Plasma Membrane Vesicles Research Articles

FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy

A current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for correlative light-electron microscopy by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a good signal-to-noise ratio and a labeling resolution of approximately 10 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits.

Abstract LB-327: Identification of novel sodium iodide symporter (NIS) interactors which modulate iodide uptake

Abstract Ablative radioiodide therapy is an effective treatment for thyroid cancer, but around a quarter of patients do not accumulate therapeutically-sufficient doses of radioiodide due to decreased expression and/or plasma membrane (PM) localization of the sodium iodide symporter (NIS). Putative use of radioiodide therapy in breast cancer is hampered by low levels of NIS membrane localisation. Currently, the regulation of NIS trafficking is ill-defined. Mass spectrometry was performed on proteins co-immunoprecipitating with lentivirally expressed NIS in whole cell and plasma membrane extracts. NIS activity was significantly altered by ADP-ribosylation factor 4 (ARF4) and valosin containing protein (VCP) in TPC1 thyroid and MDA-MB-231 breast cancer cells lentivirally-expressing NIS. ARF4 downregulation markedly decreased radioiodide uptake, and VCP downregulation increased radioiodide uptake in thyroid and breast cells. Transient overexpression of these genes significantly reversed siRNA effects on NIS function. Co-immunoprecipitation assays confirmed NIS interacts with ARF4 and VCP in vitro, and proximity ligation assays revealed the subcellular sites of interaction. Critically, human primary thyroid culture experiments confirmed that ARF4 and VCP regulate endogenous NIS function in vivo. In addressing the mechanisms of NIS trafficking we identified a C-terminal VXPX motif of NIS which is required for Arf4 regulation, whilst TIRF microscopy revealed coincident Arf4 and NIS localization to shared PM vesicles. Pharmacological inhibitor studies identified that the VCP inhibitors Eeyarastatin-1 and NMS-873 overcome VCP inhibition of NIS function. VCP, which is central to ER-associated Degradation (ERAD), selectively targeted a dimeric form of NIS, which we now identify through co-immunoprecipitation, 3D modelling and Förster resonance energy transfer (FRET) assays. TCGA data analysis of 58 matched papillary thyroid cancers revealed that Arf4 was significantly repressed and VCP highly upregulated in thyroid cancer, providing a putative explanation for repressed NIS function. Our data suggest a possible model for NIS trafficking: NIS dimerises in the ER, a process monitored by VCP and the ERAD pathway, with correctly processed and folded NIS being trafficked to the PM via C-terminal interaction with Arf4. We thus now identify two new potential therapeutic targets for enhancing radioiodide uptake in patients with radioiodide-refractory thyroid cancer. Citation Format: Alice Fletcher, Rebecca Thompson, Martin Read, Andrew Turnell, Vicki Smith, Chris J. McCabe. Identification of novel sodium iodide symporter (NIS) interactors which modulate iodide uptake [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-327.

Lipoprotein(a) Transports Diverse Host and Non-Host Small RNAs in Circulation

Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease1–4. Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.

Relation of placental alkaline phosphatase expression in human term placenta with maternal and offspring fat mass.

