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Sample records for lysosomal membrane stability

  1. Gastroprotection and lysosomal membrane stabilization by sulglicotide.

    PubMed

    Porta, R; Niada, R; Pescador, R; Mantovani, M; Prino, G

    1986-07-01

    Well-known agents that induce gastric ulcers cause a decrease in lysosomal stability, with release of lytic enzymes. Some antiulcer and cytoprotective agents have lysosomal membrane stabilizing activity when tested in vitro and ex vivo. Sulglicotide (Gliptide), a polysulfated glycopeptide with antiulcer and cytoprotective activities, was able to stabilize lysosomal membranes in vitro at concentrations between 9 and 36 micrograms/ml. The ratio of potency of sulglicotide to that of carbenoxolone was 12.2. In ex vivo experiments in rats, it was found that sulglicotide stabilized lysosomes after oral treatment. The effect was dose-dependent after intravenous treatment. Carbenoxolone, injected i.v. under the same experimental conditions, was less active (potency ratio 0.65). 16,16-dimethyl-PGE2, administered at a dose of 10 micrograms/kg orally or intravenously, had an activity equivalent to that of sulglicotide at a dose of 12.5 mg/kg i.v. or 200 mg/kg p.o. Sulglicotide (200-400 mg/kg p.o.) was also able to prevent the release of acid phosphatase from stomachs challenged for 10 min or 3 h with absolute ethanol. The same result was obtained with 200 mg/kg p.o. of carbenoxolone. These data show that sulglicotide is a potent lysosomal membrane stabilizer in vitro and ex vivo, and could explain the cytoprotective activity of this compound in different experimental models of ulcer.

  2. Enantioselective effects of methamidophos on the coelomocytes lysosomal membrane stability of Eisenia fetida.

    PubMed

    Chen, Linhua; Lu, Xianting; Ma, Yun

    2012-12-01

    Many of organophosphorous insecticides are chiral compounds. In this study, the enantioselective effects of organophosphate insecticide methamidophos on the coelomocytes lysosomal membrane stability of earthworm Eisenia fetida were studied: (1) The enantiomers of methamidophos were absolutely separated by high-performance liquid chromatography with a commercial chiral column; (2) The neutral red retention assay was used to judge the lysosomal membrane stability. The results showed that with the concentration increasing, lysosomal membranes have been significantly destroyed by individual stereoisomers and racemate of methamidophos. The neutral red retention times were significantly descended from 76.88 to 29.78 min. Both (+)- and (-)-methamidophos showed more prone to destroy the integrity of the lysosomal membrane than the racemate. However, the different effect between stereoisomers is slight.

  3. Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

    PubMed

    Okoro, Hussein K; Snyman, Reinette G; Fatoki, Olalekan S; Adekola, Folahan A; Ximba, Bhekumusa J; Slabber, Michelle Y

    2015-05-01

    The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p < 0.05) whole body TBT concentrations with concomitant significant decreases (p < 0.05) in NRRT (R(2) values of 0.85 and 0.971 for lower and higher exposure groups, respectively). The higher exposure group showed a typical dose-response curve. For the control, no TBT was detected and NRRT remained stable. It was concluded that the NRRT assay could be considered as a useful technique, and lysosomal membrane destabilization a useful early warning and cellular biomarker of stress due to TBT exposure in M. galloprovincialis.

  4. Lysosomal degradation of membrane lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2010-05-03

    The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Effects of arachidonic acid on the lysosomal ion permeability and osmotic stability.

    PubMed

    Zhang, Gu; Yi, Ya-Ping; Zhang, Guo-Jiang

    2006-02-01

    In this study, we investigated the effects of arachidonic acid, a PLA2-produced lipid metabolite, on the lysosomal permeability, osmotic sensitivity and stability. Through the measurements of lysosomal beta-hexosaminidase free activity, membrane potential, intralysosomal pH, and lysosomal latency loss in hypotonic sucrose medium, we established that arachidonic acid could increase the lysosomal permeability to both potassium ions and protons, and enhance the lysosomal osmotic sensitivity. As a result, the fatty-acid-promoted entry of potassium ions into the lysosomes via K+/H+ exchange, which could produce osmotic imbalance across their membranes and osmotically destabilize the lysosomes. In addition, the enhancement of lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shock. The results suggest that arachidonic acid may play a role in the lysosomal destabilization.

  6. Stabilization of lysosomes in anoxic myocardium by propranolol.

    PubMed Central

    Welman, E

    1979-01-01

    1. Isolated hearts of guinea-pigs were perfused aerobically and anoxically for 60 min. (+/-)-Propranolol was added to the perfusion medium in concentrations ranging from 10 ng to 10 microgram/ml. 2. Lysosome stability was assessed by measurements of latent acid hydrolase activity in homogenates of left ventricular tissue. 3. In the absence of propranolol, the integrity of the myocardial lysosomes was considerably reduced after 60 min anoxia. Lysosome stability was enhanced by the presence of (+/-)-propranolol. The optimal concentration was found to be 0.1 microgram/ml. This concentration of the (+)-isomer alone was less effective. 4. It was concluded that beta-adrenoceptor blockade was the major mechanism by which lysosome disruption was prevented but that some direct membrane effect of propranolol may also be involved. PMID:427322

  7. Membrane stabilizer

    DOEpatents

    Mingenbach, William A.

    1988-01-01

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material.

  8. DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy).

    PubMed

    Pisoni, M; Cogotzi, L; Frigeri, A; Corsi, I; Bonacci, S; Iacocca, A; Lancini, L; Mastrototaro, F; Focardi, S; Svelto, M

    2004-10-01

    The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a "cleaner" reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of beta-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r = 0.60 in 2001; r = 0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization

  9. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization.

    PubMed

    Liu, Jiazhuo; Peng, Leiwen; Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

    2016-01-26

    PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the "verge of apoptosis". When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways.

  10. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization

    PubMed Central

    Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

    2016-01-01

    PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. PMID:26716897

  11. Membrane stabilizer

    DOEpatents

    Mingenbach, W.A.

    1988-02-09

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material. 10 figs.

  12. Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT).

    PubMed

    Gutierrez, Elaine M; Seebacher, Nicole A; Arzuman, Laila; Kovacevic, Zaklina; Lane, Darius J R; Richardson, Vera; Merlot, Angelica M; Lok, Hiu; Kalinowski, Danuta S; Sahni, Sumit; Jansson, Patric J; Richardson, Des R

    2016-07-01

    The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-β-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (p<0.001) reduced the ability of Dp44mT to inhibit cancer cell proliferation (i.e., increased the IC(50)) by 140-fold. On the other hand, cholesterol extraction using methyl-β-cyclodextrin enhanced Dp44mT-induced LMP and significantly (p<0.01) increased its anti-proliferative activity. The protective effect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.

  13. The potential role of cAMP as a pollution biomarker of terrestrial environments using the land snail Eobania vermiculata: correlation with lysosomal membrane stability.

    PubMed

    Itziou, A; Dimitriadis, V K

    2009-09-01

    The present study investigates the role of the signal transduction molecule cAMP, and the lysosomal membrane stability (LMS), as biomarkers of terrestrial environmental pollution using the land snail Eobania vermiculata. Snails were exposed to different concentrations of heavy metals (Cd, Pb and Cu) and organic pollutants (chlorpyrifos, parathion-methyl and PAHs) in laboratory conditions for 25 days. In addition, snails were collected from various sites located at different distances away from two polluted areas in northern Greece (the road Agiou Dimitriou in Thessaloniki city and a lignite power station in the district of Kozani). The results of the current investigation showed significantly increased levels of cAMP in the digestive gland of snails, as well as decreased LMS values in all experimental groups compared to control animals. In support of our data, cAMP levels were significantly negatively correlated with the conventional biomarker LMS, thus encouraging the use of cAMP as a new potential stress index in terrestrial pollution biomonitoring studies.

  14. Principles of lysosomal membrane degradation: Cellular topology and biochemistry of lysosomal lipid degradation.

    PubMed

    Schulze, Heike; Kolter, Thomas; Sandhoff, Konrad

    2009-04-01

    Cellular membranes enter the lysosomal compartment by endocytosis, phagocytosis, or autophagy. Within the lysosomal compartment, membrane components of complex structure are degraded into their building blocks. These are able to leave the lysosome and can then be utilized for the resynthesis of complex molecules or can be further degraded. Constitutive degradation of membranes occurs on the surface of intra-endosomal and intra-lysosomal membrane structures. Many integral membrane proteins are sorted to the inner membranes of endosomes and lysosome after ubiquitinylation. In the lysosome, proteins are degraded by proteolytic enzymes, the cathepsins. Phospholipids originating from lipoproteins or cellular membranes are degraded by phospholipases. Water-soluble glycosidases sequentially cleave off the terminal carbohydrate residues of glycoproteins, glycosaminoglycans, and glycosphingolipids. For glycosphingolipids with short oligosaccharide chains, the additional presence of membrane-active lysosomal lipid-binding proteins is required. The presence of lipid-binding proteins overcomes the phase problem of water soluble enzymes and lipid substrates by transferring the substrate to the degrading enzyme or by solubilizing the internal membranes. The lipid composition of intra-lysosomal vesicles differs from that of the plasma membrane. To allow at least glycosphingolipid degradation by hydrolases and activator proteins, the cholesterol content of these intraorganellar membranes decreases during endocytosis and the concentration of bis(monoacylglycero)phosphate, a stimulator of sphingolipid degradation, increases. A considerable part of our current knowledge about mechanism and biochemistry of lysosomal lipid degradation is derived from a class of human diseases, the sphingolipidoses, which are caused by inherited defects within sphingolipid and glycosphingolipid catabolism.

  15. Using lysosomal membrane stability of haemocytes in Ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin.

    PubMed

    Aguirre-Martínez, Gabriela V; Buratti, Sara; Fabbr, Elena; DelValls, Angel T; Martín-Díaz, M Laura

    2013-07-01

    Although pharmaceuticals have been detected in the environment only in the range from ng/L to microg/L, it has been demonstrated that they can adversely affect the health status of aquatic organisms. Lysosomal membrane stability (LMS) has previously been applied as an indicator of cellular well-being to determine health status in bivalve mussels. The objective of this study is to evaluate LMS in Ruditapes philippinarum haemolymph using the neutral red retention assay (NRRA). Clams were exposed in laboratory conditions to caffeine (0.1, 5, 15, 50 microg/L), ibuprofen (0.1, 5, 10, 50 microg/L), carbamazepine and novobiocin (both at 0.1, 1, 10, 50 microg/L) for 35 days. Results show a dose-dependent effect of the pharmaceuticals. The neutral red retention time measured at the end of the bioassay was significantly reduced by 50% after exposure to environmental concentrations (p < 0.05) (caffeine = 15 microg/L; ibuprofen = 10 microg/L; carbamazepine = 1 microg/L and novobiocin = 1 microg/L), compared to controls. Clams exposed to these pharmaceuticals were considered to present a diminished health status (retention time < 45 min), significantly worse than controls (96 min) (p < 0.05). The predicted no environmental effect concentration (PNEC) results showed that these pharmaceuticals are very toxic at the environmental concentrations tested. Measurement of the alteration of LMS has been found to be a sensitive technique that enables evaluation of the health status of clams after exposure to pharmaceuticals under laboratory conditions, thus representing a robust Tier-1 screening biomarker.

  16. Methods for the quantification of lysosomal membrane permeabilization: a hallmark of lysosomal cell death.

    PubMed

    Aits, Sonja; Jäättelä, Marja; Nylandsted, Jesper

    2015-01-01

    Lysosomal cell death is triggered by lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal hydrolases from the lysosomal lumen into the cytosol. Once released into the cytosol, the lysosomal cathepsin proteases act as executioner proteases for the subsequent cell death-either autonomously without caspase activation or in concert with the classical apoptotic machinery. Lysosomal cell death usually remains functional in apoptosis-resistant cancer cells and thus holds great potential as a therapeutic strategy for circumventing apoptosis deficiency in cancers. Notably, lysosomal cell death also plays an important role in normal physiology, e.g., during the regression of the mammary gland. Here we present four complementary methods for the quantification and visualization of LMP during the onset of death: (1) enzymatic activity measurements of released lysosomal hydrolases in the cytosol after digitonin extraction, (2) direct visualization of LMP by monitoring the release of fluorescent dextran from lysosomes into the cytosol, (3) immunocytochemistry to detect cathepsins released into the cytosol, and (4) detection of the translocation of galectins to damaged lysosomes. The methods presented here can ideally be combined as needed to provide solid evidence for LMP after a given cytotoxic stimuli.

  17. Action of low-energy monochromatic coherent light on the stability of retinal lysosomes

    NASA Astrophysics Data System (ADS)

    Metelitsina, Irina P.; Leus, N. F.

    1995-05-01

    The data had been obtained during the experiment in vitro by irradiation of solubilized lysosomal enzymes, retinal homogenates and native lysosomes enabled us to conclude that the laser beam ((lambda) equals 632.8 nm, power density from 0.1 to 15.0 mWt/cm2) acts on the level of membranous structures of lysosomes. During irradiation of rabbits eyes in vitro with an unfocused laser beam (power density on the cornea aur face from 0.01 to 15.0 mWt/cm2 was shown, that low-energy, ranged from 0.01 to 1.0 mWt/cm2 promotes stabilization of lysosomal membranes. Irradiation with laser beam of 8.0 mWt/cm2 and more power induces destabilization of lysosomal membranes. We have also shown that vitamins A and E effecting membranotropic on lysosomes may be corrected by low-energy radiation of helium-neon laser. It is substantiated experimentally that the stabilizing effect of vitamin E may be intensified in case of the combined action of laser radiation on lysosomes. The labilizing effect of vitamin A on membranes of organelles, as was studied, may be weakened by application of laser radiation of low intensities.

  18. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

    PubMed Central

    Aits, Sonja; Kricker, Jennifer; Liu, Bin; Ellegaard, Anne-Marie; Hämälistö, Saara; Tvingsholm, Siri; Corcelle-Termeau, Elisabeth; Høgh, Søren; Farkas, Thomas; Holm Jonassen, Anna; Gromova, Irina; Mortensen, Monika; Jäättelä, Marja

    2015-01-01

    Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation. PMID:26114578

  19. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay.

    PubMed

    Aits, Sonja; Kricker, Jennifer; Liu, Bin; Ellegaard, Anne-Marie; Hämälistö, Saara; Tvingsholm, Siri; Corcelle-Termeau, Elisabeth; Høgh, Søren; Farkas, Thomas; Holm Jonassen, Anna; Gromova, Irina; Mortensen, Monika; Jäättelä, Marja

    2015-01-01

    Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation.

  20. Lysosomal membrane stability, phagocytosis and tolerance to emersion in the mussel Perna viridis (Bivalvia: Mytilidae) following exposure to acute, sublethal, copper.

    PubMed

    Nicholson, S

    2003-08-01

    The mytilid mussel Perna viridis is distributed throughout the Indo-Pacific region and is potentially a suitable candidate for biological effects (biomarker) monitoring in the subtropics. A suite of cytological and physiological responses to acute (48-72 h) copper exposures of 50-200 microgl(-1) were assessed in order to determine the suitability of P. viridis for marine pollution monitoring. Copper elicited significant destabilisation of the haemocyte lysosomal membranes and also impaired phagocytosis. Survival during emersion following exposure to copper was not related to the experimental copper exposures suggesting that higher metal concentrations may be required to interfere with anaerobic enzymes responsible for suppression of metabolism. Based on this preliminary study, cytological biomarkers evaluated in the haemocytes extracted from P. viridis should prove an effective non-destructive means of assessing metal pollution throughout the mussels subtropical range.

  1. Lysosomal membrane permeability stimulates protein aggregate formation in neurons of a lysosomal disease.

    PubMed

    Micsenyi, Matthew C; Sikora, Jakub; Stephney, Gloria; Dobrenis, Kostantin; Walkley, Steven U

    2013-06-26

    Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2(-/-) mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin-proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2(-/-) neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation.

  2. Membrane proteins of dense lysosomes from Chinese hamster ovary cells

    SciTech Connect

    Chance, S.C.

    1987-01-01

    In this work membrane proteins from lysosomes were studied in order to gain more information on the biogenesis and intracellular sorting of this class of membrane proteins. Membrane proteins were isolated from a purified population of lysosomes. These proteins were then examined for various co- and post-translational modifications which could serve as potential intracellular sorting signals. Biochemical analysis using marker enzymatic activities detected no plasma membrane, Golgi, endoplasmic reticulum, peroxisomes, mitochondria, or cytosol. Analysis after incorporation of ({sup 3}H)thymidine or ({sup 3}H)uridine detected no nuclei or ribosomes. A fraction containing integral membrane proteins was obtained from the dense lysosomes by extraction with Triton X-114. Twenty-three polypeptides which incorporated both ({sup 35}S)methionine and ({sup 3}H)leucine were detected by SDS PAGE in this membrane fraction, and ranged in molecular weight from 30-130 kDa. After incorporation by cells of various radioactive metabolic precursors, the membrane fraction from dense lysosomes was examined and was found to be enriched in mannose, galactose, fucose, palmitate, myristate, and sulfate, but was depleted in phosphate. The membrane fraction from dense lysosomes was then analyzed by SDS PAGE to determine the apparent molecular weights of modified polypepties.

  3. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

    PubMed Central

    Bharadwaj, Rajnish; Cunningham, Kathleen M.; Zhang, Ke; Lloyd, Thomas E.

    2016-01-01

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. PMID:26662798

  4. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

    PubMed

    Bharadwaj, Rajnish; Cunningham, Kathleen M; Zhang, Ke; Lloyd, Thomas E

    2016-02-15

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS.

  5. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells.

    PubMed

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth; Petersen, Nikolaj H T; Nylandsted, Jesper; Jäättelä, Marja

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.

  6. Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export

    PubMed Central

    Li, Jian; Pfeffer, Suzanne R

    2016-01-01

    LAMP1 and LAMP2 proteins are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon’s disease, an X-linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone-mediated autophagy, and its expression improves tissue function in models of aging. We show here that human LAMP1 and LAMP2 bind cholesterol in a manner that buries the cholesterol 3β-hydroxyl group; they also bind tightly to NPC1 and NPC2 proteins that export cholesterol from lysosomes. Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly. DOI: http://dx.doi.org/10.7554/eLife.21635.001 PMID:27664420

  7. Properties of the membrane proteins of rat liver lysosomes. The majority of lysosomal membrane proteins are exposed to the cytoplasm.

    PubMed Central

    Schneider, D L; Burnside, J; Gorga, F R; Nettleton, C J

    1978-01-01

    Rat liver lysosomes were lysed and subfractionated by differential centrifugation through 0.2M-NaCl to yield a membranous pellet. This membrane fraction contains less than 20% of the lysosomal protein, adenosine triphosphatase activity of about 1.2mumol/min per mg of protein, 120nmol of thiol groups/mg of protein and at least 16 protein and glycoprotein bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The gel patterns of membranes isolated from lysosomes after treatment with (1) [125I]iodidehydrogen peroxide-lactoperoxidase, (2) toluene 2,4-di-isocyanate-activated bovine serum albumin, (3) trypsin and (4) subtilisin indicate that most of the membrane proteins are exposed to the cytoplasm. These exposed proteins are candidates for intracellular receptors which recognize either substances that are to be degraded or vesicles containing those substances. PMID:153136

  8. Passive diffusion of non-electrolytes across the lysosome membrane.

    PubMed Central

    Iveson, G P; Bird, S J; Lloyd, J B

    1989-01-01

    An osmotic-protection method has been used to study the permeability of rat liver lysosomes to 43 organic non-electrolytes of formula weights ranging from 62 to 1000. A lysosome-rich centrifugal fraction of rat liver homogenate was resuspended in an unbuffered 0.25 M solution of test solute, pH 7.0, and incubated at 25 degrees C for 60 min. The free and total activities of 4-methylumbelliferyl N-acetyl-beta-D-glucosaminidase were measured after incubation for 0, 30 and 60 min. Three patterns of results were seen. In pattern A the percentage free activity remained low throughout the 60 min incubation, indicating little or no solute entry into the lysosomes. In pattern B, the percentage free activity was initially low, but rose substantially during the incubation, indicating solute entry. In pattern C there was not even initial osmotic protection, indicating very rapid solute entry. The rapidity of solute entry into the lysosomes showed no correlation with the formula weight, but a perfect inverse correlation with the hydrogen-bonding capacity of the solutes. The results, which can be used to predict the ability of further compounds to cross the lysosome membrane by unassisted diffusion, are discussed in the context of metabolite and drug release from lysosomes in vivo. PMID:2775227

  9. Discriminating lysosomal membrane protein types using dynamic neural network.

    PubMed

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  10. Selective Lysosomal Transporter Degradation by Organelle Membrane Fusion.

    PubMed

    McNally, Erin Kate; Karim, Mahmoud Abdul; Brett, Christopher Leonard

    2017-01-23

    Lysosomes rely on their resident transporter proteins to return products of catabolism to the cell for reuse and for cellular signaling, metal storage, and maintaining the lumenal environment. Despite their importance, little is known about the lifetime of these transporters or how they are regulated. Using Saccharomyces cerevisiae as a model, we discovered a new pathway intrinsic to homotypic lysosome membrane fusion that is responsible for their degradation. Transporter proteins are selectively sorted by the docking machinery into an area between apposing lysosome membranes, which is internalized and degraded by lumenal hydrolases upon organelle fusion. These proteins have diverse lifetimes that are regulated in response to protein misfolding, changing substrate levels, or TOR activation. Analogous to endocytosis for controlling surface protein levels, the "intralumenal fragment pathway" is critical for lysosome membrane remodeling required for organelle function in the context of cellular protein quality control, ion homeostasis, and metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

    PubMed Central

    Mundy, Dorothy I.; Li, Wei Ping; Luby-Phelps, Katherine; Anderson, Richard G. W.

    2012-01-01

    Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking. PMID:22238363

  12. The Chaperone-Mediated Autophagy Receptor Organizes in Dynamic Protein Complexes at the Lysosomal Membrane ▿ †

    PubMed Central

    Bandyopadhyay, Urmi; Kaushik, Susmita; Varticovski, Lyuba; Cuervo, Ana Maria

    2008-01-01

    Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane. PMID:18644871

  13. The influence of oxidation of membrane thiol groups on lysosomal proton permeability.

    PubMed Central

    Wan, F Y; Wang, Y N; Zhang, G J

    2001-01-01

    The influence of oxidation of membrane thiol groups on lysosomal proton permeability was studied by measuring lysosomal pH with FITC-conjugated dextran, determining the membrane potential with 3,3'-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Residual membrane thiol groups were measured with 5,5'-dithiobis-(2-nitrobenzoic acid). The lysosomal membrane thiol groups were modified by treatment with diamide and dithiothreitol. SDS/PAGE revealed aggregations of the membrane proteins induced by the treatment of lysosomes with diamide. The cross-linkage of proteins could be abolished by subsequent treatment with dithiothreitol, indicating that the proteins were linked via disulphide bonds. Treating the lysosomes with diamide decreased their membrane thiol groups and caused increases in lysosomal pH, membrane potential and proton leakage, which could be reversed by treatment of the lysosomes with dithiothreitol. This indicates that the lysosomal proton permeability can be increased by oxidation of the membrane thiol groups and restored to the normal level by reduction of the groups. Treatment of the lysosomes with N-ethylmaleimide reduced their membrane thiol groups but did not change the lysosomal pH or their degree of proton leakage. It suggests that protein aggregation may be an important mechanism for the increase in lysosomal proton permeability. The results raise the possibility that the proton permeability of lysosomes in vivo may be affected by the redox states of their membrane thiol groups. PMID:11716763

  14. Cholesterol transport through lysosome-peroxisome membrane contacts.

    PubMed

    Chu, Bei-Bei; Liao, Ya-Cheng; Qi, Wei; Xie, Chang; Du, Ximing; Wang, Jiang; Yang, Hongyuan; Miao, Hong-Hua; Li, Bo-Liang; Song, Bao-Liang

    2015-04-09

    Cholesterol is dynamically transported among organelles, which is essential for multiple cellular functions. However, the mechanism underlying intracellular cholesterol transport has remained largely unknown. We established an amphotericin B-based assay enabling a genome-wide shRNA screen for delayed LDL-cholesterol transport and identified 341 hits with particular enrichment of peroxisome genes, suggesting a previously unappreciated pathway for cholesterol transport. We show dynamic membrane contacts between peroxisome and lysosome, which are mediated by lysosomal Synaptotagmin VII binding to the lipid PI(4,5)P2 on peroxisomal membrane. LDL-cholesterol enhances such contacts, and cholesterol is transported from lysosome to peroxisome. Disruption of critical peroxisome genes leads to cholesterol accumulation in lysosome. Together, these findings reveal an unexpected role of peroxisome in intracellular cholesterol transport. We further demonstrate massive cholesterol accumulation in human patient cells and mouse model of peroxisomal disorders, suggesting a contribution of abnormal cholesterol accumulation to these diseases. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Para-toluenesulfonamide induces tongue squamous cell carcinoma cell death through disturbing lysosomal stability.

    PubMed

    Liu, Zhe; Liang, Chenyuan; Zhang, Zhuoyuan; Pan, Jian; Xia, Hui; Zhong, Nanshan; Li, Longjiang

    2015-11-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability, invasion, and cell death were determined. PTS significantly inhibited Tca-8113 cells' viability and invasive ability with increased cancer cell death. Flow cytometric analysis and the lactate dehydrogenase release assay showed that PTS induced cancer cell death by activating apoptosis and necrosis simultaneously. Morphological changes, such as cellular shrinkage, nuclear condensation as well as formation of apoptotic body and secondary lysosomes, were observed, indicating that PTS might induce cell death through disturbing lysosomal stability. Lysosomal integrity assay and western blot showed that PTS increased lysosomal membrane permeabilization associated with activation of lysosomal cathepsin B. Finally, PTS was shown to inhibit ATP biosynthesis and induce the release of mitochondrial cytochrome c. Therefore, our findings provide a novel insight into the use of PTS in cancer therapy.

  16. TM7SF1 (GPR137B): a novel lysosome integral membrane protein.

    PubMed

    Gao, Jialin; Xia, Libin; Lu, Meiqing; Zhang, Binhua; Chen, Yueping; Xu, Rang; Wang, Lizhuo

    2012-09-01

    In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane.

  17. Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis

    PubMed Central

    1989-01-01

    A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn- 78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with

  18. Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation.

    PubMed

    Appelqvist, Hanna; Nilsson, Cathrine; Garner, Brett; Brown, Andrew J; Kågedal, Katarina; Ollinger, Karin

    2011-02-01

    In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.

  19. A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair

    PubMed Central

    Encarnação, Marisa; Mateus, Denisa; Michelet, Xavier; Santarino, Inês; Hsu, Victor W.; Brenner, Michael B.

    2016-01-01

    Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis. PMID:27325790

  20. Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl-Glucosaminidase Activity Measurements.

    PubMed

    Jäättelä, Marja; Nylandsted, Jesper

    2015-11-02

    Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or β-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).

  1. Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

    PubMed Central

    Hissa, Bárbara; Duarte, Jacqueline G.; Kelles, Ludmila F.; Santos, Fabio P.; del Puerto, Helen L.; Gazzinelli-Guimarães, Pedro H.; de Paula, Ana M.; Agero, Ubirajara; Mesquita, Oscar N.; Guatimosim, Cristina; Chiari, Egler; Andrade, Luciana O.

    2012-01-01

    Background Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. Methodology/Principal Finding In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. Conclusion/Significance Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are

  2. Synergistic Anticancer Action of Lysosomal Membrane Permeabilization and Glycolysis Inhibition.

    PubMed

    Kosic, Milica; Arsikin-Csordas, Katarina; Paunovic, Verica; Firestone, Raymond A; Ristic, Biljana; Mircic, Aleksandar; Petricevic, Sasa; Bosnjak, Mihajlo; Zogovic, Nevena; Mandic, Milos; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

    2016-10-28

    We investigated the in vitro and in vivo anticancer effect of combining lysosomal membrane permeabilization (LMP)-inducing agent N-dodecylimidazole (NDI) with glycolytic inhibitor 2-deoxy-d-glucose (2DG). NDI-triggered LMP and 2DG-mediated glycolysis block synergized in inducing rapid ATP depletion, mitochondrial damage, and reactive oxygen species production, eventually leading to necrotic death of U251 glioma cells but not primary astrocytes. NDI/2DG-induced death of glioma cells was partly prevented by lysosomal cathepsin inhibitor E64 and antioxidant α-tocopherol, suggesting the involvement of LMP and oxidative stress in the observed cytotoxicity. LMP-inducing agent chloroquine also displayed a synergistic anticancer effect with 2DG, whereas glucose deprivation or glycolytic inhibitors iodoacetate and sodium fluoride synergistically cooperated with NDI, thus further indicating that the anticancer effect of NDI/2DG combination was indeed due to LMP and glycolysis block. The two agents synergistically induced ATP depletion, mitochondrial depolarization, oxidative stress, and necrotic death also in B16 mouse melanoma cells. Moreover, the combined oral administration of NDI and 2DG reduced in vivo melanoma growth in C57BL/6 mice by inducing necrotic death of tumor cells, without causing liver, spleen, or kidney toxicity. Based on these results, we propose that NDI-triggered LMP causes initial mitochondrial damage that is further increased by 2DG due to the lack of glycolytic ATP required to maintain mitochondrial health. This leads to a positive feedback cycle of mitochondrial dysfunction, ATP loss, and reactive oxygen species production, culminating in necrotic cell death. Therefore, the combination of LMP-inducing agents and glycolysis inhibitors seems worthy of further exploration as an anticancer strategy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Alterations in membrane trafficking and pathophysiological implications in lysosomal storage disorders.

    PubMed

    Kuech, Eva-Maria; Brogden, Graham; Naim, Hassan Y

    2016-11-01

    Lysosomal storage disorders are a heterogeneous group of more than 50 distinct inborn metabolic diseases affecting about 1 in 5000 to 7000 live births. The diseases often result from mutations followed by functional deficiencies of enzymes or transporters within the acidic environment of the lysosome, which mediate the degradation of a wide subset of substrates, including glycosphingolipids, glycosaminoglycans, cholesterol, glycogen, oligosaccharides, peptides and glycoproteins, or the export of the respective degradation products from the lysosomes. The progressive accumulation of uncleaved substrates occurs in multiple organs and finally causes a broad spectrum of different pathologies including visceral, neurological, skeletal and hematologic manifestations. Besides deficient lysosomal enzymes and transporters other defects may lead to lysosomal storage disorders, including activator defects, membrane defects or defects in modifier proteins. In this review we concentrate on four different lysosomal storage disorders: Niemann-Pick type C, Fabry disease, Gaucher disease and Pompe disease. While the last three are caused by defective lysosomal hydrolases, Niemann-Pick type C is caused by the inability to export LDL-derived cholesterol out of the lysosome. We want to emphasise potential implications of membrane trafficking defects on the pathology of these diseases, as many mutations interfere with correct lysosomal protein trafficking and alter cellular lipid homeostasis. Current therapeutic strategies are summarised, including substrate reduction therapy as well as pharmacological chaperone therapy which directly aim to improve folding and lysosomal transport of misfolded mutant proteins.

  4. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

    PubMed Central

    Corrotte, M.; Castro-Gomes, T.; Koushik, A.B.; Andrews, N.W.

    2016-01-01

    Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane. PMID:25665445

  5. A quantitative model of traffic between plasma membrane and secondary lysosomes: evaluation of inflow, lateral diffusion, and degradation

    PubMed Central

    1988-01-01

    We present here a mathematical model that accounts for the various proportions of plasma membrane constituents occurring in the lysosomal membrane of rat fibroblasts (Draye, J.-P., J. Quintart, P. J. Courtoy, and P. Baudhuin. 1987. Eur. J. Biochem. 170: 395-403; Draye, J.-P., P. J. Courtoy, J. Quintart, and P. Baudhuin. 1987. Eur. J. Biochem. 170:405-411). It is based on contents of plasma membrane markers in purified lysosomal preparations, evaluations of their half-life in lysosomes and measurements of areas of lysosomal and plasma membranes by morphometry. In rat fibroblasts, structures labeled by a 2-h uptake of horseradish peroxidase followed by a 16-h chase (i.e., lysosomes) occupy 3% of the cellular volume and their total membrane area corresponds to 30% of the pericellular membrane area. Based on the latter values, the model predicts the rate of inflow and outflow of plasma membrane constituents into lysosomal membrane, provided their rate of degradation is known. Of the bulk of polypeptides iodinated at the cell surface, only 4% reach the lysosomes every hour, where the major part (integral of 83%) is degraded with a half-life in lysosomes of integral to 0.8 h. For specific plasma membrane constituents, this model can further account for differences in the association to the lysosomal membrane by variations in the rate either of lysosomal degradation, of inflow along the pathway from the pericellular membrane to the lysosomes, or of lateral diffusion. PMID:2848849

  6. β2-microglobulin amyloid fibrils are nanoparticles that disrupt lysosomal membrane protein trafficking and inhibit protein degradation by lysosomes.

    PubMed

    Jakhria, Toral; Hellewell, Andrew L; Porter, Morwenna Y; Jackson, Matthew P; Tipping, Kevin W; Xue, Wei-Feng; Radford, Sheena E; Hewitt, Eric W

    2014-12-26

    Fragmentation of amyloid fibrils produces fibrils that are reduced in length but have an otherwise unchanged molecular architecture. The resultant nanoscale fibril particles inhibit the cellular reduction of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), a substrate commonly used to measure cell viability, to a greater extent than unfragmented fibrils. Here we show that the internalization of β2-microglobulin (β2m) amyloid fibrils is dependent on fibril length, with fragmented fibrils being more efficiently internalized by cells. Correspondingly, inhibiting the internalization of fragmented β2m fibrils rescued cellular MTT reduction. Incubation of cells with fragmented β2m fibrils did not, however, cause cell death. Instead, fragmented β2m fibrils accumulate in lysosomes, alter the trafficking of lysosomal membrane proteins, and inhibit the degradation of a model protein substrate by lysosomes. These findings suggest that nanoscale fibrils formed early during amyloid assembly reactions or by the fragmentation of longer fibrils could play a role in amyloid disease by disrupting protein degradation by lysosomes and trafficking in the endolysosomal pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Lysosomal exocytosis in response to subtle membrane damage following nanosecond pulse exposure

    NASA Astrophysics Data System (ADS)

    Dalzell, Danielle R.; Roth, Caleb C.; Bernhard, Joshua A.; Payne, Jason A.; Wilmink, Gerald J.; Ibey, Bennett L.

    2011-03-01

    The cellular response to subtle membrane damage following exposure to nanosecond electric pulses (nsEP) is not well understood. Recent work has shown that when cells are exposed to nsEP, ion permeable nanopores (< 2nm) are created in the plasma membrane in contrast to larger diameter pores (> 2nm) created by longer micro and millisecond duration pulses. Macroscopic damage to a plasma membrane by a micropipette has been shown to cause internal vesicles (lysosomes) to undergo exocytosis to repair membrane damage, a calcium mediated process called lysosomal exocytosis. Formation of large pores in the plasma membrane by electrical pulses has been shown to elicit lysosomal exocytosis in a variety of cell types. Our research objective is to determine whether lysosomal exocytosis will occur in response to nanopores formed by exposure to nsEP. In this paper we used propidium iodide (PI) and Calcium Green-1 AM ester (CaGr) to differentiate between large and small pores formed in CHO-K1 cells following exposure to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm. This information was compared to changes in membrane organization observed by increases in FM1-43 fluorescence, both in the presence and absence of calcium ions in the outside buffer. In addition, we monitored the real time migration of lysosomes within the cell using Cellular Lights assay to tag LAMP-1, a lysosomal membrane protein. Both 1 and 20 pulses elicited a large influx of extracellular calcium, while little PI uptake was observed following a single pulse exposure. Statistically significant increases in FM1-43 fluorescence were seen in samples containing calcium suggesting that calcium-triggered membrane repair may be occurring. Lastly, density of lysosomes within cells, specifically around the nucleus, appeared to change rapidly upon nsEP stimulation suggesting lysosomal migration.

  8. Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system.

    PubMed

    Kornhuber, Johannes; Henkel, Andreas W; Groemer, Teja W; Städtler, Sven; Welzel, Oliver; Tripal, Philipp; Rotter, Andrea; Bleich, Stefan; Trapp, Stefan

    2010-07-01

    Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.

  9. Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

    PubMed Central

    2016-01-01

    Objectives There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. Methods It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. Results The result showed 14 up-regulated (1.5-fold) and 13 down-regulated (2.0-fold) spots in relation to melanin exposure. Conclusions It has been found that lysosomal membrane proteins are associated with melanin to decolorize and quantity through cellular activation of lysosome. PMID:27158002

  10. Lysosomal Membrane Permeabilization Induces Cell Death in a Mitochondrion-dependent Fashion

    PubMed Central

    Boya, Patricia; Andreau, Karine; Poncet, Delphine; Zamzami, Naoufal; Perfettini, Jean-Luc; Metivier, Didier; Ojcius, David M.; Jäättelä, Marja; Kroemer, Guido

    2003-01-01

    A number of diseases are due to lysosomal destabilization, which results in damaging cell loss. To investigate the mechanisms of lysosomal cell death, we characterized the cytotoxic action of two widely used quinolone antibiotics: ciprofloxacin (CPX) or norfloxacin (NFX). CPX or NFX plus UV light (NFX*) induce lysosomal membrane permeabilization (LMP), as detected by the release of cathepsins from lysosomes. Inhibition of the lysosomal accumulation of CPX or NFX suppresses their capacity to induce LMP and to kill cells. CPX- or NFX-triggered LMP results in caspase-independent cell death, with hallmarks of apoptosis such as chromatin condensation and phosphatidylserine exposure on the plasma membrane. LMP triggers mitochondrial membrane permeabilization (MMP), as detected by the release of cytochrome c. Both CPX and NFX* cause Bax and Bak to adopt their apoptotic conformation and to insert into mitochondrial membranes. Bax−/− Bak−/− double knockout cells fail to undergo MMP and cell death in response to CPX- or NFX-induced LMP. The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum–targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death. Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization. PMID:12756268

  11. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation.

    PubMed

    Chen, Fang; Zhang, Bing; Sun, Yong; Xiong, Zeng-Xing; Peng, Han; Deng, Ze-Yuan; Hu, Jiang-Ning

    2016-04-25

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05) in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  12. Ubiquitin-dependent sorting of integral membrane proteins for degradation in lysosomes

    PubMed Central

    Piper, Robert C.

