been associated with virtually all amyloid deposits. Soluble sulfated GAGs are known for their propensity to promote the self-assembly of numerous amyloidogenic proteins and to modulate their cytotoxicity. Nonetheless, although GAGs are prevalent on the outer leaflet of eukaryotic cell plasma membrane as part of proteoglycans, their contributions in the perturbation of lipid bilayer induced by amyloid polypeptides remain unknown. Herein, we investigate the roles of GAGs in the cytotoxicity and plasma membrane perturbation induced by the islet amyloid polypeptide (IAPP), whose deposition in the pancreatic islets is associated with type II diabetes. Cellular assays using GAG-deficient cells reveal that GAGs exacerbate IAPP-induced cytotoxicity and permeabilization of the plasma membrane. Confocal microscopy and flow cytometry analyses show that IAPP sequestration at the cell surface is dependent of GAGs and of the aggregation propensity of the peptide. Using giant plasma membrane vesicles (GPMVs) prepared from GAG-deficient cells, we investigate the direct contributions of membrane-embedded proteoglycans in IAPP-induced membrane disassembly. In sharp contrast to soluble sulfated GAGs, kinetics of amyloid self-assembly expose that the presence of GAGs on GPMVs does not significantly modulate in vitro amyloid formation. Overall, this study indicates that cell surface GAGs increase the local concentration of IAPP in the vicinity of the plasma membrane, promoting lipid bilayer perturbation and cell death.

C-Terminal Peptide Modifications Reveal Direct and Indirect Roles of Hedgehog Morphogen Cholesteroylation.

Hedgehog (Hh) morphogens are involved in embryonic development and stem cell biology and, if misregulated, can contribute to cancer. One important post-translational modification with profound impact on Hh biofunction is its C-terminal cholesteroylation during biosynthesis. The current hypothesis is that the cholesterol moiety is a decisive factor in Hh association with the outer plasma membrane leaflet of producing cells, cell-surface Hh multimerization, and its transport and signaling. Yet, it is not decided whether the cholesterol moiety is directly involved in all of these processes, because their functional interdependency raises the alternative possibility that the cholesterol initiates early processes directly and that these processes can then steer later stages of Hh signaling independent of the lipid. We generated variants of the C-terminal Hh peptide and observed that these cholesteroylated peptides variably impaired several post-translational processes in producing cells and Hh biofunction in Drosophila melanogaster eye and wing development. We also found that substantial Hh amounts separated from cholesteroylated peptide tags in vitro and in vivo and that tagged and untagged Hh variants lacking their C-cholesterol moieties remained bioactive. Our approach thus confirms that Hh cholesteroylation is essential during the early steps of Hh production and maturation but also suggests that it is dispensable for Hh signal reception at receiving cells.

Impact of Intrinsic and Extrinsic Factors on Cellular Sphingomyelin Imaging with Specific Reporter Proteins.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells. Although SM is enriched in the outer leaflet of the cell plasma membrane, lipids are also observed in the inner leaflet of the plasma membrane and intracellular organelles such as endolysosomes, the Golgi apparatus and nuclei. SM is postulated to form clusters with glycosphingolipids (GSLs), cholesterol (Chol), and other SM molecules through hydrophobic interactions and hydrogen bonding. Thus, different clusters composed of SM, SM/Chol, SM/GSL and SM/GSL/Chol with different stoichiometries may exist in biomembranes. In addition, SM monomers may be located in the glycerophospholipid-rich areas of membranes. Recently developed SM-binding proteins (SBPs) distinguish these different SM assemblies. Here, we summarize the effects of intrinsic factors regulating the lipid-binding specificity of SBPs and extrinsic factors, such as the lipid phase and lipid density, on SM recognition by SBPs. The combination of different SBPs revealed the heterogeneity of SM domains in biomembranes.

RNA sequencing of whole blood in dogs with primary immune-mediated hemolytic anemia (IMHA) reveals novel insights into disease pathogenesis.

Immune-mediated hemolytic anemia (IMHA) is a life-threatening autoimmune disorder characterized by a self-mediated attack on circulating red blood cells. The disease occurs naturally in both dogs and humans, but is significantly more prevalent in dogs. Because of its shared features across species, dogs offer a naturally occurring model for studying IMHA in people. In this study, we used RNA sequencing of whole blood from treatment-naïve dogs to study transcriptome-wide changes in gene expression in newly diagnosed animals compared to healthy controls. We found many overexpressed genes in pathways related to neutrophil function, coagulation, and hematopoiesis. In particular, the most highly overexpressed gene in cases was a phospholipase scramblase, which mediates the externalization of phosphatidylserine from the inner to the outer leaflet of cell membranes. This family of genes has been shown to be critically important for programmed cell death of erythrocytes as well as the initiation of the clotting cascade. Unexpectedly, we found marked underexpression of many genes related to lymphocyte function. We also identified groups of genes that are highly associated with the inflammatory response and red blood cell regeneration in affected dogs. We did not find any genes that distinguished dogs that lived vs. those that died at 30 days following diagnosis, nor did we find any relevant genomic signatures of microbial organisms in the blood of affected animals. Future studies are warranted to validate these findings and assess their implication in developing novel therapeutic approaches for dogs and humans with IMHA.