Alkaline phosphatase is implicated in intestinal lipid transport and in the development of obesity. Placental alkaline phosphatase is localised to the microvillous plasma membrane of the placental syncytiotrophoblast at the maternal-fetal interface, but its role is unclear. We investigated the relations of placental alkaline phosphatase activity and mRNA expression with maternal body composition and offspring fat mass in humans. Term human placentas from the UK Birthright cohort (n = 52) and the Southampton Women's Survey (SWS) (n = 95) were studied. In the Birthright cohort, alkaline phosphatase activity was measured in placental microvillous plasma membrane vesicles. In the SWS, alkaline phosphatase mRNA was measured using Nanostring. Alkaline phosphatase gene expression was compared to other lipid-related genes. In Birthright samples placental microvillous plasma membrane alkaline phosphatase activity was positively associated with maternal triceps skinfold thickness and BMI (β = 0.04 (95% CI: 0.01-0.06) and β = 0.02 (0.00-0.03) µmol/mg protein/min per SD, P = 0.002 and P = 0.05, respectively) after adjusting for potential confounders. In SWS samples placental alkaline phosphatase mRNA expression in term placenta was positively associated with maternal triceps skinfold (β = 0.24 (0.04, 0.44) SD/SD, P = 0.02), had no association with neonatal %fat mass (β = 0.01 (-0.20 to 0.21) SD/SD, P = 0.93) and was negatively correlated with %fat mass at ages 4 (β = -0.28 (-0.52 to -0.04) SD/SD, P = 0.02), 6-7 (β = -0.25 (-0.49 to -0.02) SD/SD, P = 0.03) years. When compared with placental expression of other genes, alkaline phosphatase expression was positively related to genes including the lysophosphatidylcholine transporter MFSD2A (major facilitator superfamily domain containing 2A, P < 0.001) and negatively related to genes including the fatty acid transport proteins 2 and 3 (P = 0.001, P < 0.001). Our findings suggest relationships between placental alkaline phosphatase and both maternal and childhood adiposity. The inverse relationship between placental alkaline phosphatase gene expression and childhood %fat mass suggests that placental alkaline phosphatase may help to protect the foetus from the adverse effects of maternal obesity.

Cysteine: A multifaceted amino acid involved in signaling, plant resistance and antifungal development

Early effects induced by cysteine were monitored using the model of Mimosa pudica pulvinar cells. Rapid dose-dependent membrane depolarization (within seconds) and modification of proton secretion (within minutes) were triggered at cysteine concentrations higher than 0.1 mM. These effects did not result from a modification of the plasma membrane H+-ATPase activity nor from a protonophore effect as shown by assays on plasma membrane vesicles isolated from pulvinar tissues. In a 0.5–10 mM range, cysteine inhibited the ion-driven turgor-mediated seismonastic reaction of Mimosa pudica primary pulvini and the dark-induced movement of Cassia fasciculata leaflets. At concentrations higher than 1 mM, it induced a long-lasting leaflet necrosis dependent on the concentration and treatment duration. Electron microscopy showed that cysteine induced important damage in the nucleus, mitochondria, endoplasmic reticulum and Golgi of the M. pudica motor cell. Cysteine inhibited in a concentration-dependent manner, from 0.5 to 20 mM, both the mycelial growth and the spore germination of the fungal pathogens Phaeomoniella chlamydospora and Phaeoacremonium minimum implicated in esca disease of grapevines. Using [35S] cysteine, we showed that the amino acid was absorbed following leaf spraying, translocated from leaves to other parts of grapevine cuttings and accumulated within trunks and roots. Therefore, cysteine showed relevant properties to be a candidate able to control fungal diseases either by acting as an early signal directing plant host reaction or/and by acting directly on fungal development.

Determination of the Membrane Environment of CD59 in Living Cells.

The organization and dynamics of proteins and lipids in the plasma membrane, and their role in membrane functionality, have been subject of a long-lasting debate. Specifically, it is unclear to what extent membrane proteins are affected by their immediate lipid environment and vice versa. Studies on model membranes and plasma membrane vesicles indicated preferences of proteins for lipid phases characterized by different acyl chain order; however, whether such phases do indeed exist in live cells is still not known. Here, we refine a previously developed micropatterning approach combined with single molecule tracking to quantify the influence of the glycosylphosphatidylinositol-anchored (GPI-anchored) protein CD59 on its molecular environment directly in the live cell plasma membrane. We find that locally enriched and immobilized CD59 presents obstacles to the diffusion of fluorescently labeled lipids with a different phase-partitioning behavior independent of cell cholesterol levels and type of lipid. Our results give no evidence for either specific binding of the lipids to CD59 or the existence of nanoscopic ordered membrane regions associated with CD59.