    2007-01-01

    Summary The pathways that deliver newly synthesized proteins that reside in lysosomes are well understood by comparison with our knowledge of how integral membrane proteins are sorted and delivered to the lysosome for degradation. Many membrane proteins are sorted to lysosomes following ubiquitination, which provides a sorting signal that can operate for sorting at the TGN (trans-Golgi network), at the plasma membrane or at the endosome for delivery into lumenal vesicles. Candidate multicomponent machines that can potentially move ubiquitinated integral membrane cargo proteins have been identified, but much work is still required to ascertain which of these candidates directly recognizes ubiquitinated cargo and what they do with cargo after recognition. In the case of the machinery required for sorting into the lumenal vesicles of endosomes, other functions have also been determined including a link between sorting and movement of endosomes along microtubules. PMID:17689064

  13. Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane

    PubMed Central

    1992-01-01

    We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail. PMID:1560028

  14. Intracellular target for alpha-terthienyl photosensitization: involvement of lysosomal membrane damage.

    PubMed

    Sasaki, M; Koyama, S; Tokiwa, K; Fujita, H

    1993-05-01

    Intracellular targets for the photosensitizer alpha-terthienyl (alpha T) were examined by fluorescence microscopy and microfluorospectrometry using human nonkeratinized buccal cells. Intracellular distribution of alpha T was observed as fluorescent patches widely dispersed in the cytoplasm. The distribution of the fluorescent patches was compared with that of acid phosphatase activity visualized as an azo dye produced by the fast garnet 2-methyl-4-[(2-methyl-phenyl)azo]benzenediasonium sulfate reaction. Because both the distribution sites coincided, lysosomes were the likely sites of intracellular affinity of alpha T. However, because acid phosphatase is not a specific lysosomal marker, we tried to detect another lysosomal enzyme, beta-galactosidase, to confirm if the fluorescent patches were lysosomes, using fluorescein-di-(beta-D-galactopyranoside) (FDG) as a fluorogenic substrate. Without UV-A (320-400 nm) irradiation of the cells after uptake of alpha T and FDG, no significant fluorescence was observed. In contrast, with prior UV-A irradiation in the presence of alpha T and FDG, the bright yellow fluorescence of fluorescein, which is the digested product of FDG, was clearly detected in the cells by fluorescence microscopy. This observation implied that inflow of external FDG into the lysosomes is caused by lysosomal membrane damage on alpha T photosensitization. The present results indicated that lysosomes are the primary photosensitization site of alpha T.

  15. [Cestode lysosomes].

    PubMed

    Smirnov, L P; Bogdan, V V

    1989-01-01

    By differential centrifugation method a lysosomal fraction was obtained from five species of cestodes, which possesses the highest specific activity of acidic phosphatases as compared to other subcellular fractions. By isopyknic centrifugation in the density gradient of saccharose the lysosomal fraction is divided into primary and secondary lysosomes. Lysosomes of cestodes are similar to those of vertebrate animals in the character of fractional distribution of acidic phosphatase, sedimentation abilities and sensitivity of membranes to triton X-100.

  16. Lysosome stability during lytic infection by simian virus 40.

    PubMed

    Einck, K H; Norkin, L C

    1979-01-01

    By 48 h postinfection, 40--80% of SV40-infected CV-1 cells have undergone irreversible injury as indicated by trypan blue staining. Nevertheless, at this time the lysosomes of these cells appear as discrete structures after vital staining with either acridine orange or neutral red. Lysosomes, vitally stained with neutral red at 24 h postinfection, were still intact in cells stained with trypan blue at 48 h. Acid phosphatase activity is localized in discrete cytoplasmic particles at 48 h, as indicated by histochemical staining of both fixed and unfixed cells.

  17. Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3.

    PubMed

    Li, Caiyue; Ma, Wenbo; Yin, Shikui; Liang, Xin; Shu, Xiaodong; Pei, Duanqing; Egan, Terrance M; Huang, Jufang; Pan, Aihua; Li, Zhiyuan

    2016-05-01

    The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

    SciTech Connect

    Magini, Alessandro; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

    2013-10-18

    Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes, but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.

  19. Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

    PubMed

    Rood, Ilse M; Merchant, Michael L; Wilkey, Daniel W; Zhang, Terry; Zabrouskov, Vlad; van der Vlag, Johan; Dijkman, Henry B; Willemsen, Brigith K; Wetzels, Jack F; Klein, Jon B; Deegens, Jeroen K

    2015-11-01

    Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

  20. Mechanism of Aloe Vera extract protection against UVA: shelter of lysosomal membrane avoids photodamage.

    PubMed

    Rodrigues, Daniela; Viotto, Ana Cláudia; Checchia, Robert; Gomide, Andreza; Severino, Divinomar; Itri, Rosangela; Baptista, Maurício S; Martins, Waleska Kerllen

    2016-03-01

    The premature aging (photoaging) of skin characterized by wrinkles, a leathery texture and mottled pigmentation is a well-documented consequence of exposure to sunlight. UVA is an important risk factor for human cancer also associated with induction of inflammation, immunosuppression, photoaging and melanogenesis. Although herbal compounds are commonly used as photoprotectants against the harmful effects of UVA, the mechanisms involved in the photodamage are not precisely known. In this study, we investigated the effects of Aloe Vera (Aloe barbadensis mil) on the protection against UVA-modulated cell killing of HaCaT keratinocytes. Aloe Vera exhibited the remarkable ability of reducing both in vitro and in vivo photodamage, even though it does not have anti-radical properties. Interestingly, the protection conferred by Aloe Vera was associated with the maintenance of membrane integrity in both mimetic membranes and intracellular organelles. The increased lysosomal stability led to a decrease in lipofuscinogenesis and cell death. This study explains why Aloe Vera extracts offer protection against photodamage at a cellular level in both the UV and visible spectra, leading to its beneficial use as a supplement in protective dermatological formulations.

  1. Ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells

    SciTech Connect

    Chung, Hyewon; Yoon, Young Hee; Hwang, Jung Jin; Cho, Kyung Sook; Koh, Jae Young; Kim, June-Gone

    2009-03-01

    Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death.

  2. A new approach on valproic acid induced hepatotoxicity: involvement of lysosomal membrane leakiness and cellular proteolysis.

    PubMed

    Pourahmad, Jalal; Eskandari, Mohammad Reza; Kaghazi, Amineh; Shaki, Fatemeh; Shahraki, Jafar; Fard, Javad Khalili

    2012-06-01

    Although valproic acid (VPA) a proven anticonvulsant agent thought to have relatively few side-effects VPA has been referred as the third most common xenobiotic suspected of causing death due to liver injury. In this study the cellular pathways involved in VPA hepatotoxicity were investigated in isolated rat hepatocytes. Accelerated cytotoxicity mechanism screening (ACMS) techniques using fluorescent probes including, ortho-phthalaldehyde, rhodamine 123 and acridine orange were applied for measurement of ROS formation, glutathione depletion, mitochondrial membrane potential and Lysosomal membrane damage, respectively. Our results showed that cytotoxic action of VPA is mediated by lysosomal membrane leakiness along with reactive oxygen species (ROS) formation and decline of mitochondrial membrane potential before cell lysis ensued. Incubation of hepatocytes with VPA also caused rapid hepatocyte glutathione (GSH) depletion which is another marker of cellular oxidative stress. Most of the VPA induced GSH depletion could be attributed to the expulsion of GSSG. Our results also showed that CYP2EI is involved in the mechanism of VPA cytotoxicity. We finally concluded that VPA hepatotoxicity is a result of metabolic activation by CYP2E1 and ROS formation, leading to lysosomal labialization, mitochondrial/lysosomal toxic cross-talk and finally general cellular proteolysis in the rat hepatocytes. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

    PubMed

    Leow, San Min; Chua, Shu Xian Serene; Venkatachalam, Gireedhar; Shen, Liang; Luo, Le; Clement, Marie-Veronique

    2016-12-18

    Here we provide evidence to link sub-lethal oxidative stress to lysosomal biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of a lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosomes biogenesis under conditions of sub-lethal oxidative stress.

  4. Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages.

    PubMed

    Ghosh, Moumita; Carlsson, Fredrik; Laskar, Amit; Yuan, Xi-Ming; Li, Wei

    2011-02-18

    Moderate lysosomal membrane permeabilization (LMP) is an important inducer of apoptosis. Macrophages are professional scavengers and are rich in hydrolytic enzymes and iron. In the present study, we found that LMP by lysosomotropic detergent MSDH resulted in early up-regulation of lysosomal cathepsins, oxidative stress and ferritin up-regulation, and cell death. Lysosomotropic base NH(4)Cl reduced the ferritin induction and oxidative stress in apoptotic cells induced by MSDH. Cysteine cathepsin inhibitors significantly protected cell death and oxidative stress, but had less effect on ferritin induction. We conclude that oxidative stress induced by lysosomal rupture causes ferritin induction with concomitant mitochondrial damage, which are the potential target for prevention of cellular oxidative stress and cell death induced by typical lysosomotropic substances in different disorders.

  5. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas.

    PubMed

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G; Kristensen, Bjarne

    2013-01-01

    Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm.

  6. Evidence for a cholesterol transport pathway from lysosomes to endoplasmic reticulum that is independent of the plasma membrane.

    PubMed

    Underwood, K W; Jacobs, N L; Howley, A; Liscum, L

    1998-02-13

    We have studied the movement of low density lipoprotein (LDL)-derived cholesterol in cultured Chinese hamster ovary cells. Our hypothesis is that when LDL cholesterol is effluxed from lysosomes, the bulk of LDL cholesterol is mobilized to the plasma membrane, while another pathway delivers LDL cholesterol from lysosomes to acyl-CoA/cholesterol acyltransferase (ACAT) in the endoplasmic reticulum. Three lines of evidence support this model. First, LDL cholesterol transport to ACAT can be blocked without inhibiting the movement of cholesterol from lysosomes to plasma membrane or from plasma membrane to endoplasmic reticulum. Second, LDL cholesterol transport to ACAT is normal in a Chinese hamster ovary mutant with defective plasma membrane-to-ACAT movement. Third, LDL cholesterol is not diluted by the plasma membrane cholesterol pool before reaching ACAT. Our evidence supports a vesicular model of cholesterol transport from lysosomes to the endoplasmic reticulum that is independent of the plasma membrane.

  7. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    SciTech Connect

    Bame, K.J.

    1986-01-01

    Acetyl-CoA:..cap alpha..-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal ..cap alpha..-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism.

  8. Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

    PubMed Central

    Naroeni, Aroem; Jouy, Nicolas; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Porte, Françoise

    2001-01-01

    Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments. PMID:11119541

  9. How and why intralumenal membrane fragments form during vacuolar lysosome fusion

    PubMed Central

    Mattie, Sevan; McNally, Erin K.; Karim, Mahmoud A.; Vali, Hojatollah; Brett, Christopher L.

    2017-01-01

    Lysosomal membrane fusion mediates the last step of the autophagy and endocytosis pathways and supports organelle remodeling and biogenesis. Because fusogenic proteins and lipids concentrate in a ring at the vertex between apposing organelle membranes, the encircled area of membrane can be severed and internalized within the lumen as a fragment upon lipid bilayer fusion. How or why this intralumenal fragment forms during fusion, however, is not entirely clear. To better understand this process, we studied fragment formation during homotypic vacuolar lysosome membrane fusion in Saccharomyces cerevisiae. Using cell-free fusion assays and light microscopy, we find that GTPase activation and trans-SNARE complex zippering have opposing effects on fragment formation and verify that this affects the morphology of the fusion product and regulates transporter protein degradation. We show that fragment formwation is limited by stalk expansion, a key intermediate of the lipid bilayer fusion reaction. Using electron microscopy, we present images of hemifusion diaphragms that form as stalks expand and propose a model describing how the fusion machinery regulates fragment formation during lysosome fusion to control morphology and protein lifetimes. PMID:27881666

  10. Phase coexistence in a triolein-phosphatidylcholine system. Implications for lysosomal membrane properties.

    PubMed

    Pakkanen, Kirsi I; Duelund, Lars; Vuento, Matti; Ipsen, John Hjort

    2010-02-01

    The effects of tri- and monoglycerides on phospholipid (POPC) membranes were studied using spectroscopical methods. Triolein was found to form two types of POPC-rich membranes, both with POPC or as a three-component system with monopalmitin. These two membrane types were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. Marked differences were seen in the physical properties of these phases, even though only minor compositional variation was detected. The light, less polar phase was found to be less ordered and more fluid and seemed to allow significantly lower amount of water penetration into the membrane-water interface than pure POPC membrane. The heavy phase, apart from their slightly altered water penetration, resembled more a pure POPC membrane. As triglycerides are present in lysosomal membranes, the present results can be seen as an implication for polarity-based water permeability barrier possibly contributing to the integrity of lysosomes.

  11. Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

    PubMed Central

    2012-01-01

    Background Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Results Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Conclusions Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death

  12. Tropomyosin modulates erythrocyte membrane stability

    PubMed Central

    An, Xiuli; Salomao, Marcela; Guo, Xinhua; Gratzer, Walter; Mohandas, Narla

    2007-01-01

    The ternary complex of spectrin, actin, and 4.1R (human erythrocyte protein 4.1) defines the nodes of the erythrocyte membrane skeletal network and is inseparable from membrane stability under mechanical stress. These junctions also contain tropomyosin (TM) and the other actin-binding proteins, adducin, protein 4.9, tropomodulin, and a small proportion of capZ, the functions of which are poorly defined. Here, we have examined the consequences of selective elimination of TM from the membrane. We have shown that the mechanical stability of the membranes of resealed ghosts devoid of TM is grossly, but reversibly, impaired. That the decreased membrane stability of TM-depleted membranes is the result of destabilization of the ternary complex of the network junctions is demonstrated by the strongly facilitated entry into the junctions in situ of a β-spectrin peptide, containing the actin- and 4.1R-binding sites, after extraction of the TM. The stabilizing effect of TM is highly specific, in that it is only the endogenous isotype, and not the slightly longer muscle TM that can bind to the depleted membranes and restore their mechanical stability. These findings have enabled us identify a function for TM in elevating the mechanical stability of erythrocyte membranes by stabilizing the spectrin-actin-4.1R junctional complex. PMID:17008534

  13. 58-F, a flavanone from Ophiopogon japonicus, prevents hepatocyte death by decreasing lysosomal membrane permeability

    PubMed Central

    Yan, Xiaofeng; Ye, Tingjie; Hu, Xudong; Zhao, Pei; Wang, Xiaoling

    2016-01-01

    Lysosome membrane permeabilization (LMP) has been implicated in cell death. In the present study, we investigated the relationship between cell death and H2O2-/CCl4-induced LMP in hepatocytes in vitro and following acute liver injury in vivo. The key finding was that H2O2 triggered LMP by oxidative stress, as evidenced by a suppression of LAMP1 expression, a reduction in LysoTracker Green and AO staining, and the leakage of proton and cathepsin B/D from the lysosome to the cytoplasm, resulting in cell death. CCl4 also triggered hepatocyte death by decreasing lysosome LAMP1 expression and by inducing the accumulation of products of peroxidative lipids and oxidized proteins. Furthermore, a novel compound 5,8-dimethoxy-6-methyl-7-hydroxy-3-3(2-hydroxy-4-methoxybenzyl) chroman-4-one (58-F) was extracted from Ophiopogon japonicus and served as a potential therapeutic drug. In vivo and in vitro results showed that 58-F effectively rescued hepatocytes by decreasing LMP and by inducing lysosomal enzyme translocation to the cytosol. PMID:27306715

  14. P2X4 Forms Functional ATP-activated Cation Channels on Lysosomal Membranes Regulated by Luminal pH*

    PubMed Central

    Huang, Peng; Zou, Yuanjie; Zhong, Xi Zoë; Cao, Qi; Zhao, Kexin; Zhu, Michael X.; Murrell-Lagnado, Ruth; Dong, Xian-Ping

    2014-01-01

    P2X receptors are commonly known as plasma membrane cation channels involved in a wide variety of cell functions. The properties of these channels have been extensively studied on the plasma membrane. However, studies in amoeba suggest that P2X receptors are also present intracellularly and involved in vesicle fusion with the plasma membrane. Recently, it was shown that in addition to plasma membrane expression, mammalian P2X4 was also localized intracellularly in lysosomes. However, it was not clear whether the lysosomal P2X4 receptors function as channels and how they are activated and regulated. In this paper, we show that both P2X4 and its natural ligand, ATP, are enriched in lysosomes of COS1 and HEK293 cells. By directly recording membrane currents from enlarged lysosomal vacuoles, we demonstrated that lysosomal P2X4 formed channels activated by ATP from the luminal side in a pH-dependent manner. While the acidic pH at the luminal side inhibited P2X4 activity, increasing the luminal pH in the presence of ATP caused P2X4 activation. We further showed that, as for the plasma membrane P2X4, the lysosomal P2X4 was potentiated by ivermectin but insensitive to suramin and PPADS, and it permeated the large cation N-methyl-d-glucamine upon activation. Our data suggest that P2X4 forms functional ATP-activated cation channels on lysosomal membranes regulated by luminal pH. Together with the reported fusion effect of intracellular P2X in lower organisms, we speculate that the lysosome-localized P2X4 may play specific roles in membrane trafficking of acidic organelles in mammalian cells. PMID:24817123

  15. Marine alkaloid Monanchocidin a overcomes drug resistance by induction of autophagy and lysosomal membrane permeabilization.

    PubMed

    Dyshlovoy, Sergey A; Hauschild, Jessica; Amann, Kerstin; Tabakmakher, Ksenia M; Venz, Simone; Walther, Reinhard; Guzii, Alla G; Makarieva, Tatiana N; Shubina, Larisa K; Fedorov, Sergey N; Stonik, Valentin A; Bokemeyer, Carsten; Balabanov, Stefan; Honecker, Friedemann; von Amsberg, Gunhild

    2015-07-10

    Monanchocidin A (MonA) is a novel alkaloid recently isolated from the marine sponge Monanchora pulchra. The compound reveals cytotoxic activity in genitourinary cancers including cisplatin-sensitive and -resistant germ cell tumor (GCT) cell lines, hormone-sensitive and castration-resistant prostate carcinoma cell lines and different bladder carcinoma cell lines. In contrast, non-malignant cells were significantly less sensitive. MonA is highly synergistic with cisplatin in GCT cells. Induction of autophagy at lower and lysosomal membrane permeabilization (LMP) at higher concentrations were identified as the dominating modes of action. Cytotoxicity and protein degradation could be inhibited by 3-methyladenine, an inhibitor of autophagy. LMP was confirmed by loss of acridine orange staining of lysosoms and by release of cathepsin B. In conclusion, MonA exerts cytotoxic activity by mechanisms different from "classical" apoptosis, and could be a promising new compound to overcome resistance to standard therapies in genitourinary malignancies.

  16. Kinetic evidence that newly-synthesized endogenous lysosome-associated membrane protein-1 (LAMP-1) first transits early endosomes before it is delivered to lysosomes.

    PubMed

    Ebrahim, Roshan; Thilo, Lutz

    2011-05-01

    After de novo synthesis of lysosome-associated membrane proteins (LAMPs), they are sorted in the trans-Golgi network (TGN) for delivery to lysosomes. Opposing views prevail on whether LAMPs are targeted to lysosomes directly, or indirectly via prelysosomal stages of the endocytic pathway, in particular early endosomes. Conflicting evidence is based on kinetic measurements with too limited quantitative data for sufficient temporal and organellar resolution. Using cells of the mouse macrophage cell line, P338D(1), this study presents detailed kinetic data that describe the extent of, and time course for, the appearance of newly-synthesized LAMP-1 in organelles of the endocytic pathway, which had been loaded selectively with horse-radish peroxidase (HRP) by appropriate periods of endocytosis. After a 5-min pulse of metabolic labelling, LAMP-1 was trapped in the respective organelles by HRP-catalyzed crosslinking with membrane-permeable diaminobenzidine (DAB). These kinetic observations provide sufficient quantitative evidence that in P338D(1) cells the bulk of newly-synthesized endogenous LAMP-1 first appeared in early endosomes, before it was delivered to late endosomes and lysosomes about 25 min later.

  17. Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

    PubMed

    Granger, B L; Green, S A; Gabel, C A; Howe, C L; Mellman, I; Helenius, A

    1990-07-15

    lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

  18. [Application of lysosomal detection in marine pollution monitoring: research progress].

    PubMed

    Weng, You-Zhu; Fang, Yong-Qiang; Zhang, Yu-Sheng

    2013-11-01

    Lysosome is an important organelle existing in eukaryotic cells. With the development of the study on the structure and function of lysosome in recent years, lysosome is considered as a target of toxic substances on subcellular level, and has been widely applied abroad in marine pollution monitoring. This paper summarized the biological characteristics of lysosomal marker enzyme, lysosome-autophagy system, and lysosomal membrane, and introduced the principles and methods of applying lysosomal detection in marine pollution monitoring. Bivalve shellfish digestive gland and fish liver are the most sensitive organs for lysosomal detection. By adopting the lysosomal detection techniques such as lysosomal membrane stability (LMS) test, neutral red retention time (NRRT) assay, morphological measurement (MM) of lysosome, immunohistochemical (Ih) assay of lysosomal marker enzyme, and electron microscopy (EM), the status of marine pollution can be evaluated. It was suggested that the lysosome could be used as a biomarker for monitoring marine environmental pollution. The advantages and disadvantages of lysosomal detection and some problems worthy of attention were analyzed, and the application prospects of lysosomal detection were discussed.

  19. Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation.

    PubMed

    Bewley, Martin A; Naughton, Michael; Preston, Julie; Mitchell, Andrea; Holmes, Ashleigh; Marriott, Helen M; Read, Robert C; Mitchell, Timothy J; Whyte, Moira K B; Dockrell, David H

    2014-10-07

    Intracellular killing of Streptococcus pneumoniae is complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY's ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation. Importance: Streptococcus

  20. Impact of the Sea Empress oil spill on lysosomal stability in mussel blood cells.

    PubMed

    Fernley, P W; Moore, M N; Lowe, D M; Donkin, P; Evans, S

    2000-01-01

    Coastal zones are among the most productive and vulnerable areas on the planet. An example of impact on these fragile environments was shown in the case of the "Sea Empress" oil tanker, which ran aground in the Bristol Channel in 1996, spilling 72,000 tons of "Forties" crude oil. The objective was to investigate the sub-lethal cellular pathology and tissue hydrocarbon contamination in marine mussel populations, 4 months after the initial spill, using the neutral red retention (NRR) assay for lysosomal stability in blood cells. NRR was reduced in mussels, and indicative of cell injury, from the two sites closest to the spill in comparison with more distant and reference sites. Lysosomal stability was inversely correlated with polycyclic aromatic hydrocarbon concentrations in mussel tissues. Reduced lysosomal stability has previously been shown to contribute to impaired immunocompetence and to autophagic loss of body tissues. The use of this type of technique is discussed in the context of cost-effective, ecotoxicological tools for Integrated Coastal Zone Management.

  1. LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium

    PubMed Central

    Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E.; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

    2016-01-01

    Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation. PMID:27329040

  2. LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium.

    PubMed

    Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

    2016-07-01

    Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

  3. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    PubMed

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed.

  4. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    SciTech Connect

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

  5. Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

    PubMed Central

    Roma, Paula Magda S.; Alves, Ana Paula; Rocha, Carolina D.; Valverde, Thalita M.; Aguiar, Pedro Henrique N.; Almeida, Fernando P.; Guimarães, Allan J.; Guatimosim, Cristina; Silva, Aristóbolo M.; Fernandes, Maria C.; Andrews, Norma W.; Viana, Nathan B.; Mesquita, Oscar N.; Agero, Ubirajara; Andrade, Luciana O.

    2013-01-01

    In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal. PMID:24376622

  6. Effect of aloe vera leaf gel extract on membrane bound phosphatases and lysosomal hydrolases in rats with streptozotocin diabetes.

    PubMed

    Rajasekaran, S; Sriram, N; Arulselvan, P; Subramanian, S

    2007-03-01

    Diabetes mellitus is known to promote deterioration of membrane function and impair intra cellular metabolism in the organism. The aim of the present study was to examine the effect of the ethanolic extract from Aloe vera leaf gel on membrane bound phosphatases and lysosomal hydrolases in the liver and kidney of streptozotocin (STZ)-induced diabetic rats. The rats treated with STZ showed significant alterations in the activities of membrane bound phosphatases and lysosomal hydrolases in the liver and kidney. Oral administration of Aloe vera gel extract at a dose of 300 mg/kg body weight/day to STZ-induced diabetic rats for a period of 21 days significantly restored the alterations in enzymes activity to near normalcy. These results were compared with glibenclamide, a reference drug. Thus, the present study confirms that Aloe vera gel extract possesses a significant beneficial effect on membrane bound phosphatases and lysosomal hydrolases.

  7. Membrane Stability Testing

    SciTech Connect

    Hobbs, D.T.

    1997-09-30

    The Electrosynthesis Co. Inc. (ESC) was contracted by the Westinghouse Savannah River Company to investigate the long term performance and durability of cell components (anode, membrane, cathode) in an electrochemical caustic recovery process using a simulated SRC liquid waste as anolyte solution. This report details the results of two long-term studies conducted using an ICI FM01 flow cell. This cell is designed and has previously been demonstrated to scale up directly into the commercial scale ICI FM21 cell.

  8. Lysosomal integral membrane protein 2 (LIMP-2) restricts the invasion of Trypanosoma cruzi extracellular amastigotes through the activity of the lysosomal enzyme β-glucocerebrosidase.

    PubMed

    Gonçalves, Viviane Martinelli; D'Almeida, Vânia; Müller, Karen Barbosa; Real, Fernando; Mortara, Renato Arruda

    2014-03-01

    Lysosomal integral membrane protein 2 (LIMP-2, SCARB2) is directly linked to β-glucocerebrosidase enzyme (βGC) and mediates the transport of this enzyme from the Golgi complex to lysosomes. Active βGC cleaves the β-glycosidic linkages of glucosylceramide, an intermediate in the metabolism of sphingoglycolipids, generating ceramide. In this study we used mouse embryonic fibroblasts (MEFs) deficient for LIMP-2 and observed that these cells were more susceptible to infection by extracellular amastigotes of the protozoan parasite Trypanosoma cruzi when compared to wild-type (WT) fibroblasts. The absence of LIMP-2 decreases the activity of βGC measured in fibroblast extracts. Replacement of βGC enzyme in LIMP-2 deficient fibroblasts restores the infectivity indices to those of WT cells in T. cruzi invasion assays. Considering the participation of βGC in the production of host cell ceramide, we propose that T. cruzi extracellular amastigotes are more invasive to cells deficient in this membrane component. These results contribute to our understanding of the role of host cell lysosomal components in T. cruzi invasion.

  9. APOL1 risk variants enhance podocyte necrosis through compromising lysosomal membrane permeability

    PubMed Central

    Lan, Xiqian; Jhaveri, Aakash; Cheng, Kang; Wen, Hongxiu; Saleem, Moin A.; Mathieson, Peter W.; Mikulak, Joanna; Aviram, Sharon; Malhotra, Ashwani; Skorecki, Karl

    2014-01-01

    Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent. PMID:24899058

  10. Membrane-active antimicrobial peptides and human placental lysosomal extracts are highly active against mycobacteria.

    PubMed

    Jena, Prajna; Mishra, Bibhuti; Leippe, Matthias; Hasilik, Andrej; Griffiths, Gareth; Sonawane, Avinash

    2011-05-01

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manifests discreet strategies to subvert host immune responses, which enable the pathogen to survive and multiply inside the macrophages. This problem is further worsened by the emergence of multidrug resistant mycobacterial strains, which make most of the anti-tuberculous drugs ineffective. It is thus imperative to search for and design better therapeutic strategies, including employment of new antibiotics. Recently, naturally produced antimicrobial molecules such as enzymes, peptides and their synthetic analogs have emerged as compounds with potentially significant therapeutical applications. Although, many antimicrobial peptides have been identified only very few of them have been tested against mycobacteria. A major limitation in using peptides as therapeutics is their sensitivity to enzymatic degradation or inactivity under certain physiological conditions such as relatively high salt concentration. Here, we show that NK-2, a peptide representing the cationic core region of the lymphocytic effector protein NK-lysin, and Ci-MAM-A24, a synthetic salt-tolerant peptide derived from immune cells of Ciona intestinalis, efficiently kill Mycobacterium smegmatis and Mycobacterium bovis-BCG. In addition, NK-2 and Ci-MAM-A24 showed a synergistic killing effect against M. smegmatis, no cytotoxic effect on mouse macrophages at bactericidal concentrations, and were even found to kill mycobacteria residing inside the macrophages. We also show that human placental lysosomal contents exert potent killing effect against mycobacteria under acidic and reducing growth conditions. Electron microscopic studies demonstrate that the lysosomal extract disintegrate bacterial cell membrane resulting in killing of mycobacteria.

  11. Lipids regulate the hydrolysis of membrane bound glucosylceramide by lysosomal β-glucocerebrosidase.

    PubMed

    Abdul-Hammed, Misbaudeen; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2017-03-01

    Glucosylceramide (GlcCer) is the primary storage lipid in the lysosomes of Gaucher patients and a secondary one in Niemann-Pick disease types A, B, and C. The regulatory roles of lipids on the hydrolysis of membrane bound GlcCer by lysosomal β-glucocerebrosidase (GBA1) was probed using a detergent-free liposomal assay. The degradation rarely occurs at uncharged liposomal surfaces in the absence of saposin (Sap) C. However, anionic lipids stimulate GlcCer hydrolysis at low pH by up to 1,000-fold depending on the nature and position of the negative charges in their head groups while cationic lipids inhibit the degradation, thus showing the importance of electrostatic interactions between the polycationic GBA1 and the negatively charged vesicle surfaces at low pH. Ceramide, fatty acids, monoacylglycerol, and diacylglycerol also stimulate GlcCer hydrolysis while SM, sphingosine, and sphinganine play strong inhibitory roles, thereby explaining the secondary storage of GlcCer in Niemann-Pick diseases. Surprisingly, cholesterol stimulates GlcCer degradation in the presence of bis(monoacylglycero)phosphate (BMP). Sap C strongly stimulates GlcCer hydrolysis even in the absence of BMP and the regulatory roles of the intraendolysosomal lipids on its activity is discussed. Our data suggest that these strong modifiers of GlcCer hydrolysis affect the genotype-phenotype correlation in several cases of Gaucher patients independent of the types. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  12. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    PubMed

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin.

    PubMed

    Li, Yanyan; Chen, Man; Xu, Yanyan; Yu, Xiao; Xiong, Ting; Du, Min; Sun, Jian; Liu, Liegang; Tang, Yuhan; Yao, Ping

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

  14. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

    PubMed Central

    Li, Yanyan; Chen, Man; Xu, Yanyan; Yu, Xiao; Xiong, Ting; Du, Min; Sun, Jian; Liu, Liegang; Tang, Yuhan; Yao, Ping

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD. PMID:27057276

  15. Relationship between lysosomal membrane destabilization and chemical body burden in eastern oysters (Crassostrea virginica) from Galveston Bay, Texas, USA.

    PubMed

    Hwang, Hyun-Min; Wade, Terry L; Sericano, Jose L

    2002-06-01

    Lysosomal destabilization was measured by using hemocytes of eastern oysters (Crassostrea virginica) collected along a chemical concentration gradient in Galveston Bay, Texas, USA. Results of the lysosomal response were compared to concentrations of organic compounds and trace elements in oyster tissue. Concentrations (on a dry-wt basis) ranged from 288 to 2,390 ng/g for polycyclic aromatic hydrocarbons (PAHs), 38 to 877 ng Sn/g for tri-n-butyltin (TBT), 60 to 562 ng/g for polyclorinated biphenyls (PCBs), and 7 to 71 ng/g for total DDT. Trace element concentrations (on a dry-wt basis) ranged from 1.1 to 4.0 microg/g for Cd, 105 to 229 microg/g for Cu, 212 to 868 microg/g for Al, and 1,200 to 8,180 microg/g for Zn. The percentage of destabilized lysosomes ranged from 34 to 81%. A significant positive correlation (p < 0.05) was observed between lysosomal destabilization and body burden of organic compounds (PAHs, PCBs, TBT, and chlorinated pesticides). No significant correlation was found between metal concentrations and lysosomal destabilization. Based on lysosomal destabilization, the study sites in Galveston Bay can be placed in one of three groups: healthy (Hanna Reef and Confederate Bay), moderately damaged (Offats Bayou and Todd's Dump), and highly damaged (Yacht Club and Ship Channel). Lysosomal destabilization that is consistent with toxic chemical body burdens supports previous observations that lysosomal membranes are damaged by toxic chemicals and indicates that this method can serve as an early screening tool to assess overall ecosystem health by using oysters.

  16. The Arabidopsis tonoplast is almost devoid of glycoproteins with complex N-glycans, unlike the rat lysosomal membrane

    PubMed Central

    Pedrazzini, Emanuela; Caprera, Andrea; Fojadelli, Ilaria; Stella, Alessandra; Rocchetti, Alessandra; Bassin, Barbara; Martinoia, Enrico; Vitale, Alessandro

    2016-01-01

    The distribution of the N-glycoproteome in integral membrane proteins of the vacuolar membrane (tonoplast) or the plasma membrane of Arabidopsis thaliana and, for further comparison, of the Rattus norvegicus lysosomal and plasma membranes, was analyzed. In silico analysis showed that potential N-glycosylation sites are much less frequent in tonoplast proteins. Biochemical analysis of Arabidopsis subcellular fractions with the lectin concanavalin A, which recognizes mainly unmodified N-glycans, or with antiserum against Golgi-modified N-glycans confirmed the in silico results and showed that, unlike the plant plasma membrane, the tonoplast is almost or totally devoid of N-glycoproteins with Golgi-modified glycans. Lysosomes share with vacuoles the hydrolytic functions and the position along the secretory pathway; however, our results indicate that their membranes had a divergent evolution. We propose that protection against the luminal hydrolases that are abundant in inner hydrolytic compartments, which seems to have been achieved in many lysosomal membrane proteins by extensive N-glycosylation of the luminal domains, has instead been obtained in the vast majority of tonoplast proteins by limiting the length of such domains. PMID:26748395

  17. Kinetic stability of membrane proteins.

    PubMed

    González Flecha, F Luis

    2017-09-18

    Although membrane proteins constitute an important class of biomolecules involved in key cellular processes, study of the thermodynamic and kinetic stability of their structures is far behind that of soluble proteins. It is known that many membrane proteins become unstable when removed by detergent extraction from the lipid environment. In addition, most of them undergo irreversible denaturation, even under mild experimental conditions. This process was found to be associated with partial unfolding of the polypeptide chain exposing hydrophobic regions to water, and it was proposed that the formation of kinetically trapped conformations could be involved. In this review, we will describe some of the efforts toward understanding the irreversible inactivation of membrane proteins. Furthermore, its modulation by phospholipids, ligands, and temperature will be herein discussed.

  18. Enhanced thermal stability of lysosomal beta-D-galactosidase in parenchymal cells of tumour bearing mice.

    PubMed Central

    Lenti, L.; Lipari, M.; Lombardi, D.; Zicari, A.; Dotta, A.; Pontieri, G. M.

    1986-01-01

    The thermal stability of the enzyme beta-D-galactosidase varies among different organs in normal C57Bl/6 mice, and increases in the same organs in mice with Lewis Lung carcinoma. Thermal stability of this enzyme is also increased by treatment of the mice with cell-free extracts of tumour cells or with inflammatory compounds such as carrageenan or orosomucoid. After desialylation, orosomucoid more effectively increases the heat stability of the enzyme. By contrast talc, which has no galactosyl groups, is without effect on the stability of the enzyme in vivo. Macrophages of tumour bearing mice release into the culture medium a more heat resistant enzyme than macrophages from control mice. In both cases the heat resistance of the secreted enzyme is higher when fetal calf serum is present in the culture medium. Bovine serum does not modify the thermal stability of beta-D-galactosidase in this system. Incubation of lysosomal fractions of various organs with the synthetic beta-D-galactosidase substrate, p-nitrophenyl-galactopyranoside, also strongly increases the heat resistance of the enzyme. The results suggest that one factor influencing the heat resistance of this enzyme may be complex formation between the enzyme and its substrates, an example of substrate protection of the enzyme. This may not be the only factor involved in enzyme stabilization in vivo. PMID:3099822

  19. An approach to the assessment of membrane stability of cultured cells.

    PubMed

    Thaw, H H; Lukinius, A; Brunk, U T; Collins, V P

    1983-01-01

    A simple method for assessing the combined stability of the plasma and lysosomal membranes of cultured cells is described. Monolayers of normal, human glial cells were incubated in situ in an isotonic, buffered sucrose solution (pH 5.0) containing the acid phosphatase (AP) enzyme substrate p-nitrophenyl phosphate (PNPP). The rate of appearance, in the solution, of the reaction product p-nitrophenol (PNP) was measured spectrophotometrically, curves then plotted, and fitted by computer. "Lag time" (LT) was calculated, and an index of membrane lability constructed, termed "fragility index" (FI). Transmission electron microscopy (TEM), "vital" staining of the cells with fluorescein diacetate (FDA) and Evans Blue (EB), and use of a Gomori-type cytochemical technique, indicate that the data reflects the combined stability of lysosomal and plasma membranes. The latter playing the more critical role. Cell cultures pre-incubated with various membrane labilizing or stabilizing agents were compared. Control, 0.3 M sucrose, and normal saline treated cells demonstrated similar stability. Distilled water decreased AP latency (increased fragility), and the magnitude of this effect was time dependent. Cells fixed in glutaraldehyde (GA) retained much of their osmotic reactivity, as confirmed by distilled water treatment. Oxygen derived free radicals caused pronounced fragility, while dexamethasone, a membrane stabilizing agent, decreased membrane fragility. Triton X-100 abolished latency completely, and total AP activity was very rapidly recovered outside the cells in the surrounding incubation medium. These results suggest this technique yields a measure of membrane stability which is sensitive enough to differentiate between known stabilizers and labilizers of membranes. Hence, this may prove an easy and useful aid for the assessment of how various substances and environments modulate the lysosomal and plasma membrane stability of cultured cells.

  20. Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors

    PubMed Central

    1995-01-01

    Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein- dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp- containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella- infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes. PMID

  1. Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1.

    PubMed Central

    Damoiseaux, J G; Döpp, E A; Calame, W; Chao, D; MacPherson, G G; Dijkstra, C D

    1994-01-01

    The monoclonal antibody (mAb) ED1 is being used widely as a marker for rat macrophages. The distribution of the recognized antigen in tissues and isolated cells strongly supports this use as a macrophage marker, since the majority of macrophages are recognized and only seldomly are other cell types stained by mAb ED1. In the present study we further characterized the recognized antigen by a detailed description of the localization of the antigen and by determining biochemical and functional properties. We show that the antigen is expressed on the membranes of cytoplasmic granules, like phagolysosomes, as well as on the cell surface. The amount of ED1 expression in a single cell can be correlated to phagocytic activity of the respective cell type, but the mAb ED1 is not able to block latex phagocytosis or bacterial killing. The mAb ED1 appears to recognize a heavily glycosylated protein of 90,000-110,000 MW, depending on the cell type used as antigen source. A possible relation with other known lysosomal glycoproteins with a similar molecular weight is discussed. Images Figure 1 Figure 3 Figure 4 Figure 5 PMID:7821959

  2. Lysosomal trafficking regulator Lyst links membrane trafficking to toll-like receptor–mediated inflammatory responses

    PubMed Central

    Krautkrämer, Martina

    2017-01-01

    Subcellular compartmentalization of receptor signaling is an emerging principle in innate immunity. However, the functional integration of receptor signaling pathways into membrane trafficking routes and its physiological relevance for immune responses is still largely unclear. In this study, using Lyst-mutant beige mice, we show that lysosomal trafficking regulator Lyst links endolysosomal organization to the selective control of toll-like receptor 3 (TLR3)– and TLR4-mediated proinflammatory responses. Consequently, Lyst-mutant mice showed increased susceptibility to bacterial infection and were largely resistant to endotoxin-induced septic shock. Mechanistic analysis revealed that Lyst specifically controls TLR3- and TLR4-induced endosomal TRIF (TIR domain–containing adapter-inducing interferon β) signaling pathways. Loss of functional Lyst leads to dysregulated phagosomal maturation, resulting in a failure to form an activation-induced Rab7+ endosomal/phagosomal compartment. This specific Rab7+ compartment was further demonstrated to serve as a major site for active TRIF signaling events, thus linking phagosomal maturation to specific TLR signaling pathways. The immunoregulatory role of Lyst on TLR signaling pathways was confirmed in human cells by CRISPR/Cas9-mediated gene inactivation. As mutations in LYST cause human Chédiak-Higashi syndrome, a severe immunodeficiency, our findings also contribute to a better understanding of human disease mechanisms. PMID:27881733

  3. Lysosomal trafficking regulator Lyst links membrane trafficking to toll-like receptor-mediated inflammatory responses.