Age-dependent membrane release and degradation of full-length glycosylphosphatidylinositol-anchored proteins in rats

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are associated with the surface of eucaryotic cells only through a covalently coupled carboxy-terminal GPI glycolipid structure which is anchored at the outer leaflet of plasma membranes. This mode of membrane association may be responsible for the recent observations that full-length GPI-APs harbouring the complete GPI anchor are (i) released from isolated rat adipocytes in vitro and (ii) expressed in rat and human serum. The upregulation of the adipocyte release in response to increased cell size and blood glucose/insulin levels of the donor rats and downregulation of the expression in serum of insulin resistant and diabetic rats have been reconciled with enhanced degradation of the full-length GPI-APs released into micelle-like complexes together with (lyso) phospholipids and cholesterol by serum GPI-specific phospholipase D (GPI-PLD).Here by using a sensitive and reliable sensing method for full-length GPI-APs, which relies on surface acoustic waves propagating over microfluidic chips, the upregulation of (i) the release of the full-length GPI-APs CD73, alkaline phosphatase and CD55 from isolated adipocyte plasma membranes monitored in a “lab-on-the-chip” configuration, (ii) their release from isolated rat adipocytes into the incubation medium and (iii) the lipolytic cleavage of their GPI anchors in serum was demonstrated to increase with age (3–16 weeks) and body weight (87−477 g) of (healthy) donor rats. In contrast, the amount of full-length GPI-APs in rat serum, as determined by chip-based sensing, turned out to decline with age/body weight. These correlations suggest that age-/weight-induced alterations (in certain biophysical/biochemical characteristics) of plasma membranes are responsible for the release of full-length GPI-APs which becomes counteracted by elevated GPI-PLD activity in serum. Thus, sensitive and specific measurement of these GPI-AP-relevant parameters may be useful for monitoring of age-related cell surface changes, in general, and diseases, in particular.

Phosphatidylinositol 4,5-bisphosphate is localized in the plasma membrane outer leaflet and regulates cell adhesion and motility

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.

Nanodomains can persist at physiologic temperature in plasma membrane vesicles and be modulated by altering cell lipids

The formation and properties of liquid-ordered (Lo) lipid domains (rafts) in the plasma membrane are still poorly understood. This limits our ability to manipulate ordered lipid domain-dependent biological functions. Giant plasma membrane vesicles (GPMVs) undergo large-scale phase separations into coexisting Lo and liquid-disordered lipid domains. However, large-scale phase separation in GPMVs detected by light microscopy is observed only at low temperatures. Comparing Förster resonance energy transfer-detected versus light microscopy-detected domain formation, we found that nanodomains, domains of nanometer size, persist at temperatures up to 20°C higher than large-scale phases, up to physiologic temperature. The persistence of nanodomains at higher temperatures is consistent with previously reported theoretical calculations. To investigate the sensitivity of nanodomains to lipid composition, GPMVs were prepared from mammalian cells in which sterol, phospholipid, or sphingolipid composition in the plasma membrane outer leaflet had been altered by cyclodextrin-catalyzed lipid exchange. Lipid substitutions that stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on the thermal stability of nanodomains and large-scale phase separation in GPMVs, with nanodomains persisting at higher temperatures than large-scale phases for a wide range of lipid compositions. This indicates that it is likely that plasma membrane nanodomains can form under physiologic conditions more readily than large-scale phase separation. We also conclude that membrane lipid substitutions carried out in intact cells are able to modulate the propensity of plasma membranes to form ordered domains. This implies lipid substitutions can be used to alter biological processes dependent upon ordered domains.

Mass spectrometry in combination with a chiral column and multichannel-MRM allows comprehensive analysis of glycosphingolipid molecular species from mouse brain

Glycosphingolipids (GSLs) exist exclusively in the outer leaflet of plasma membrane in mammalian cells and have diverse structures including different classes of sugars and various molecular species of ceramide moieties. Establishing methods that measure each molecular species in GSL classes should aid functional characterization of GSLs and reveal details about the mechanism of pathogenesis in glycosphingolipidoses. Using an IF-3 chiral column that has never been used for lipid analyses, we developed a liquid chromatography-mass spectrometry (LC-MS) method to separate various GSLs based on sugar and ceramide moieties. To examine GSLs in detail a multichannel-multiple reaction monitoring (multichannel-MRM) mode was used and covered a range of 500–2000 Da. Common fragment ions detected with higher collision energy in the positive ion mode were m/z 264 and 292, and are derived from d18:1 and d20:1 ions, respectively. Both species were used as product ions in the multichannel-MRM for the simultaneous measurement of neutral GSLs, gangliosides and sulfatides. Comprehensive analysis of GSLs in mouse brain using this method revealed that for gangliosides and LacCer, d18:1–C18:0 and d20:1–C18:0 were the major molecular species, whereas d18:1–C24:0 and d18:1–C24:1 were the major molecular species of sulfatides. The results revealed a diverse GSL fatty acid profile. In conclusion, by combining IF-3 chiral column and the multichannel-MRM method various molecular species of GSLs were detected successfully, and a metabolomics approach based on this LC-MS method should facilitate functional analysis of GSLs and the discovery of early biomarkers of glycosphingolipidoses at the molecular level.