P-363 - Cytochrome c stimulates the superoxide anion production by cytochrome b5 reductase in neuronal synaptic plasma membrane vesicles

The plasma membrane NADH oxidase activity of synaptosomes and cerebellar granule neurons releases superoxide anion (O2•-) as a collateral product. In this work, we have measured the effect of cytochrome c (Cyt c) on the NADH-dependent O2•- production by rat neuronal synaptic plasma membrane vesicles. In these membranes, the Cyt c stimulated O2•- production was inhibited by antibodies against cytochrome b5 reductase (Cb5R). O2•- production by Cb5R was confirmed using purified soluble and membrane recombinant enzymes. Addition of Cyt c to the assay induced a burst of O2•- concomitant to the reduction of Cyt c by Cb5R. The low dissociation constant measured for Cyt c/Cb5R complex (0.40 ±0.05 and 0.38 ±0.02 μM for soluble and membrane Cb5R, respectively), support that Cb5R is a main molecular partner of Cyt c upon its release from mitochondria during apoptosis. O2•- production and Cyt c reduction are consequences of the stimulated NADH consumption by Cb5R upon complex formation with Cyt c. Participation of Cb5R in the apoptosis signalling network is suggested.

Bioinspired nanocarriers for an effective chemotherapy of hepatocellular carcinoma.

Drug-loaded nanoparticles have been widely researched in the antitumor. However, some of them are unsatisfactory in the long blood circulation and controlled drug release. Red blood cell (RBC) membrane vesicles (RV)-coated nanoparticles have gained more and more attention in drug delivery for their many unique advantages, such as excellent stability, long blood circulation, and reduced the macrophage cells uptake. Herein, by utilizing the advantages of RV, we fabricated RV-coated poly(lactide- co-glycolide) (PLGA)-docetaxel (RV/PLGA/DTX) nanoparticles to enhance the antitumor efficiency in vivo. The RV/PLGA/DTX showed spherical morphology with particle size of about 100 nm and zeta potential at -12.63 mV, which could maintain stability for a long time. The RV/PLGA/DTX significantly enhanced cellular uptake of DTX compared to PLGA/DTX in HepG2 cells. Moreover, RV/PLGA/DTX showed the strongest antitumor effect in vitro. Prolonged blood circulation and enhanced DTX accumulation at the tumor site through enhanced permeability and retention (EPR) effect were achieved by RV/PLGA/DTX, which eventually obtained satisfactory antitumor effect and depressed system toxicity on mice bearing HepG2 xenografts mouse models when compared with free DTX. The hematoxylin and eosin (H&E) and immunofluorescence assays further proved the advantages of RV/PLGA/DTX in vivo antitumor. These RV-coated nanoparticles provide a mimetic therapy, completely inhibited the growth of the HepG2 cells, and with simple compositions, suggesting it to be an ideal strategy for improving the antitumor effect of drug-loaded nanoparticles.

Determinants of Raft Partitioning of the Helicobacter pylori Pore-Forming Toxin VacA.

Helicobacter pylori, a Gram-negative bacterium, is a well-known risk factor for gastric cancer. H. pylori vacuolating cytotoxin A (VacA) is a secreted pore-forming toxin that induces a wide range of cellular responses. Like many other bacterial toxins, VacA has been hypothesized to utilize lipid rafts to gain entry into host cells. Here, we used giant plasma membrane vesicles (GPMVs) as a model system to understand the preferential partitioning of VacA into lipid rafts. We show that a wild-type (WT) toxin predominantly associates with the raft phase. Acid activation of VacA enhances binding of the toxin to GPMVs but is not required for raft partitioning. VacA mutant proteins with alterations at the amino terminus (resulting in impaired membrane channel formation) and a nonoligomerizing VacA mutant protein retain the ability to preferentially associate with lipid rafts. Consistent with these results, the isolated VacA p55 domain was capable of binding to lipid rafts. We conclude that the affinity of VacA for rafts is independent of its capacity to oligomerize or form membrane channels.