    PubMed

    Westphal, Andreas; Cheng, Weijia; Yu, Jinbo; Grassl, Guntram; Krautkrämer, Martina; Holst, Otto; Föger, Niko; Lee, Kyeong-Hee

    2017-01-01

    Subcellular compartmentalization of receptor signaling is an emerging principle in innate immunity. However, the functional integration of receptor signaling pathways into membrane trafficking routes and its physiological relevance for immune responses is still largely unclear. In this study, using Lyst-mutant beige mice, we show that lysosomal trafficking regulator Lyst links endolysosomal organization to the selective control of toll-like receptor 3 (TLR3)- and TLR4-mediated proinflammatory responses. Consequently, Lyst-mutant mice showed increased susceptibility to bacterial infection and were largely resistant to endotoxin-induced septic shock. Mechanistic analysis revealed that Lyst specifically controls TLR3- and TLR4-induced endosomal TRIF (TIR domain-containing adapter-inducing interferon β) signaling pathways. Loss of functional Lyst leads to dysregulated phagosomal maturation, resulting in a failure to form an activation-induced Rab7(+) endosomal/phagosomal compartment. This specific Rab7(+) compartment was further demonstrated to serve as a major site for active TRIF signaling events, thus linking phagosomal maturation to specific TLR signaling pathways. The immunoregulatory role of Lyst on TLR signaling pathways was confirmed in human cells by CRISPR/Cas9-mediated gene inactivation. As mutations in LYST cause human Chédiak-Higashi syndrome, a severe immunodeficiency, our findings also contribute to a better understanding of human disease mechanisms. © 2016 Westphal et al.

  4. Engineered nanomaterial-induced lysosomal membrane permeabilization and anti-cathepsin agents.

    PubMed

    Bunderson-Schelvan, Melisa; Holian, Andrij; Hamilton, Raymond F

    2017-01-01

    Engineered nanomaterials (ENMs), or small anthropogenic particles approximately < 100 nm in size and of various shapes and compositions, are increasingly incorporated into commercial products and used for industrial and medical purposes. There is an exposure risk to both the population at large and individuals in the workplace with inhalation exposures to ENMs being a primary concern. Further, there is increasing evidence to suggest that certain ENMs may represent a significant health risk, and many of these ENMs exhibit distinct similarities with other particles and fibers that are known to induce adverse health effects, such as asbestos, silica, and particulate matter (PM). Evidence regarding the importance of lysosomal membrane permeabilization (LMP) and release of cathepsins in ENM toxicity has been accumulating. The aim of this review was to describe our current understanding of the mechanisms leading to ENM-associated pathologies, including LMP and the role of cathepsins with a focus on inflammation. In addition, anti-cathepsin agents, some of which have been tested in clinical trials and may prove useful for ameliorating the harmful effects of ENM exposure, are examined.

  5. Lysosomal integral membrane protein Sidt2 plays a vital role in insulin secretion.

    PubMed

    Gao, Jialin; Yu, Cui; Xiong, Qianyin; Zhang, Yao; Wang, Lizhuo

    2015-01-01

    Abnormal insulin secretion results in impaired glucose tolerance and is one of the causal factors in the etiology of type 2 diabetes mellitus. Sidt2, a lysosomal integral membrane protein, plays a critical role in insulin secretion. Here, we further investigate its regulation in insulin secretion. We show that Sidt2(-/-) mice exhibit weight loss, decreased postnatal survival rate with aging, increased fasting glucose and impaired glucose tolerance. After loading high levels of glucose in their diet, Sidt2(-/-) mice produce notably lower insulin levels at the first-phase secretion compared with Sidt2(+/+) mice. Consistent with the in vivo study, INS-1 cells treated with Sidt2 siRNA produced less insulin when loaded with 16.7 mM of glucose. Only 2 of the 13 genes, synap1 and synap3 which encode soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, showed significantly decreased expression in Sidt2(-/-) mice. In conclusion, Sdit2 may play a vital role in the regulation of insulin secretion via two SNARE proteins synap1 and syanp3.

  6. Niemann-Pick C1 functions independently of Niemann-Pick C2 in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo.

    PubMed

    Goldman, Stephen D B; Krise, Jeffrey P

    2010-02-12

    The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

  7. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) assemble via distinct modes.

    PubMed

    Terasawa, Kazue; Tomabechi, Yuri; Ikeda, Mariko; Ehara, Haruhiko; Kukimoto-Niino, Mutsuko; Wakiyama, Motoaki; Podyma-Inoue, Katarzyna A; Rajapakshe, Anupama R; Watabe, Tetsuro; Shirouzu, Mikako; Hara-Yokoyama, Miki

    2016-10-21

    Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) have a large, heavily glycosylated luminal domain composed of two subdomains, and are the most abundant protein components in lysosome membranes. LAMP-1 and LAMP-2 have distinct functions, and the presence of both proteins together is required for the essential regulation of autophagy to avoid embryonic lethality. However, the structural aspects of LAMP-1 and LAMP-2 have not been elucidated. In the present study, we demonstrated that the subdomains of LAMP-1 and LAMP-2 adopt the unique β-prism fold, similar to the domain structure of the dendritic cell-specific-LAMP (DC-LAMP, LAMP-3), confirming the conserved aspect of this family of lysosome-associated membrane proteins. Furthermore, we evaluated the effects of the N-domain truncation of LAMP-1 or LAMP-2 on the assembly of LAMPs, based on immunoprecipitation experiments. We found that the N-domain of LAMP-1 is necessary, whereas that of LAMP-2 is repressive, for the organization of a multimeric assembly of LAMPs. Accordingly, the present study suggests for the first time that the assembly modes of LAMP-1 and LAMP-2 are different, which may underlie their distinct functions.

  8. Bosutinib, an SRC inhibitor, induces caspase-independent cell death associated with permeabilization of lysosomal membranes in melanoma cells.

    PubMed

    Noguchi, S; Shibutani, S; Fukushima, K; Mori, T; Igase, M; Mizuno, T

    2017-03-28

    SRC kinase (SRC proto-oncogene, non-receptor tyrosine kinase) is a promising target for the treatment of solid cancers including human melanoma. Bosutinib (Bosu), a SRC inhibitor, has already been applied to the treatment of human chronic myelogenous leukemia and also has been assessed its safety in dogs. The aim of this study was to clarify a novel anti-tumour mechanism of Bosu in canine and human melanoma cells. The canine and human melanoma cells were treated with Bosu and its effects were evaluated by the cell viability, the protein expression levels such as caspase-3 and LC3, Annexin V/Propidium iodide staining, and confocal immunostaining. Bosu induced the massive caspase-independent cell death, and blocked autophagy flux, which resulted from lysosomal dysfunction. Lysosomal dysfunction caused by Bosu was due to lysosomal membrane permeabilization (LMP), which resulted in the release of lysosomal hydrolases including cathepsin B. Our data suggest that Bosu induces the cell death through induction of LMP in melanoma cells and is a promising therapeutic agent for treatment of melanoma in both dogs and humans. © 2017 John Wiley & Sons Ltd.

  9. The potential role of lysosome-associated membrane protein 3 (LAMP3) on cardiac remodelling

    PubMed Central

    Jiang, Ding-Sheng; Yi, Xin; Huo, Bo; Liu, Xin-Xin; Li, Rui; Zhu, Xue-Hai; Wei, Xiang

    2016-01-01

    Lysosome-associated membrane protein 3 (LAMP3) was first identified as a cell surface marker of mature dendritic cells and specifically expressed in lung tissues. Recently studies demonstrated that LAMP3 plays a critical role in several cancers, and regulated by hypoxia. However, whether LAMP3 expressed in the heart and cardiomyocytes and changed its expression level in the hearts with cardiac remodelling was largely unknown. In this study, we first cultured H9C2 (a clonal muscle cell line from rat heart) and stimulated with 1 μM angiotensin II (Ang II), or 100 μM isoproterenol (ISO), or 100 μM phenylephrine (PE) for indicated times. We found that LAMP3 expression level was significantly increased after these stimulation. Next, the pressure overload-induced cardiac remodelling mouse model was performed in the wild type C57BL/6J mice. After 4 and 8 weeks of transverse aortic constriction (TAC), obvious cardiac remodelling was observed in the wild type mice compared with sham group. Importantly, LAMP3 expression level was gradually elevated from 2 weeks to 8 weeks after TAC surgery. Furthermore, in human dilated cardiomyopathy (DCM) hearts, severe cardiac remodelling was observed, as evidenced by remarkably increased cardiomyocytes cross sectional area and collagen deposition. Notably, the mRNA and protein level of LAMP3 were significantly increased in the DCM hearts compared with donor hearts. Immunohistochemistry assay showed that LAMP3 was expression in the cardiomyocytes and responsible for its increased expression in the hearts. Our data indicated that LAMP3 might have a potential role in the process of cardiac remodelling. PMID:27069538

  10. MiR-207/352 regulate lysosomal-associated membrane proteins and enzymes following ischemic stroke.

    PubMed

    Tao, J; Liu, W; Shang, G; Zheng, Y; Huang, J; Lin, R; Chen, L

    2015-10-01

    The role of microRNAs (miRNAs) in lysosome-mediated neuronal death and survival following ischemic stroke remains unknown. Herein, using miRNA and mRNA gene expression profiling microarrays, we identified the differentially expressed 24 miRNAs and 494 genes in the cortical peri-infarct area, respectively. Integrating the miRNA targets and mRNA expression profiles, we found 47 genes of miRNA targets, including lysosomal-associated membrane protein 2 (LAMP2), Hexb, Bcl2, etc. MiR-207 and miR-352 were mainly downregulated after ischemic stroke, followed by a slight return to baseline during post-middle cerebral artery occlusion (MCAO) 1d to 7d. Furthermore, the luciferase reporter assay demonstrated that LAMP2 and Hexb were the direct targets of miR-207 and miR-352, respectively. After lateral ventricle injection with miR-207 agonist mimics, the neurological deficit scores and infarct volumes were attenuated, and the structure of mitochondria ridges was improved. In addition, miR-207 mimics could reduce the number of cellular lysosome and autophagosome, whereas increase the number of autophagic vacuoles, indicating miR-207 might affect the latter part of lysosomal-autophagy pathway and mitochondria-induced apoptosis. These results suggested that miR-207 and miR-352 were involved in lysosomal pathway for mediating ischemic injury and spontaneous recovery. MiR-207 mimics as potential target drugs could protect against autophagic cell death after ischemic stroke. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. Lysosomal membrane permeabilization: Carbon nanohorn-induced reactive oxygen species generation and toxicity by this neglected mechanism

    SciTech Connect

    Yang, Mei; Zhang, Minfang; Tahara, Yoshio; Chechetka, Svetlana; Miyako, Eijiro; Iijima, Sumio; Yudasaka, Masako

    2014-10-01

    Understanding the molecular mechanisms responsible for the cytotoxic effects of carbon nanomaterials is important for their future biomedical applications. Carbon nanotubular materials induce the generation of reactive oxygen species (ROS), which causes cell death; however, the exact details of this process are still unclear. Here, we identify a mechanism of ROS generation that is involved in the apoptosis of RAW264.7 macrophages caused by excess uptake of carbon nanohorns (CNHs), a typical type of carbon nanotubule. CNH accumulated in the lysosomes, where they induced lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteases, such as cathepsins, which in turn caused mitochondrial dysfunction and triggered the generation of ROS in the mitochondria. The nicotinamide adenine dinucleotide phosphate oxidase was not directly involved in CNH-related ROS production, and the ROS generation cannot be regulated by mitochondrial electron transport chain. ROS fed back to amplify the mitochondrial dysfunction, leading to the subsequent activation of caspases and cell apoptosis. Carbon nanotubules commonly accumulate in the lysosomes after internalization in cells; however, lysosomal dysfunction has not attracted much attention in toxicity studies of these materials. These results suggest that LMP, a neglected mechanism, may be the primary reason for carbon nanotubule toxicity. - Highlights: • We clarify an apoptotic mechanism of RAW264.7 cells caused by carbon nanohorns. • In the meantime, the mechanism of CNH-induced ROS generation is identified. • LMP is the initial factor of CNH-induced ROS generation and cell death. • Cathepsins work as mediators that connect LMP and mitochondrial dysfunction.

  12. Application and evaluation of the neutral red retention (NRR) assay for lysosomal stability in mussel populations along the Iberian Mediterranean coast.

    PubMed

    Martínez-Gómez, Concepción; Benedicto, José; Campillo, Juan Antonio; Moore, Michael

    2008-04-01

    The neutral red retention (NRR) assay to determine lysosomal membrane stability (LMS) was applied to wild mussels (Mytilus galloprovincialis) sampled from fourteen sites with different degrees of chemical pollution along the Iberian Mediterranean coast in 2002 and 2003. Total body burdens for a range of contaminants were measured in pooled samples (n = 50) of whole soft tissues in order to explore possible causality. Mean LMS values (n = 15) were significantly greater in mussels from one of the selected two reference sites (San Diego) than in chemically well-characterized hotspot sites. Mean LMS values were inversely correlated with contaminant concentrations in mussel tissues (except for Zn). The multidimensional scaling ordination technique classified the sampling sites into three groups according to their chemical-physical-biological similarities and the results were statistically confirmed using ANOSIM analysis. The results show that lysosomal stability is an effective indicator of health status in mussels along the Iberian Mediterranean coast and that it is related to body burden of contaminants, particularly polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs).

  13. Lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) is a substrate of cathepsin-F, a cysteine protease mutated in type-B-Kufs-disease.

    PubMed

    Peters, Judith; Rittger, Andrea; Weisner, Rebecca; Knabbe, Johannes; Zunke, Friederike; Rothaug, Michelle; Damme, Markus; Berkovic, Samuel F; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2015-02-13

    The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase β-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

  14. Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

    PubMed Central

    1995-01-01

    In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo. PMID:7490281

  15. Interaction of Salmonella enterica Serotype Typhimurium with Dendritic Cells Is Defined by Targeting to Compartments Lacking Lysosomal Membrane Glycoproteins

    PubMed Central

    García-Del Portillo, Francisco; Jungnitz, Heidrun; Rohde, Manfred; Guzmán, Carlos A.

    2000-01-01

    Dendritic cells (DCs) play a central role in the generation of acquired immunity to infections by pathogenic microorganisms. Salmonella enterica serotype Typhimurium is known to survive and proliferate intracellularly within macrophages and nonphagocytic cells, but no data exist on how this pathogen interacts with DCs. In this report, we show the capacity of serotype Typhimurium to survive within the established mouse DC line CB1. In contrast to the case for the macrophage model, the compartments of DCs containing serotype Typhimurium are devoid of lysosomal membrane glycoproteins and the PhoPQ two-component regulatory system is not essential for pathogen intracellular survival. PMID:10768999

  16. Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2.

    PubMed

    Zunke, Friederike; Andresen, Lisa; Wesseler, Sophia; Groth, Johann; Arnold, Philipp; Rothaug, Michelle; Mazzulli, Joseph R; Krainc, Dimitri; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2016-04-05

    The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2-derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies.

  17. Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2

    PubMed Central

    Zunke, Friederike; Andresen, Lisa; Wesseler, Sophia; Groth, Johann; Arnold, Philipp; Rothaug, Michelle; Mazzulli, Joseph R.; Krainc, Dimitri; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2016-01-01

    The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2–derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies. PMID:27001828

  18. (-)-Epigallocatechin-3-gallate induces non-apoptotic cell death in human cancer cells via ROS-mediated lysosomal membrane permeabilization.

    PubMed

    Zhang, Yin; Yang, Nai-Di; Zhou, Fan; Shen, Ting; Duan, Ting; Zhou, Jing; Shi, Yin; Zhu, Xin-Qiang; Shen, Han-Ming

    2012-01-01

    (-)-Epigallocatechin-3-gallate (EGCG) is the most extensive studied tea polyphenol for its anti-cancer function. In this study, we report a novel mechanism of action for EGCG-mediated cell death by identifying the critical role of lysosomal membrane permeabilization (LMP). First, EGCG-induced cell death in human cancer cells (both HepG2 and HeLa) was found to be caspase-independent and accompanied by evident cytosolic vacuolization, only observable when cells were treated in serum-free medium. The cytosolic vacuolization observed in EGCG-treated cells was most probably caused by lysosomal dilation. Interestingly, EGCG was able to disrupt autophagic flux at the degradation stage by impairment of lysosomal function, and EGCG-induced cell death was independent of Atg5 or autophagy. The key finding of this study is that EGCG is able to trigger LMP, as evidenced by Lyso-Tracker Red staining, cathepsin D cytosolic translocation and cytosolic acidification. Consistently, a lysosomotropic agent, chloroquine, effectively rescues the cell death via suppressing LMP-caused cytosolic acidification. Lastly, we found that EGCG promotes production of intracellular ROS upstream of LMP and cell death, as evidenced by increased level of ROS in cells treated with EGCG and the protective effects of antioxidant N-acetylcysteine (NAC) against EGCG-mediated LMP and cell death. Taken together, data from our study reveal a novel mechanism underlying EGCG-induced cell death involving ROS and LMP. Therefore, understanding this lysosome-associated cell death pathway shed new lights on the anti-cancer effects of EGCG.

  19. DNA damage, acetylcholinesterase activity and lysosomal stability in native and transplanted mussels (Mytilus edulis) in areas close to coastal chemical dumping sites in Denmark.

    PubMed

    Rank, Jette; Lehtonen, Kari K; Strand, Jakob; Laursen, Martha

    2007-08-15

    Biomarkers of genotoxicity (DNA damage, measured as tail moment in the Comet assay), neurotoxicity (acetylcholinesterase inhibition, AChE) and general stress (lysosomal membrane stability, LMS) were studied in native and transplanted blue mussels (Mytilus edulis) in coastal areas of western Denmark potentially affected by anthropogenic pollution originating from chemical dumping sites. The results indicate responses to pollution in all the biomarkers applied at the suspected areas, but the results were not consistent. Seasonal fluctuations in exposure situations at the study sites make interpretation of chemical pollution complex, as seen especially in the variability in results on DNA damage, and also in regard to AChE activity. These investigations further stress the importance of understanding the effects of natural factors (salinity, temperature, water levels, rain and storm events) in correct interpretation of the biomarker data obtained. In addition, adaptation of populations to local contamination may play a role in some of the response patterns observed.

  20. Ubiquitination and dynactin regulate TMEPAI lysosomal trafficking

    PubMed Central

    Luo, Shenheng; Jing, Lei; Zhao, Tian; Li, Yuyin; Liu, Zhenxing; Diao, Aipo

    2017-01-01

    The transmembrane prostate androgen-induced protein (TMEPAI) has been reported to be elevated in various tumor cells, is localized to the lysosome and promotes lysosome stability. The molecular mechanism of TMEPAI trafficking however to the lysosome is unknown. Here we report that clathrin and CI-M6PR mediate TMEPAI transport from the Golgi directly into the endo-lysosomal pathway. TMEPAI is ubiquitinated at its C-terminal region and ubiquitination modification of TMEPAI is a signal for its lysosomal trafficking. Moreover, TMEPAI binds the ubiquitin binding proteins Hrs and STAM which is required for its lysosomal transport. In addition, TMEPAI interacts with the dynactin pointed-end complex subunits dynactin 5 and dynactin 6. The aa 132–155 domain is essential for specific TMEPAI binding and deletion of this binding site leads to mis-trafficking of TMEPAI to the plasma membrane. These results reveal the pathway and mechanism regulating transport of TMEPAI to the lysosome, which helps to further understand the role of TMEPAI in tumorigenesis. PMID:28218281

  1. Morphological alteration, lysosomal membrane fragility and apoptosis of the cells of Indian freshwater sponge exposed to washing soda (sodium carbonate).

    PubMed

    Mukherjee, Soumalya; Ray, Mitali; Dutta, Manab Kumar; Acharya, Avanti; Mukhopadhyay, Sandip Kumar; Ray, Sajal

    2015-12-01

    Washing soda is chemically known as sodium carbonate and is a component of laundry detergent. Domestic effluent, drain water and various anthropogenic activities have been identified as major routes of sodium carbonate contamination of the freshwater ecosystem. The freshwater sponge, Eunapius carteri, bears ecological and evolutionary significance and is considered as a bioresource in aquatic ecosystems. The present study involves estimation of morphological damage, lysosomal membrane integrity, activity of phosphatases and apoptosis in the cells of E. carteri under the environmentally realistic concentrations of washing soda. Exposure to washing soda resulted in severe morphological alterations and damages in cells of E. carteri. Fragility and destabilization of lysosomal membranes of E. carteri under the sublethal exposure was indicative to toxin induced physiological stress in sponge. Prolonged exposure to sodium carbonate resulted a reduction in the activity of acid and alkaline phosphatases in the cells of E. carteri. Experimental concentration of 8 mg/l of washing soda for 192 h yielded an increase in the physiological level of cellular apoptosis among the semigranulocytes and granulocytes of E. carteri, which was suggestive to possible shift in apoptosis mediated immunoprotection. The results were indicative of an undesirable shift in the immune status of sponge. Contamination of the freshwater aquifers by washing soda thus poses an alarming ecotoxicological threat to sponges.

  2. Membrane Stability during Biopreservation of Blood Cells

    PubMed Central

    Stoll, Christoph; Wolkers, Willem F.

    2011-01-01

    Summary Storage methods, which can be taken into consideration for red blood cells and platelets, include liquid storage, cryopreservation and freeze-drying. Red blood cells can be hypothermically stored at refrigerated temperatures, whereas platelets are chilling sensitive and therefore cannot be stored at temperatures below 20 °C. Here we give an overview of available cryopreservation and freeze-drying procedures for blood cells and discuss the effects of these procedures on cells, particularly on cellular membranes. Cryopreservation and freeze-drying may result in chemical and structural modifications of cellular membranes. Membranes undergo phase and permeability changes during freezing and drying. Cryo- and lyoprotective agents prevent membrane damage by different mechanisms. Cryoprotective agents are preferentially excluded from membrane surfaces. They decrease the activation energy for water transport during freezing and control the rate of cellular dehydration. Lyoprotectants are thought to stabilize membranes during drying by forming direct hydrogen bonding interactions with phospholipid head groups. In addition, lyoprotectants can form a glassy state at room temperature. Recently liposomes have been investigated to stabilize blood cells during freezing and freeze-drying. Liposomes modify the composition of cellular membranes by lipid and cholesterol transfer, which can stabilize or destabilize the low temperature response of cells. PMID:21566710

  3. Stabilization of Erythrocyte Membranes by Polyamines

    NASA Astrophysics Data System (ADS)

    Ballas, Samir K.; Mohandas, Narla; Marton, Laurence J.; Shohet, Stephen B.

    1983-04-01

    Using a laser diffraction technique, we have studied the effects of putrescine, spermidine, and spermine, the three physiologic polyamines, on the deformability and mechanical stability of human erythrocyte membranes. Ghosts resealed with polyamines were subjected to high fluid shear stress in an ektacytometer. All polyamines increased the membrane shear modulus (decreased deformability) in a concentration- and time-dependent manner. The order of effectiveness was spermine > spermidine > putrescine. At 10 μ M, spermine appreciably decreased membrane deformability. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytometer and deformability was continuously recorded as the deformable ghosts fragmented into rigid spherical vesicles. Polyamines, especially spermine, caused a noticeable increase in the t1/2 for fragmentation. These effects could not be ascribed to proteolysis or Ca2+-induced transglutamination. That the effects of polyamines were specific and not simply due to their positive charge was demonstrated by the finding that Ca2+ and Mg2+ destabilized the erythrocyte membrane as evidenced by decreasing the t1/2 for fragmentation. Extracellular polyamines were not effective except under conditions that caused significant accumulation inside the cell. The data indicate that intracellular physiologic polyamines, especially spermine, decrease erythrocyte membrane deformability and stabilize the membrane skeleton, making it more resistant to fragmentation.

  4. The AP-3 adaptor complex mediates sorting of yeast and mammalian PQ-loop-family basic amino acid transporters to the vacuolar/lysosomal membrane

    PubMed Central

    Llinares, Elisa; Barry, Abdoulaye Oury; André, Bruno

    2015-01-01

    The limiting membrane of lysosomes in animal cells and that of the vacuole in yeast include a wide variety of transporters, but little is known about how these proteins reach their destination membrane. The mammalian PQLC2 protein catalyzes efflux of basic amino acids from the lysosome, and the similar Ypq1, −2, and −3 proteins of yeast perform an equivalent function at the vacuole. We here show that the Ypq proteins are delivered to the vacuolar membrane via the alkaline phosphatase (ALP) trafficking pathway, which requires the AP-3 adaptor complex. When traffic via this pathway is deficient, the Ypq proteins pass through endosomes from where Ypq1 and Ypq2 properly reach the vacuolar membrane whereas Ypq3 is missorted to the vacuolar lumen via the multivesicular body pathway. When produced in yeast, PQLC2 also reaches the vacuolar membrane via the ALP pathway, but tends to sort to the vacuolar lumen if AP-3 is defective. Finally, in HeLa cells, inhibiting the synthesis of an AP-3 subunit also impairs sorting of PQLC2 to lysosomes. Our results suggest the existence of a conserved AP-3-dependent trafficking pathway for proper delivery of basic amino acid exporters to the yeast vacuole and to lysosomes of human cells. PMID:26577948

  5. The AP-3 adaptor complex mediates sorting of yeast and mammalian PQ-loop-family basic amino acid transporters to the vacuolar/lysosomal membrane.

    PubMed

    Llinares, Elisa; Barry, Abdoulaye Oury; André, Bruno

    2015-11-18

    The limiting membrane of lysosomes in animal cells and that of the vacuole in yeast include a wide variety of transporters, but little is known about how these proteins reach their destination membrane. The mammalian PQLC2 protein catalyzes efflux of basic amino acids from the lysosome, and the similar Ypq1, -2, and -3 proteins of yeast perform an equivalent function at the vacuole. We here show that the Ypq proteins are delivered to the vacuolar membrane via the alkaline phosphatase (ALP) trafficking pathway, which requires the AP-3 adaptor complex. When traffic via this pathway is deficient, the Ypq proteins pass through endosomes from where Ypq1 and Ypq2 properly reach the vacuolar membrane whereas Ypq3 is missorted to the vacuolar lumen via the multivesicular body pathway. When produced in yeast, PQLC2 also reaches the vacuolar membrane via the ALP pathway, but tends to sort to the vacuolar lumen if AP-3 is defective. Finally, in HeLa cells, inhibiting the synthesis of an AP-3 subunit also impairs sorting of PQLC2 to lysosomes. Our results suggest the existence of a conserved AP-3-dependent trafficking pathway for proper delivery of basic amino acid exporters to the yeast vacuole and to lysosomes of human cells.

  6. The proteome of lysosomes.

    PubMed

    Schröder, Bernd A; Wrocklage, Christian; Hasilik, Andrej; Saftig, Paul

    2010-11-01

    Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from co-purifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.

  7. Steviol stabilizes polycystin 1 expression and promotes lysosomal degradation of CFTR and β-catenin proteins in renal epithelial cells.

    PubMed

    Yuajit, Chaowalit; Muanprasat, Chatchai; Homvisasevongsa, Sureeporn; Chatsudthipong, Varanuj

    2017-08-09

    Malfunction of polycystin 1 (PC1) is linked to abnormally high epithelial cell proliferation and fluid secretion, eventually leading to renal cyst development and declined renal function as found in autosomal dominant polycystic kidney disease (ADPKD). Currently, there is no effective therapy for ADPKD. Recent studies report PC1 regulates CFTR chloride channels and β-catenin levels in normal renal epithelial cells. Concurrently, our previous study found steviol retarded renal cyst enlargement in an in vitro and in an in vivo models by reducing CFTR expression and activity. Therefore, a potential relationship between steviol and PC1 is worthy of exploration. The present study was aimed to determine the effect of steviol on PC1, CFTR, and β-catenin levels in renal epithelial cells with defective PC1 biogenesis and expression (Prkcsh(-/-) cell) and postnatal Pkd1 homozygous cell (Pkd1(-/-) cells). Using western blot analysis, it was found that steviol treatment at 100μM for 24-48h substantially enhanced and stabilized PC1 C-terminal expression, while decreasing CFTR and β-catenin protein expression in both Prkcsh(-/-) and Pkd1(-/-) cells. In addition, steviol promoted LAMP2 expression, a lysosomal enzyme marker. Interestingly, hydroxychloroquine (a lysosome inhibitor) treatment abolished steviol's effect in reducing CFTR and β-catenin protein expression. Taken together, these findings suggest steviol slows cyst progression in cells and animal models of PKD, in part, by enhancing and stabilizing PC1 protein expression as well as by promoting lysosomal degradation of CFTR and β-catenin. Therefore, steviol may represent a promising compound for treatment of polycystic kidney disease. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  8. Ceramic membranes with enhanced thermal stability

    DOEpatents

    Anderson, Marc A.; Xu, Qunyin; Bischoff, Brian L.

    1993-01-01

    A method of creating a ceramic membrane with enhanced thermal stability is disclosed. The method involves combining quantities of a first metal alkoxide with a second metal, the quantities selected to give a preselected metal ratio in the resultant membrane. A limited amount of water and acid is added to the combination and stirred until a colloidal suspension is formed. The colloid is dried to a gel, and the gel is fired at a temperature greater than approximately 400.degree. C. The porosity and surface area of ceramic membranes formed by this method are not adversely affected by this high temperature firing.

  9. Formation and Stability of Lipid Membrane Nanotubes.

    PubMed

    Bahrami, Amir Houshang; Hummer, Gerhard

    2017-09-26

    Lipid membrane nanotubes are abundant in living cells, even though tubules are energetically less stable than sheet-like structures. According to membrane elastic theory, the tubular endoplasmic reticulum (ER), with its high area-to-volume ratio, appears to be particularly unstable. We explore how tubular membrane structures can nevertheless be induced and why they persist. In Monte Carlo simulations of a fluid-elastic membrane model subject to thermal fluctuations and without constraints on symmetry, we find that a steady increase in the area-to-volume ratio readily induces tubular structures. In simulations mimicking the ER wrapped around the cell nucleus, tubules emerge naturally as the membrane area increases. Once formed, a high energy barrier separates tubules from the thermodynamically favored sheet-like membrane structures. Remarkably, this barrier persists even at large area-to-volume ratios, protecting tubules against shape transformations despite enormous driving forces toward sheet-like structures. Molecular dynamics simulations of a molecular membrane model confirm the metastability of tubular structures. Volume reduction by osmotic regulation and membrane area growth by lipid production and by fusion of small vesicles emerge as powerful factors in the induction and stabilization of tubular membrane structures.

  10. A LAPF/phafin1-like protein regulates TORC1 and lysosomal membrane permeabilization in response to endoplasmic reticulum membrane stress

    PubMed Central

    Kim, Adam; Cunningham, Kyle W.

    2015-01-01

    Lysosomal membrane permeabilization (LMP) is a poorly understood regulator of programmed cell death that involves leakage of luminal lysosomal or vacuolar hydrolases into the cytoplasm. In Saccharomyces cerevisiae, LMP can be induced by antifungals and endoplasmic reticulum stressors when calcineurin also has been inactivated. A genome-wide screen revealed Pib2, a relative of LAPF/phafin1 that regulates LMP in mammals, as a pro-LMP protein in yeast. Pib2 associated with vacuolar and endosomal limiting membranes in unstressed cells in a manner that depended on its FYVE domain and on phosphatidylinositol 3-phosphate (PI(3)P) biosynthesis. Genetic experiments suggest that Pib2 stimulates the activity of TORC1, a vacuole-associated protein kinase that is sensitive to rapamycin, in a pathway parallel to the Ragulator/EGO complex containing the GTPases Gtr1 and Gtr2. A hyperactivating mutation in the catalytic subunit of TORC1 restored LMP to the gtr1∆ and pib2∆ mutants and also prevented the synthetic lethality of the double mutants. These findings show novel roles of PI(3)P and Pib2 in the regulation of TORC1, which in turn promoted LMP and nonapoptotic death of stressed cells. Rapamycin prevented the death of the pathogenic yeast Candida albicans during exposure to fluconazole plus a calcineurin inhibitor, suggesting that TORC1 broadly promotes sensitivity to fungistats in yeasts. PMID:26510498

  11. Metallothionein protects against oxidative stress-induced lysosomal destabilization

    PubMed Central

    Baird, Sarah K.; Kurz, Tino; Brunk, Ulf T.

    2005-01-01

    The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions. PMID:16236025

  12. Regulators of Lysosome Function and Dynamics in Caenorhabditis elegans

    PubMed Central

    Gee, Kevin; Zamora, Danniel; Horm, Teresa; George, Laeth; Upchurch, Cameron; Randall, Justin; Weaver, Colby; Sanford, Caitlin; Miller, Austin; Hernandez, Sebastian; Dang, Hope; Fares, Hanna

    2017-01-01

    Lysosomes, the major membrane-bound degradative organelles, have a multitude of functions in eukaryotic cells. Lysosomes are the terminal compartments in the endocytic pathway, though they display highly dynamic behaviors, fusing with each other and with late endosomes in the endocytic pathway, and with the plasma membrane during regulated exocytosis and for wound repair. After fusing with late endosomes, lysosomes are reformed from the resulting hybrid organelles through a process that involves budding of a nascent lysosome, extension of the nascent lysosome from the hybrid organelle, while remaining connected by a membrane bridge, and scission of the membrane bridge to release the newly formed lysosome. The newly formed lysosomes undergo cycles of homotypic fusion and fission reactions to form mature lysosomes. In this study, we used a forward genetic screen in Caenorhabditis elegans to identify six regulators of lysosome biology. We show that these proteins function in different steps of lysosome biology, regulating lysosome formation, lysosome fusion, and lysosome degradation. PMID:28122949

  13. Membrane chemical stability and seed longevity.

    PubMed

    Golovina, Elena A; Van As, Henk; Hoekstra, Folkert A

    2010-03-01

    Here, we investigate the relationships between the chemical stability of the membrane surface and seed longevity. Dry embryos of long-lived tomato and short-lived onion seeds were labeled with 5-doxyl-stearic acid (5-DS). Temperature-induced loss of the electron spin resonance signal caused by chemical conversion of 5-DS to nonparamagnetic species was used to characterize the membrane surface chemical stability. No difference was found between temperature plots of 5-DS signal intensity in dry onion and tomato below 345 K. Above this temperature, the 5-DS signal remained unchanged in tomato embryos and irreversibly disappeared in onion seeds. The role of the physical state and chemical status of the membrane environment in the chemical stability of membrane surfaces was estimated for model systems containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dried alone or in the presence of trehalose or glucose. Fourier transform infrared spectroscopy was used to follow temperature-induced structural changes in dry POPC. Spin-label technique was used to relate the chemical stability of 5-DS with the dynamic properties of the bilayer and 5-DS motion behavior. In all the models, the decrease in 5-DS signal intensity was always observed above T(m) for the membrane surface. The 5-DS signal was irreversibly lost at high temperature when dry POPC was embedded in a glucose matrix. The loss of 5-DS signal was moderate when POPC was dried alone or in the presence of trehalose. Comparison of model and in vivo data shows that the differences in longevity between onion and tomato seeds are caused by differences in the chemical status of the membrane surface rather than the degree of its immobilization.

  14. α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

    PubMed Central

    Wiens, Mayim E.

    2017-01-01

    ABSTRACT α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5) blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV) infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses. PMID:28119475

  15. Thrombospondin expression in myofibers stabilizes muscle membranes

    PubMed Central

    Vanhoutte, Davy; Schips, Tobias G; Kwong, Jennifer Q; Davis, Jennifer; Tjondrokoesoemo, Andoria; Brody, Matthew J; Sargent, Michelle A; Kanisicak, Onur; Yi, Hong; Gao, Quan Q; Rabinowitz, Joseph E; Volk, Talila; McNally, Elizabeth M; Molkentin, Jeffery D

    2016-01-01

    Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs’ as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy. DOI: http://dx.doi.org/10.7554/eLife.17589.001 PMID:27669143

  16. Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

    PubMed Central

    Bartee, Eric; Eyster, Craig A.; Viswanathan, Kasinath; Mansouri, Mandana; Donaldson, Julie G.; Früh, Klaus

    2010-01-01

    Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins. PMID:21151997

  17. Membrane-Associated RING-CH proteins associate with Bap31 and target CD81 and CD44 to lysosomes.

    PubMed

    Bartee, Eric; Eyster, Craig A; Viswanathan, Kasinath; Mansouri, Mandana; Donaldson, Julie G; Früh, Klaus

    2010-12-02

    Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

  18. [Experimental models of lysosomal phase reactivity in blood leukocytes exposed to low doses of potassium cyanide].

    PubMed

    Dolgushin, M V; Khomuev, G D

    2013-01-01

    Cytochemical analysis of acid phosphatase was used to evaluate lysosomal membranes stability under oral intake of potassium cyanide by rats over one month in daily doses of 1.30 mg/kg (1/10 LD50) and 0.65 mg/kg (1/20 LD50). The authors demonstrated phase-related dose-dependent changes in the lysosomal state, and the main response feature was associated with functional activation that usually followed the membrane alteration.

  19. Polycyclic aromatic hydrocarbon body residues and lysosomal membrane destabilization in mussels exposed to the Dubai Star bunker fuel oil (intermediate fuel oil 380) spill in San Francisco Bay.

    PubMed

    Hwang, Hyun-Min; Stanton, Beckye; McBride, Toby; Anderson, Michael J

    2014-05-01

    Following the spill of bunker fuel oil (intermediate fuel oil 380, approximately 1500-3000 L) into San Francisco Bay in October 2009, polycyclic aromatic hydrocarbon (PAH) concentrations in mussels from moderately oiled areas increased up to 87 554 ng/g (dry wt) and, 3 mo later, decreased to concentrations found in mussels collected prior to oiling, with a biological half-life of approximately 16 d. Lysosomal membrane destabilization increased in mussels with higher PAH body burdens.