Nanodomains Persist to much Higher Temperatures than Large Scale Phase Separation in Giant Plasma Membrane Vesicles and Can Respond Differently to Alterations of Plasma Membrane Lipid Composition

The formation and properties of liquid ordered (Lo) lipid domains (rafts) in the plasma membrane is still poorly understood. Giant plasma membrane vesicles (GPMV) derived from mammalian cells can undergo large scale phase separations into co-existing Lo and disordered lipid (Ld) domains with properties similar to domains in artificial lipid vesicles containing simple lipid mixtures. However, large scale phase separation in GPMV detected by light microscopy only occurs at low temperature. We compared large domain formation detected by light microscopy to nanodomain formation in GPMV using a FRET assay. We found that nanodomains can persist to physiological temperatures, much higher temperatures than large scale phase separation. Altering sterol or phospholipid structure in the plasma membrane outer leaflet of intact mammalian cells by cyclodextrin-catalyzed lipid exchange altered both GPMV lipid composition and the ability of GPMV to form ordered domains. Phospholipids and sterols that either stabilize or destabilize ordered domain formation in artificial lipid vesicles had a similar effect on ordered domain stability in GPMV. Nanodomain and large scale domain formation were generally stabilized or destabilized by lipid substitutions to a similar (but not identical) extent. These results show it is possible to alter the lipid composition and properties of membrane vesicles derived from plasma membrane. Other experiments found that after transfection and expression of soluble GFP in mammalian cells, GPMVs contained GFP in their lumen. Because membrane vesicles can be used to deliver biomolecules to cells, combining an analogous expression step with lipid exchange might allow preparation of plasma membrane vesicles with improved abilities to deliver cytosolic molecules to other cells.

Upregulated phospholipase D activity toward glycosylphosphatidylinositol-anchored proteins in micelle-like serum complexes in metabolically deranged rats and humans.

Glycosylphosphatidylinositol-anchored proteins (GPI-AP) with the complete glycolipid anchor attached have previously been shown to be released from the outer plasma membrane leaflet of rat adipocytes in positive correlation to cell size and blood glucose/insulin levels of the donor rats. Furthermore, they are present in rat and human serum, however, at amounts that are lower in insulin-resistant/obese rats compared with normal ones. These findings prompted further evaluation of the potential of full-length GPI-AP for the prediction and stratification of metabolically deranged states. A comparison of the signatures of horizontal surface acoustic waves that were generated by full-length GPI-AP in the course of their specific capture by and subsequent dissociation from a chip-based sensor between those from rat serum and those reconstituted into lipidic structures strongly argues for expression of full-length GPI-AP in serum in micelle-like complexes in concert with phospholipids, lysophospholipids, and cholesterol. Both the reconstituted and the rat serum complexes were highly sensitive toward mechanical forces, such as vibration. Furthermore, full-length GPI-AP reconstituted into micelle-like complexes represented efficient substrates for cleavage by serum glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). These findings raised the possibility that the upregulated release of full-length GPI-AP into micelle-like serum complexes from metabolically deranged cells is compensated by elevated GPI-PLD activity. In fact, serum GPI-PLD activity toward full-length GPI-AP in micelle-like complexes, but not in detergent micelles, was positively correlated to early states of insulin resistance and obesity in genetic and diet-induced rat models as well as to the body weight in humans. Moreover, the differences in the degradation of GPI-AP in micelle-like complexes were found to rely in part on the interaction of serum GPI-PLD with an activating serum factor. These data suggest that serum GPI-PLD activity measured with GPI-AP in micelle-like complexes is indicative of enhanced release of full-length GPI-AP from relevant tissues into the circulation as a consequence of early metabolic derangement in rats and humans.

Imaging mass spectrometry allows for neuroanatomic-specific detection of gangliosides in the healthy and diseased brain.

Gangliosides have a wide variety of biological functions due to their location on the outer leaflet of plasma membranes. They form a critical component of membrane rafts, or ganglioside-enriched microdomains, where they influence the physical properties of the membrane as well as its function. Gangliosides can change their structure to meet their external and internal environmental demands. This ability to change structure makes gangliosides both fascinating and technologically challenging targets to identify and understand. A full understanding on how gangliosides are regulated within the central nervous system (CNS) is critical, as ganglioside dysregulation is observed in the aging brain as well as in several neurodegenerative injuries and diseases such as stroke, Alzheimer's disease, Parkinson's disease, Huntington's disease and several lysosomal storage disorders diseases, including Tay Sach's disease. Mass spectrometry (MS) has become a useful means to better understand ganglioside composition and function. Imaging mass spectrometry (IMS) provides the added benefit of placing analytical information within an anatomical context. This review article will discuss recent advances in MS-based detection methods, with a focus on IMS-based approaches to help understand the spatial-specific role gangliosides in the healthy brain as in CNS injuries and disease.