Intravesicular Acidification Regulates Lipopolysaccharide Inflammation and Tolerance through TLR4 Trafficking

TLRs recognize pathogen components and drive innate immune responses. They localize at either the plasma membrane or intracellular vesicles such as endosomes and lysosomes, and proper cellular localization is important for their ligand recognition and initiation of signaling. In this study, we disrupted ATP6V0D2, a component of vacuolar-type H+ adenosine triphosphatase (V-ATPase) that plays a central role in acidification of intracellular vesicles, in a macrophage cell line. ATP6V0D2-deficient cells exhibited reduced cytokine production in response to endosome-localized, nucleic acid-sensing TLR3, TLR7, and TLR9, but enhanced inflammatory cytokine production and NF-κB activation following stimulation with LPS, a TLR4 agonist. Moreover, they had defects in internalization of cell surface TLR4 and exhibited enhanced inflammatory cytokine production after repeated LPS stimulation, thereby failing to induce LPS tolerance. A component of the V-ATPase complex interacted with ARF6, the small GTPase known to regulate TLR4 internalization, and ARF6 deficiency resulted in prolonged TLR4 expression on the cell surface. Taken together, these findings suggest that ATP6V0D2-dependent intravesicular acidification is required for TLR4 internalization, which is associated with prevention from excessive LPS-triggered inflammation and induction of tolerance.

Leukocyte-derived microparticles exaggerate endothelial senescence and vascular dysfunction induced by high glucose

Microparticles (MPs) are plasma membrane vesicles and vascular effectors. High levels of pro-inflammatory cytokines and procoagulant endothelial-derived MPs circulate in diabetic patients. We have shown that (i) leukocyte-derived MPs shed in response to stress are pro-inflammatory, procoagulant and prosenescent endothelial effectors; (ii) high glucose induces premature endothelial senescence. To determine the possibility that leukocyte-derived MPs affect endothelial senescence and vascular function in response to high glucose. Leukocyte MPs were isolated from rat splenocytes with either 5 mg/ml LPS (MPLPS), 25 ng/ml PMA/1 mM A23187 ionophore (MPPMAi), or vehicle (MPCTL). Porcine coronary artery endothelial cells (ECs) at passage 1 were incubated for 48 h with 1–30 nM MPs in high or low glucose concentration (HG 25 mM, NG 5.5 mM). Senescence-associated β-galactosidase (SA-ß-GAL) activity was assessed by C12FDG, protein expression by Western blot analysis. Pig coronary artery rings were pre-incubated with HG or NG for 12 h prior to addition of 1–30 nM MPs for 12 h. Bradykinin (BK)-induced endothelium-dependent relaxations were assessed in organ chambers, and staining of target proteins by confocal microscopy. At 10 nM, MPLPS and MPPMAi enhanced SA-b-GAL activity both by about 2-fold in NG, and respectively 3 and 3.7-fold in HG. The expression of senescence markers p21, p16 doubled and that of eNOS decreased 2-fold. MPPMAi and MPLPS induced a concentration-dependent inhibition of BK-induced relaxation, inhibition by respectively 10 nM and 30 nM,being 65% in NG, amounting to about 85% in HG, whereas 30 nM MPCTL had no effect. MPPMAi and MPLPS reduced eNOS expression by 60% in NG and 80% in HG. Conversely, VCAM-1, COX-2 were up-regulated. Leukocyte-derived MPs enhance HG-induced alteration of the endothelial function by inducing premature senescence and might contribute to vascular dysfunction in diabetes patients.

Arginine-Rich Cell-Penetrating Peptides Require Nucleolin and Cholesterol-Poor Subdomains for Translocation across Membranes.