  20. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

    PubMed

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

    2014-12-19

    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

  1. The histone deacetylase inhibitor trichostatin A reduces lysosomal pH and enhances cisplatin-induced apoptosis.

    PubMed

    Eriksson, I; Joosten, M; Roberg, K; Ollinger, K

    2013-01-01

    High activity of histone deacetylases (HDACs) has been documented in several types of cancer and may be associated with survival advantage. In a head and neck squamous cell carcinoma cell line, cisplatin-induced apoptosis was augmented by pretreatment with the HDAC inhibitor trichostatin A. Apoptosis was accompanied by lysosomal membrane permeabilization (LMP), as shown by immunoblotting of the lysosomal marker protease cathepsin B in extracted cytosol and by immunofluorescence. Moreover, LAMP-2 (lysosomal associated membrane protein-2) was translocated from lysosomal membranes and found in a digitonin extractable fraction together with cytosolic proteins and pretreatment with trichostatin A potentiated the release. Overall, protein level of LAMP-2 was decreased during cell death and, interestingly, inhibition of cysteine cathepsins, by the pan-cysteine cathepsin inhibitor zFA-FMK, prevented loss of LAMP-2. The importance of LAMP-2 for lysosomal membrane stability, was confirmed by showing that LAMP-2 knockout MEFs (mouse embryonic fibroblasts) were more sensitive to cisplatin as compared to the corresponding wildtype cells. Trichostatin A reduced lysosomal pH from 4.46 to 4.25 and cell death was prevented when lysosomal pH was increased by NH(4)Cl, or when inhibiting the activity of lysosomal proteases. We conclude that trichostatin A enhances cisplatin induced cell death by decreasing lysosomal pH, which augments cathepsin activity resulting in reduced LAMP-2 level, and might promote LMP.

  2. The Antioxidant Profiles, Lysosomal and Membrane Enzymes Activity in Patients with Acute Pancreatitis

    PubMed Central

    Milnerowicz, Halina; Bukowski, Radosław; Jabłonowska, Monika; Ściskalska, Milena; Milnerowicz, Stanisław

    2014-01-01

    Oxidative stress and inflammatory mediators, such as IL-6, play an important role in the pathophysiology of acute pancreatitis. The study was aimed to assess the degree of the pro/antioxidative imbalance and estimate which antioxidant plays a role in the maintenance of pro/antioxidative balance during acute pancreatitis. The study was investigated in the blood of 32 patients with acute pancreatitis and 37 healthy subjects. IL-6 concentration as early marker of inflammation was determinated. The intensity of oxidative stress was assessed by TBARS concentration. To investigate antioxidative status, the GPx and Cu/Zn SOD activities and the levels of GSH, MT, SH groups, and TRAP were measured. The concentrations of Cu and Zn as ions participating in the maintenance of antioxidant enzymes stability and playing a role in the course of disease were determinated. The activities of GGT, AAP, NAG, and β-GD as markers of tissue damage were also measured. An increase in IL-6 concentration, which correlated with Ranson criteria, and an increase in GPx activity, levels of MT, TBARS, or GGT, and NAG activities in patients group compared to healthy subjects were demonstrated. A decrease in GSH level in patients group compared to control group was noted. The studies suggest that GPx/GSH and MT play the role of the first line of defence against oxidative stress and pro/antioxidant imbalance in the course of acute pancreatitis. PMID:25298618

  3. Synergistic salubrious effect of ferulic acid and ascorbic acid on membrane-bound phosphatases and lysosomal hydrolases during experimental myocardial infarction in rats.

    PubMed

    Yogeeta, Surinder Kumar; Gnanapragasam, Arunachalam; Senthilkumar, Subramanian; Subhashini, Rajakannu; Devaki, Thiruvengadam

    2006-12-23

    Altered membrane integrity has been suggested as a major factor in the development of cellular injury during myocardial necrosis. The present study was designed to investigate the effect of the combination of ferulic acid (FA) and ascorbic acid (AA) on lysosomal hydrolases and membrane-bound phosphatases during isoproterenol (ISO) induced myocardial necrosis in rats. Induction of rats with 1SO (150 mg/kg b.wt, i.p.) for 2 days resulted in a significant increase in the activities of lysosomal hydrolases (beta-D-glucuronidase, beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase and cathepsin-D) in the heart and serum. A significant increase in plasma lactate level, cardiac levels of sodium, calcium and a decrease in cardiac level of potassium was also observed, which was paralleled by abnormal activities of membrane-bound phosphatases (Na(+)-K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase) in the heart of ISO-administered rats. Pre-co-treatment with the combination of FA (20 mg/kg b.wt) and AA (80 mg/kg b.wt) orally for 6 days significantly attenuated these abnormalities and restored the levels to near normalcy when compared to individual drug treated groups. The combination of FA and AA preserved the membrane integrity by mitigating the oxidative stress and associated cellular damage more effectively when compared to individual treatment groups. In our study, the protection conferred by FA and AA might be through the nitric oxide pathway and by their ability of quenching free radicals. In conclusion, these findings indicate the synergistic modulation of lysosomal hydrolases and membrane phosphatases by the combination of FA and AA.

  4. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

    PubMed Central

    Thelin, William R.; Chen, Yun; Gentzsch, Martina; Kreda, Silvia M.; Sallee, Jennifer L.; Scarlett, Cameron O.; Borchers, Christoph H.; Jacobson, Ken; Stutts, M. Jackson; Milgram, Sharon L.

    2007-01-01

    The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation. PMID:17235394

  5. Lysosomal cell death mechanisms in aging.

    PubMed

    Gómez-Sintes, Raquel; Ledesma, María Dolores; Boya, Patricia

    2016-12-01

    Lysosomes are degradative organelles essential for cell homeostasis that regulate a variety of processes, from calcium signaling and nutrient responses to autophagic degradation of intracellular components. Lysosomal cell death is mediated by the lethal effects of cathepsins, which are released into the cytoplasm following lysosomal damage. This process of lysosomal membrane permeabilization and cathepsin release is observed in several physiopathological conditions and plays a role in tissue remodeling, the immune response to intracellular pathogens and neurodegenerative diseases. Many evidences indicate that aging strongly influences lysosomal activity by altering the physical and chemical properties of these organelles, rendering them more sensitive to stress. In this review we focus on how aging alters lysosomal function and increases cell sensitivity to lysosomal membrane permeabilization and lysosomal cell death, both in physiological conditions and age-related pathologies.

  6. Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

    PubMed Central

    Kong, Xiang Y.; Nesset, Cecilie Kasi; Damme, Markus; Løberg, Else-Marit; Lübke, Torben; Mæhlen, Jan; Andersson, Kristin B.; Lorenzo, Petra I.; Roos, Norbert; Thoresen, G. Hege; Rustan, Arild C.; Kase, Eili T.; Eskild, Winnie

    2014-01-01

    Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage. PMID:24487409

  7. Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA

    PubMed Central

    1988-01-01

    LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes. PMID:3339090

  8. (−)-Epigallocatechin-3-Gallate Induces Non-Apoptotic Cell Death in Human Cancer Cells via ROS-Mediated Lysosomal Membrane Permeabilization

    PubMed Central

    Zhang, Yin; Yang, Nai-Di; Zhou, Fan; Shen, Ting; Duan, Ting; Zhou, Jing; Shi, Yin; Zhu, Xin-Qiang; Shen, Han-Ming

    2012-01-01

    (−)-Epigallocatechin-3-gallate (EGCG) is the most extensive studied tea polyphenol for its anti-cancer function. In this study, we report a novel mechanism of action for EGCG-mediated cell death by identifying the critical role of lysosomal membrane permeabilization (LMP). First, EGCG-induced cell death in human cancer cells (both HepG2 and HeLa) was found to be caspase-independent and accompanied by evident cytosolic vacuolization, only observable when cells were treated in serum-free medium. The cytosolic vacuolization observed in EGCG-treated cells was most probably caused by lysosomal dilation. Interestingly, EGCG was able to disrupt autophagic flux at the degradation stage by impairment of lysosomal function, and EGCG-induced cell death was independent of Atg5 or autophagy. The key finding of this study is that EGCG is able to trigger LMP, as evidenced by Lyso-Tracker Red staining, cathepsin D cytosolic translocation and cytosolic acidification. Consistently, a lysosomotropic agent, chloroquine, effectively rescues the cell death via suppressing LMP-caused cytosolic acidification. Lastly, we found that EGCG promotes production of intracellular ROS upstream of LMP and cell death, as evidenced by increased level of ROS in cells treated with EGCG and the protective effects of antioxidant N-acetylcysteine (NAC) against EGCG-mediated LMP and cell death. Taken together, data from our study reveal a novel mechanism underlying EGCG-induced cell death involving ROS and LMP. Therefore, understanding this lysosome-associated cell death pathway shed new lights on the anti-cancer effects of EGCG. PMID:23056433

  9. Ceria nanoparticles stabilized by organic surface coatings activate the lysosome-autophagy system and enhance autophagic clearance.

    PubMed

    Song, Wensi; Soo Lee, Seung; Savini, Marzia; Popp, Lauren; Colvin, Vicki L; Segatori, Laura

    2014-10-28

    Cerium oxide nanoparticles (nanoceria) are widely used in a variety of industrial applications including UV filters and catalysts. The expanding commercial scale production and use of ceria nanoparticles have inevitably increased the risk of release of nanoceria into the environment as well as the risk of human exposure. The use of nanoceria in biomedical applications is also being currently investigated because of its recently characterized antioxidative properties. In this study, we investigated the impact of ceria nanoparticles on the lysosome-autophagy system, the main catabolic pathway that is activated in mammalian cells upon internalization of exogenous material. We tested a battery of ceria nanoparticles functionalized with different types of biocompatible coatings (N-acetylglucosamine, polyethylene glycol and polyvinylpyrrolidone) expected to have minimal effect on lysosomal integrity and function. We found that ceria nanoparticles promote activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and induce upregulation of genes of the lysosome-autophagy system. We further show that the array of differently functionalized ceria nanoparticles tested in this study enhance autophagic clearance of proteolipid aggregates that accumulate as a result of inefficient function of the lysosome-autophagy system. This study provides a mechanistic understanding of the interaction of ceria nanoparticles with the lysosome-autophagy system and demonstrates that ceria nanoparticles are activators of autophagy and promote clearance of autophagic cargo. These results provide insights for the use of nanoceria in biomedical applications, including drug delivery. These findings will also inform the design of engineered nanoparticles with safe and precisely controlled impact on the environment and the design of nanotherapeutics for the treatment of diseases with defective autophagic function and accumulation of lysosomal storage material.

  10. Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and the Bay of Biscay: lysosomal responses.

    PubMed

    Garmendia, Larraitz; Izagirre, Urtzi; Cajaraville, Miren P; Marigómez, Ionan

    2011-04-01

    In order to assess the long-term lysosomal responses to the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities from Galicia and the Bay of Biscay (North Iberian peninsula) in July, and September 2003, April, July, and October 2004-2005 and April 2006. Lysosomal membrane stability (labilisation period, LP) and lysosomal structural changes (lysosomal volume density, Vv(L) and lysosomal surface-to-volume ratio, S/V(L)) were measured as general stress biomarkers. The most remarkable long-term effects after the POS were drastic changes in lysosomal size (lysosomal enlargement) and membrane stability (extremely low LP values) up to April-04. Later on, a recovery trend was envisaged all along the studied area after July-04, albeit membrane stability continued to be below 20 min throughout the studied period up to April-06, which indicates a "distress-to-moderate-stress" condition. Lysosomal Response Index (LRI) revealed that environmental stress was more marked in Galicia than in the Bay of Biscay, mainly in the first sampling year, although a "moderate-to-high-stress" condition persisted until July-05. Overall, although lysosomal size returned to reference values, membrane stability was not fully recovered indicating a stress situation throughout the studied period.

  11. Beta-glucuronidase and hexosaminidase are marker enzymes for different compartments of the endo-lysosomal system in mussel digestive cells.

    PubMed

    Izagirre, U; Angulo, E; Wade, S C; ap Gwynn, I; Marigómez, I

    2009-02-01

    In environmental toxicology, the most commonly used techniques used to visualise lysosomes in order to determine their responses to pollutants (LSC test: lysosomal structural changes test; LMS test: lysosomal membrane stability test) are based on the histochemical application of lysosomal marker enzymes. In mussel digestive cells, the marker enzymes used are beta-glucuronidase (beta-Gus) and hexosaminidase (Hex). The present work has been aimed at determining the distribution of these lysosomal marker enzymes in the various compartments of the endo-lysosomal system (ELS) of mussel digestive cells and at exploring whether intercellular transfer of lysosomal enzymes occurs between digestive and basophilic cells. Immunogold cytochemistry has allowed us to conclude that beta-Gus is present in every compartment of the digestive cell ELS, whereas Hex is not so widely distributed. Moreover, Hex is intimately linked to the lysosomal membrane, whereas beta-Gus appears to be not necessarily membrane-bound. Therefore, two populations of heterolysosomes with different enzyme load and membrane stability have been distinguished in the digestive cell. In addition, heterolysosomes of different electron density have been commonly observed merging together by contact; we suggest that some might act as storage granules for lysosomal enzymes. On the other hand, beta-Gus seems to be released to the digestive alveolar lumen in secretory lysosomes produced by basophilic cells and endocytosed by digestive cells. Regarding the implications of the present study on the interpretation of lysosomal biomarkers, we conclude that beta-Gus, but not Hex, histochemistry provides an appropriate marker for the LSC test and that, although both lysosomal marker enzymes can be employed in the LMS test, different values would be obtained depending on the marker enzyme employed.

  12. LYSOSOMAL DISRUPTION BY BACTERIAL TOXINS

    PubMed Central

    Bernheimer, Alan W.; Schwartz, Lois L.

    1964-01-01

    Bernheimer, Alan W. (New York University School of Medicine, New York), and Lois L. Schwartz. Lysosomal disruption by bacterial toxins. J. Bacteriol. 87:1100–1104. 1964.—Seventeen bacterial toxins were examined for capacity (i) to disrupt rabbit leukocyte lysosomes as indicated by decrease in turbidity of lysosomal suspensions, and (ii) to alter rabbit liver lysosomes as measured by release of β-glucuronidase and acid phosphatase. Staphylococcal α-toxin, Clostridium perfringens α-toxin, and streptolysins O and S affected lysosomes in both systems. Staphylococcal β-toxin, leucocidin and enterotoxin, Shiga neurotoxin, Serratia endotoxin, diphtheria toxin, tetanus neurotoxin, C. botulinum type A toxin, and C. perfringens ε-toxin were not active in either system. Staphylococcal δ-toxin, C. histolyticum collagenase, crude C. perfringens β-toxin, and crude anthrax toxin caused lysosomal damage in only one of the test systems. There is a substantial correlation between the hemolytic property of a toxin and its capacity to disrupt lysosomes, lending support to the concept that erythrocytes and lysosomes are bounded by similar membranes. PMID:5874534

  13. Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump

    PubMed Central

    1995-01-01

    Endocytosed proteins are sorted in early endosomes to be recycled to the plasma membrane or transported further into the degradative pathway. We studied the role of endosomes acidification on the endocytic trafficking of the transferrin receptor (TfR) as a representative for the recycling pathway, the cation-dependent mannose 6-phosphate receptor (MPR) as a prototype for transport to late endosomes, and fluid-phase endocytosed HRP as a marker for transport to lysosomes. Toward this purpose, bafilomycin A1 (Baf), a specific inhibitor of the vacuolar proton pump, was used to inhibit acidification of the vacuolar system. Microspectrofluorometric measurement of the pH of fluorescein-rhodamine-conjugated transferrin (Tf)-containing endocytic compartments in living cells revealed elevated endosomal pH values (pH > 7.0) within 2 min after addition of Baf. Although recycling of endocytosed Tf to the plasma membrane continued in the presence of Baf, recycled Tf did not dissociate from its receptor, indicating failure of Fe3+ release due to a neutral endosomal pH. In the presence of Baf, the rates of internalization and recycling of Tf were reduced by a factor of 1.40 +/- 0.08 and 1.57 +/- 0.25, respectively. Consequently, little if any in TfR expression at the cell surface was measured during Baf treatment. Sorting between endocytosed TfR and MPR was analyzed by the HRP-catalyzed 3,3'- diaminobenzidine cross-linking technique, using transferrin conjugated to HRP to label the endocytic pathway of the TfR. In the absence of Baf, endocytosed surface 125I-labeled MPR was sorted from the TfR pathway starting at 10 min after uptake, reaching a plateau of 40% after 45 min. In the presence of Baf, sorting was initiated after 20 min of uptake, reaching approximately 40% after 60 min. Transport of fluid-phase endocytosed HRP to late endosomes and lysosomes was measured using cell fractionation and immunogold electron microscopy. Baf did not interfere with transport of HRP to MPR

  14. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    PubMed

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  15. Protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

    PubMed

    Roy, Subhro Jyoti; Stanely Mainzen Prince, Ponnian

    2012-11-01

    In the pathology of myocardial infarction, lysosomal lipid peroxidation and resulting enzyme release play an important role. We evaluated the protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats. Male Wistar rats were treated with sinapic acid (12 mg/kg body weight) orally daily for 10 days and isoproterenol (100 mg/kg body weight) was injected twice at an interval of 24 h (9th and 10th day). Then, lysosomal lipid peroxidation, lysosomal enzymes in serum, heart homogenate, lysosomal fraction and myocardial infarct size were measured. Isoproterenol induced myocardial infarcted rats showed a significant increase in serum creatine kinase-MB and lysosomal lipid peroxidation. The activities of β-glucuronidase, β-galactosidase, cathepsin-B and D were significantly increased in serum, heart and the activities of β-glucuronidase and cathepsin-D were significantly decreased in lysosomal fraction of myocardial infarcted rats. Pre-and-co-treatment with sinapic acid normalized all the biochemical parameters and reduced myocardial infarct size in myocardial infarcted rats. In vitro studies confirmed the free radical scavenging effects of sinapic acid. The possible mechanisms for the observed effects are attributed to sinapic acid's free radical scavenging and membrane stabilizing properties. Thus, sinapic acid has protective effects on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

  16. Pickering Membranes Stabilized by Saturn Particles.

    PubMed

    Krejca, Matthias Michael; Wüstner, Cornell; Goedel, Werner A

    2017-09-07

    We report on a novel method to synthesize particles -called by us Saturn particles - having two hydrophobic caps that oppose each other and are separated from each other by a hydro-philic belt that encircles the particle. Mixtures of these particles with water and air, without the usage of low molar mass surfactants, easily form Pickering foams and Pickering mem-branes that are stable for days. These Pickering membranes are composed of a thin film of water into which the particles are embedded in such a way that the belt is surrounded by the water and the caps protrude out of the water into the air at the top and bottom side of the water film. As expected for a liquid membrane, these Pickering membranes are permeable for gases - the permeance being proportional to the solubility and diffusion coefficient of the gas con-sidered. Experimentally obtained permeance values agree reasonably well with theoretical calculations.

  17. Shifting the paradigm: the putative mitochondrial protein ABCB6 resides in the lysosomes of cells and in the plasma membrane of erythrocytes.

    PubMed

    Kiss, Katalin; Brozik, Anna; Kucsma, Nora; Toth, Alexandra; Gera, Melinda; Berry, Laurence; Vallentin, Alice; Vial, Henri; Vidal, Michel; Szakacs, Gergely

    2012-01-01

    ABCB6, a member of the adenosine triphosphate-binding cassette (ABC) transporter family, has been proposed to be responsible for the mitochondrial uptake of porphyrins. Here we show that ABCB6 is a glycoprotein present in the membrane of mature erythrocytes and in exosomes released from reticulocytes during the final steps of erythroid maturation. Consistent with its presence in exosomes, endogenous ABCB6 is localized to the endo/lysosomal compartment, and is absent from the mitochondria of cells. Knock-down studies demonstrate that ABCB6 function is not required for de novo heme biosynthesis in differentiating K562 cells, excluding this ABC transporter as a key regulator of porphyrin synthesis. We confirm the mitochondrial localization of ABCB7, ABCB8 and ABCB10, suggesting that only three ABC transporters should be classified as mitochondrial proteins. Taken together, our results challenge the current paradigm linking the expression and function of ABCB6 to mitochondria.

  18. The membrane-stabilizing action of zinc carnosine (Z-103) in stress-induced gastric ulceration in rats

    SciTech Connect

    Cho, C.H.; Luk, C.T.; Ogle, C.W. )

    1991-01-01

    Zinc compounds have been shown to antagonize various types of gastric ulceration in rats. Zinc carnosine (Z-103), a newly developed agent was, therefore, examined for its antiulcer effect in stress-induced ulceration and also its membrane stabilizing action in rat stomachs. Cold-restraint stress induced severe hemorrhagic lesions together with increased mast cell degranulation and {beta}-glucuronidase release in the gastric glandular mucosa. A-103 pretreatment with a single oral dose reversed these actions in a dose-dependent manner. When the compound was incubated in concentrations of 10{sup {minus}7}, 10{sup {minus}6}, 10{sup {minus}5} or 10{sup {minus}4} M, with isolated hepatic lysosomes, it significantly reduced the spontaneous release of {beta}-glucuronidase in the medium. The present study not only demonstrates the antiulcer effect of Z-103 but also indicates that the protective action is likely to be mediated by its membrane-stabilizing action on mast cells and lysosomes in the gastric glandular mucosa.

  19. Stabilization of laminar boundary layers by compliant membranes

    NASA Astrophysics Data System (ADS)

    Domaradzki, J. Andrzej; Metcalfe, Ralph W.

    1987-03-01

    Direct numerical simulations have been performed to study boundary layer flow over a compliant membrane. While coupling directly to the fluid dynamics code through the wall boundary conditions, this membrane model displays many of the important features of other compliant coatings. While membrane parameters are identified that increase the critical Reynolds number by a factor of 2 compared with the rigid wall, this is mitigated by a significant increase in the growth rates in the unstable region. A detailed analysis of the temporal evolution and spatial structure of the terms in the kinetic energy balance equation shows significant differences in the behavior of two different classes of modes of the system: class A waves, which are destabilized by increased dissipation in the membrane, and class B waves, which are stabilized by membrane dissipation. For class A waves, the dominant dissipative mechanism is viscous damping in the fluid augmented by negative energy production, the principal stabilization mechanism seen in the 'smart wall' algorithm simulations. For class B waves, direct transfer of energy to, and dissipation by, the membrane dominates stabilization of the flow. For class A waves, the membrane stabilizes the flow not by dissipating energy, but by modifying the flow to decrease energy production and enhance viscous dissipation in the fluid.

  20. Brief exposure to copper activates lysosomal exocytosis.

    PubMed

    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

    Copper (Cu) is essential mineral, but its toxicity necessitates existence of powerful machinery responsible for the extraction of excess Cu from the cell. Cu exposure was recently shown to induce the translocation of Cu pump ATP7B to the lysosomes followed by lysosomal exocytosis. Here we sought to investigate the mechanisms underlying the effect of Cu on lysosomal exocytosis. We found that brief exposure to Cu activates lysosomal exocytosis, which was measured as a release of the lysosomal digestive enzyme β-hexosaminidase (β-hex) into the extracellular medium and by the presence lysosomal protein LAMP1 at the plasma membrane. Such release depends on calcium (Ca) and on the lysosomal SNARE VAMP7. ATP7B knockdown using RNAi suppressed the basal lysosomal exocytosis, but did not affect the ability of Cu to activate it. ATP7B knockdown was associated with sustained oxidative stress. The removal of Ca from the extracellular medium suppressed the Cu-dependent component of the lysosomal exocytosis. We propose that Cu promotes lysosomal exocytosis by facilitating a Ca-dependent step of the lysosomal exocytosis.

  1. Lysosomal fusion and SNARE function are impaired by cholesterol accumulation in lysosomal storage disorders

    PubMed Central

    Fraldi, Alessandro; Annunziata, Fabio; Lombardi, Alessia; Kaiser, Hermann-Josef; Medina, Diego Luis; Spampanato, Carmine; Fedele, Anthony Olind; Polishchuk, Roman; Sorrentino, Nicolina Cristina; Simons, Kai; Ballabio, Andrea

    2010-01-01

    The function of lysosomes relies on the ability of the lysosomal membrane to fuse with several target membranes in the cell. It is known that in lysosomal storage disorders (LSDs), lysosomal accumulation of several types of substrates is associated with lysosomal dysfunction and impairment of endocytic membrane traffic. By analysing cells from two severe neurodegenerative LSDs, we observed that cholesterol abnormally accumulates in the endolysosomal membrane of LSD cells, thereby reducing the ability of lysosomes to efficiently fuse with endocytic and autophagic vesicles. Furthermore, we discovered that soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors (SNAREs), which are key components of the cellular membrane fusion machinery are aberrantly sequestered in cholesterol-enriched regions of LSD endolysosomal membranes. This abnormal spatial organization locks SNAREs in complexes and impairs their sorting and recycling. Importantly, reducing membrane cholesterol levels in LSD cells restores normal SNARE function and efficient lysosomal fusion. Our results support a model by which cholesterol abnormalities determine lysosomal dysfunction and endocytic traffic jam in LSDs by impairing the membrane fusion machinery, thus suggesting new therapeutic targets for the treatment of these disorders. PMID:20871593

  2. Behaviour of Steel Arch Stabilized by a Textile Membrane

    NASA Astrophysics Data System (ADS)

    Svoboda, O.; Machacek, J.

    2015-11-01

    Behaviour of the slender steel arch supporting textile membranes in a membrane structure with respect to in-plane and out-of plane stability is investigated in the paper. In the last decades the textile membranes have been widely used to cover both common and exclusive structures due to progress in new membrane materials with eminent properties. Nevertheless, complex analysis of such membranes in interaction with steel structure (carbon/stainless steel perimeter or supporting elements) is rather demanding, even with specialized software. Laboratory model of a large membrane structure simulating a shelter roof of a concert stage was tested and the resulting stress/deflection values are presented. The model of a reasonable size was provided with prestressed membrane of PVC coated polyester fabric Ferrari® Précontraint 702S and tested under various loadings. The supporting steel structure consisted of two steel arch tubes from S355 grade steel and perimeter prestressed cables. The stability behaviour of the inner tube was the primary interest of the investigation. The SOFiSTiK software was used to analyse the structural behaviour in 3D. Numerical non-linear analysis of deflections and internal forces of the structure under symmetrical and asymmetrical loadings covers various membrane prestressing and specific boundary conditions. The numerical results are validated using test results. Finally, the preliminary recommendations for appropriate numerical modelling and stability design of the supporting structure are presented.

  3. Effects of phospholipase A2 on the lysosomal ion permeability and osmotic sensitivity.

    PubMed

    Wang, Jiong-Wei; Sun, Lin; Hu, Jin-Shan; Li, Ying-Bin; Zhang, Guo-Jiang

    2006-01-01

    In this study, we investigated the mechanism of PLA(2)-induced lysosomal destabilization. Through the measurements of lysosomal beta-hexosaminidase free activity, their membrane potential, the intra-lysosomal pH and the lysosomal latency loss in hypotonic sucrose medium, we established that PLA(2) could increase the lysosomal membrane permeability to both potassium ions and protons. The enzyme could also enhance the organelle osmotic sensitivity. The increases in the lysosomal ion permeability promoted influx of potassium ions into the lysosomes via K(+)/H(+) exchange. The resulted osmotic imbalance across the lysosomal membranes osmotically destabilized the lysosomes. In addition, the enhancement of the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in the osmotic stress. The results explain how PLA(2) destabilized the lysosomes.

  4. Stability and dynamics of membrane-spanning DNA nanopores

    NASA Astrophysics Data System (ADS)

    Maingi, Vishal; Burns, Jonathan R.; Uusitalo, Jaakko J.; Howorka, Stefan; Marrink, Siewert J.; Sansom, Mark S. P.

    2017-03-01

    Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine.

  5. Stability and dynamics of membrane-spanning DNA nanopores

    PubMed Central

    Maingi, Vishal; Burns, Jonathan R.; Uusitalo, Jaakko J.; Howorka, Stefan; Marrink, Siewert J.; Sansom, Mark S. P.

    2017-01-01

    Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine. PMID:28317903

  6. Role of cardiolipin in stability of integral membrane proteins.

    PubMed

    Musatov, Andrej; Sedlák, Erik

    2017-08-23

    Cardiolipin (CL) is a unique phospholipid with a dimeric structure having four acyl chains and two phosphate groups found almost exclusively in certain membranes of bacteria and of mitochondria of eukaryotes. CL interacts with numerous proteins and has been implicated in function and stabilization of several integral membrane proteins (IMPs). While both functional and stabilization roles of CL in IMPs has been generally acknowledged, there are, in fact, only limited number of quantitative analysis that support this function of CL. This is likely caused by relatively complex determination of parameters characterizing stability of IMPs and particularly intricate assessment of role of specific PLs such as CL in IMPs stability. This review aims to summarize quantitative findings regarding stabilization role of CL in IMPs reported up to now. Copyright © 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

  7. RADIATION STABILITY OF NAFION MEMBRANES USED FOR ISOTOPE SEPARATION BY PROTON EXCHANGE MEMBRANE ELECTROLYSIS

    SciTech Connect

    Fox, E

    2009-05-15

    Proton Exchange Membrane Electrolyzers have potential interest for use for hydrogen isotope separation from water. In order for PEME to be fully utilized, more information is needed on the stability of Nafion when exposed to radiation. This work examines Nafion 117 under varying exposure conditions, including dose rate, total dosage and atmospheric condition. Analytical tools, such as FT-IR, ion exchange capacity, DMA and TIC-TOC were used to characterize the exposed membranes. Analysis of the water from saturated membranes can provide important data on the stability of the membranes during radiation exposure. It was found that the dose rate of exposure plays an important role in membrane degradation. Potential mechanisms for membrane degradation include peroxide formation by free radicals.

  8. A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation

    PubMed Central

    Li, Xinran; Rydzewski, Nicholas; Hider, Ahmad; Zhang, Xiaoli; Yang, Junsheng; Wang, Wuyang; Gao, Qiong; Cheng, Xiping; Xu, Haoxing

    2016-01-01

    To mediate the degradation of bio-macromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosome storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region, where autophagosomes accumulate, upon autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end directed dynactin-dynein motor, while PI(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PI(3,5)P2-TRPML1-ALG-2-dynein signaling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Collectively, Ca2+ release from lysosomes provides an on-demand mechanism regulating lysosome motility, positioning, and tubulation. PMID:26950892

  9. Ankyrin protein networks in membrane formation and stabilization

    PubMed Central

    Cunha, Shane R; Mohler, Peter J

    2009-01-01

    In eukaryotic cells, ankyrins serve as adaptor proteins that link membrane proteins to the underlying cytoskeleton. These adaptor proteins form protein complexes consisting of integral membrane proteins, signalling molecules and cytoskeletal components. With their modular architecture and ability to interact with many proteins, ankyrins organize and stabilize these protein networks, thereby establishing the infrastructure of membrane domains with specialized functions. To this end, ankyrin collaborates with a number of proteins including cytoskeletal proteins, cell adhesion molecules and large structural proteins. This review addresses the targeting and stabilization of protein networks related to ankyrin interactions with the cytoskeletal protein β-spectrin, L1-cell adhesion molecules and the large myofibrillar protein obscurin. The significance of these interactions for differential targeting of cardiac proteins and neuronal membrane formation is also presented. Finally, this review concludes with a discussion about ankyrin dysfunction in human diseases such as haemolytic anaemia, cardiac arrhythmia and neurological disorders. PMID:19840192

  10. Regulated lysosomal exocytosis mediates cancer progression

    PubMed Central

    Machado, Eda; White-Gilbertson, Shai; van de Vlekkert, Diantha; Janke, Laura; Moshiach, Simon; Campos, Yvan; Finkelstein, David; Gomero, Elida; Mosca, Rosario; Qiu, Xiaohui; Morton, Christopher L.; Annunziata, Ida; d’Azzo, Alessandra

    2015-01-01

    Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf−⁄− mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance. PMID:26824057

  11. Liquid permeation and chemical stability of anodic alumina membranes

    PubMed Central

    Buldakov, Dmitrii A; Tishkin, Alexey A; Lukashin, Alexey V; Eliseev, Andrei A

    2017-01-01

    A study on the chemical stability of anodic alumina membranes and their performance in long-term water and organic solvent permeation experiments is reported. Anodic alumina possesses high stability for both protonic and aprotonic organic solvents. However, serious degradation of the membrane occurs in pure water, leading to a drastic decrease of permeance (over 20% of the initial value after the passing of 0.250 m3/m2 of pure water). The drying of the membrane induces further permeance drop-off. The rate of membrane degradation strongly depends on the pH of the penetrant solution and increases in basic media. According to 27Al NMR and thermogravimetry results, the degradation of the membranes is associated with the dissolution of water-soluble [Al13O4(OH)24(H2O)12]7+ polyhydroxocomplexes and their further redeposition in the form of [Al(OH)4]−, resulting in channels blocking. This process intensifies in basic pH due to the high positive charge of the anodic alumina surface. An approach for improving anodic aluminum oxide stability towards dissolution in water by carbon CVD coating of the membrane walls is suggested. PMID:28382245

  12. Stabilization of porous glass reverse-osmosis membranes

    NASA Technical Reports Server (NTRS)

    Ballou, E. V.; Leban, M. I.; Wydeven, T.

    1972-01-01

    Application of porous glass in form of capillary tubes for low capacity ion exchange in hyperfiltration experiments is discussed. Efficiency of desalination by process of reverse osmosis is described. Stabilization of porous glass membrane by presence of aluminum chloride is analyzed.

  13. Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

    NASA Astrophysics Data System (ADS)

    Socaciu, Carmen; Jessel, Robert; Diehl, Horst A.

    2000-12-01

    The carotenoids β-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

  14. Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins

    PubMed Central

    Picard, Martin; Duval-Terrié, Caroline; Dé, Emmanuelle; Champeil, Philippe

    2004-01-01

    Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C10, was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrié et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca2+-dependent ATPase and Ca2+-pumping activity. We found that 18C10-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions. PMID:15459343

  15. Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins.

    PubMed

    Picard, Martin; Duval-Terrié, Caroline; Dé, Emmanuelle; Champeil, Philippe

    2004-11-01

    Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C(10), was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrie et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca(2+)-dependent ATPase and Ca(2+)-pumping activity. We found that 18C(10)-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions.

  16. Phosphatidic acid osmotically destabilizes lysosomes through increased permeability to K+ and H+.

    PubMed

    Yi, Y-P; Wang, X; Zhang, G; Fu, T-S; Zhang, G-J

    2006-06-01

    Lysosomal destabilization is a critical event not only for the organelle but also for living cells. However, what factors can affect lysosomal stability is not fully studied. In this work, the effects of phosphatidic acid (PA) on the lysosomal integrity were investigated. Through the measurements of lysosomal beta-hexosaminidase free activity, intralysosomal pH, leakage of lysosomal protons and lysosomal latency loss in hypotonic sucrose medium, we established that PA could increase the lysosomal permeability to K+ and H+, and enhance the lysosomal osmotic sensitivity. Treatment of lysosomes with PA promoted entry of K+ into the organelle via K+/H+ exchange, which could produce osmotic stresses and osmotically destabilize the lysosomes. In addition, PA-induced increase in the lysosomal osmotic sensitivity caused the lysosomes to become more liable to destabilization in osmotic shocks. The results suggest that PA may play a role in the lysosomal destabilization.

  17. A novel class of autoantigens of anti-neutrophil cytoplasmic antibodies in necrotizing and crescentic glomerulonephritis: the lysosomal membrane glycoprotein h-lamp-2 in neutrophil granulocytes and a related membrane protein in glomerular endothelial cells

    PubMed Central

    1995-01-01

    Necrotizing and crescentic glomerulonephritis (NCGN) is frequently associated with circulating antineutrophil cytoplasmic autoantibodies (ANCA). It is established that ANCA are specific for soluble enzymes of granules of polymorphonuclear neutrophil granulocytes (PMN), such as myeloperoxidase (MPO) or protease 3 (PR3). The purpose of this study was to identify membrane proteins of PMNs, and/or glomerular cells, as additional autoantigenic ANCA targets. When membrane protein fractions were prepared from PMNs and isolated human glomeruli, and immunoblotted with ANCA sera of NCGN patients, two bands with apparent molecular masses of 170 and 80-110 kD (gp170/80-110) were labeled in PMNs, and a 130-kD glycoprotein (gp130) in glomeruli. Gp130 was purified, and monoclonal and rabbit antibodies (Abs) were produced which showed the same double specificity as the patient's ANCA. Using these probes, evidence was provided that gp170/80-110 is identical with human lysosomal-associated membrane protein 2 (h-lamp-2), because both proteins were immunologically cross-reactive and screening of a cDNA expression library from human promyelocytic leukemia cells with anti- gp130 Ab yielded a clone derived from h-lamp-2. Gp170/80-110 was localized primarily in granule membranes of resting PMNs, and was translocated to the cell surfaces by activation with FMLP. By contrast, gp130 was localized in the surface membranes of endothelial cells of human glomerular and renal interstitial capillaries, rather than in lysosomes, as found for h-lamp-2. Potential clinical relevance of autoantibodies to gp170/80-110 and gp130 was assessed in a preliminary trial, in which ANCA sera of patients (n = 16) with NCGN were probed with purified or recombinant antigens. Specific reactivity was detected in approximately 90% of cases with active phases of NCGN, and frequently also in combination with autoantibodies specific for PR3 or MPO. Collectively, these data provide evidence that h-lamp-2 in PMNs and a

  18. Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels.

    PubMed

    Múgica, M; Izagirre, U; Marigómez, I

    2015-07-01

    The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

  19. A cation counterflux supports lysosomal acidification

    PubMed Central

    Steinberg, Benjamin E.; Huynh, Kassidy K.; Brodovitch, Alexandre; Jabs, Sabrina; Stauber, Tobias; Jentsch, Thomas J.

    2010-01-01

    The profound luminal acidification essential for the degradative function of lysosomes requires a counter-ion flux to dissipate an opposing voltage that would prohibit proton accumulation. It has generally been assumed that a parallel anion influx is the main or only counter-ion transport that enables acidification. Indeed, defective anion conductance has been suggested as the mechanism underlying attenuated lysosome acidification in cells deficient in CFTR or ClC-7. To assess the individual contribution of counter-ions to acidification, we devised means of reversibly and separately permeabilizing the plasma and lysosomal membranes to dialyze the cytosol and lysosome lumen in intact cells, while ratiometrically monitoring lysosomal pH. Replacement of cytosolic Cl− with impermeant anions did not significantly alter proton pumping, while the presence of permeant cations in the lysosomal lumen supported acidification. Accordingly, the lysosomes were found to acidify to the same pH in both CFTR- and ClC-7–deficient cells. We conclude that cations, in addition to chloride, can support lysosomal acidification and defects in lysosomal anion conductance cannot explain the impaired microbicidal capacity of CF phagocytes. PMID:20566682

  20. CDw78 defines MHC class II-peptide complexes that require Ii chain-dependent lysosomal trafficking, not localization to a specific tetraspanin membrane microdomain.

    PubMed

    Poloso, Neil J; Denzin, Lisa K; Roche, Paul A

    2006-10-15

    MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.

  1. Capture-stabilize approach for membrane protein SPR assays.

    PubMed

    Chu, Ruiyin; Reczek, David; Brondyk, William

    2014-12-08

    Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.

  2. Stability properties of elementary dynamic models of membrane transport.

    PubMed

    Hernández, Julio A

    2003-01-01

    Living cells are characterized by their capacity to maintain a stable steady state. For instance, cells are able to conserve their volume, internal ionic composition and electrical potential difference across the plasma membrane within values compatible with the overall cell functions. The dynamics of these cellular variables is described by complex integrated models of membrane transport. Some clues for the understanding of the processes involved in global cellular homeostasis may be obtained by the study of the local stability properties of some partial cellular processes. As an example of this approach, I perform, in this study, the neighborhood stability analysis of some elementary integrated models of membrane transport. In essence, the models describe the rate of change of the intracellular concentration of a ligand subject to active and passive transport across the plasma membrane of an ideal cell. The ligand can be ionic or nonionic, and it can affect the cell volume or the plasma membrane potential. The fundamental finding of this study is that, within the physiological range, the steady states are asymptotically stable. This basic property is a necessary consequence of the general forms of the expressions employed to describe the active and passive fluxes of the transported ligand.

  3. The role of interfacial lipids in stabilizing membrane protein oligomers.

    PubMed

    Gupta, Kallol; Donlan, Joseph A C; Hopper, Jonathan T S; Uzdavinys, Povilas; Landreh, Michael; Struwe, Weston B; Drew, David; Baldwin, Andrew J; Stansfeld, Phillip J; Robinson, Carol V

    2017-01-19

    Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na(+)/H(+) antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

  4. Structuring detergents for extracting and stabilizing functional membrane proteins.

    PubMed

    Matar-Merheb, Rima; Rhimi, Moez; Leydier, Antoine; Huché, Frédéric; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W; Falson, Pierre

    2011-03-31

    Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. Anionic calix[4]arene based detergents (C4Cn, n=1-12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. These compounds seem promising to extract in a functional state membrane proteins obeying the positive inside rule. In that context, they may

  5. Structuring Detergents for Extracting and Stabilizing Functional Membrane Proteins

    PubMed Central

    Matar-Merheb, Rima; Galián, Carmen; Desuzinges-Mandon, Elodie; Ficheux, Damien; Flot, David; Aghajari, Nushin; Kahn, Richard; Di Pietro, Attilio; Jault, Jean-Michel; Coleman, Anthony W.; Falson, Pierre

    2011-01-01

    Background Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. Methodology/Principal Findings Anionic calix[4]arene based detergents (C4Cn, n = 1–12) were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5–24 nm, with the critical micellar concentration (CMC) being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein), a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM). They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux) much more efficiently than SDS (sodium dodecyl sulphate), FC12 (Foscholine 12) or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. Conclusion/Significance These compounds seem promising to extract in a functional state

  6. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis.

    PubMed

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan

    2014-04-01

    In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24h at 18°C and 26°C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18°C and 26°C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution monitoring programmes and, vice versa, the presence of pollutants may condition the capacity of mussels to respond against thermal stress in a climate change scenario.

  7. Lysosomes in apoptosis.

    PubMed

    Ivanova, Saska; Repnik, Urska; Bojic, Lea; Petelin, Ana; Turk, Vito; Turk, Boris

    2008-01-01

    Lysosomes are specialized organelles for protein recycling and as such are involved in the terminal steps of autophagy. However, it has become evident that lysosomes also play an important role in the progression of apoptosis. This latter function seems to be dependent on lysosomal proteases, which need to be released into the cytosol for apoptosis to be efficient. Among the lysosomal proteases, the most abundant are the cysteine cathepsins and the aspartic protease cathepsin D, which seem to be the major apoptosis mediators. This chapter reviews the methods used to study lysosomes and lysosomal proteases.

  8. [Lysosomal system in hormonal mechanisms. Review].

    PubMed

    Duran Reyes, G; González Macías, G; Hicks, J J

    1995-02-01

    The role of lysosomes in the intracellular mechanism of action of several steroid an proteic hormones has been demonstrated. In presence of the specific hormone the target cell induce membranal changes and the lysosomes are moved toward the nucleus; after this the lysosomal enzymes are released in the perinuclear space. For the moment it is not possible to know the biochemical role of this enzymatic activities upon the nucleic acids function and des-repretion process of specific genes, but the inhibition of lysosomes movement utilizing hormone antagonist or dexamethasone inhibits some reproductive process like the implantation of the mammalian egg. We present herein a review related with the mode action of some hormones through the lysosomes in reproductive processes.

  9. Stabilization of concentration fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Palmieri, Benoit; Safran, Samuel

    2012-02-01

    Finite-size domains have been observed at the surface of cells. These lipids ``rafts'' are stable nanodomains enriched in saturated lipids and cholesterol. While line tension favors macrodomains, one explanation for raft stabilization suggests that the membrane composition is tuned close to a spinodal temperature. From this point of view, rafts are long-lived concentration fluctuations in the mixed phase. We propose a ternary mixture model for the cell membrane that includes hybrid lipids which have one saturated and one unsaturated hydrocarbon chain. Finite amount of hybrid lipids reduces the packing incompatibility at the saturated/unsaturated lipid interface and stabilizes the concentration fluctuations. Hybrid-Hybrid interactions are included in the model and further increase the life-time of the rafts and decrease their length-scales. Moreover, the hybrid has extra orientational degrees of freedom that may lead to modulated phases.

  10. Effects of contaminant exposure and food restriction on hepatic autophagic lysosomal parameters in Herring Gull (Larus argentatus) chicks.

    PubMed

    Hegseth, Marit Nøst; Gorbi, Stephania; Bocchetti, Raffaella; Camus, Lionel; Gabrielsen, Geir Wing; Regoli, Francesco

    2014-08-01

    Lysosomal autophagic responses, such as lysosomal membrane stability, neutral lipids (NL), lipofuscin (LF), and malondialdehyde (MDA) levels, are valuable measures of cellular early-onset effects induced by environmental stress factors, such as contaminant exposure and fasting. In this study, these parameters were analysed and related to levels of halogenated organic contaminants (HOCs) in 40 Herring Gull (Larus argentatus) chicks. Chicks were experimentally exposed to HOCs through diet and went through a period of nutrient deprivation at the end of the experiment. HOC exposure and fasting were conducted separately and in combination. NL storages were depleted, and lysosomal membranes were destabilised after HOC exposure and nutrient deprivation. These responses were not related specifically to one type of stress or the extent of the treatment. No synergistic or additive effects from the combination of HOC exposure and fasting were observed. LF accumulated, and MDA levels increased as a result of fasting, but were unaffected by HOC exposure. LF accumulation was strongly associated with the percent weight change in the chicks. Large weight loss was associated with high LF levels, and slight weight gain was associated with low LF levels. Hence, food deprivation affected all the measured parameters, and HOC exposure decreased NL levels and lysosomal membrane stability in HG chick liver. Furthermore, autophagic lysosomal parameters have frequently been applied as biomarkers of cellular health status in previous studies of marine and terrestrial invertebrates, and this study suggests that these parameters may be good candidates for biomarkers of cellular health status in seabirds as well.

  11. Lysosomes as mediators of drug resistance in cancer.

    PubMed

    Zhitomirsky, Benny; Assaraf, Yehuda G

    2016-01-01

    Drug resistance remains a leading cause of chemotherapeutic treatment failure and cancer-related mortality. While some mechanisms of anticancer drug resistance have been well characterized, multiple mechanisms remain elusive. In this respect, passive ion trapping-based lysosomal sequestration of multiple hydrophobic weak-base chemotherapeutic agents was found to reduce the accessibility of these drugs to their target sites, resulting in a markedly reduced cytotoxic effect and drug resistance. Recently we have demonstrated that lysosomal sequestration of hydrophobic weak base drugs triggers TFEB-mediated lysosomal biogenesis resulting in an enlarged lysosomal compartment, capable of enhanced drug sequestration. This study further showed that cancer cells with an increased number of drug-accumulating lysosomes are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. In addition to passive drug sequestration of hydrophobic weak base chemotherapeutics, other mechanisms of lysosome-mediated drug resistance have also been reported; these include active lysosomal drug sequestration mediated by ATP-driven transporters from the ABC superfamily, and a role for lysosomal copper transporters in cancer resistance to platinum-based chemotherapeutics. Furthermore, lysosomal exocytosis was suggested as a mechanism to facilitate the clearance of chemotherapeutics which highly accumulated in lysosomes, thus providing an additional line of resistance, supplementing the organelle entrapment of chemotherapeutics away from their target sites. Along with these mechanisms of lysosome-mediated drug resistance, several approaches were recently developed for the overcoming of drug resistance or exploiting lysosomal drug sequestration, including lysosomal photodestruction and drug-induced lysosomal membrane permeabilization. In this review we explore the current literature addressing the role of lysosomes in mediating cancer drug

  12. Stabilizing factors of phospholipid asymmetry in the erythrocyte membrane.

    PubMed

    Dressler, V; Haest, C W; Plasa, G; Deuticke, B; Erusalimsky, J D

    1984-08-22

    Transbilayer reorientation (flip) of exogenous lysophospholipids and changes of the transbilayer distribution of endogenous phospholipids were studied in human erythrocytes and membrane vesicles. (1) Exogenous lysophosphatidylserine irreversibly accumulates in the inner membrane layer of resealed ghosts of human erythrocytes. (2) This accumulation even occurs after complete loss of asymmetric distribution of endogenous phosphatidylethanolamine and partial loss of phosphatidylserine asymmetry in diamide-treated cells. (3) Formation of inside-out and right-side-out vesicles from erythrocyte membranes results in a loss of endogenous phospholipid asymmetry as well as of the ability to establish asymmetry of exogenous lysophosphatidylserine. Rates of transbilayer reorientation of lysophospholipids for the vesicles, however, are comparable to those for intact cells. (4) Loss of endogenous asymmetry of phosphatidylserine is also observed in vesicles isolated from erythrocytes after heat denaturation of spectrin. The asymmetry in the residual cells is maintained. (5) In contrast to the loss of asymmetry of phosphatidylethanolamine and of phosphatidylserine, the asymmetry of sphingomyelin is completely maintained in the vesicles. (6) The stability of phospholipid asymmetry in the native cell is discussed in terms of a limitation of access of phospholipids to hypothetical reorientation sites. Such a limitation may either be the result of interaction of phospholipids with the membrane skeleton as in case of phosphatidylserine and phosphatidylethanolamine, or the result of lipid-lipid interactions as in case of sphingomyelin.

  13. The role of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes

    PubMed Central

    Lokar, Maruša; Kabaso, Doron; Resnik, Nataša; Sepčić, Kristina; Kralj-Iglič, Veronika; Veranič, Peter; Zorec, Robert; Iglič, Aleš

    2012-01-01

    Intercellular membrane nanotubes (ICNs) are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant) urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are discussed. PMID:22605937

  14. Emulsion liquid membrane for textile dye removal: Stability study

    NASA Astrophysics Data System (ADS)

    Kusumastuti, Adhi; Syamwil, Rodia; Anis, Samsudin

    2017-03-01

    Although textile dyes is basically available in very low concentration; it should be removed due to the toxicity to human body and environment. Among the existing methods, emulsion liquid membrane (ELM) is a promising method by providing high interfacial area and the ability to remove a very low concentration of the solute. The optimal emulsions were produced using commercially supplied homogeniser. The drop size was measured by the aid of microscope and image J software. Initially, methylene blue in simulated wastewater was extracted using a stirrer. Methylene blue concentration was determined using spectrophotometer. The research obtained optimal emulsion at surfactant concentration of 4 wt. %, kerosene as diluent, emulsification time of 30 min, emulsification speed of 2000 rpm. The lowest membrane breakage and the longest stability time were about 0.11% and 150 min, respectively.

  15. Podocytes Degrade Endocytosed Albumin Primarily in Lysosomes

    PubMed Central

    Carson, John M.; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B.; Blaine, Judith

    2014-01-01

    Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, p<0.05). Cytokine production and cell death were significantly increased in HUPECs exposed to albumin and chloroquine alone, and these effects were potentiated by exposure to albumin plus chloroquine. Compared to wild-type mice, glomerular staining of LAMP-1 was significantly increased in Denys-Drash mice and appeared to be most prominent in podocytes. These data suggest lysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and

  16. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    PubMed

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  17. Sickled Erythrocytes Reversal and Membrane Stabilizing Compounds in Telfairia occidentalis

    PubMed Central

    Atabo, Samuel; Umar, Ismaila Alhaji; James, Dorcas Bolanle; Mamman, Aisha Indo

    2016-01-01

    Background and Purpose. Traditional management of sickle cell disease (SCD) is ubiquitous in Africa. In south-eastern Nigeria, Telfairia occidentalis (T. occidentalis) is strongly recommended for consumption by SCD patients, owing to its presumed therapeutic effect. This study investigates the antisickling and membrane regenerative potentials of T. occidentalis in sickled erythrocytes. Experimental Approach. Sickled erythrocytes obtained from SCD patients were treated with sodium metabisulphite (2%) to induce further sickling. Heat and hypotonic-induced lyses of red blood cells' membranes were also carried out. The RBCs were treated with varying concentration (10.0, 1.0, and 0.1 mg mL−1 and 0.5, 1.0, 1.5, 2.0, and 2.5 mg mL−1, resp.) of T. occidentalis extracts as treatment regimen for in vitro antisickling and membrane stabilizing assays. Extract with peak activity was purified and reused in antisickling assay. Key Results. The antisickling activity of aqueous and methanolic extracts of leaves, seeds, and stem of Telfairia occidentalis at 10.0, 1.0, and 0.1 mg mL−1 revealed that the aqueous leaves extract (10 mg mL−1) exhibited the highest antisickling activity (64.03%) which was significantly (p < 0.05) higher than that of the stem (47.30%) and seeds (37.50%). Partially purified fractions recorded improved antisickling effect (peak activity of 70%). Characterization (using GC-MS) of the most active fraction revealed some bioactive compounds. In the membrane stabilizing assay, methanolic and aqueous stem extracts of T. occidentalis showed the highest effect of 71.85% and 61.29%, respectively. Conclusions and Implications. The results provide scientific evidence for ethnopharmacological use of T. occidentalis in the management of SCD. PMID:27433373

  18. Stabilized phospholipid membranes in chromatography: toward membrane protein-functionalized stationary phases.

    PubMed

    Gallagher, Elyssia S; Mansfield, Elisabeth; Aspinwall, Craig A

    2014-04-01

    Transmembrane protein (TMP)-functionalized materials have resulted in powerful new methods in chemical analysis. Of particular interest is the development of high-throughput, TMP-functionalized stationary phases for affinity chromatography of complex mixtures of analytes. Several natural and synthetic phospholipids and lipid mimics have been used for TMP reconstitution, although the resulting membranes often lack the requisite chemical and temporal stability for long-term use, a problem that is exacerbated in flowing separation systems. Polymerizable lipids with markedly increased membrane stability and TMP functionality have been developed over the past two decades. More recently, these lipids have been incorporated into a range of analytical methods, including separation techniques, and are now poised to have a significant impact on TMP-based separations. Here, we describe current methods for preparing TMP-containing stationary phases and examine the potential utility of polymerizable lipids in TMP affinity chromatography.

  19. Solid-Supported Lipid Membranes: Formation, Stability and Applications

    NASA Astrophysics Data System (ADS)

    Goh, Haw Zan

    This thesis presents a comprehensive investigation of the formation of supported lipid membranes with vesicle hemifusion, their stability under detergents and organic solvents and their applications in molecular biology. In Chapter 3, we describe how isolated patches of DOPC bilayers supported on glass surfaces are dissolved by various detergents (decyl maltoside, dodecyl maltoside, CHAPS, CTAB, SDS, TritonX-100 and Tween20) at their CMC, as investigated by fluorescence video microscopy. In general, detergents partition into distal leaflets of bilayers and lead to the expansion of the bilayers through a rolling motion of the distal over the proximal leaflets, in agreement with the first stage of the established 3-stage model of lipid vesicle solubilization by detergents. Subsequently, we study the partitioning of organic solvents (methanol, ethanol, isopropanol, propanol, acetone and chloroform) into isolated bilayer patches on glass in Chapter 4 with fluorescence microscopy. The area expansion of bilayers due to the partitioning of organic solvents is measured. From the titration of organic solvents, we measured the rate of area expansion as a function of the volume fraction of organic solvents, which is proposed to be a measure of strength of interactions between solvents and membranes. From the same experiments, we also measure the maximum expansion of bilayers (or the maximum binding stoichiometry between organic solvents and lipids) before structural breakdown, which depends on the depth of penetration of solvents to the membranes. In Chapter 5, we investigate the formation of sparsely-tethered bilayer lipid membranes (stBLMs) with vesicle hemifusion. In vesicle hemifusion, lipid vesicles in contact with a hydrophobic alkyl-terminated self-assembled monolayer (SAM) deposit a lipid monolayer to the SAM surface, thus completing the bilayer. Electrical Impedance Spectroscopy and Neutron Reflectivity are used to probe the integrity of stBLMs in terms of their

  20. Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity

    PubMed Central

    2012-01-01

    Background Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polystyrene particles (CPS) of different sizes (20-500 nm) on lysosomes of the endothelial cell line EAhy926 after short (24h) and long (72h-96h) exposure times. Lysosomal localization of CPS, as well as lysosomal pH, lysosomal membrane integrity, morphology of the endosomal-lysosomal system and activities of the lysosomal enzymes,cathepsin B and sulfatases, upon exposure to CPS were recorded. Results CPS in sizes ≤100 nm showed high co-localization with lysosomes already after 4h, larger CPS after 24h. None of the particles at non-cytotoxic concentrations caused marked changes in lysosomal pH or destroyed lysosomal membrane integrity. At 24h of exposure, 20 nm CPS induced significant dilatation of the endosomal-lysosomal system and reduced activity of lysosomal sulfatases. After 72h, these alterations were less pronounced. Conclusions Despite accumulation in lysosomes CPS induced only small changes in lysosomes. Upon longer contact, these changes are even less pronounced. The presented panel of assays may serve to identify effects on lysosomes also for other NPs. PMID:22789069

  1. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

    PubMed

    Solberg, L B; Stang, E; Brorson, S-H; Andersson, G; Reinholt, F P

    2015-02-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

  2. Lysosome-associated membrane glycoprotein 1 predicts fratricide amongst T cell receptor transgenic CD8+ T cells directed against tumor-associated antigens

    PubMed Central

    Kirschner, Andreas; Thiede, Melanie; Blaeschke, Franziska; Richter, Günther H.S.; Gerke, Julia S.; Baldauf, Michaela C.; Grünewald, Thomas G.P.; Busch, Dirk H.; Burdach, Stefan; Thiel, Uwe

    2016-01-01

    Aim Autologous as well as allogeneic CD8+ T cells transduced with tumor antigen specific T cell receptors (TCR) may cause significant tumor lysis upon adoptive transfer. Besides unpredictable life-threatening off-target effects, these TCRs may unexpectedly commit fratricide. We hypothesized lysosome-associated membrane glycoprotein 1 (LAMP1, CD107a) to be a marker for fratricide in TCR transgenic CD8+ T cells. Methods We identified HLA-A*02:01/peptide-restricted T cells directed against ADRB3295. After TCR identification, we generated HLA-A*02:01/peptide restricted TCR transgenic T cells by retroviral transduction and tested T cell expansion rates as well as A*02:01/peptide recognition and ES killing in ELISpot and xCELLigence assays. Expansion arrest was analyzed via Annexin and CD107a staining. Results were compared to CHM1319-TCR transgenic T cells. Results Beta-3-adrenergic receptor (ADRB3) as well as chondromodulin-1 (CHM1) are over-expressed in Ewing Sarcoma (ES) but not on T cells. TCR transgenic T cells demonstrated HLA-A*02:01/ADRB3295 mediated ES recognition and killing in ELISpot and xCELLigence assays. 24h after TCR transduction, CD107a expression correlated with low expansion rates due to apoptosis of ADRB3 specific T cells in contrast to CHM1 specific transgenic T cells. Amino-acid exchange scans clearly indicated the cross-reactive potential of HLA-A*02:01/ADRB3295- and HLA-A*02:01/CHM1319-TCR transgenic T cells. Comparison of peptide motive binding affinities revealed extended fratricide among ADRB3295 specific TCR transgenic T cells in contrast to CHM1319. Conclusion Amino-acid exchange scans alone predict TCR cross-reactivity with little specificity and thus require additional assessment of potentially cross-reactive HLA-A*02:01 binding candidates. CD107a positivity is a marker for fratricide of CD8+ TCR transgenic T cells. PMID:27447745

  3. Stability of membrane potential in heart mitochondria: Single mitochondrion imaging

    SciTech Connect

    Uechi, Yukiko; Yoshioka, Hisashi; Morikawa, Daisuke; Ohta, Yoshihiro . E-mail: ohta@cc.tuat.ac.jp

    2006-06-16

    Mitochondrial membrane potential ({delta}{psi} {sub m}) plays an important role in cellular activity. Although {delta}{psi} {sub m} of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of {delta}{psi} {sub m}. The current study is performed to investigate the factors that stabilize {delta}{psi} {sub m} in cells by observing {delta}{psi} {sub m} of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of {delta}{psi} {sub m} in cells.

  4. Stabilization of composition fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Safran, Samuel; Palmieri, Benoit

    2013-03-01

    A ternary mixture model is proposed to describe composition fluctuations in mixed membranes composed of saturated, unsaturated and hybrid lipids. The asymmetric hybrid lipid has one saturated and one unsaturated hydrocarbon chain and it can reduce the packing incompatibility between saturated and unsaturated lipids. A methodology to recast the free-energy of the lattice in terms of a continuous isotropic field theory is proposed and used to analyze composition fluctuations above the critical temperature. The effect of hybrid lipids on fluctuations domains rich in saturated/unsaturated lipids is predicted. The correlation length of such fluctuations decreases significantly with increasing amounts of hybrids even if the temperature is maintained close to the critical temperature. This provides an upper bound for the domain sizes expected in rafts stabilized by hybrids, above the critical temperature. When the hybrid composition of the membrane is increased further, a crossover value is found above which ``stripe-like'' fluctuations are observed. The wavelength of these fluctuations decreases with increasing hybrid fraction and tends toward a molecular size in a membrane that contains only hybrids.

  5. Lysosomes, cholesterol and atherosclerosis

    PubMed Central

    Jerome, W Gray

    2011-01-01

    Cholesterol-engorged macrophage foam cells are a critical component of the atherosclerotic lesion. Reducing the sterol deposits in lesions reduces clinical events. Sterol accumulations within lysosomes have proven to be particularly hard to mobilize out of foam cells. Moreover, excess sterol accumulation in lysosomes has untoward effects, including a complete disruption of lysosome function. Recently, we demonstrated that treatment of sterol-engorged macrophages in culture with triglyceride-containing particles can reverse many of the effects of cholesterol on lysosomes and dramatically reduce the sterol burden in these cells. This article describes what is known about lysosomal sterol engorgement, discusses the possible mechanisms by which triglyceride could produce its effects, and evaluates the possible positive and negative effects of reducing the lysosomal cholesterol levels in foam cells. PMID:21643524

  6. Membrane potential stabilizes the O intermediate in liposomes containing bacteriorhodopsin.

    PubMed

    Kalaidzidis, I V; Belevich, I N; Kalaidzidis, Y L; Kaulen, A D

    1999-10-01

    In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple-blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed. In the wild-type bacteriorhodopsin liposomes, the bathointermediate formation is pH-independent within the pH 5-7 range. The efficiency of the long-lived O intermediate formation increases at a low pH. In the wild-type as well as in the E204Q mutant purple membrane, the O intermediate decay is slowed down at slightly higher pH values than that of the purple-blue transition. It is suggested that the membrane potential affects the equilibrium between the bacteriorhodopsin ground state (Glu-204 is protonated and Asp-85 is deprotonated) and the O intermediate (Asp-85 is protonated and Glu-204 is deprotonated), stabilizing the latter by changing the relative affinity of Asp-85 and Glu-204 to H(+). At a low pH, protonation of a proton-releasing group (possibly Glu-194) in the bacteriorhodopsin ground state seems to prevent deprotonation of the Glu-204 during the photocycle. Thus, all protonatable residues of the outward proton pathway should be protonated in the O intermediate. Under such conditions, membrane potential stabilization of the O intermediate in the liposomes can be attributed to the direct effect of the potential on the pK value of Asp-85.

  7. Nanoporous membranes with electrochemically switchable, chemically stabilized ionic selectivity.

    PubMed

    Small, Leo J; Wheeler, David R; Spoerke, Erik D

    2015-10-28

    Nanopore size, shape, and surface charge all play important roles in regulating ionic transport through nanoporous membranes. The ability to control these parameters in situ provides a means to create ion transport systems tunable in real time. Here, we present a new strategy to address this challenge, utilizing three unique electrochemically switchable chemistries to manipulate the terminal functional group and control the resulting surface charge throughout ensembles of gold plated nanopores in ion-tracked polycarbonate membranes 3 cm(2) in area. We demonstrate the diazonium mediated surface functionalization with (1) nitrophenyl chemistry, (2) quinone chemistry, and (3) previously unreported trimethyl lock chemistry. Unlike other works, these chemistries are chemically stabilized, eliminating the need for a continuously applied gate voltage to maintain a given state and retain ionic selectivity. The effect of surface functionalization and nanopore geometry on selective ion transport through these functionalized membranes is characterized in aqueous solutions of sodium chloride at pH = 5.7. The nitrophenyl surface allows for ionic selectivity to be irreversibly switched in situ from cation-selective to anion-selective upon reduction to an aminophenyl surface. The quinone-terminated surface enables reversible changes between no ionic selectivity and a slight cationic selectivity. Alternatively, the trimethyl lock allows ionic selectivity to be reversibly switched by up to a factor of 8, approaching ideal selectivity, as a carboxylic acid group is electrochemically revealed or hidden. By varying the pore shape from cylindrical to conical, it is demonstrated that a controllable directionality can be imparted to the ionic selectivity. Combining control of nanopore geometry with stable, switchable chemistries facilitates superior control of molecular transport across the membrane, enabling tunable ion transport systems.

  8. Alpha-lipoic acid and alpha-lipoamide prevent oxidant-induced lysosomal rupture and apoptosis.

    PubMed

    Persson, H L; Svensson, A I; Brunk, U T

    2001-01-01

    Alpha-lipoic acid (LA) and its corresponding derivative, alpha-lipoamide (LM), have been described as antioxidants, but the mechanisms of their putative antioxidant effects remain largely uncharacterised. The vicinal thiols present in the reduced forms of these compounds suggest that they might possess metal chelating properties. We have shown previously that cell death caused by oxidants may be initiated by lysosomal rupture and that this latter event may involve intralysosomal iron which catalyzes Fenton-type chemistry and resultant peroxidative damage to lysosomal membranes. Here, using cultured J774 cells as a model, we show that both LA and LM stabilize lysosomes against oxidative stress, probably by chelating intralysosomal iron and, consequently, preventing intralysosomal Fenton reactions. In preventing oxidant-mediated apoptosis, LM is significantly more effective than LA, as would be expected from their differing capacities to enter cells and concentrate within the acidic lysosomal compartment. As previously reported, the powerful iron-chelator, desferrioxamine (Des) (which also locates within the lysosomal compartment), also provides protection against oxidant-mediated cell death. Interestingly, although Des enhances the partial protection afforded by LA, it confers no additional protection when added with LM. Therefore, the antioxidant actions of LA and LM may arise from intralysosomal iron chelation, with LM being more effective in this regard.

  9. Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis

    PubMed Central

    Sreetama, S C; Takano, T; Nedergaard, M; Simon, S M; Jaiswal, J K

    2016-01-01

    Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca2+-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca2+-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte – Syt XI – suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair. PMID:26450452

  10. Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis.

    PubMed

    Sreetama, S C; Takano, T; Nedergaard, M; Simon, S M; Jaiswal, J K

    2016-04-01

    Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca(2+)-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca(2+)-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte--Syt XI--suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

  11. Aspects of the digestive gland cells of the mussel Mytilus galloprovincialis, in relation to lysosomal enzymes, lipofuscin presence and shell size: contribution in the assessment of marine pollution biomarkers.

    PubMed

    Raftopoulou, E K; Dimitriadis, V K

    2012-02-01

    The present study investigates the histochemical localization of N-acetyl-β-hexozaminidase (Hex), acid phosphatase (AcP) and β-glucuronidase (β-Gus) in the digestive gland of mussels Mytilus galloprovincialis, as well as the clarification of suitable enzyme for biomarkers' application dealing with lysosomes. The results show more intense and homogenous localization of Hex, in relation to AcP and β-Gus and, thus, Hex histochemistry is supported as more suitable procedure for the evaluation of "lysosomal membrane stability" and "morphometrical alterations of lysosomes". The affection of lipofuscin granules on lysosomal enzymes' activity is also discussed. Additionally, the present study examines the response of small- and large-sized mussels M. galloprovincialis by assessing the "lysosomal membrane stability", "morphometrical alterations of lysosomes", "lysosomal response index (LRI)" and "structural epithelial changes in digestive tubules". The results indicate appreciable alterations of the above parameters in large-sized mussels, supporting their greater influence by the environmental factors, in relation to small-sized ones.

  12. Dynamical Stabilization of Large Pores in Bilayer Membranes

    NASA Astrophysics Data System (ADS)

    Nelson, Philip

    1997-03-01

    I will review two recent results involving large stable pores in lipid bilayer membranes. Needham and Zhelev have created long-lived, dynamically stabilized pores in vesicles using micropipette suction. We explain the long lifetime of these pores, and their very sudden closure. (2) Bar-Ziv, Frisch, and Moses have induced vesicles to expel interior objects after irradiation with laser light. This process also involves the formation of a long-lived exit pore in the outer vesicle. We report new experimental results and sketch a theory to explain them. The main hypothesis is that the laser trap pulls in lipid and ejects it in the form of submicron objects, whose osmotic activity then drives the expulsion.

  13. Lysosomal and tissue-level biomarkers in mussels cross-transplanted among four estuaries with different pollution levels.

    PubMed

    Lekube, Xabier; Izagirre, Urtzi; Soto, Manu; Marigómez, Ionan

    2014-02-15

    A 3-4 wk cross-transplantation experiment was carried out in order to investigate the sensitivity, rapidity, durability and reversibility of lysosomal and tissue-level biomarkers in the digestive gland of mussels. Four localities in the Basque coast with different levels of chemical pollution and environmental stress were selected. Lysosomal membrane stability (LP) and lysosomal structural changes (VvL; S/VL; NvL) and changes in cell-type composition in digestive gland epithelium (VvBAS) were investigated to determine short (2d) and mid-term (3-4 wk) responses after cross-transplantation. Mussels from Txatxarramendi presented VvBAS<0.1 μm(3)/μm(3) (unstressed) whilst VvBAS>0.12 μm(3)/μm(3) was recorded in mussels from Plentzia (moderate stress) and VvBAS>0.2 μm(3)/μm(3) in Arriluze and Muskiz (high stress). Accordingly, LP<10 min (high stress) was recorded in mussels from Muskiz and Arriluze and LP~15 min (low-to-moderate stress) in those from Plentzia and Txatxarramendi. According to the VvL, S/VL and NvL data, a certain lysosomal enlargement was envisaged in mussels from Arriluze in comparison with those from Txatxarramendi and Plentzia. Mussels from Muskiz exhibited a peculiar endo-lysosomal system made of abundant tiny lysosomes (low VvL and high S/VL and NvL values). Lysosomal and tissue-level biomarkers were responsive after 2d cross-transplantation between the reference and the polluted localities, which indicated that these biomarkers were quickly induced and, to a large extent, reversible. Moreover, the tissue-level biomarker values were maintained during the entire period (3-4 wk) of cross-transplantation, which evidenced the durability of the responsiveness. In contrast, comparisons in the mid-term were unfeasible for lysosomal biomarkers as these exhibited a seasonal winter attenuation resulting from low food availability and low temperatures. In conclusion, lysosomal enlargement and membrane stability and changes in cell-type composition were

  14. Cell biology in China: Focusing on the lysosome.

    PubMed

    Yang, Chonglin; Wang, Xiaochen

    2017-06-01

    The view that lysosomes are merely the recycling bins of the cell has changed greatly during recent years. Lysosomes are now known to play a central role in signal transduction, cellular adaptation, plasma membrane repair, immune responses and many other fundamental cellular processes. In conjunction with the seminal discoveries made by international colleagues, many important questions regarding lysosomes are being addressed by Chinese scientists. In this review, we briefly summarize recent exciting findings in China on lysosomal signaling, biogenesis, integrity and physiological functions. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Effect of antimalarials treatment on rat liver lysosomal function-Anin vivo study.

    PubMed

    Patel, Samir P; Katewa, Subhash D; Katyare, Surendra S

    2005-01-01

    Effects of treatmentin vivo with the antimalarials:chloroquine (CQ), primaquine (PQ) and quinine(Q) on lysosomal enzymes and lysosomal membrane integrity were examined. Treatment with the three antimalarials showed an apparent increase in the membrane stability. CQ treatment resulted in increase in both the 'free' and 'total' activities of all the enzymes i.e. acid phosphatase, RNase II, DNase II and cathepsin D. PQ treatment lowered the 'free' and 'total' activities of acid phosphatase and cathepsin D, but the DNase II activities increased. Treatment with Q resulted in increased 'free' and 'total' activities of RNase II and DNase II. While 'free' activities of acid phosphatase and cathepsin D were low; the 'total' activities increased significantly. Our results suggest that a generalized increase in free nucleases activities following prolonged treatment with antimalarials may lead to cell damage and/or necrosis.

  16. Lysosomal disruption preferentially targets acute myeloid leukemia cells and progenitors

    PubMed Central

    Sukhai, Mahadeo A.; Prabha, Swayam; Hurren, Rose; Rutledge, Angela C.; Lee, Anna Y.; Sriskanthadevan, Shrivani; Sun, Hong; Wang, Xiaoming; Skrtic, Marko; Seneviratne, Ayesh; Cusimano, Maria; Jhas, Bozhena; Gronda, Marcela; MacLean, Neil; Cho, Eunice E.; Spagnuolo, Paul A.; Sharmeen, Sumaiya; Gebbia, Marinella; Urbanus, Malene; Eppert, Kolja; Dissanayake, Dilan; Jonet, Alexia; Dassonville-Klimpt, Alexandra; Li, Xiaoming; Datti, Alessandro; Ohashi, Pamela S.; Wrana, Jeff; Rogers, Ian; Sonnet, Pascal; Ellis, William Y.; Corey, Seth J.; Eaves, Connie; Minden, Mark D.; Wang, Jean C.Y.; Dick, John E.; Nislow, Corey; Giaever, Guri; Schimmer, Aaron D.

    2012-01-01

    Despite efforts to understand and treat acute myeloid leukemia (AML), there remains a need for more comprehensive therapies to prevent AML-associated relapses. To identify new therapeutic strategies for AML, we screened a library of on- and off-patent drugs and identified the antimalarial agent mefloquine as a compound that selectively kills AML cells and AML stem cells in a panel of leukemia cell lines and in mice. Using a yeast genome-wide functional screen for mefloquine sensitizers, we identified genes associated with the yeast vacuole, the homolog of the mammalian lysosome. Consistent with this, we determined that mefloquine disrupts lysosomes, directly permeabilizes the lysosome membrane, and releases cathepsins into the cytosol. Knockdown of the lysosomal membrane proteins LAMP1 and LAMP2 resulted in decreased cell viability, as did treatment of AML cells with known lysosome disrupters. Highlighting a potential therapeutic rationale for this strategy, leukemic cells had significantly larger lysosomes compared with normal cells, and leukemia-initiating cells overexpressed lysosomal biogenesis genes. These results demonstrate that lysosomal disruption preferentially targets AML cells and AML progenitor cells, providing a rationale for testing lysosomal disruption as a novel therapeutic strategy for AML. PMID:23202731

  17. Involvement of lysosomes in the early stages of axon degeneration.

    PubMed

    Zheng, Jin; Yan, Tingting; Feng, Yan; Zhai, Qiwei

    2010-02-01

    Axon degeneration is a common hallmark of many neurodegenerative diseases, and the underlying mechanism remains largely unknown. Lysosomes are involved in some neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Whether lysosomes are involved in axon degeneration is yet to be elucidated. In this study, we found only about 10% lysosomes remained in axons of cultured superior cervical ganglia (SCGs) after transection for 4h when stained with LysoTracker. Furthermore, we found that lysosomal disruption occurred earlier than morphological changes and loss of mitochondrial membrane potential. In addition, the well-known axon-protective protein Wld(S) delayed injury-induced axon degeneration from both morphological changes and lysosomal disruption. Lysosomal inhibitors including chloroquine and ammonium chloride induced axon degeneration in cultured SCGs, and Wld(S) also slowed down the axon degeneration induced by lysosomal inhibitors. All these data suggest that lysosomal disruption is an early marker of axon degeneration, and inhibition of lysosome induces axon degeneration in a Wld(S)-protectable way. Thus, maintenance of normal lysosomal function might be an important approach to delay axon degeneration in neurodegenerative diseases.

  18. Regulation of lysosomal ion homeostasis by channels and transporters.

    PubMed

    Xiong, Jian; Zhu, Michael X

    2016-08-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H(+), Ca(2+), Na(+), K(+), and Cl(-) across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease.

  19. Regulation of lysosomal ion homeostasis by channels and transporters

    PubMed Central

    Xiong, Jian; Zhu, Michael X.

    2016-01-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H+, Ca2+, Na+, K+, and Cl− across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease. PMID:27430889

  20. Preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in experimentally induced myocardial infarction.

    PubMed

    Jyoti Roy, Abhro; Stanely Mainzen Prince, P

    2013-01-15

    The present study was designed to evaluate the preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in isoproterenol induced myocardial infarcted rats. Male albino Wistar rats were pretreated with p-coumaric acid (8 mg/kg body weight) daily for a period of 7 days after which isoproterenol (100mg/kg body weight) was injected subcutaneously into rats twice at an interval of 24h (8th and 9th day).The activity/levels of serum cardiac diagnostic markers, heart lysosomal lipid peroxidation products and the activities of lysosomal enzymes (β-glucuronidase, β-galactosidase, cathepsin-B and cathepsin-D) were significantly (P<0.05) increased in the serum and heart of isoproterenol induced myocardial infarcted rats. Isoproterenol also lowered the activities of β-glucuronidase and cathepsin-D in the lysosomal fraction. The pretreatment with p-coumaric acid significantly (P<0.05) prevented the changes in the levels of lysosomal lipid peroxidation products and the activities of lysosomal enzymes. In addition, p-coumaric acid greatly reduced myocardial infarct size. p-Coumaric acid pretreatment (8 mg/kg body weight) to normal rats did not show any significant effect. Thus, this study showed that p-coumaric acid prevents lysosomal dysfunction against cardiac damage induced by isoproterenol and brings back the levels of lipid peroxidation products and activities of lysosomal enzymes to near normal levels. The in vitro study also revealed the free radical scavenging activity of p-coumaric acid. Thus, the observed effects are due to p-coumaric acid's free radical scavenging and membrane stabilizing properties.