Proficient transport vectors called cell-penetrating peptides (CPPs) internalize into eukaryotic cells mostly via endocytic pathways and facilitate the uptake of various cargo molecules attached to them. However, some CPPs are able to induce disturbances in the plasma membrane and translocate through it seemingly in an energy-independent manner. For understanding this phenomenon, giant plasma membrane vesicles (GPMVs) derived from the cells are a beneficial model system, since GPMVs have a complex membrane composition comparable to the cells yet lack cellular energy-dependent mechanisms. We investigated the translocation of arginine-rich CPPs into GPMVs with different membrane compositions. Our results demonstrate that lower cholesterol content favors accumulation of nona-arginine and, additionally, sequestration of cholesterol increases the uptake of the CPPs in vesicles with higher cholesterol packing density. Furthermore, the proteins on the surface of vesicles are essential for the uptake of arginine-rich CPPs: downregulation of nucleolin decreases the accumulation and digestion of proteins on the membrane suppresses translocation even more efficiently.

The formation of giant plasma membrane vesicles enable new insights into the regulation of cholesterol efflux

Aberrant cellular cholesterol accumulation contributes to the pathophysiology of many diseases including neurodegenerative disorders such as Niemann-Pick Type C (NPC) and Alzheimer's Disease1–4. Many aspects of cholesterol efflux from cells remain elusive. Here we describe the utility of cholesterol-rich giant plasma membrane vesicles (GPMVs) as a means to monitor cholesterol that is translocated to the plasma membrane for secretion. We demonstrate that small molecules known to enhance lipid efflux, including those in clinical trials for lipid storage disorders, enhance this GPMV formation. Conversely, pharmacological inhibition of cholesterol efflux blocks GPMV formation. We show that microtubule stabilization via paclitaxel treatment and increased tubulin acetylation via HDAC6 inhibition promotes the formation of GPMVs with concomitant reduction in cellular cholesterol in a cell model of NPC disease. The pan-deacetylase inhibitor panobinostat, which has been shown to reduce the severity of cholesterol storage in NPC, elicited a similar response. Further, the disruption of actin polymerization inhibits the formation of GPMVs, whereas the small GTP-binding protein Arl4c promotes actin remodeling at sites overlapping with GPMV formation. Thus, monitoring the formation of GPMVs provides a new avenue to better understand diseases whose pathology may be sensitive to alterations in cellular cholesterol.

Erythrocyte membrane bioinspired near-infrared persistent luminescence nanocarriers for in vivo long-circulating bioimaging and drug delivery

Combination of biological entities with functional nanostructure would produce the excellent systemic drug-delivery vehicles that possess the ability to cross the biological barriers. Herein, from a biomimetic point of view, erythrocyte membrane bioinspired optical nanocarrier is fabricated by integrating Red blood cell (RBC) membrane vesicle with near-infrared persistent luminescence nanophosphors (PLNPs). The triple-doped zinc gallogermanate nanostructures with super-long near-infrared persistent luminescence (ZGGO) are used as optical emission center, mesoporous silica coated on the PLNPs (ZGGO@mSiO2) is employed for drug delivery, and the RBC membrane vesicle is introduced for biomimetic functionalization to ensure the developed nanocarriers bypass macrophage uptake and systemic clearance. Owing to the coating of natural erythrocyte membrane along with membrane lipids and associated membrane proteins, the proposed bioinspired nanocarriers have exhibited cell-mimicking property. Retaining the applicability of PLNPs core that favored in vitro excitation, the developed RBC-ZGGO@mSiO2 biomimetic nanocarriers have demonstrated intense fluorescence, super-long persistent luminescence, monodispersed nanosize, red light renewability, and excellent biocompatibility. In vivo mice bioimaging and biodistribution study demonstrate the erythrocyte membrane bioinspired nanoprobe loaded with doxorubicin as ideal nanocarriers for long-circulating bioimaging, in situ real-time monitoring and drug delivery. We believe the PLNPs-based biomimetic nanocarriers offer a promising nano-platform for diagnostics and therapeutics application.