  1. Endothelial Nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis.

    PubMed

    Chen, Yang; Li, Xiang; Boini, Krishna M; Pitzer, Ashley L; Gulbins, Erich; Zhang, Yang; Li, Pin-Lan

    2015-02-01

    Inflammasomes play a critical role in the development of vascular diseases. However, the molecular mechanisms activating the inflammasome in endothelial cells and the relevance of this inflammasome activation is far from clear. Here, we investigated the mechanisms by which an Nlrp3 inflammasome is activated to result in endothelial dysfunction during coronary arteritis by Lactobacillus casei (L. casei) cell wall fragments (LCWE) in a mouse model for Kawasaki disease. Endothelial dysfunction associated with increased vascular cell adhesion protein 1 (VCAM-1) expression and endothelial-leukocyte adhesion was observed during coronary arteritis in mice treated with LCWE. Accompanied with these changes, the inflammasome activation was also shown in coronary arterial endothelium, which was characterized by a marked increase in caspase-1 activity and IL-1β production. In cultured endothelial cells, LCWE induced Nlrp3 inflammasome formation, caspase-1 activation and IL-1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasone, and ceramide. However, a potassium channel blocker glibenclamide or an oxygen free radical scavenger N-acetyl-l-cysteine had no effects on LCWE-induced inflammasome activation. LCWE also increased endothelial cell lysosomal membrane permeability and triggered lysosomal cathepsin B release into cytosol. Silencing cathepsin B blocked LCWE-induced Nlrp3 inflammasome formation and activation in endothelial cells. In vivo, treatment of mice with cathepsin B inhibitor also abolished LCWE-induced inflammasome activation in coronary arterial endothelium. It is concluded that LCWE enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the endothelial Nlrp3 inflammasome, which may contribute to the development of coronary arteritis.

  2. A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB.

    PubMed

    Settembre, Carmine; Zoncu, Roberto; Medina, Diego L; Vetrini, Francesco; Erdin, Serkan; Erdin, SerpilUckac; Huynh, Tuong; Ferron, Mathieu; Karsenty, Gerard; Vellard, Michel C; Facchinetti, Valeria; Sabatini, David M; Ballabio, Andrea

    2012-03-07

    The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB-/- cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.

  3. Porous polybenzimidazole membranes with excellent chemical stability and ion conductivity for direct borohydride fuel cells

    NASA Astrophysics Data System (ADS)

    Chen, Dongju; Yu, Shanshan; Liu, Xue; Li, Xianfeng

    2015-05-01

    Porous membranes based on polybenzimidazole (PBI) are firstly introduced in direct borohydride fuel cell application (DBFC). Membranes with different thicknesses and porosity are successfully fabricated via water vapor phase inversion process. The prepared membranes show excellent ion conductivity and chemical stability under DBFC operating condition. Compare with Nafion 115, the prepared membranes show higher ion conductivity, as a result, much higher peak power density. No weight loss is observed after immersing the prepared membranes in a 3 M NaOH solution for 30 days, indicating the excellent chemical stability of porous PBI membranes. And the DBFC cells assembled with prepared membranes could discharge at 200 mA cm-2 for more than 250 h without voltage decay, which is the longest time reported by far. This work provides a totally new idea for fabricating versatile DBFC membranes.

  4. Endosome-lysosomes and neurodegeneration.

    PubMed

    Mayer, R J; Tipler, C; Laszlo, L; Arnold, J; Lowe, J; Landon, M

    1994-01-01

    A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins. Although our knowledge concerning these diseases is increasing, they remain largely untreatable. Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials. These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Common features of the disease processes give new direction to therapeutic intervention.

  5. Insights into thermophilic archaebacterial membrane stability from simplified models of lipid membranes

    NASA Astrophysics Data System (ADS)

    Davis, Charles H.; Nie, Huifen; Dokholyan, Nikolay V.

    2007-05-01

    Lipid aggregation into fluid bilayers is an essential process for sustaining life. Simplified models of lipid structure, which allow for long time scales or large length scales not obtainable with all-atom simulations, have recently been developed and show promise for describing lipid dynamics in biological systems. Here, we describe two simplified models, a reduced-lipid model and a bola-lipid model for thermophilic bacterial membranes, developed for use with the rapid discrete molecular dynamics simulation method. In the reduced-lipid model, we represent the lipid chain by a series of three beads interacting through pairwise discrete potentials that model hydrophobic attractions between hydrocarbon tails in implicit solvent. Our phase diagram recapitulates those produced by continuous potential models with similar coarse-grained lipid representations. We also find that phase transition temperatures for our reduced-lipid model are dependent upon the flexibility of the lipid chain, giving an insight into archaebacterial membrane stability and prompting development of a bola-lipid model specific for archaebacteria lipids. With both the reduced-lipid and bola-lipid model, we find that the reduced flexibility inherent in archaebacteria lipids yields more stable bilayers as manifested by increased phase transition temperatures. The results of these studies provide a simulation methodology for lipid molecules in biological systems and show that discrete molecular dynamics is applicable to lipid aggregation and dynamics.

  6. TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity.

    PubMed

    Soyombo, Abigail A; Tjon-Kon-Sang, Sandra; Rbaibi, Youssef; Bashllari, Enkelejda; Bisceglia, Jill; Muallem, Shmuel; Kiselyov, Kirill

    2006-03-17

    Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles, which was hypothesized to be caused by the altered membrane fusion and fission events. How mutations in TRP-ML1 lead to aberrant lipolysis is not known. Here we present evidence that MLIV is a metabolic disorder that is not associated with aberrant membrane fusion/fission events. Thus, measurement of lysosomal pH revealed that the lysosomes in TRP-ML1(-/-) cells obtained from the patients with MLIV are over-acidified. TRP-ML1 can function as a H(+) channel, and the increased lysosomal acidification in TRP-ML1(-/-) cells is likely caused by the loss of TRP-ML1-mediated H(+) leak. Measurement of lipase activity using several substrates revealed a marked reduction in lipid hydrolysis in TRP-ML1(-/-) cells, which was rescued by the expression of TRP-ML1. Cell fractionation indicated specific loss of acidic lipase activity in TRP-ML1(-/-) cells. Furthermore, dissipation of the acidic lysosomal pH of TRP-ML1(-/-) cells by nigericin or chloroquine reversed the lysosomal storage disease phenotype. These findings provide a new mechanism to account for the pathogenesis of MLIV.

  7. P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

    PubMed

    Kaur, Jasber; Cutler, Daniel F

    2002-03-22

    The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.

  8. A lysosome-centered view of nutrient homeostasis.

    PubMed

    Mony, Vinod K; Benjamin, Shawna; O'Rourke, Eyleen J

    2016-01-01

    Lysosomes are highly acidic cellular organelles traditionally viewed as sacs of enzymes involved in digesting extracellular or intracellular macromolecules for the regeneration of basic building blocks, cellular housekeeping, or pathogen degradation. Bound by a single lipid bilayer, lysosomes receive their substrates by fusing with endosomes or autophagosomes, or through specialized translocation mechanisms such as chaperone-mediated autophagy or microautophagy. Lysosomes degrade their substrates using up to 60 different soluble hydrolases and release their products either to the cytosol through poorly defined exporting and efflux mechanisms or to the extracellular space by fusing with the plasma membrane. However, it is becoming evident that the role of the lysosome in nutrient homeostasis goes beyond the disposal of waste or the recycling of building blocks. The lysosome is emerging as a signaling hub that can integrate and relay external and internal nutritional information to promote cellular and organismal homeostasis, as well as a major contributor to the processing of energy-dense molecules like glycogen and triglycerides. Here we describe the current knowledge of the nutrient signaling pathways governing lysosomal function, the role of the lysosome in nutrient mobilization, and how lysosomes signal other organelles, distant tissues, and even themselves to ensure energy homeostasis in spite of fluctuations in energy intake. At the same time, we highlight the value of genomics approaches to the past and future discoveries of how the lysosome simultaneously executes and controls cellular homeostasis.

  9. TFEB regulates lysosomal proteostasis.

    PubMed

    Song, Wensi; Wang, Fan; Savini, Marzia; Ake, Ashley; di Ronza, Alberto; Sardiello, Marco; Segatori, Laura

    2013-05-15

    Loss-of-function diseases are often caused by destabilizing mutations that lead to protein misfolding and degradation. Modulating the innate protein homeostasis (proteostasis) capacity may lead to rescue of native folding of the mutated variants, thereby ameliorating the disease phenotype. In lysosomal storage disorders (LSDs), a number of highly prevalent alleles have missense mutations that do not impair the enzyme's catalytic activity but destabilize its native structure, resulting in the degradation of the misfolded protein. Enhancing the cellular folding capacity enables rescuing the native, biologically functional structure of these unstable mutated enzymes. However, proteostasis modulators specific for the lysosomal system are currently unknown. Here, we investigate the role of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and function, in modulating lysosomal proteostasis in LSDs. We show that TFEB activation results in enhanced folding, trafficking and lysosomal activity of a severely destabilized glucocerebrosidase (GC) variant associated with the development of Gaucher disease (GD), the most common LSD. TFEB specifically induces the expression of GC and of key genes involved in folding and lysosomal trafficking, thereby enhancing both the pool of mutated enzyme and its processing through the secretory pathway. TFEB activation also rescues the activity of a β-hexosaminidase mutant associated with the development of another LSD, Tay-Sachs disease, thus suggesting general applicability of TFEB-mediated proteostasis modulation to rescue destabilizing mutations in LSDs. In summary, our findings identify TFEB as a specific regulator of lysosomal proteostasis and suggest that TFEB may be used as a therapeutic target to rescue enzyme homeostasis in LSDs.

  10. Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model

    PubMed Central

    Bagh, Maria B.; Peng, Shiyong; Chandra, Goutam; Zhang, Zhongjian; Singh, Satya P.; Pattabiraman, Nagarajan; Liu, Aiyi; Mukherjee, Anil B.

    2017-01-01

    Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1−/− mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1−/− mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases. PMID:28266544

  11. Control of lipid membrane stability by cholesterol content.

    PubMed Central

    Raffy, S; Teissié, J

    1999-01-01

    Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content. PMID:10096902

  12. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    PubMed Central

    Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2016-01-01

    ABSTRACT Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy. PMID:27046251

  13. Hematopoietic protein-1 regulates the actin membrane skeleton and membrane stability in murine erythrocytes.

    PubMed

    Chan, Maia M; Wooden, Jason M; Tsang, Mark; Gilligan, Diana M; Hirenallur-S, Dinesh K; Finney, Greg L; Rynes, Eric; Maccoss, Michael; Ramirez, Julita A; Park, Heon; Iritani, Brian M

    2013-01-01

    Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1⁻/⁻ erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1⁻/⁻ erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes.

  14. Hematopoietic Protein-1 Regulates the Actin Membrane Skeleton and Membrane Stability in Murine Erythrocytes

    PubMed Central

    Chan, Maia M.; Wooden, Jason M.; Tsang, Mark; Gilligan, Diana M.; Hirenallur-S, Dinesh K.; Finney, Greg L.; Rynes, Eric; MacCoss, Michael; Ramirez, Julita A.; Park, Heon; Iritani, Brian M.

    2013-01-01

    Hematopoietic protein-1 (Hem-1) is a hematopoietic cell specific member of the WAVE (Wiskott-Aldrich syndrome verprolin-homologous protein) complex, which regulates filamentous actin (F-actin) polymerization in many cell types including immune cells. However, the roles of Hem-1 and the WAVE complex in erythrocyte biology are not known. In this study, we utilized mice lacking Hem-1 expression due to a non-coding point mutation in the Hem1 gene to show that absence of Hem-1 results in microcytic, hypochromic anemia characterized by abnormally shaped erythrocytes with aberrant F-actin foci and decreased lifespan. We find that Hem-1 and members of the associated WAVE complex are normally expressed in wildtype erythrocyte progenitors and mature erythrocytes. Using mass spectrometry and global proteomics, Coomassie staining, and immunoblotting, we find that the absence of Hem-1 results in decreased representation of essential erythrocyte membrane skeletal proteins including α- and β- spectrin, dematin, p55, adducin, ankyrin, tropomodulin 1, band 3, and band 4.1. Hem1−/− erythrocytes exhibit increased protein kinase C-dependent phosphorylation of adducin at Ser724, which targets adducin family members for dissociation from spectrin and actin, and subsequent proteolysis. Increased adducin Ser724 phosphorylation in Hem1−/− erythrocytes correlates with decreased protein expression of the regulatory subunit of protein phosphatase 2A (PP2A), which is required for PP2A-dependent dephosphorylation of PKC targets. These results reveal a novel, critical role for Hem-1 in the homeostasis of structural proteins required for formation and stability of the actin membrane skeleton in erythrocytes. PMID:23424621

  15. Stabilized composite membranes and membrane electrode assemblies for high temperature/low relative humidity polymer electrolyte fuel cell operation

    NASA Astrophysics Data System (ADS)

    Ramani, Vijay Krishna

    Polymer electrolyte membrane fuel cells (PEMFCs) have a variety of applications in the stationary power, mobile power and automotive power sectors. Existing membrane technology presently permits fuel cell operation at temperatures less than 100°C under fully saturated conditions. However, several advantages such as easier heat rejection rates and improved impurities tolerance by the anode electrocatalyst result by operating a PEMFC at elevated temperatures (above 100°C) and lower relative humidities. In an attempt to extend the operating range of the polymer electrolyte membrane, perfluorosulfonic acid (NafionRTM) based organic/inorganic (heteropolyacid) composite membranes were investigated in terms of thermal and electrochemical stability, additive stability and conductivity. Tungsten based heteropolyacids (HPAs) were found to be electrochemically stable as opposed to molybdenum based additives. The stability of the inorganic heteropolyacid additive in aqueous environments was enhanced by ion exchanging the protons of the HPAs with larger counter ions. An additional stabilization technique developed involved improving the interaction of HPA with NafionRTM by linking the particles to the sulfonic acid clusters via a sol-gel induced metal oxide linkage. The proton conductivity of the composite membranes was found to depend on the particle size of the HPA additive. A two order of magnitude change in additive particle size was attained by modification of the membrane preparation technique. This modification resulted in a nearly 50% increase in conductivity. The membranes prepared were characterized by thermal analysis, spectroscopy and microscopy. A technique was developed to incorporate existing MEA preparation and HPA stabilization techniques to the composite membranes with small HPA particles. All MEAs prepared were evaluated at high temperatures (120°C) and low relative humidities (35%) in an operating fuel cell, with membrane resistance and hence conductivity

  16. Stability of antibody-conjugated gold nanoparticles in the endo-lysosomal nanoenvironment: Implications for non-invasive radiofrequency-based cancer therapy

    PubMed Central

    Raoof, Mustafa; Corr, Stuart J.; Kaluarachchi, Warna D.; Massey, Katheryn L.; Briggs, Katrina; Zhu, Cihui; Cheney, Matthew A.; Wilson, Lon J.; Curley, Steven A.

    2012-01-01

    The use of non-invasive radiofrequency (RF) electric fields as an energy source for thermal activation of nanoparticles within cancer cells could be a valuable addition to the emerging field of nano-mediated cancer therapies. Based on investigations of cell death through hyperthermia, and offering the ability for total body penetration by RF fields, this technique is thought to compliment and possibly out-perform existing nano-heat-treatments that utilize alternative heat production via optical or magnetic stimuli. However, it remains a challenge to understand fully the complex RF-nanoparticle-intracellular interactions before full system optimization can be engineered. Herein we have shown that liver cancer cells can selectively internalize antibody-conjugated gold nanoparticles (AuNPs) through receptor-mediated endocytosis, with the nanoparticles predominantly accumulating and aggregating within cytoplasmic endo-lysosomes. After exposure to an external RF field, non-aggregated AuNPs absorbed and dissipated energy as heat causing thermal damage to the targeted cancer cells. We also observed that RF absorption and heat dissipation is dependent on solubility of AuNPs in the colloid, which is pH dependent. Furthermore, by modulating endo-lysosomal pH it is possible to prevent intracellular AuNP aggregation and enhance thermal cytotoxicity in hepatocellular cancer cells. PMID:22349096

  17. Reactivation of Lysosomal Ca2+ Efflux Rescues Abnormal Lysosomal Storage in FIG4-Deficient Cells.

    PubMed

    Zou, Jianlong; Hu, Bo; Arpag, Sezgi; Yan, Qing; Hamilton, Audra; Zeng, Yuan-Shan; Vanoye, Carlos G; Li, Jun

    2015-04-29

    Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.

  18. A Model for Prediction of Heat Stability of Photosynthetic Membranes

    USDA-ARS?s Scientific Manuscript database

    A previous study has revealed a positive correlation between heat-induced damage to photosynthetic membranes (thylakoid membranes) and chlorophyll loss. In this study, we exploited this correlation and developed a model for prediction of thermal damage to thylakoids. Prediction is based on estimat...

  19. Stabilization of membrane necks by adhesive particles, substrate surfaces, and constriction forces.

    PubMed

    Agudo-Canalejo, Jaime; Lipowsky, Reinhard

    2016-10-21

    Membrane remodelling processes involving the formation and fission of small buds require the formation and closure of narrow membrane necks, both for biological membranes and for model membranes such as lipid bilayers. The conditions required for the stability of such necks are well understood in the context of budding of vesicles with bilayer asymmetry and/or intramembrane domains. In many cases, however, the necks form in the presence of an adhesive surface, such as a solid particle or substrate, or the cellular cortex itself. Examples of such processes in biological cells include endocytosis, exocytosis and phagocytosis of solid particles, the formation of extracellular and outer membrane vesicles by eukaryotic and prokaryotic cells, as well as the closure of the cleavage furrow in cytokinesis. Here, we study the interplay of curvature elasticity, membrane-substrate adhesion, and constriction forces to obtain generalized stability conditions for closed necks which we validate by numerical energy minimization. We then explore the consequences of these stability conditions in several experimentally accessible systems such as particle-filled membrane tubes, supported lipid bilayers, giant plasma membrane vesicles, bacterial outer membrane vesicles, and contractile rings around necks. At the end, we introduce an intrinsic engulfment force that directly describes the interplay between curvature elasticity and membrane-substrate adhesion.

  20. Lysosomes serve as a platform for hepatitis A virus particle maturation and nonlytic release.

    PubMed

    Seggewiß, Nicole; Paulmann, Dajana; Dotzauer, Andreas

    2016-01-01

    Early studies on hepatitis A virus (HAV) in cell culture demonstrated the inclusion of several viral particles in an intracellular lipid-bilayer membrane. However, the origin of these virus-associated membranes and the mechanism for the non-lytic release of HAV into bile are still unknown. Analyzing the association of this virus with cell organelles, we found that newly synthesized HAV particles accumulate in lysosomal organelles and that lysosomal enzymes are involved in the maturation cleavage of the virion. Furthermore, by inhibiting the processes of fusion of lysosomes with the plasma membrane, we found that the nonlytic release of HAV from infected cells occurs via lysosome-related organelles.

  1. Investigation of the anti-inflammatory and membrane-stabilizing potential of spiramycin in vitro.

    PubMed

    Theron, A J; Feldman, C; Anderson, R

    2000-08-01

    The effects of the 16-member macrolide spiramycin (2.5-80 mg/L) and the 14-member agent clarithromycin on the production of superoxide by activated human neutrophils were compared in vitro and related to membrane-stabilizing activity. Superoxide production was measured by lucigenin-enhanced chemiluminescence with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 microM) as the stimulus, and membrane-stabilizing activity was measured by a haemolytic procedure. Clarithromycin, but not spiramycin, caused dose-related inhibition of superoxide production by activated neutrophils and also protected erythrocytes against haemolysis, while spiramycin possessed only weak membrane-stabilizing activity. These observations underscore the apparent association between the anti-inflammatory and membrane-stabilizing properties of macrolides.

  2. Hydrocarbon-Based Polymer Electrolyte Membranes: Importance of Morphology on Ion Transport and Membrane Stability.

    PubMed

    Shin, Dong Won; Guiver, Michael D; Lee, Young Moo

    2017-03-03

    A fundamental understanding of polymer microstructure is important in order to design novel polymer electrolyte membranes (PEMs) with excellent electrochemical performance and stabilities. Hydrocarbon-based polymers have distinct microstructure according to their chemical structure. The ionic clusters and/or channels play a critical role in PEMs, affecting ion conductivity and water transport, especially at medium temperature and low relative humidity (RH). In addition, physical properties such as water uptake and dimensional swelling behavior depend strongly on polymer morphology. Over the past few decades, much research has focused on the synthetic development and microstructural characterization of hydrocarbon-based PEM materials. Furthermore, blends, composites, pressing, shear field, electrical field, surface modification, and cross-linking have also been shown to be effective approaches to obtain/maintain well-defined PEM microstructure. This review summarizes recent work on developments in advanced PEMs with various chemical structures and architecture and the resulting polymer microstructures and morphologies that arise for potential application in fuel cell, lithium ion battery, redox flow battery, actuators, and electrodialysis.

  3. The antimicrobial peptide microcin J25 stabilizes the gel phase of bacterial model membranes.

    PubMed

    Rintoul, M R; Morero, R D; Dupuy, F G

    2015-05-01

    The bacterial membrane interaction of the antimicrobial peptide microcin J25 was studied with the probe-free techniques Langmuir monolayers and infrared spectroscopy. Membrane model systems composed by phosphatidylethanolamine:phosphatidylglycerol 7:3, which mimic the cytoplasmic membrane of Gram negative bacteria, were used in both monolayer and bilayer approaches. The peptide reduced the transition surface pressure of the expanded-to-condensed lipid monolayer states, as well as increased the gel-to-liquid crystalline transition temperature in bilayers, indicating a stabilization of membrane ordered state. In addition, a reduction of the surface pressure at which condensed domains appeared was observed upon mixed monolayers compression after microcin J25 adsorption. The results indicate a favorable interaction of microcin J25 with bacterial membrane model systems. Also, the effects on the ordered phases stabilization are discussed in terms of the biological effects observed in membranes of sensitive cells.

  4. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    PubMed Central

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  5. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    PubMed

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  6. Protein kinase C-δ isoform mediates lysosome labilization in DNA damage-induced apoptosis

    PubMed Central

    PARENT, NICOLAS; SCHERER, MAX; LIEBISCH, GERHARD; SCHMITZ, GERD; BERTRAND, RICHARD

    2013-01-01

    A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highly-enriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-δ (PKC-δ). In this study, we show that the PKC-δ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-δ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-δ or ASM activity reduces both the CPT-mediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-δ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. PMID:21174057

  7. A mechanism for overcoming P-glycoprotein-mediated drug resistance: novel combination therapy that releases stored doxorubicin from lysosomes via lysosomal permeabilization using Dp44mT or DpC

    PubMed Central

    Seebacher, Nicole A; Richardson, Des R; Jansson, Patric J

    2016-01-01

    The intracellular distribution of a drug can cause significant variability in both activity and selectivity. Herein, we investigate the mechanism by which the anti-cancer agents, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and the clinically trialed, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), re-instate the efficacy of doxorubicin (DOX), in drug-resistant P-glycoprotein (Pgp)-expressing cells. Both Dp44mT and DpC potently target and kill Pgp-expressing tumors, while DOX effectively kills non-Pgp-expressing cancers. Thus, the combination of these agents should be considered as an effective rationalized therapy for potently treating advanced and resistant tumors that are often heterogeneous in terms of Pgp-expression. These studies demonstrate that both Dp44mT and DpC are transported into lysosomes via Pgp transport activity, where they induce lysosomal-membrane permeabilization to release DOX trapped within lysosomes. This novel strategy of loading lysosomes with DOX, followed by permeabilization with Dp44mT or DpC, results in the relocalization of stored DOX from its lysosomal 'safe house' to its nuclear targets, markedly enhancing cellular toxicity against resistant tumor cells. Notably, the combination of Dp44mT or DpC with DOX showed a very high level of synergism in multiple Pgp-expressing cell types, for example, cervical, breast and colorectal cancer cells. These studies revealed that the level of drug synergy was proportional to Pgp activity. Interestingly, synergism was ablated by inhibiting Pgp using the pharmacological inhibitor, Elacridar, or by inhibiting Pgp-expression using Pgp-silencing, demonstrating the importance of Pgp in the synergistic interaction. Furthermore, lysosomal-membrane stabilization inhibited the relocalization of DOX from lysosomes to the nucleus upon combination with Dp44mT or DpC, preventing synergism. This latter observation demonstrated the importance of lysosomal-membrane

  8. Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles.

    PubMed

    Jimeno-Romero, A; Oron, M; Cajaraville, M P; Soto, M; Marigómez, I

    2016-10-01

    The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO2 nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10 mg Ti/L either as TiO2 NPs (60 and 180 nm) or bulk TiO2, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO2 NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO2 NPs of 60 nm were internalised within digestive cell lysosomes to a higher extent than TiO2 NPs of 180 nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO2 NPs although more markedly after exposure to TiO2-60 NPs. Exposure to bulk TiO2 and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO2 and DSLS.

  9. The effects of hydrocortisone and glycyrrhizine on the enzyme releases of arylsulfatase and hyaluronidase from lysosomes of liver.

    PubMed

    Ozeki, T; Tokawa, Y; Ogasawara, T; Sato, K; Kan, M

    1978-03-15

    Hydrocortisone and glycyrrhizine act as both stabilizers and labilizers of the lysosomes of liver. The effect of both agents on the lysosomes is changeable according to the duration of their administration.

  10. Biogenesis of lysosomes in marshall cells and in cells of the male reproductive system.

    PubMed

    Morales, C R; Fuska, J; Zhao, Q; Lefrancois, S

    2001-05-01

    The mechanism of plasma membrane trafficking and degradation is still poorly understood. This investigation deals with the biogenesis of lysosomes during endocytic flow in Marshall cells and in various cell types of the male reproductive system. Marshall cells were exposed to ammonium chloride (NH4Cl) and leupeptin after labeling with cationic ferritin. In some experiments, the treated cells were immunogold labeled with anti-prosaposin antibody. NH4Cl and leupeptin are lysosomotropic agents that affect the endosomal-lysosomal progression. Testes, efferent ducts and epididymis from mouse mutants with defects affecting plasma membrane degradation were also used to analyze this process. NH4Cl produced a retention of cationic ferritin in endosomes and hindered the endosomal/lysosomal progression. Leupeptin did not affect this process. NH4Cl decreased the labeling of prosaposin in endosomes and lysosomes, while leupeptin increased the labeling of prosaposin in lysosomes. The number of lysosomes per cytoplasmic area was higher in treated cells than in controls. These findings suggest that leupeptin affected lysosomes whereas NH4Cl affected both endosomes and lysosomes. The endosomal and lysosomal accumulation of prosaposin induced by the treatment with NH4Cl and leupeptin indicated that the site of entry of prosaposinwas both the lysosome and endosome. Electron microscopy (EM) of tissues from mouse mutants with defects affecting plasma membrane degradation substantiated these observations. The EM analysis revealed a selective accumulation of multivesicular bodies (MVBs) and the disappearance of lysosomes, in testicular fibroblasts, nonciliated cells of the efferent ducts and principal cells of the epididymis, suggesting that MVBs are precursors of lysosomes. (1) endosomes and MVBs are a required steps for degradation of membranes; (2) endosomes and MVBs are precursors of lysosomes; and (3) endosomes, MVBs, and lysosomes appear to be transient organelles. Copyright 2001

  11. Enhanced thermal stability of the thylakoid membranes from spruce. A comparison with selected angiosperms.

    PubMed

    Karlický, Václav; Kurasová, Irena; Ptáčková, Božena; Večeřová, Kristýna; Urban, Otmar; Špunda, Vladimír

    2016-12-01

    Recently, we have found that thermal stability of photosystem II (PSII) photochemistry in spruce needles is higher than in other plants (barley, beech) cultivated under the same temperatures. In this work, temperature dependences of various characteristics of PSII organization were studied in order to obtain complex information on the thermal stability of PSII function and organization in spruce. Temperature dependency of circular dichroism spectra revealed by about 6 °C higher thermal stability of macrodomain organization in spruce thylakoid membranes in comparison with Arabidopsis and barley ones; however, thermal disintegration of light-harvesting complex of PSII did not significantly differ among the species studied. These results thus indicate that thermal stability of PSII macro-organization in spruce thylakoid membranes is enhanced to a similar extent as thermal stability of PSII photochemistry. Clear-native polyacrylamide gel electrophoresis of preheated thylakoids demonstrated that among the separated pigment-protein complexes, only PSII supercomplexes (SCs) revealed considerably higher thermal stability in spruce thylakoids as compared to Arabidopsis and barley ones. Hence we suggest that higher thermal stability of PSII macro-organization of spruce is influenced by the maintenance of PSII SCs in the thylakoid membrane. In addition, we discuss possible effects of different PSII organizations and lipid compositions on the thermal stability of spruce thylakoid membranes.

  12. Activation of peroxisome proliferator-activated receptor α induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders.

    PubMed

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J; Sims, Katherine B; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-04-17

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role.

  13. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

    PubMed

    Ögmundsdóttir, Margrét H; Heublein, Sabine; Kazi, Shubana; Reynolds, Bruno; Visvalingam, Shivanthy M; Shaw, Michael K; Goberdhan, Deborah C I

    2012-01-01

    Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+)-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

  14. Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability.

    PubMed

    Levi, Valeria; Villamil Giraldo, Ana M; Castello, Pablo R; Rossi, Juan P F C; González Flecha, F Luis

    2008-11-15

    Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.

  15. The release of lysosomal arylsulfatase from liver lysosomes exposed to 2-chloroethylethyl sulfide.

    PubMed

    Shin, S; Choi, D S; Kim, Y B; Cha, S H; Sok, D E

    1995-08-18

    Treatment of a lysosome-rich fraction from liver with 2-chloroethylethyl sulfide resulted in a dose-dependent release of arylsulfatase. The inclusion of Ca2+ enhanced the enzyme release by approximately 2.3-fold. The enhancing effect of Ca2+, showing an EC50 value of 30 mM, was mimicked by neither Mg2+ nor Mn2+. Studies on a structural requirement and a time-dependent release suggest that the Ca(2+)-dependent release proceeds via a specific process involving the alkylation of lysosomal membranes by 2-chloroethylethyl sulfide. Furthermore, the Ca(2+)-dependent process was prevented partially by either leupeptin or gentamycin, but neither pepstatin nor PMSF, implying that the enzyme release may be partially mediated by lysosomal cysteine-protease or phospholipase. Meanwhile, the Ca(2+)-independent release seems to be expressed non-specifically by various compounds.

  16. Impact of Lysosome Status on Extracellular Vesicle Content and Release

    PubMed Central

    Eitan, Erez; Suire, Caitlin; Zhang, Shi; Mattson, Mark P.

    2016-01-01

    Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells. PMID:27238186

  17. Subcellular Trafficking of Mammalian Lysosomal Proteins: An Extended View

    PubMed Central

    Staudt, Catherine; Puissant, Emeline; Boonen, Marielle

    2016-01-01

    Lysosomes clear macromolecules, maintain nutrient and cholesterol homeostasis, participate in tissue repair, and in many other cellular functions. To assume these tasks, lysosomes rely on their large arsenal of acid hydrolases, transmembrane proteins and membrane-associated proteins. It is therefore imperative that, post-synthesis, these proteins are specifically recognized as lysosomal components and are correctly sorted to this organelle through the endosomes. Lysosomal transmembrane proteins contain consensus motifs in their cytosolic regions (tyrosine- or dileucine-based) that serve as sorting signals to the endosomes, whereas most lysosomal acid hydrolases acquire mannose 6-phosphate (Man-6-P) moieties that mediate binding to two membrane receptors with endosomal sorting motifs in their cytosolic tails. These tyrosine- and dileucine-based motifs are tickets for boarding in clathrin-coated carriers that transport their cargo from the trans-Golgi network and plasma membrane to the endosomes. However, increasing evidence points to additional mechanisms participating in the biogenesis of lysosomes. In some cell types, for example, there are alternatives to the Man-6-P receptors for the transport of some acid hydrolases. In addition, several “non-consensus” sorting motifs have been identified, and atypical transport routes to endolysosomes have been brought to light. These “unconventional” or “less known” transport mechanisms are the focus of this review. PMID:28036022

  18. Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes.

    PubMed

    Tanaka, Yoshinori; Suzuki, Genjiro; Matsuwaki, Takashi; Hosokawa, Masato; Serrano, Geidy; Beach, Thomas G; Yamanouchi, Keitaro; Hasegawa, Masato; Nishihara, Masugi

    2017-01-10

    Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases.

  19. Graphene Oxide Membranes with Strong Stability in Aqueous Solutions and Controllable Lamellar Spacing.

    PubMed

    Xi, Yue-Heng; Hu, Jia-Qi; Liu, Zhuang; Xie, Rui; Ju, Xiao-Jie; Wang, Wei; Chu, Liang-Yin

    2016-06-22

    Graphene oxide (GO) membranes become emerging efficient filters for molecular or ionic separation due to their well-defined two-dimensional nanochannels formed by closely spaced GO sheets and tunable physicochemical properties. The stability of GO membranes in aqueous solutions is a prerequisite for their applications. Here we show a novel and easy strategy for fabricating GO membranes with strong stability in aqueous solutions and controllable lamellar spacing by simply doping with partially reduced graphene oxide (prGO) sheets. With our prGO-doping strategy, the interlayer stabilizing force in GO membranes is enhanced due to the weakened repulsive hydration and enhanced π-π attraction between GO sheets; as a result, the fabricated GO membranes are featured with controllable lamellar spacing and extraordinary stability in water or even strong acid and base solutions as well as strong mechanical properties, which will expand the application scope of GO membranes and provide ever better performances in their applications with aqueous solution environments.

  20. Hydrophobic asymmetric ultrafiltration PVDF membranes: an alternative separator for VFB with excellent stability.

    PubMed

    Wei, Wenping; Zhang, Huamin; Li, Xianfeng; Zhang, Hongzhang; Li, Yun; Vankelecom, Ivo

    2013-02-14

    Polyvinylidene fluoride (PVDF) ultrafiltration membranes were investigated for the first time in vanadium redox flow battery (VFB) applications. Surprisingly, PVDF ultrafiltration membranes with hydrophobic pore walls and relatively large pore sizes of several tens of nanometers proved able to separate vanadium ions and protons efficiently, thus being suitable as a VFB separator. The ion selectivity of this new type of VFB membrane could be tuned readily by controlling the membrane morphology via changes in the composition of the membrane casting solution, and the casting thickness. The results showed that the PVDF membranes offered good performances and excellent stability in VFB applications, where it could, performance-wise, truly substitute Nafion in VFB applications, but at a much lower cost.

  1. Mechanical and thermal stability of adhesive membranes with nonzero bending rigidity

    NASA Astrophysics Data System (ADS)

    Tallinen, Tuomas; Astrom, Jan; Kekalainen, Pekka; Timonen, Jussi

    2011-03-01

    Membranes at a microscopic scale are affected by thermal fluctuations and self-adhesion due to Van der Waals forces. Methods to prepare membranes of even molecular scale, e.g. graphene, have been recently developed, and the question of their mechanical and thermal stability is of crucial importance. To this end we modeled microscopic membranes with a short-range attractive interaction and applied Langevin dynamics. Their behavior was also analyzed under external loading. Even though these membranes folded during isotropic compression as a result of energy minimization, the process at high confinement did not differ much from crumpling of macroscopic thin sheets. The main difference appeared when the external load was released. In such cases, for membranes of sufficiently large size L , folded or scrolled conformations emerged. At high enough temperature T entropic effects made such conformations unfavorable, however. Possible conformations of free-standing membranes (``phase diagrams'') were determined in the TL -plane.

  2. The relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells.

    PubMed

    Meng, Su-Fang; Mao, Wei-Ping; Wang, Fang; Liu, Xiao-Qian; Shao, Luan-Luan

    2015-11-01

    This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL-68). The expression of LC3B-II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd-exposed WRL-68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL-68 cells. The lysosomal activation was markedly decreased when the cells were co-treated with 3-MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome-lysosome fusion. The colocalization of lysosome-associated membrane protein-2 (LAMP2) and GFP-LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome-lysosome fusion). We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome-lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL-68 cells. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca(2+) stores and the intracellular Ca(2+) channels or pumps were possibly pH-dependent.

  3. Erythrocyte membrane stability to hydrogen peroxide is decreased in Alzheimer disease.

    PubMed

    Gilca, Marilena; Lixandru, Daniela; Gaman, Laura; Vîrgolici, Bogdana; Atanasiu, Valeriu; Stoian, Irina

    2014-01-01

    The brain and erythrocytes have similar susceptibility toward free radicals. Therefore, erythrocyte abnormalities might indicate the progression of the oxidative damage in Alzheimer disease (AD). The aim of this study was to investigate erythrocyte membrane stability and plasma antioxidant status in AD. Fasting blood samples (from 17 patients with AD and 14 healthy controls) were obtained and erythrocyte membrane stability against hydrogen peroxide and 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), serum Trolox equivalent antioxidant capacity (TEAC), residual antioxidant activity or gap (GAP), erythrocyte catalase activity (CAT), erythrocyte superoxide dismutase (SOD) activity, erythrocyte nonproteic thiols, and total plasma thiols were determined. A significant decrease in erythrocyte membrane stability to hydrogen peroxide was found in AD patients when compared with controls (P<0.05). On the contrary, CAT activity (P<0.0001) and total plasma thiols (P<0.05) were increased in patients with AD compared with controls. Our results indicate that the most satisfactory measurement of the oxidative stress level in the blood of patients with AD is the erythrocyte membrane stability to hydrogen peroxide. Reduced erythrocyte membrane stability may be further evaluated as a potential peripheral marker for oxidative damage in AD.

  4. EMC1-dependent stabilization drives membrane penetration of a partially destabilized non-enveloped virus

    PubMed Central

    Bagchi, Parikshit; Inoue, Takamasa; Tsai, Billy

    2016-01-01

    Destabilization of a non-enveloped virus generates a membrane transport-competent viral particle. Here we probe polyomavirus SV40 endoplasmic reticulum (ER)-to-cytosol membrane transport, a decisive infection step where destabilization initiates this non-enveloped virus for membrane penetration. We find that a member of the ER membrane protein complex (EMC) called EMC1 promotes SV40 ER membrane transport and infection. Surprisingly, EMC1 does so by using its predicted transmembrane residue D961 to bind to and stabilize the membrane-embedded partially destabilized SV40, thereby preventing premature viral disassembly. EMC1-dependent stabilization enables SV40 to engage a cytosolic extraction complex that ejects the virus into the cytosol. Thus EMC1 acts as a molecular chaperone, bracing the destabilized SV40 in a transport-competent state. Our findings reveal the novel principle that coordinated destabilization-stabilization drives membrane transport of a non-enveloped virus. DOI: http://dx.doi.org/10.7554/eLife.21470.001 PMID:28012275

  5. Stability studies of immobilized lipase on rice husk and eggshell membrane

    NASA Astrophysics Data System (ADS)

    Abdulla, R.; Sanny, S. A.; Derman, E.

    2017-06-01

    Lipase immobilization for biodiesel production is gaining importance day by day. In this study, lipase from Burkholderia cepacia was immobilized on activated support materials namely rice husk and egg shell membrane. Both rice husk and eggshell membrane are natural wastes that holds a lot of potential as immobilization matrix. Rice husk and eggshell membrane were activated with glutaraldehyde. Lipase was immobilized on the glutaraldehyde-activated support material through adsorption. Immobilization efficiency together with enzyme activity was observed to choose the highest enzyme loading for further stability studies. Immobilization efficiency of lipase on rice husk was 81 as compared to an immobilization efficiency of 87 on eggshell membrane. Immobilized lipase on eggshell membrane exhibited higher enzyme activity as compared to immobilized lipase on rice husk. Eggshell membrane also reported higher stability than rice husk as immobilization matrix. Both types of immobilized lipase retatined its activity after ten cycles of reuse. In short, eggshell membrane showed to be a better immobilization platform for lipase as compared to rice husk. However, with further improvement in technique of immobilization, the stability of both types of immobilized lipase can be improved to a greater extent.

  6. The Unsaturation of Membrane Lipids Stabilizes Photosynthesis against Heat Stress.

    PubMed Central

    Gombos, Z.; Wada, H.; Hideg, E.; Murata, N.

    1994-01-01

    The effect of the unsaturation of glycerolipids of thylakoid membranes on the heat tolerance of the photosynthetic evolution of oxygen was studied in vivo by mutation and transformation of fatty-acid desaturases in the cyanobacterium Synechocystis PCC6803. The experimental results indicate that elimination of dienoic lipid molecules decreases, to a small but distinct extent, the heat tolerance of photosynthetic oxygen evolution, but that elimination of trienoic lipid molecules has no effect on the heat tolerance. This conclusion contrasts with the previous hypothesis that the heat tolerance of photosynthesis is enhanced upon an increase in the level of saturation of membrane lipids. It is also shown that light does not affect the nature of the effect of lipid unsaturation on the heat tolerance of photosynthesis. PMID:12232106

  7. Cholesterol expels ibuprofen from the hydrophobic membrane core and stabilizes lamellar phases in lipid membranes containing ibuprofen.

    PubMed

    Alsop, Richard J; Armstrong, Clare L; Maqbool, Amna; Toppozini, Laura; Dies, Hannah; Rheinstädter, Maikel C

    2015-06-28

    There is increasing evidence that common drugs, such as aspirin and ibuprofen, interact with lipid membranes. Ibuprofen is one of the most common over the counter drugs in the world, and is used for relief of pain and fever. It interacts with the cyclooxygenase pathway leading to inhibition of prostaglandin synthesis. From X-ray diffraction of highly oriented model membranes containing between 0 and 20 mol% ibuprofen, 20 mol% cholesterol, and dimyristoylphosphatidylcholine (DMPC), we present evidence for a non-specific interaction between ibuprofen and cholesterol in lipid bilayers. At a low ibuprofen concentrations of 2 mol%, three different populations of ibuprofen molecules were found: two in the lipid head group region and one in the hydrophobic membrane core. At higher ibuprofen concentrations of 10 and 20 mol%, the lamellar bilayer structure is disrupted and a lamellar to cubic phase transition was observed. In the presence of 20 mol% cholesterol, ibuprofen (at 5 mol%) was found to be expelled from the membrane core and reside solely in the head group region of the bilayers. 20 mol% cholesterol was found to stabilize lamellar membrane structure and the formation of a cubic phase at 10 and 20 mol% ibuprofen was suppressed. The results demonstrate that ibuprofen interacts with lipid membranes and that the interaction is strongly dependent on the presence of cholesterol.

  8. Vacuolar ATPase in Phagosome-Lysosome Fusion

    PubMed Central

    Kissing, Sandra; Hermsen, Christina; Repnik, Urska; Nesset, Cecilie Kåsi; von Bargen, Kristine; Griffiths, Gareth; Ichihara, Atsuhiro; Lee, Beth S.; Schwake, Michael; De Brabander, Jef; Haas, Albert; Saftig, Paul

    2015-01-01

    The vacuolar H+-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes. PMID:25903133

  9. Poloxamer-188 and citicoline provide neuronal membrane integrity and protect membrane stability in cortical spreading depression.

    PubMed

    Yıldırım, Timur; Eylen, Alpaslan; Lule, Sevda; Erdener, Sefik Evren; Vural, Atay; Karatas, Hulya; Ozveren, Mehmet Faik; Dalkara, Turgay; Gursoy-Ozdemir, Yasemin

    2015-01-01

    Under pathological conditions such as brain trauma, subarachnoid hemorrhage and stroke, cortical spreading depression (CSD) or peri-infarct depolarizations contribute to brain damage in animal models of neurological disorders as well as in human neurological diseases. CSD causes transient megachannel opening on the neuronal membrane, which may compromise neuronal survival under pathological conditions. Poloxamer-188 (P-188) and citicoline are neuroprotectants with membrane sealing properties. The aim of this study is to investigate the effect of P-188 and citicoline on the neuronal megachannel opening induced by CSD in the mouse brain. We have monitored megachannel opening with propidium iodide, a membrane impermeable fluorescent dye and, demonstrate that P-188 and citicoline strikingly decreased CSD-induced neuronal PI influx in cortex and hippocampal dentate gyrus. Therefore, these agents may be providing neuroprotection by blocking megachannel opening, which may be related to their membrane sealing action and warrant further investigation for treatment of traumatic brain injury and ischemic stroke.

  10. Thermal stability of lipid-depleted purple membranes at neutral and low pH values.

    PubMed

    Taneva, S G; Koynova, R; Tenchov, B

    1994-05-30

    Differential scanning calorimetry was used to compare the thermal behavior of native and delipidated purple membrane fragments at pH values corresponding to purple, blue and acid-purple forms. At neutral pH, delipidation results in a 2.5- to 3-times increase in the cooperativity of the denaturational transition, accompanied by a minor increase in its temperature. At pH values below 5 the delipidated membranes exhibit considerably higher thermal stability than the native membranes. The reversible predenaturational transition observed in the native state is not detectable upon delipidation. There is no strict correlation between color changes upon acidification and deionization of either native or delipidated purple membranes and their thermal stability.

  11. The biogenesis of lysosomes and lysosome-related organelles.

    PubMed

    Luzio, J Paul; Hackmann, Yvonne; Dieckmann, Nele M G; Griffiths, Gillian M

    2014-09-02

    Lysosomes were once considered the end point of endocytosis, simply used for macromolecule degradation. They are now recognized to be dynamic organelles, able to fuse with a variety of targets and to be re-formed after fusion events. They are also now known to be the site of nutrient sensing and signaling to the cell nucleus. In addition, lysosomes are secretory organelles, with specialized machinery for regulated secretion of proteins in some cell types. The biogenesis of lysosomes and lysosome-related organelles is discussed, taking into account their dynamic nature and multiple roles. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  12. Effect of L-carnitine and acetyl-L-carnitine on the human erythrocyte membrane stability and deformability.

    PubMed

    Arduini, A; Rossi, M; Mancinelli, G; Belfiglio, M; Scurti, R; Radatti, G; Shohet, S B

    1990-01-01

    In this study we examined the effect of carnitine and acetylcarnitine on the human erythrocyte membrane stability and membrane deformability. Since erythrocyte membranes are impermeable to these compounds, we resealed erythrocyte ghosts in the presence of different concentrations of carnitine or acetylcarnitine. Resealed ghosts can be adequately studied in their cellular deformability and membrane stability properties by means of ektacytometry. Both carnitine and acetylcarnitine alter the membrane stability but not membrane deformability of the red cell membrane. Resealed ghosts containing 20, 50, 150, and 300 microM carnitine had 1.1, 1.6, 0.9, and 0.7 times the normal stability. While resealed ghosts containing 20, 50, 150, and 300 microM acetylcarnitine had 1.1, 1.5, 1.3, and 1.2 times the normal stability. Such changes were found to be reversible. We also conducted SDS PAGE of cytoskeletal membrane proteins from membrane fragments and residual membranes produced during membrane stability analysis, and unsheared resealed membranes in those samples where we observed an increase or a decrease of membrane stability. No changes in the cytoskeletal membrane proteins were noticed, even when the samples, prior SDS PAGE analysis, were treated with or without dithiothreitol. In addition, fluorescence steady state anisotropy of DPH in the erythrocyte membrane treated with carnitine or acetylcarnitine shows no modification of the lipid order parameter. Our results would suggest that both carnitine and its acetyl-ester, at physiological concentrations, may increase membrane stability in mature erythrocytes, most likely via a specific interaction with one or more cytoskeletal proteins, and that this effect would manifest when the erythrocytes are subjected to high shear stress.

  13. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

    PubMed

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

  14. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

    PubMed Central

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function. PMID:26674091

  15. Lysosomal photodamage induces cell death via mitochondrial apoptotic pathway

    NASA Astrophysics Data System (ADS)

    Liu, Lei; Wang, Xian-wang; Li, Hui

    2009-11-01

    Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using real-time single-cell analysis. In this study, the fluorescence probes Cyto c-GFP and DsRed-Mit were used to detect the spatial and temporal changes of cytochrome c in real-time in sub-cell level; the Rhodamine 123 dyes were used to monitor the changes of mitochondrial membrane potential. The results showed that, after PDT treatment,the mitochondrial membrane potential decreased, and cytochrome c released from mitochondria; The caspase-3 was activated obviously. These results suggested that lysosomal photodamage activates mitochondrial apoptotic pathway to induce cell death.

  16. Cell wall polysaccharide biosynthesis by membrane fragments from Streptococcus pyogenes and stabilized L-form.

    PubMed

    Cohen, M; Panos, C

    1971-05-01

    The formation and composition of a cell wall rhamnose-containing polysaccharide by membrane fragments from Streptococcus pyogenes and its stabilized L-form were compared. Also, the effect of prior treatment on the ability of coccal whole-cell and membrane fragments to incorporate radioactivity from thymidine diphosphate-(14)C-rhamnose, and the results of subsequent attempts to remove labeled polysaccharide from such membranes are given. L-form membrane fragments were capable of only 10% uptake of (14)C-rhamnose from this nucleotide as compared with streptococcal membranes. However, once bound, both membrane fragments polymerized rhamnose to the same extent. These findings tend to negate the almost complete lack of polymeric rhamnose within the intact L-form as being due to the absence of membrane enzymes necessary for the transfer of rhamnose from a suitable precursor to membrane acceptor sites or enzymes responsible for rhamnose polymerization. Degradation of labeled rhamnose polysaccharide after isolation from coccal membranes by mild acid hydrolysis showed muramic acid and glucosamine to be attached. This same polysaccharide from L-form membrane fragments was devoid of amino sugars. These data suggest the possible involvement of amino sugars in the attachment of cell wall polymeric rhamnose to the streptococcal cytoplasmic membrane. The absence of attached amino sugars to rhamnose polysaccharide from L-form membrane fragments is discussed in terms of this organism's continued inability for new cell wall formation. The isolation, from streptococcal membrane fragments, of a polysaccharide containing rhamnose and amino sugars common to at least two different streptococcal cell wall-type polymers was demonstrated.

  17. Stability and Degradation Mechanisms of Radiation-Grafted Polymer Electrolyte Membranes for Water Electrolysis.

    PubMed

    Albert, Albert; Lochner, Tim; Schmidt, Thomas J; Gubler, L

    2016-06-22

    Radiation-grafted membranes are a promising alternative to commercial membranes for water electrolyzers, since they exhibit lower hydrogen crossover and area resistance, better mechanical properties, and are of potentially lower cost than perfluoroalkylsulfonic acid membranes, such as Nafion. Stability is an important factor in view of the expected lifetime of 40 000 h or more of an electrolyzer. In this study, combinations of styrene (St), α-methylstyrene (AMS), acrylonitrile (AN), and 1,3-diisopropenylbenzene (DiPB) are cografted into 50 μm preirradiated poly(ethylene-co-tetrafluoroethylene) (ETFE) base film, followed by sulfonation to produce radiation-grafted membranes. The stability of the membranes with different monomer combinations is compared under an accelerated stress test (AST), and the degradation mechanisms are investigated. To mimic the conditions in an electrolyzer, in which the membrane is always in contact with liquid water at elevated temperature, the membranes are immersed in water for 5 days at 90 °C, so-called thermal stress test (TST). In addition to testing in air atmosphere tests are also carried out under argon to investigate the effect of the absence of oxygen. The water is analyzed with UV-vis spectroscopy and ion chromatography. The ion exchange capacity (IEC), swelling degree, and Fourier transform infrared (FTIR) spectra of the membranes are compared before and after the test. Furthermore, energy-dispersive X-ray (EDX) spectroscopic analysis of the membrane cross-section is performed. Finally, the influence of the TST to the membrane area resistance and hydrogen crossover is measured. The stability increases along the sequence St/AN, St/AN/DiPB, AMS/AN, and AMS/AN/DiPB grafted membrane. The degradation at the weak-link, oxygen-induced degradation, and hydrothermal degradation are proposed in addition to the "swelling-induced detachment" reported in the literature. By mitigating the possible paths of degradation, the AMS

  18. Preparation and characterization of titania-deposited silica composite hollow fiber membranes with high hydrothermal stability.

    PubMed

    Kwon, Young-Nam; Kim, In-Chul

    2013-11-01

    Hydrothermal stability of a porous nickel-supported silica membrane was successfully improved by deposition of titania multilayers on colloidal silica particles embedded in the porous nickel fiber support. Porous nickel-supported silica membranes were prepared by means of a dipping-freezing-fast drying (DFF) method. The titania layers were deposited on colloidal silica particles by repeating hydrolysis and condensation reactions of titanium isopropoxide on the silica particle surfaces. The deposition of thin titania layers on the nickel-supported silica membrane was verified by various analytical tools. The water flux and the solute rejection of the porous Ni fiber-supported silica membranes did not change after titania layer deposition, indicating that thickness of titania layers deposited on silica surface is enough thin not to affect the membrane performance. Moreover, improvement of the hydrothermal stability in the titania-deposited silica membranes was confirmed by stability tests, indicating that thin titania layers deposited on silica surface played an important role as a diffusion barrier against 90 degrees C water into silica particles.

  19. Prostaglandins, Lysosomes, and Radiation Injury

    DTIC Science & Technology

    1980-01-01

    SCIENTIFIC REPORT Prostaglandins, lysosomes, and radiation injury 7 P. J. Trocha <G. N. Catravas DTI.C A DEFENSE NUCLEAR AGENCY -LL ARMED FORCES...been shown to be altered with tissue injury . Yet no studies have been performed to monitor lysosomal enzyme activi- ties and PG levels simultaneously...nd R. Pi olm. Raven Pra& New Yort- D 980. Prostaglandins, Lysosomes, and Radiation Injury Paul J. Trocha and George N. Catravas Armed Forces

  20. The spectrin-based membrane skeleton stabilizes mouse megakaryocyte membrane systems and is essential for proplatelet and platelet formation

    PubMed Central

    Patel-Hett, Sunita; Wang, Hongbei; Begonja, Antonija J.; Thon, Jonathan N.; Alden, Eva C.; Wandersee, Nancy J.; An, Xiuli; Mohandas, Narla; Hartwig, John H.

    2011-01-01

    Megakaryocytes generate platelets by remodeling their cytoplasm first into proplatelets and then into preplatelets, which undergo fission to generate platelets. Although the functions of microtubules and actin during platelet biogenesis have been defined, the role of the spectrin cytoskeleton is unknown. We investigated the function of the spectrin-based membrane skeleton in proplatelet and platelet production in murine megakaryocytes. Electron microscopy revealed that, like circulating platelets, proplatelets have a dense membrane skeleton, the main fibrous component of which is spectrin. Unlike other cells, megakaryocytes and their progeny express both erythroid and nonerythroid spectrins. Assembly of spectrin into tetramers is required for invaginated membrane system maturation and proplatelet extension, because expression of a spectrin tetramer–disrupting construct in megakaryocytes inhibits both processes. Incorporation of this spectrin-disrupting fragment into a novel permeabilized proplatelet system rapidly destabilizes proplatelets, causing blebbing and swelling. Spectrin tetramers also stabilize the “barbell shapes” of the penultimate stage in platelet production, because addition of the tetramer-disrupting construct converts these barbell shapes to spheres, demonstrating that membrane skeletal continuity maintains the elongated, pre-fission shape. The results of this study provide evidence for a role for spectrin in different steps of megakaryocyte development through its participation in the formation of invaginated membranes and in the maintenance of proplatelet structure. PMID:21566095

  1. Deficiency of skeletal membrane protein band 4.1 in homozygous hereditary elliptocytosis. Implications for erythrocyte membrane stability.

    PubMed Central

    Tchernia, G; Mohandas, N; Shohet, S B

    1981-01-01

    Erythrocytes from three patients with severe hemolytic anemia, marked erythrocyte fragmentation, and elliptocytic poikilocytosis, were studied in terms of both their membrane protein composition and their mechanical characteristics. Erythrocytes from the patients' parents and one minimally affected and one normal sibling were also studied. Morphologic observations implied that the severely affected patients suffered from homozygous hereditary elliptocytosis because erythrocytes of both parents and the one minimally affected sibling showed moderate elliptocytosis on smear, whereas those of an unaffected sibling had normal morphology. The parallel findings of markedly reduced levels of band 4.1 in the erythrocyte membrane proteins of the patients and an intermediate reduction in the cells of the parents and the putative heterozygous sibling, suggest that the elliptocytic shape of the cells was related to the reduced levels of band 4.1. Additional studies showed marked abnormalities in cellular deformability and membrane fragility in the erythrocytes from the homozygous patients. Importantly, these changes were also closely proportional to the reduced levels of band 4.1, suggesting a central role for this protein in the maintenance of normal membrane stability and normal cell shape. It seems likely that this role for band 4.1 is intimately related to its known biochemical connection to the "membrane skeleton" through its linkage with spectrin and actin. Images PMID:6894932

  2. What lysosomes actually tell us about Parkinson's disease?

    PubMed

    Bourdenx, Mathieu; Dehay, Benjamin

    2016-12-01

    Parkinson's disease is a common neurodegenerative disorder of unknown origin mainly characterized by the loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta and the presence of intraneuronal proteinaceous inclusions called Lewy bodies. Lysosomes are dynamic organelles that degrade, in a controlled manner, cellular components delivered via the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Increasing amounts of evidence suggest a central role of lysosomal impairment in PD aetiology. This review provides an update on how genetic evidence support this connection and highlights how the neuropathologic and mechanistic evidence might relate to the disease process in sporadic forms of Parkinson's disease. Finally, we discuss the influence of ageing on lysosomal impairment and PD aetiology and therapeutic strategies targeting lysosomal function.

  3. Structural and mechanical heterogeneity of the erythrocyte membrane reveals hallmarks of membrane stability.

    PubMed

    Picas, Laura; Rico, Félix; Deforet, Maxime; Scheuring, Simon

    2013-02-26

    The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosphorylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

  4. Mechanical and Thermal Stability of Adhesive Membranes with Nonzero Bending Rigidity

    NASA Astrophysics Data System (ADS)

    Tallinen, T.; Åström, J. A.; Kekäläinen, P.; Timonen, J.

    2010-07-01

    Membranes at a microscopic scale are affected by thermal fluctuations and self-adhesion due to van der Waals forces. Methods to prepare membranes of even molecular scale, e.g., graphene, have recently been developed, and the question of their mechanical and thermal stability is of crucial importance. To this end we modeled microscopic membranes with an attractive interaction and applied Langevin dynamics. Their behavior was also analyzed under external loading. Even though these membranes folded during isotropic compression as a result of energy minimization, the process at high confinement was similar to crumpling of macroscopic nonadhesive sheets. The main difference appeared when the compression was released. In such cases, for membranes of sufficiently large size, folded or scrolled conformations emerged. At high temperature entropic effects made such conformations unfavorable, however.

  5. Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability.

    PubMed

    Chen, Guan-Jie; Kuo, Chia-Hung; Chen, Chih-I; Yu, Chung-Cheng; Shieh, Chwen-Jen; Liu, Yung-Chuan

    2012-02-01

    In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    SciTech Connect

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-02-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts.

  7. Kinetics of lysosomal storage of indigestible matter.

    PubMed Central

    Hurley, J; Alward, J

    1975-01-01

    In lysosomal storage diseases and in accumulation of lipofusion in the lysosomes there is a gradual eroding of the lysosomal system due to overloading the lysosomes by molecules which cannot be digested or expelled. The kinetics of this accumulation is examined for tissue cultures in terms of the cell growth rate, lysosomal production rate, and of generation of the indigestible element. PMID:1125388

  8. Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling and freezing.

    PubMed

    De Leeuw, F E; De Leeuw, A M; Den Daas, J H; Colenbrander, B; Verkleij, A J

    1993-02-01

    In this study attempts were made to improve the survival rates of bull spermatozoa after freezing/thawing and to clarify the importance of certain agents to the cryopreservation of spermatozoa. For that purpose the standard freezing extender was modified by the addition of different concentrations of various cryoprotectants and membrane-stabilizing agents: glycerol, 1,2-propanediol, polyvinylpyrrolidone, sucrose, egg yolk, lipid vesicles, and bovine serum albumin (BSA). Sperm membrane impermeability toward H33258 was employed as the parameter for sperm integrity during cooling and after freezing/thawing. Exclusion of glycerol from the extender did not significantly affect sperm integrity. Replacing 6% glycerol by 6% 1,2-propanediol resulted in reduced sperm survival, whereas replacement of glycerol by 62.5 mM sucrose slightly improved survival rates. Addition of 5 or 10% polyvinylpyrrolidone (either or not in combination with 0.5 M sucrose) significantly reduced sperm integrity. Excluding egg yolk from the extender caused a serious decrease of sperm survival after both cooling and freezing. The cryoprotection offered by egg yolk could not be mimicked by dioleoylphosphatidylcholine (DOPC) vesicles or DOPC/phosphatidic acid/cholesterol vesicles in concentrations up to 29 or 9 mM, respectively. However, the freezing extender containing 6.5 mM DOPC vesicles in combination with 6% BSA yielded results which did not significantly differ from those obtained with the standard extender; higher vesicle concentrations combined with BSA might produce even better results. Further research on the cryopreservation of bovine spermatozoa should focus on membrane stabilization since the membrane-stabilizing compounds yield more promising results than the ice-preventing agents.

  9. TFEB-mediated increase in peripheral lysosomes regulates store-operated calcium entry

    PubMed Central

    Sbano, Luigi; Bonora, Massimo; Marchi, Saverio; Baldassari, Federica; Medina, Diego L.; Ballabio, Andrea; Giorgi, Carlotta; Pinton, Paolo

    2017-01-01

    Lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca2+) signaling. Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. Given these observations, we investigated the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis. PMID:28084445

  10. BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

    PubMed Central

    Zhong, Xi Zoë; Sun, Xue; Cao, Qi; Dong, Gaofeng; Schiffmann, Raphael; Dong, Xian-Ping

    2016-01-01

    Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs. PMID:27670435

  11. Extracellular lysosome-associated membrane protein-1 (LAMP-1) mediates autoimmune disease progression in the NOD model of type 1 diabetes.

    PubMed

    De Carvalho Bittencourt, Marcelo; Herren, Suzanne; Graber, Pierre; Vilbois, Francis; Pasquali, Christian; Berney, Claude; Plitz, Thomas; Nicoletti, Ferdinando; Kosco-Vilbois, Marie H

    2005-05-01

    Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4(+)CD25(+)) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-gamma-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-gamma-driven responses.

  12. Lysosomal adaptation: how the lysosome responds to external cues.

    PubMed

    Settembre, Carmine; Ballabio, Andrea

    2014-05-05

    Recent evidence indicates that the importance of the lysosome in cell metabolism and organism physiology goes far beyond the simple disposal of cellular garbage. This dynamic organelle is situated at the crossroad of the most important cellular pathways and is involved in sensing, signaling, and transcriptional mechanisms that respond to environmental cues, such as nutrients. Two main mediators of these lysosomal adaptation mechanisms are the mTORC1 kinase complex and the transcription factor EB (TFEB). These two factors are linked in a lysosome-to-nucleus signaling pathway that provides the lysosome with the ability to adapt to extracellular cues and control its own biogenesis. Modulation of lysosomal function by acting on TFEB has a profound impact on cellular clearance and energy metabolism and is a promising therapeutic target for a large variety of disease conditions. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

  13. Membrane nanotubes induced by aqueous phase separation and stabilized by spontaneous curvature

    PubMed Central

    Li, Yanhong; Lipowsky, Reinhard; Dimova, Rumiana

    2011-01-01

    Tubular membrane structures are widespread in eukaryotic cells, but the mechanisms underlying their formation and stability are not well understood. Previous work has focused on tube extrusion from cells and model membranes under the application of external forces. Here, we present novel membrane/polymer systems, where stable tubes form in the absence of externally applied forces. Solutions of two water-soluble polymers, polyethylene glycol and dextran, were encapsulated in giant lipid vesicles, cell-size model systems. Hypertonic deflation induced phase separation of the enclosed solution. The excess membrane area created during the deflation process was stored in a large number of membrane nanotubes inside the vesicle. The tubes had a diameter below optical resolution and became visible only when fluorescently labeled. The tubes were rather stable: In the absence of external forces, they existed for several days. A theoretical analysis of the shapes of the deflated vesicles reveals that these shapes would be unstable if the membranes had no spontaneous curvature. Using the large separation of length scales between the tube diameter and the overall size of the vesicles, the spontaneous curvature can be calculated and is found to be about -1/(240 nm) for a certain range of polymer concentrations. The nanotubes could also be retracted back into the mother vesicle by increasing the membrane tension via micropipette aspiration of the vesicle. Membrane tubes, which can form and be retracted easily, should be relevant for lipid storage in cells. PMID:21383120

  14. Documentation of an Imperative To Improve Methods for Predicting Membrane Protein Stability.

    PubMed

    Kroncke, Brett M; Duran, Amanda M; Mendenhall, Jeffrey L; Meiler, Jens; Blume, Jeffrey D; Sanders, Charles R

    2016-09-13

    There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of <0.4 for membrane proteins. Rosetta and PROVEAN showed a modest ability to classify mutations as destabilizing (ΔΔG < -0.5 kcal/mol), with a 7 in 10 chance of correctly discriminating a randomly chosen destabilizing variant from a randomly chosen stabilizing variant. However, even this performance is significantly worse than for soluble proteins. This study highlights the need for further development of reliable and reproducible methods for predicting thermodynamic folding stability in membrane proteins.

  15. Early stabilization of traumatic aortic transection and mitral valve regurgitation with extracorporeal membrane oxygenation.

    PubMed

    Lambrechts, David L; Wellens, Francis; Vercoutere, Rik A; De Geest, Raf

    2003-01-01

    We report a case of life-threatening aortic transection with concomitant mitral papillary muscle rupture and severe lung contusion caused by a failed parachute jump. This blunt thoracic injury was treated by early stabilization with extracorporeal membrane oxygenation followed by successful delayed graft repair of the descending aorta and mitral valve replacement with a mechanical prosthesis.

  16. Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures

    PubMed Central

    Sonoda, Yo; Newstead, Simon; Hu, Nien-Jen; Alguel, Yilmaz; Nji, Emmanuel; Beis, Konstantinos; Yashiro, Shoko; Lee, Chiara; Leung, James; Cameron, Alexander D.; Byrne, Bernadette; Iwata, So; Drew, David

    2011-01-01

    Summary Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures. PMID:21220112

  17. Genetics Home Reference: lysosomal acid lipase deficiency

    MedlinePlus

    ... Home Health Conditions lysosomal acid lipase deficiency lysosomal acid lipase deficiency Printable PDF Open All Close All ... to view the expand/collapse boxes. Description Lysosomal acid lipase deficiency is an inherited condition characterized by ...

  18. Methane to syngas conversion. Part I. Equilibrium conditions and stability requirements of membrane materials

    NASA Astrophysics Data System (ADS)

    Frade, J. R.; Kharton, V. V.; Yaremchenko, A.; Naumovich, E.

    Thermodynamic data have been used to predict the dependence of methane conversion on temperature and oxygen partial pressure in mixed conducting membrane reactors, and the corresponding fractions of water vapor, H 2, CO and CO 2. The relations between methane conversion, gas composition and oxygen partial pressure were also used to formulate the oxygen balance in mixed conducting membrane reactors, with tubular reactor and continuous stirred tank reactor (CSTR) configurations. A single dimensionless parameter accounts for the combined effects of geometric parameters of the membrane reactor, the permeability of the membrane material, and flow rate at the entry of the reactor. Selected examples were calculated to illustrate the effects of steam to methane and inert to methane ratios in the gas entering the reactor. The values of oxygen partial pressure required to attain the highest yield of CO and H 2 were also used to estimate the stability requirements to be met by mixed conducting membrane materials. Suitable membrane designs might be needed to bridge the difference between the conditions inside the reactors and the stability limits of known mixed conductors.

  19. Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

    PubMed Central

    Pascua-Maestro, Raquel

    2017-01-01

    Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular

  20. Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress.

    PubMed

    Pascua-Maestro, Raquel; Diez-Hermano, Sergio; Lillo, Concepción; Ganfornina, Maria D; Sanchez, Diego

    2017-02-01

    Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular

  1. Maturation of the Gag core decreases the stability of retroviral lipid membranes

    PubMed Central

    Davidoff, Candice; Payne, Riley; Willis, Sharon H.; Doranz, Benjamin J.; Rucker, Joseph B.

    2012-01-01

    To better understand how detergents disrupt enveloped viruses, we monitored the biophysical stability of murine leukemia virus (MLV) virus-like particles (VLPs) against a panel of commonly used detergents using real-time biosensor measurements. Although exposure to many detergents, such as Triton X-100 and Empigen, results in lysis of VLP membranes, VLPs appeared resistant to complete membrane lysis by a significant number of detergents, including Tween 20, Tween 80, Lubrol, and Saponin. VLPs maintained their structural integrity after exposure to Tween 20 at concentrations up to 500-fold above its CMC. Remarkably, VLPs containing immature cores composed of unprocessed (uncleaved) Gag polyprotein were significantly more resistant to detergent lysis than VLPs with mature cores. Although the maturity of retroviral Gag is known to influence the stability of the protein core structure itself, our studies suggest that the maturity of the Gag core also influences the stability of the lipid bilayer surrounding the core. PMID:22995186

  2. A Study of Lipid Bilayer Membrane Stability Using Precise Measurements of Specific Capacitance

    PubMed Central

    White, Stephen H.

    1970-01-01

    A method is described for measuring the specific capacitance (Cm) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10-2 M KCl show that Cm depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), Cm increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm2 with a time constant of ∼15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. Cm of final-state membranes depends upon applied voltage (Va) and obeys the equation Cm = C0 + βVa2 where Va ≃ VDC + VrmsAC. C0 and β depend upon temperature; C0 decreases linearly with temperature while β increases linearly. At 20°C, C0 = 0.559 ±0.01 (SD) μF/cm2 and β = 0.0123 ±0.0036 (SD) (μF/cm2)/(mv2) and at 34°C, C0 = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in Cm are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves. ImagesFigure 2 PMID:5489777

  3. A time course of orchestrated endophilin action in sensing, bending, and stabilizing curved membranes

    PubMed Central

    Poudel, Kumud R.; Dong, Yongming; Yu, Hang; Su, Allen; Ho, Thuong; Liu, Yan; Schulten, Klaus; Bai, Jihong

    2016-01-01

    Numerous proteins act in concert to sculpt membrane compartments for cell signaling and metabolism. These proteins may act as curvature sensors, membrane benders, and scaffolding molecules. Here we show that endophilin, a critical protein for rapid endocytosis, quickly transforms from a curvature sensor into an active bender upon membrane association. We find that local membrane deformation does not occur until endophilin inserts its amphipathic helices into lipid bilayers, supporting an active bending mechanism through wedging. Our time-course studies show that endophilin continues to drive membrane changes on a seconds-to-minutes time scale, indicating that the duration of endocytosis events constrains the mode of endophilin action. Finally, we find a requirement of coordinated activities between wedging and scaffolding for endophilin to produce stable membrane tubules in vitro and to promote synaptic activity in vivo. Together these data demonstrate that endophilin is a multifaceted molecule that precisely integrates activities of sensing, bending, and stabilizing curvature to sculpt membranes with speed. PMID:27170174

  4. High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

    NASA Astrophysics Data System (ADS)

    Miyagi, Atsushi; Chipot, Christophe; Rangl, Martina; Scheuring, Simon

    2016-09-01

    Annexins are abundant cytoplasmic proteins that can bind to negatively charged phospholipids in a Ca2+-dependent manner, and are known to play a role in the storage of Ca2+ and membrane healing. Little is known, however, about the dynamic processes of protein-Ca2+-membrane assembly and disassembly. Here we show that high-speed atomic force microscopy (HS-AFM) can be used to repeatedly induce and disrupt annexin assemblies and study their structure, dynamics and interactions. Our HS-AFM set-up is adapted for such biological applications through the integration of a pumping system for buffer exchange and a pulsed laser system for uncaging caged compounds. We find that biochemically identical annexins (annexin V) display different effective Ca2+ and membrane affinities depending on the assembly location, providing a wide Ca2+ buffering regime while maintaining membrane stabilization. We also show that annexin is membrane-recruited and forms stable supramolecular assemblies within ˜5 s in conditions that are comparable to a membrane lesion in a cell. Molecular dynamics simulations provide atomic detail of the role played by Ca2+ in the reversible binding of annexin to the membrane surface.

  5. Can Stabilization and Inhibition of Aquaporins Contribute to Future Development of Biomimetic Membranes?

    PubMed Central

    To, Janet; Torres, Jaume

    2015-01-01

    In recent years, the use of biomimetic membranes that incorporate membrane proteins, i.e., biomimetic-hybrid membranes, has increased almost exponentially. Key membrane proteins in these systems have been aquaporins, which selectively permeabilize cellular membranes to water. Aquaporins may be incorporated into synthetic lipid bilayers or to more stable structures made of block copolymers or solid-state nanopores. However, translocation of aquaporins to these alien environments has adverse consequences in terms of performance and stability. Aquaporins incorporated in biomimetic membranes for use in water purification and desalination should also withstand the harsh environment that may prevail in these conditions, such as high pressure, and presence of salt or other chemicals. In this respect, modified aquaporins that can be adapted to these new environments should be developed. Another challenge is that biomimetic membranes that incorporate high densities of aquaporin should be defect-free, and this can only be efficiently ascertained with the availability of completely inactive mutants that behave otherwise like the wild type aquaporin, or with effective non-toxic water channel inhibitors that are so far inexistent. In this review, we describe approaches that can potentially be used to overcome these challenges. PMID:26266425

  6. A time course of orchestrated endophilin action in sensing, bending, and stabilizing curved membranes.

    PubMed

    Poudel, Kumud R; Dong, Yongming; Yu, Hang; Su, Allen; Ho, Thuong; Liu, Yan; Schulten, Klaus; Bai, Jihong

    2016-07-01

    Numerous proteins act in concert to sculpt membrane compartments for cell signaling and metabolism. These proteins may act as curvature sensors, membrane benders, and scaffolding molecules. Here we show that endophilin, a critical protein for rapid endocytosis, quickly transforms from a curvature sensor into an active bender upon membrane association. We find that local membrane deformation does not occur until endophilin inserts its amphipathic helices into lipid bilayers, supporting an active bending mechanism through wedging. Our time-course studies show that endophilin continues to drive membrane changes on a seconds-to-minutes time scale, indicating that the duration of endocytosis events constrains the mode of endophilin action. Finally, we find a requirement of coordinated activities between wedging and scaffolding for endophilin to produce stable membrane tubules in vitro and to promote synaptic activity in vivo. Together these data demonstrate that endophilin is a multifaceted molecule that precisely integrates activities of sensing, bending, and stabilizing curvature to sculpt membranes with speed. © 2016 Poudel et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  7. Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

    PubMed

    Kelly, B M; Yu, C Z; Chang, P L

    1989-02-01

    Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.

  8. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

    PubMed Central

    Tomczak, Melanie M; Hincha, Dirk K; Estrada, Sergio D; Wolkers, Willem F; Crowe, Lois M; Feeney, Robert E; Tablin, Fern; Crowe, John H

    2002-01-01

    Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants. PMID:11806929

  9. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes

    NASA Astrophysics Data System (ADS)

    Chen, Fan; Gerber, Sabina; Korkhov, Volodymyr M.; Mireku, Samantha; Bucher, Monika; Locher, Kaspar P.; Zenobi, Renato

    2015-03-01

    We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC2D2. We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

  10. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

    PubMed

    Tomczak, Melanie M; Hincha, Dirk K; Estrada, Sergio D; Wolkers, Willem F; Crowe, Lois M; Feeney, Robert E; Tablin, Fern; Crowe, John H

    2002-02-01

    Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants.

  11. Signals for the lysosome: a control center for cellular clearance and energy metabolism

    PubMed Central

    Settembre, Carmine; Fraldi, Alessandro; Medina, Diego L.

    2015-01-01

    Preface For a long time lysosomes were considered merely to be cellular “incinerators” involved in the degradation and recycling of cellular waste. However, there is now compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signaling and energy metabolism. Furthermore, the essential role of lysosomes in the autophagic pathway puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master gene, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy, has revealed how the lysosome adapts to environmental cues, such as starvation, and suggests novel therapeutic strategies for modulating lysosomal function in human disease. PMID:23609508

  12. MCOLN1 is a ROS sensor in lysosomes that regulates autophagy

    PubMed Central

    Zhang, Xiaoli; Cheng, Xiping; Yu, Lu; Yang, Junsheng; Calvo, Raul; Patnaik, Samarjit; Hu, Xin; Gao, Qiong; Yang, Meimei; Lawas, Maria; Delling, Markus; Marugan, Juan; Ferrer, Marc; Xu, Haoxing

    2016-01-01

    Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes ‘host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca2+-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell. PMID:27357649

  13. Ceramic membrane fouling during ultrafiltration of oil/water emulsions: roles played by stabilization surfactants of oil droplets.

    PubMed

    Lu, Dongwei; Zhang, Tao; Ma, Jun

    2015-04-07

    Oil/water (O/W) emulsion stabilized by surfactants is the part of oily wastewater that is most difficult to handle. Ceramic membrane ultrafiltration presently is an ideal process to treat O/W emulsions. However, little is known about the fouling mechanism of the ceramic membrane during O/W emulsion treatment. This paper investigated how stabilization surfactants of O/W emulsions influence the irreversible fouling of ceramic membranes during ultrafiltration. An unexpected phenomenon observed was that irreversible fouling was much less when the charge of the stabilization surfactant of O/W emulsions is opposite to the membrane. The less ceramic membrane fouling in this case was proposed to be due to a synergetic steric effect and demulsification effect which prevented the penetration of oil droplets into membrane pores and led to less pore blockage. This proposed mechanism was supported by cross section images of fouled and virgin ceramic membranes taken with scanning electron microscopy, regression results of classical fouling models, and analysis of organic components rejected by the membrane. Furthermore, this mechanism was also verified by the existence of a steric effect and demulsification effect. Our finding suggests that ceramic membrane oppositely charged to the stabilization surfactant should be applied in ultrafiltration of O/W emulsions to alleviate irreversible membrane fouling. It could be a useful rule for ceramic membrane ultrafiltration of oily wastewater.