Hepatic Transport of 25-Hydroxyvitamin D3 Conjugates: A Mechanism of 25-Hydroxyvitamin D3 Delivery to the Intestinal Tract.

Vitamin D3 is an important prohormone critical for maintaining calcium and phosphate homeostasis in the body and regulating drug-metabolizing enzymes and transporters. 25-Hydroxyvitamin D3 (25OHD3), the most abundant circulating metabolite of vitamin D3, is further transformed to the biologically active metabolite 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) by CYP27B1 in the kidney and extrarenal tissues, and to nonactive metabolites by other cytochrome P450 enzymes. In addition, 25OHD3 undergoes sulfation and glucuronidation in the liver, forming two major conjugative metabolites, 25OHD3-3-O-sulfate (25OHD3-S) and 25OHD3-3-O-glucuronide (25OHD3-G), both of which were detected in human blood and bile. Considering that the conjugates excreted into the bile may be circulated to and reabsorbed from the intestinal lumen, deconjugated to 25OHD3, and then converted to 1α,25-(OH)2D3, exerting local intestinal cellular effects, it is crucial to characterize enterohepatic transport mechanisms of 25OHD3-S and 25OHD3-G, and thereby understand and predict mechanisms of interindividual variability in mineral homeostasis. In the present study, with plasma membrane vesicle and cell-based transport studies, we showed that 25OHD3-G is a substrate of multidrug resistance proteins 2 and 3, OATP1B1, and OATP1B3, and that 25OHD3-S is probably a substrate of breast cancer resistance protein, OATP2B1, and OATP1B3. We also demonstrated sinusoidal and canalicular efflux of both conjugates using sandwich-cultured human hepatocytes. Given substantial expression of these transporters in liver hepatocytes and intestinal enterocytes, this study demonstrates for the first time that transporters could play important roles in the enterohepatic circulation of 25OHD3 conjugates, providing an alternative pathway of 25OHD3 delivery to the intestinal tract, which could be critical for vitamin D receptor-dependent gene regulation in enterocytes.

Liposomal Treatment of Cancer Cells Modulates Uptake Pathway of Polymeric Nanoparticles by Altering Membrane Stiffness.

Nanomedicines can be taken up by cells via nonspecific and dynamin-dependent (energy-dependent) clathrin and caveolae-mediated endocytosis. While significant effort has focused on targeting pathway-specific transporters, the role of nanobiophysics in the cell lipid bilayer nanoparticle uptake pathway remains largely unexplored. In this study, it is demonstrated that stiffness of lipid bilayer is a key determinant of uptake of liposomes by mammalian cells. Dynamin-mediated endocytosis (DME) of liposomes is found to correlate with its phase behavior, with transition toward solid phase promoting DME, and transition toward fluidic phase resulting in dynamin-independent endocytosis. Since liposomes can transfer lipids to cell membrane, it is sought to engineer the biophysical properties of the membrane of breast epithelial tumor cells (MD-MBA-231) by treatment with phosphatidylcholine liposomes, and elucidate its effect on the uptake of polymeric nanoparticles. Analysis of the giant plasma membrane vesicles derived from treated cells using flicker spectroscopy reveals that liposome treatment alters membrane stiffness and DME of nanoparticles. Since liposomes have a history of use in drug delivery, localized priming of tumors with liposomes may present a hitherto unexploited means of targeting tumors based on biophysical interactions.

Plasma membrane aquaporins mediates vesicle stability in broccoli.

The use of in vitro membrane vesicles is attractive because of possible applications in therapies. Here we aimed to compare the stability and functionality of plasma membrane vesicles extracted from control and salt-treated broccoli. The impact of the amount of aquaporins was related to plasma membrane osmotic water permeability and the stability of protein secondary structure. Here, we describe for first time an increase in plant aquaporins acetylation under high salinity. Higher osmotic water permeability in NaCl vesicles has been related to higher acetylation, upregulation of aquaporins, and a more stable environment to thermal denaturation. Based on our findings, we propose that aquaporins play an important role in vesicle stability.

Lateral Distribution and Mobility of Transmembrane Proteins in Plasma Membrane Vesicles

Lateral Distribution and Mobility of Transmembrane Proteins in Plasma Membrane Vesicles

Cell-Like Mechanical Response in Passive Plasma Membrane Vesicles

Giant plasma membrane vesicles (GPMVs) which are isolated from adherent cells are an emerging model system for the study of lipid-lipid and lipid-protein interactions. The membrane composition of GPMVs closely resembles the plasma membrane lipidome and proteome, but lacks active remodeling and cytoskeletal support. In addition to the complex composition, GPMVs are expected to retain the plasma membrane structural characteristics, thus allowing access to material parameters otherwise concealed in the active cellular system. Here, we report bending rigidity values for GPMVs at varying GPMV isolation conditions. Further, we correlate them with the membrane viscosity and order assessed from Laurdan general polarization and fluorescent lifetimes of molecular rotors. When aspirated in micropipettes, GPMVs show unexpected mechanical properties, which are not found in simple lipid bilayers. GPMVs exhibit droplet-like instabilities upon increasing aspiration pressures. We find that the apparent elastic modulus matches closely reported values of cellular cortical tension. To understand how the passive GPMV membrane gives rise to cell-like responses, we employed fluorescent lipid analogs, nanotube pulling experiments and electron microscopy. We detect membrane reservoirs exhibiting lipid sorting and investigate their role in the observed soft elastic response. Additionally, fluorescent lifetime imaging of a membrane order sensitive dye helps to clarify the GPMV membrane structural response to mechanical tension. The results contribute to the understanding of the plasma membrane structure and enable synthetic reconstitution of plasma membrane like comportments.

Lipid-Anchored Ras Proteins Sense/Modulate Plasma Membrane Curvature in an Isoform- Specific Manner

Ras proteins, including isoforms H-, N- and K-Ras, are lipid-anchored small GTPases and play key roles in cell growth, division, proliferation, and migration. Constitutively active Ras mutants are oncogenic and found in 20% of all human tumors. Ras signaling is highly compartmentalized to the plasma membrane (PM), making their trafficking critical to their biological function. Ras trafficking is still poorly understood. Ras C-termini are lipid-modified, which potentially allows direct sensing and modulation of PM curvature, a key ultrastructural event in membrane trafficking. We measured Ras lateral oligomerization, an essential step in Ras signaling, on giant plasma membrane vesicles (GPMVs) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FLIM-FRET). Nanometer-scaled membrane curvature changes in the form of thermal undulations were manipulated via altering osmotic pressure. Hypotonic conditions disrupted local clustering of H-Ras anchor but enhanced K-Ras oligomerization, suggesting that H-Ras favors curved membrane and conversely for K-Ras. Super resolution electron microscopy-spatial mapping was used to quantify the lateral oligomerization of Ras on intact cell PM. Expression of positive curvature-inducing amphiphysin 2 BAR domain (BARamph2) enhanced H-Ras lateral oligomerization, but disrupted K-Ras oligomerization. On the other hand, expressing negative curvature-inducing IRS53p BAR domain (BARIRS53p) almost completely abolished H-Ras oligomerization, while having little effect on K-Ras. EM-bivariate co-localization analysis shows that H- or K-Ras co-localized extensively with both BAR domains. This was correlated with elevated mitogenic signaling (preferentially regulated by K-Ras) but diminished PI3K signaling (preferentially regulated by H-Ras) when exposed to hypotonic medium. Atomic force microscopy shows that expressing H-Ras anchor induced more PM protrusions and decreased the elastic Young's modulus of apical PM of live kidney cells, suggesting that H-Ras anchor induces PM curvature. Our data suggest that Ras senses and modulates membrane curvature in an isoform-specific manner.