  14. Cryoprotectin: a plant lipid-transfer protein homologue that stabilizes membranes during freezing.

    PubMed Central

    Hincha, Dirk K

    2002-01-01

    Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non-freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold-regulated proteins is not yet known. We summarize our knowledge of those cold-regulated proteins that are able to stabilize membranes during a freeze-thaw cycle. Special emphasis is placed on cryoprotectin, a lipid-transfer protein homologue that was isolated from cold-acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze-thaw damage. PMID:12171654

  15. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1*

    PubMed Central

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-01-01

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1. PMID:26797118

  16. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1.

    PubMed

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-03-18

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes.

    PubMed

    Krumova, Sashka Boychova; Laptenok, Sergey Petrovich; Kovács, László; Tóth, Tünde; van Hoek, Arie; Garab, Gyozo; van Amerongen, Herbert

    2010-09-01

    We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type-WT) occur at 4-7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.

  18. Influence of environmental conditions on the stability of oil in water emulsions containing droplets stabilized by lecithin-chitosan membranes.

    PubMed

    Ogawa, Satoshi; Decker, Eric A; McClements, D Julian

    2003-08-27

    Oil-in-water emulsions containing cationic droplets stabilized by lecithin-chitosan membranes were produced using a two-stage process. A primary emulsion containing anionic lecithin-coated droplets was prepared by homogenizing oil and emulsifier solution using a high-pressure valve homogenizer (5 wt % corn oil, 1 wt % lecithin, 100 mM acetic acid, pH 3.0). A secondary emulsion containing cationic lecithin-chitosan-coated droplets was formed by diluting the primary emulsion with an aqueous chitosan solution (1 wt % corn oil, 0.2 wt % lecithin, 100 mM acetic acid, and 0.036 wt % chitosan). The stabilities of the primary and secondary emulsions with the same oil concentration to thermal processing, freeze-thaw cycling, high calcium chloride concentrations, and lipid oxidation were determined. The results showed that the secondary emulsions had better stability to droplet aggregation during thermal processing (30-90 degrees C for 30 min), freeze-thaw cycling (-10 degrees C for 22 h/30 degrees C for 2 h), and high calcium chloride contents (stability to environmental stresses.

  19. Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia.

    PubMed

    Lu, Yunzhe; Hanada, Toshihiko; Fujiwara, Yuko; Nwankwo, Jennifer O; Wieschhaus, Adam J; Hartwig, John; Huang, Sha; Han, Jongyoon; Chishti, Athar H

    2016-07-07

    Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin-actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin's actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate-dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex. © 2016 by The American Society of Hematology.

  20. A New Method for Measuring Edge Tensions and Stability of Lipid Bilayers: Effect of Membrane Composition

    PubMed Central

    Portet, Thomas; Dimova, Rumiana

    2010-01-01

    We report a novel and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (eggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of DOPC with cholesterol and dioleoylphosphatidylethanolamine. Our data confirm previous results for eggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of dioleoylphosphatidylethanolamine is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. It is presumed that interlipid hydrogen bonding is the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on bilayers made of stearoyloleoylphosphatidylcholine, suggesting that cholesterol influences the membrane mechanical stability in a lipid-specific manner. PMID:21081074

  1. Gene disruption of dematin causes precipitous loss of erythrocyte membrane stability and severe hemolytic anemia

    PubMed Central

    Lu, Yunzhe; Hanada, Toshihiko; Fujiwara, Yuko; Nwankwo, Jennifer O.; Wieschhaus, Adam J.; Hartwig, John; Huang, Sha; Han, Jongyoon

    2016-01-01

    Dematin is a relatively low abundance actin binding and bundling protein associated with the spectrin–actin junctions of mature erythrocytes. Primary structure of dematin includes a loosely folded core domain and a compact headpiece domain that was originally identified in villin. Dematin’s actin binding properties are regulated by phosphorylation of its headpiece domain by cyclic adenosine monophosphate–dependent protein kinase. Here, we used a novel gene disruption strategy to generate the whole body dematin gene knockout mouse model (FLKO). FLKO mice, while born at a normal Mendelian ratio, developed severe anemia and exhibited profound aberrations of erythrocyte morphology and membrane stability. Having no apparent effect on primitive erythropoiesis, FLKO mice show significant enhancement of erythroblast enucleation during definitive erythropoiesis. Using membrane protein analysis, domain mapping, electron microscopy, and dynamic deformability measurements, we investigated the mechanism of membrane instability in FLKO erythrocytes. Although many membrane and cytoskeletal proteins remained at their normal levels, the major peripheral membrane proteins spectrin, adducin, and actin were greatly reduced in FLKO erythrocytes. Our results demonstrate that dematin plays a critical role in maintaining the fundamental properties of the membrane cytoskeleton complex. PMID:27073223

  2. β-Glucuronidase, a Regulator of Lyme Arthritis Severity, Modulates Lysosomal Trafficking and MMP-9 Secretion in Response to Inflammatory Stimuli.

    PubMed

    Bramwell, Kenneth K C; Mock, Kelton; Ma, Ying; Weis, John H; Teuscher, Cory; Weis, Janis J

    2015-08-15

    The lysosomal enzyme β-glucuronidase (Gusb) is a key regulator of Lyme-associated and K/B×N-induced arthritis severity. The luminal enzymes present in lysosomes provide essential catabolic functions for the homeostatic degradation of a variety of macromolecules. In addition to this essential catabolic function, lysosomes play important roles in the inflammatory response following infection. Secretory lysosomes and related vesicles can participate in the inflammatory response through fusion with the plasma membrane and release of bioactive contents into the extracellular milieu. In this study, we show that GUSB hypomorphism potentiates lysosomal exocytosis following inflammatory stimulation. This leads to elevated secretion of lysosomal contents, including glycosaminoglycans, lysosomal hydrolases, and matrix metalloproteinase 9, a known modulator of Lyme arthritis severity. This mechanistic insight led us to test the efficacy of rapamycin, a drug known to suppress lysosomal exocytosis. Both Lyme and K/B×N-associated arthritis were suppressed by this treatment concurrent with reduced lysosomal release.

  3. beta-glucuronidase, a regulator of Lyme arthritis severity, modulates lysosomal trafficking and MMP-9 secretion in response to inflammatory stimuli

    PubMed Central

    Bramwell, Kenneth K.C.; Mock, Kelton; Ma, Ying; Weis, John H.; Teuscher, Cory; Weis, Janis J.

    2015-01-01

    The lysosomal enzyme beta-glucuronidase (Gusb) is a key regulator of Lyme-associated and K/B×N-induced arthritis severity. The luminal enzymes present in lysosomes provide essential catabolic functions for the homeostatic degradation of a variety of macromolecules. In addition to this essential catabolic function, lysosomes play important roles in the inflammatory response following infection. Secretory lysosomes and related vesicles can participate in the inflammatory response through fusion with the plasma membrane and release of bioactive contents into the extracellular milieu. Here we show that GUSB hypomorphism potentiates lysosomal exocytosis following inflammatory stimulation. This leads to elevated secretion of lysosomal contents, including glycosaminoglycans, lysosomal hydrolases, and Matrix Metalloproteinase 9, a known modulator of Lyme arthritis severity. This mechanistic insight led us to test the efficacy of Rapamycin, a drug known to suppress lysosomal exocytosis. Both Lyme and K/B×N-associated arthritis were suppressed by this treatment concurrent with reduced lysosomal release. PMID:26170381

  4. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

    PubMed

    Bao, Huan; Dalal, Kush; Wang, Victor; Rouiller, Isabelle; Duong, Franck

    2013-08-01

    The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

  5. Neuropathic Lysosomal Storage Disorders

    PubMed Central

    Pastores, Gregory M.; Maegawa, Gustavo H.B.

    2014-01-01

    The lysosomal storage disorders (LSDs) are a clinically heterogeneous group of inborn errors of metabolism, associated with the accumulation of incompletely degraded macromolecules within several cellular sites. Affected individuals present with a broad range of clinical problems, including hepatosplenomegaly and skeletal dysplasia. Onset of symptoms may range from birth to adulthood. The majority are associated with neurological features, including developmental delay, behavioral/psychiatric disturbances, seizures, acroparesthesia, motor weakness, cerebrovascular ischemic events and extra-pyramidal signs. It should be noted that later-onset forms are often misdiagnosed as symptoms, which might include psychiatric manifestations, are slowly progressive and may precede other neurologic or systemic features. Inheritance is primarily autosomal recessive. For all subtypes, diagnosis can be confirmed using a combination of biochemical and/or molecular assays. In a few LSDs, treatment with either hematopoietic stem cell transplantation, enzyme replacement or substrate reduction therapy is available. Genetic counseling is important, so patients and their families can be informed of reproductive risks, disease prognosis and therapeutic options. Investigations of disease mechanisms are providing insights into potential therapeutic approaches. Symptomatic care, which remains the mainstay for most subtypes, can lead to significant improvement in quality of life. PMID:24176423

  6. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    PubMed Central

    Chae, Pil Seok; Rasmussen, Søren G. F.; Rana, Rohini; Gotfryd, Kamil; Chandra, Richa; Goren, Michael A.; Kruse, Andrew C.; Nurva, Shailika; Loland, Claus J.; Pierre, Yves; Drew, David; Popot, Jean-Luc; Picot, Daniel; Fox, Brian G.; Guan, Lan; Gether, Ulrik; Byrne, Bernadette; Kobilka, Brian; Gellman, Samuel H.

    2011-01-01

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces displayed by native IMPs. Many proteins remain difficult to study owing to a lack of suitable detergents. We introduce a class of amphiphiles, each of which is built around a central quaternary carbon atom derived from neopentyl glycol, with hydrophilic groups derived from maltose. Representatives of this maltose-neopentyl glycol (MNG) amphiphile family display favorable behavior relative to conventional detergents, as tested on multiple membrane protein systems, leading to enhanced structural stability and successful crystallization. MNG amphiphiles are promising tools for membrane protein science because of the ease with which they may be prepared and the facility with which their structures may be varied. PMID:21037590

  7. Stability Limit of Water by Metastable Vapor-Liquid Equilibrium with Nanoporous Silicon Membranes.

    PubMed

    Chen, I-Tzu; Sessoms, David A; Sherman, Zachary; Choi, Eugene; Vincent, Olivier; Stroock, Abraham D

    2016-06-16

    Liquid can sustain mechanical tension as its pressure drops below the vapor-liquid coexistence line and becomes less than zero, until it reaches the stability limit-the pressure at which cavitation inevitably occurs. For liquid water, its stability limit is still a subject of debate: the results obtained by researchers using a variety of techniques show discrepancies between the values of the stability limit and its temperature dependence as temperature approaches 0 °C. In this work, we present a study of the stability limit of water by the metastable vapor-liquid equilibrium (MVLE) method with nanoporous silicon membranes. We also report on an experimental system which enables tests of the temperature dependence of the stability limit with MVLE. The stability limit we found increases monotonically (larger tension) as temperature approaches 0 °C; this trend contradicts the centrifugal result of Briggs but agrees with the experiments by acoustic cavitation. This result confirms that a quasi-static method can reach stability values similar to that from the dynamic stretching technique, even close to 0 °C. Nevertheless, our results fall in the range of ∼ -20 to -30 MPa, a range that is consistent with the majority of experiments but is far less negative than the limit obtained in experiments involving quartz inclusions and that predicted for homogeneous nucleation.

  8. Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling.

    PubMed

    You, Changjiang; Marquez-Lago, Tatiana T; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K Christopher; Leier, André; Piehler, Jacob

    2016-12-01

    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.

  9. Expression, Stability, and Membrane Integration of Truncation Mutants of Bovine Rhodopsin

    NASA Astrophysics Data System (ADS)

    Heymann, Jurgen A. W.; Subramaniam, Sriram

    1997-05-01

    Premature termination of protein synthesis by nonsense mutations is at the molecular origin of a number of inherited disorders in the family of G protein-coupled seven-helix receptor proteins. To understand how such truncated polypeptides are processed by the cell, we have carried out COS-1 cell expression studies of mutants of bovine rhodopsin truncated at the first 1, 1.5, 2, 3, or 5 transmembrane segments (TMS) of the seven present in wild-type opsin. Our experiments show that successful completion of different stages in the cellular processing of the protein [membrane insertion, N-linked glycosylation, stability to proteolytic degradation, and transport from the endoplasmic reticulum (ER) membrane] requires progressively longer lengths of the polypeptide chain. Thus, none of the truncations affected the ability of the polypeptides to be integral membrane proteins. C-terminal truncations that generated polypeptides with fewer than two TMS resulted in misorientation and prevented glycosylation at the N terminus, whereas truncations that generated polypeptides with fewer than five TMS greatly destabilized the protein. However, all of the truncations prevented exit of the polypeptide from the ER. We conclude that during the biogenesis of rhodopsin, proper integration into the ER membrane occurs only after the synthesis of at least two TMS is completed. Synthesis of the next three TMS confers a gradual increase in stability, whereas the presence of more than five TMS is necessary for exit from the ER.

  10. Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22

    PubMed Central

    2016-01-01

    Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot–Marie–Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT. PMID:26102530

  11. Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling

    PubMed Central

    You, Changjiang; Marquez-Lago, Tatiana T.; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K. Christopher; Leier, André; Piehler, Jacob

    2016-01-01

    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane. PMID:27957535

  12. DNA as membrane-bound ligand-receptor pairs: duplex stability is tuned by intermembrane forces.

    PubMed

    Beales, Paul A; Vanderlick, T Kyle

    2009-02-18

    We use membrane-anchored DNA as model adhesion receptors between lipid vesicles. By studying the thermal stability of DNA duplex formation, which tethers the vesicles into superstructures, we show that the melting temperature of a 10-base DNA sequence is dependent on the lipid composition of the tethered vesicles. We propose a simple model that describes how the intermembrane interactions tilt the free energy landscape for DNA binding. From our model, we estimate the area per DNA in the binding sites between vesicles and also the total area of the adhesion plaques. We find that vesicles containing a small proportion of cationic lipid that are modified with membrane-anchored DNA can be reversibly tethered by specific DNA interactions and that the DNA also induces a small attraction between these membranes, which stabilizes the DNA duplex. By increasing the equilibrium intermembrane distance on binding, we show that intermembrane interactions become negligible for the binding thermodynamics of the DNA and hence the thermal stability of vesicle aggregates becomes independent of lipid composition at large enough intervesicle separations. We discuss the implications of our findings with regards to cell adhesion and fusion receptors, and the programmable self-assembly of nano-structured materials by DNA hybridization.

  13. Inhomogeneous stability of bacteriorhodopsin in purple membrane against photobleaching at high temperature.

    PubMed

    Yokoyama, Yasunori; Sonoyama, Masashi; Mitaku, Shigeki

    2004-02-15

    Heterogeneity in the state of bacteriorhodopsin in purple membrane was studied through temperature jump experiments carried out in darkness and under illumination with visible light. The thermal denaturation, the irreversible component of spectral change at high temperature, had two decay components, suggesting that bacteriorhodopsin in purple membrane has heterogeneous stability. The temperature dependence of kinetic parameters under illumination revealed that the fast-decay component gradually increased at above 60 degrees C, indicating that the proportion of unstable bacteriorhodopsin increased. Significant change in the visible circular dichroism (CD) spectra was observed in darkness in the same temperature range as the increase of the fast-decay component under illumination. Denaturation experiments for C-terminal-cleaved bacteriorhodopsin showed that the C-terminal segment had some effect on the structural stability of bacteriorhodopsin under illumination. Dynamic and static models of the inhomogeneous stability of bacteriorhodopsin in purple membrane are discussed on the basis of the results of the denaturation kinetics and the visible CD spectra. Copyright 2004 Wiley-Liss, Inc.

  14. Enhanced Stability of Laminated Graphene Oxide Membranes for Nanofiltration via Interstitial Amide Bonding.

    PubMed

    Nam, Yoon Tae; Choi, Junghoon; Kang, Kyoung Min; Kim, Dae Woo; Jung, Hee-Tae

    2016-10-03

    Laminated graphene oxide (GO) has promising use as a membrane because of its high permeance, chemical and mechanical stability, as well as the molecular sieving effect of its interlayers. However, the hydrophilic surface of GO, which is highly decorated with oxygen groups, easily induces delamination of stacked GO films in aqueous media, thereby limiting the practical application. To stabilize GO films in aqueous media, we functionalized a polymer support with branched polyethylene-imine (BPEI). BPEI adsorbed intercalated into the stacked GO sheets via diffusion during filtration. The GO/BPEI membrane obtained exhibits high stability during sonication (>1 h duration, 40 kHz frequency) in water within a broad pH range (2-12). In contrast, the GO film spontaneously delaminated upon sonication. Furthermore, BPEI treatment did not affect the filtration performance of the GO film, as evidenced by the high rejection rates (>90%) for the dye molecules methylene blue, rose bengal, and brilliant blue and by their permeation rates of ca. 124, 34.8, 12.2, and 5.1%, respectively, relative to those of a typical GO membrane.

  15. Influence of acute exercise on the osmotic stability of the human erythrocyte membrane.

    PubMed

    Paraiso, L F; de Freitas, M V; Gonçalves-E-Oliveira, A F M; de Almeida Neto, O P; Pereira, E A; Mascarenhas Netto, R C; Cunha, L M; Bernardino Neto, M; de Agostini, G G; Resende, E S; Penha-Silva, N

    2014-12-01

    This study evaluated the effects of 2 different types of acute aerobic exercise on the osmotic stability of human erythrocyte membrane and on different hematological and biochemical variables that are associated with this membrane property. The study population consisted of 20 healthy and active men. Participants performed single sessions of 2 types of exercise. The first session consisted of 60 min of moderate-intensity continuous exercise (MICE). The second session, executed a week later, consisted of high-intensity interval exercise (HIIE) until exhaustion. The osmotic stability of the erythrocyte membrane was represented by the inverse of the salt concentration (1/H50) at the midpoint of the sigmoidal curve of dependence between the absorbance of hemoglobin and the NaCl concentration. The values of 1/H50 changed from 2.29±0.1 to 2.33±0.09 after MICE and from 2.30±0.08 to 2.23±0.12 after HIIE. During MICE mean corpuscular volume increased, probably due to in vivo lysis of older erythrocytes, with preservation of cells that were larger and more resistant to in vitro lysis. The study showed that a single bout of acute exercise affected erythrocyte stability, which increased after MICE and decreased after HIIE.

  16. Template-particle stabilized bicontinuous emulsion yielding controlled assembly of hierarchical high-flux filtration membranes.

    PubMed

    Hess, Samuel C; Kohll, A Xavier; Raso, Renzo A; Schumacher, Christoph M; Grass, Robert N; Stark, Wendelin J

    2015-01-14

    A novel solvent-evaporation-based process that exploits template-particle stabilized bicontinuous emulsions for the formation of previously unreached membrane morphologies is reported in this article. Porous membranes have a wide range of applications spanning from water filtration, pharmaceutical purification, and battery separators to scaffolds for tissue engineering. Different situations require different membrane morphologies including various pore sizes and pore gradients. However, most of the previously reported membrane preparation procedures are restricted to specific morphologies and morphology alterations require an extensive optimization process. The tertiary system presented in this article, which consists of a poly(ether sulfone)/dimethylacetamide (PES/DMAc) solution, glycerol, and ZnO-nanoparticles, allows simple and exact tuning of pore diameters ranging from sub-20 nm, up to 100 nm. At the same time, the pore size gradient is controlled from 0 up to 840%/μm yielding extreme asymmetry. In addition to structural analysis, water flux rates of over 5600 L m(-2) h(-1) are measured for membranes retaining 45 nm silica beads.

  17. The influence of plasma membrane electrostatic properties on the stability of cell ionic composition.

    PubMed Central

    Genet, S; Costalat, R; Burger, J

    2001-01-01

    An electro-osmotic model is developed to examine the influence of plasma membrane superficial charges on the regulation of cell ionic composition. Assuming membrane osmotic equilibrium, the ion distribution predicted by Gouy-Chapman-Grahame (GCG) theory is introduced into ion transport equations, which include a kinetic model of the Na/K-ATPase based on the stimulation of this ion pump by internal Na(+) ions. The algebro-differential equation system describing dynamics of the cell model has a unique resting state, stable with respect to finite-sized perturbations of various types. Negative charges on the membrane are found to greatly enhance relaxation toward steady state following these perturbations. We show that this heightened stability stems from electrostatic interactions at the inner membrane side that shift resting state coordinates along the sigmoidal activation curve of the sodium pump, thereby increasing the pump sensitivity to internal Na(+) fluctuations. The accuracy of electrostatic potential description with GCG theory is proved using an alternate formalism, based on irreversible thermodynamics, which shows that pressure contribution to ion potential energy is negligible in electrostatic double layers formed at the surfaces of biological membranes. We discuss implications of the results regarding a reliable operation of ionic process coupled to the transmembrane electrochemical gradient of Na(+) ions. PMID:11606261

  18. Stability of aneurysm solutions in a fluid-filled elastic membrane tube

    NASA Astrophysics Data System (ADS)

    Il'ichev, A. T.; Fu, Y.-B.

    2012-08-01

    When a hyperelastic membrane tube is inflated by an internal pressure, a localized bulge will form when the pressure reaches a critical value. As inflation continues the bulge will grow until it reaches a maximum size after which it will then propagate in both directions to form a hat-like profile. The stability of such bulging solutions has recently been studied by neglecting the inertia of the inflating fluid and it was shown that such bulging solutions are unstable under pressure control. In this paper we extend this recent study by assuming that the inflation is by an inviscid fluid whose inertia we take into account in the stability analysis. This reflects more closely the situation of aneurysm formation in human arteries which motivates the current series of studies. It is shown that fluid inertia would significantly reduce the growth rate of the unstable mode and thus it has a strong stabilizing effect.

  19. Salinomycin kills cancer stem cells by sequestering iron in lysosomes

    NASA Astrophysics Data System (ADS)

    Mai, Trang Thi; Hamaï, Ahmed; Hienzsch, Antje; Cañeque, Tatiana; Müller, Sebastian; Wicinski, Julien; Cabaud, Olivier; Leroy, Christine; David, Amandine; Acevedo, Verónica; Ryo, Akihide; Ginestier, Christophe; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle; Codogno, Patrice; Mehrpour, Maryam; Rodriguez, Raphaël

    2017-10-01

    Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

  20. Lysosomal Degradation of α-Synuclein in Vivo*

    PubMed Central

    Mak, Sally K.; McCormack, Alison L.; Manning-Boğ, Amy B.; Cuervo, Ana Maria; Di Monte, Donato A.

    2010-01-01

    Pathologic accumulation of α-synuclein is a feature of human parkinsonism and other neurodegenerative diseases. This accumulation may be counteracted by mechanisms of protein degradation that have been investigated in vitro but remain to be elucidated in animal models. In this study, lysosomal clearance of α-synuclein in vivo was indicated by the detection of α-synuclein in the lumen of lysosomes isolated from the mouse midbrain. When neuronal α-synuclein expression was enhanced as a result of toxic injury (i.e. treatment of mice with the herbicide paraquat) or transgenic protein overexpression, the intralysosomal content of α-synuclein was also significantly increased. This effect was paralleled by a marked elevation of the lysosome-associated membrane protein type 2A (LAMP-2A) and the lysosomal heat shock cognate protein of 70 kDa (hsc70), two essential components of chaperone-mediated autophagy (CMA). Immunofluorescence microscopy revealed an increase in punctate (lysosomal) LAMP-2A staining that co-localized with α-synuclein within nigral dopaminergic neurons of paraquat-treated and α-synuclein-overexpressing animals. The data provide in vivo evidence of lysosomal degradation of α-synuclein under normal conditions and, quite importantly, under conditions of enhanced protein burden. In the latter, increased lysosomal clearance of α-synuclein was mediated, at least in part, by CMA induction. It is conceivable that these neuronal mechanisms of protein clearance play an important role in neurodegenerative processes characterized by abnormal α-synuclein buildup. PMID:20200163

  1. Hydrogen bond stabilities in membrane-reconstituted alamethicin from amide-resolved hydrogen-exchange measurements.

    PubMed

    Dempsey, C E; Handcock, L J

    1996-04-01

    Amide-resolved hydrogen-deuterium exchange-rate constants were measured for backbone amides of alamethicin reconstituted in dioleoylphosphatidylcholine vesicles by an exchange-trapping method combined with high-resolution nuclear magnetic resonance spectroscopy. In vesicles containing alamethicin at molar ratios between 1:20 and 1:100 relative to lipid, the exchange-rate constants increased with increasing volume of the D20 buffer in which the vesicles were suspended, indicating that exchange under these conditions is dominated by partitioning of the peptide into the aqueous phase. This was supported by observation of a linear relationship between the exchange-rate constants for amides in membrane-reconstituted alamethicin and those for amides in alamethicin dissolved directly into D2O buffer. Significant protection of amides from exchange with D2O buffer in membrane-reconstituted alamethicin is interpreted in terms of stabilization by helical hydrogen bonding. Under conditions in which amide exchange occurred by partitioning of the peptide into solution, only lower limits for hydrogen-bond stabilities in the membrane were determined; all the potentially hydrogen-bonded amides of alamethicin are at least 1000-fold exchange protected in the membrane-bound state. When partitioning of alamethicin into the aqueous phase was suppressed by hydration of reconstituted vesicles in a limiting volume of water [D2O:dioleoylphosphatidylcholine:alamethicin; 220:1:0.05; (M:M:M)], the exchange-protection factors exhibited helical periodicity with highly exchange-protected, and less well-protected, amides on the nonpolar and polar helix faces, respectively. The exchange data indicate that, under the conditions studied, alamethicin adopts a stable helical structure in DOPC bilayers in which all the potentially hydrogen-bonded amides are stabilized by helical hydrogen bonds. The protection factors define the orientation of the peptide helix with respect to an aqueous phase, which is

  2. Deficiency of ATP13A2 leads to lysosomal dysfunction, α-synuclein accumulation, and neurotoxicity.

    PubMed

    Usenovic, Marija; Tresse, Emilie; Mazzulli, Joseph R; Taylor, J Paul; Krainc, Dimitri

    2012-03-21

    The autophagy-lysosomal pathway plays an important role in the clearance of long-lived proteins and dysfunctional organelles. Lysosomal dysfunction has been implicated in several neurodegenerative disorders including Parkinson's disease and related synucleinopathies that are characterized by accumulations of α-synuclein in Lewy bodies. Recent identification of mutations in genes linked to lysosomal function and neurodegeneration has offered a unique opportunity to directly examine the role of lysosomes in disease pathogenesis. Mutations in lysosomal membrane protein ATP13A2 (PARK9) cause familial Kufor-Rakeb syndrome characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While previous data suggested a role of ATP13A2 in α-synuclein misfolding and toxicity, the mechanistic link has not been established. Here we report that loss of ATP13A2 in human fibroblasts from patients with Kufor-Rakeb syndrome or in mouse primary neurons leads to impaired lysosomal degradation capacity. This lysosomal dysfunction results in accumulation of α-synuclein and toxicity in primary cortical neurons. Importantly, silencing of endogenous α-synuclein attenuated the toxicity in ATP13A2-depleted neurons, suggesting that loss of ATP13A2 mediates neurotoxicity at least in part via the accumulation of α-synuclein. Our findings implicate lysosomal dysfunction in the pathogenesis of Kufor-Rakeb syndrome and suggest that upregulation of lysosomal function and downregulation of α-synuclein represent important therapeutic strategies for this disorder.

  3. hLGDB: a database of human lysosomal genes and their regulation.

    PubMed

    Brozzi, Alessandro; Urbanelli, Lorena; Germain, Pierre Luc; Magini, Alessandro; Emiliani, Carla

    2013-01-01

    Lysosomes are cytoplasmic organelles present in almost all eukaryotic cells, which play a fundamental role in key aspects of cellular homeostasis such as membrane repair, autophagy, endocitosis and protein metabolism. The characterization of the genes and enzymes constituting the lysosome represents a central issue to be addressed toward a better understanding of the biology of this organelle. In humans, mutations that cause lysosomal enzyme deficiencies result in >50 different disorders and severe pathologies. So far, many experimental efforts using different methodologies have been carried out to identity lysosomal genes. The Human Lysosome Gene Database (hLGDB) is the first resource that provides a comprehensive and accessible census of the human genes belonging to the lysosomal system. This database was developed by collecting and annotating gene lists from many different sources. References to the studies that have identified each gene are provided together with cross databases gene related information. Special attention has been given to the regulation of the genes through microRNAs and the transcription factor EB. The hLGDB can be easily queried to retrieve, combine and analyze information on different lists of lysosomal genes and their regulation by microRNA (binding sites predicted by five different algorithms). The hLGDB is an open access dynamic project that will permit in the future to collapse in a unique publicly accessible resource all the available biological information about lysosome genes and their regulation. Database URL: http://lysosome.unipg.it/.

  4. hLGDB: a database of human lysosomal genes and their regulation

    PubMed Central

    Brozzi, Alessandro; Urbanelli, Lorena; Luc Germain, Pierre; Magini, Alessandro; Emiliani, Carla

    2013-01-01

    Lysosomes are cytoplasmic organelles present in almost all eukaryotic cells, which play a fundamental role in key aspects of cellular homeostasis such as membrane repair, autophagy, endocitosis and protein metabolism. The characterization of the genes and enzymes constituting the lysosome represents a central issue to be addressed toward a better understanding of the biology of this organelle. In humans, mutations that cause lysosomal enzyme deficiencies result in >50 different disorders and severe pathologies. So far, many experimental efforts using different methodologies have been carried out to identity lysosomal genes. The Human Lysosome Gene Database (hLGDB) is the first resource that provides a comprehensive and accessible census of the human genes belonging to the lysosomal system. This database was developed by collecting and annotating gene lists from many different sources. References to the studies that have identified each gene are provided together with cross databases gene related information. Special attention has been given to the regulation of the genes through microRNAs and the transcription factor EB. The hLGDB can be easily queried to retrieve, combine and analyze information on different lists of lysosomal genes and their regulation by microRNA (binding sites predicted by five different algorithms). The hLGDB is an open access dynamic project that will permit in the future to collapse in a unique publicly accessible resource all the available biological information about lysosome genes and their regulation. Database URL: http://lysosome.unipg.it/ PMID:23584836

  5. Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

    PubMed Central

    Aittaleb, Mohamed; Chen, Po-Ju; Akaaboune, Mohammed

    2015-01-01

    ABSTRACT Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn−/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity. PMID:26330529

  6. Palmitoylation-dependent association with CD63 targets the Ca2+ sensor synaptotagmin VII to lysosomes

    PubMed Central

    Flannery, Andrew R.; Czibener, Cecilia

    2010-01-01

    Syt VII is a Ca2+ sensor that regulates lysosome exocytosis and plasma membrane repair. Because it lacks motifs that mediate lysosomal targeting, it is unclear how Syt VII traffics to these organelles. In this paper, we show that mutations or inhibitors that abolish palmitoylation disrupt Syt VII targeting to lysosomes, causing its retention in the Golgi complex. In macrophages, Syt VII is translocated simultaneously with the lysosomal tetraspanin CD63 from tubular lysosomes to nascent phagosomes in a Ca2+-dependent process that facilitates particle uptake. Mutations in Syt VII palmitoylation sites block trafficking of Syt VII, but not CD63, to lysosomes and phagosomes, whereas tyrosine replacement in the lysosomal targeting motif of CD63 causes both proteins to accumulate on the plasma membrane. Complexes of CD63 and Syt VII are detected only when Syt VII palmitoylation sites are intact. These findings identify palmitoylation-dependent association with the tetraspanin CD63 as the mechanism by which Syt VII is targeted to lysosomes. PMID:21041449

  7. Signaling from lysosomes to mitochondria sensitizes cancer cells to photodynamic treatment

    NASA Astrophysics Data System (ADS)

    Hung, Hsin-I.; Quiogue, Geraldine; Lemasters, John J.; Nieminen, Anna-Liisa

    2011-02-01

    Previously, we showed that photosensitizers that localize to lysosomes are more effective in killing cancer cells than ones directed to mitochondria after photodynamic treatment (PDT). The photosensitizer, phthalocyanine 4 (Pc 4), localizes primarily to mitochondrial membranes in cancer cells, resulting in mitochondria-mediated cell death. However, analogues of Pc 4 (e.g., Pc 181) that primarily target lysosomes still produce mitochondria-mediated cell death, although the time course is slower compared to Pc 4-PDT. In A431 epidermoid carcinoma cells, these new analogues preferentially localized in lysosomes were highly efficient in inducing apoptotic cell death. To assess further how lysosomes contribute to PDT, we monitored cell killing of A431 cells after Pc 4-PDT in the presence and absence of bafilomycin, an inhibitor of the acidic vacuolar proton pump that collapses the pH gradient of the lysosomal/endosomal compartment. Bafilomycin by itself was not toxic but greatly enhanced Pc 4-PDT-induced mitochondrial depolarization and cell killing. Both depolarization and cell killing were substantially prevented by iron chelators. The fact that Pc 4-PDT plus bafilomycin treatment did not induce lysosomal membrane damage prior to mitochondrial depolarization suggests that bafilomycin instead induced release of redox active iron from lysosomes into the cytosol that further translocated into mitochondria, where iron-mediated free radical formation occurred. In conclusion, agents that disturb lysosomal function could potentially be used as adjuvants with mitochondrion-targeted photosensitizers to enhance phototoxicity.

  8. Removal of seminal plasma enhances membrane stability on fresh and cooled stallion spermatozoa.

    PubMed

    Barrier-Battut, I; Bonnet, C; Giraudo, A; Dubois, C; Caillaud, M; Vidament, M

    2013-02-01

    Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4 °C: motility, plasma membrane integrity as evaluated by hypo-osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty-six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.

  9. Rotenone impairs autophagic flux and lysosomal functions in Parkinson's disease.

    PubMed

    Wu, F; Xu, H-D; Guan, J-J; Hou, Y-S; Gu, J-H; Zhen, X-C; Qin, Z-H

    2015-01-22

    Rotenone is an environmental neurotoxin that induces accumulation of α-synuclein and degeneration of dopaminergic neurons in substantia nigra pars compacta (SNpc), but the molecular mechanisms are not fully understood. We investigated whether rotenone induced impairment of autophagic flux and lysosomal functions. Autophagy flux, accumulation of α-synuclein, lysosomal membrane integrity and neurodegeneration were assessed in the rotenone-treated rat model and PC12 cells, and the effects of the autophagy inducer trehalose on rotenone's cytotoxicity were also studied. Rotenone administration significantly reduced motor activity and caused a loss of tyrosine hydroxylase in SNpc of Lewis rats. The degeneration of nigral dopaminergic neurons was accompanied by the deposition of α-synuclein aggregates, autophagosomes and redistribution of cathepsin D from lysosomes to the cytosol. In cultured PC12 cells, rotenone also induced increases in protein levels of α-synuclein, microtubule-associated protein 1 light chain 3-II, Beclin 1, and p62. Rotenone increased lysosomal membrane permeability as evidenced by leakage of N-acetyl-beta-d-glucosaminidase and cathepsin D, the effects were blocked by reactive oxygen species scavenger tiron. Autophagy inducer trehalose enhanced the nuclear translocation of transcription factor EB, accelerated the clearance of autophagosomes and α-synuclein and attenuated rotenone-induced cell death of PC12 cells. Meanwhile, administration of trehalose to rats in drinking water (2%) decreased rotenone-induced dopaminergic neurons loss in SNpc. These studies indicate that the lysosomal dysfunction contributes to rotenone's neurotoxicity and restoration of lysosomal function could be a new therapeutic strategy for Parkinson's disease. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. Fructans from oat and rye: composition and effects on membrane stability during drying.

    PubMed

    Hincha, Dirk K; Livingston, David P; Premakumar, Ramaswamy; Zuther, Ellen; Obel, Nicolai; Cacela, Constança; Heyer, Arnd G

    2007-06-01

    Fructans have been implicated in the abiotic stress tolerance of many plant species, including grasses and cereals. To elucidate the possibility that cereal fructans may stabilize cellular membranes during dehydration, we used liposomes as a model system and isolated fructans from oat (Avena sativa) and rye (Secale cereale). Fructans were fractionated by preparative size exclusion chromatography into five defined size classes (degree of polymerization (DP) 3 to 7) and two size classes containing high DP fructans (DP>7 short and long). They were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The effects of the fructans on liposome stability during drying and rehydration were assessed as the ability of the sugars to prevent leakage of a soluble marker from liposomes and liposome fusion. Both species contain highly complex mixtures of fructans, with a DP up to 17. The two DP>7 fractions from both species were unable to protect liposomes, while the fractions containing smaller fructans were protective to different degrees. Protection showed an optimum at DP 4 and the DP 3, 4, and 5 fractions from oat were more protective than all other fractions from both species. In addition, we found evidence for synergistic effects in membrane stabilization in mixtures of low DP with DP>7 fructans. The data indicate that cereal fructans have the ability to stabilize membranes under stress conditions and that there are size and species dependent differences between the fructans. In addition, mixtures of fructans, as they occur in living cells may have protective properties that differ significantly from those of the purified fractions.

  11. Characterization of Drosophila Saposin-related mutants as a model for lysosomal sphingolipid storage diseases

    PubMed Central

    Schulze, Heike; Paradis, Marie; Gosejacob, Dominic; Papan, Cyrus; Shevchenko, Andrej; Psathaki, Olympia Ekaterina; Thielisch, Melanie; Sandhoff, Konrad

    2017-01-01

    ABSTRACT Sphingolipidoses are inherited diseases belonging to the class of lysosomal storage diseases (LSDs), which are characterized by the accumulation of indigestible material in the lysosome caused by specific defects in the lysosomal degradation machinery. While some LSDs can be efficiently treated by enzyme replacement therapy (ERT), this is not possible if the nervous system is affected due to the presence of the blood-brain barrier. Sphingolipidoses in particular often present as severe, untreatable forms of LSDs with massive sphingolipid and membrane accumulation in lysosomes, neurodegeneration and very short life expectancy. The digestion of intralumenal membranes within lysosomes is facilitated by lysosomal sphingolipid activator proteins (saposins), which are cleaved from a prosaposin precursor. Prosaposin mutations cause some of the severest forms of sphingolipidoses, and are associated with perinatal lethality in mice, hampering studies on disease progression. We identify the Drosophila prosaposin orthologue Saposin-related (Sap-r) as a key regulator of lysosomal lipid homeostasis in the fly. Its mutation leads to a typical spingolipidosis phenotype with an enlarged endolysosomal compartment and sphingolipid accumulation as shown by mass spectrometry and thin layer chromatography. Sap-r mutants show reduced viability with ∼50% survival to adulthood, allowing us to study progressive neurodegeneration and analyze their lipid profile in young and aged flies. Additionally, we observe a defect in sterol homeostasis with local sterol depletion at the plasma membrane. Furthermore, we find that autophagy is increased, resulting in the accumulation of mitochondria in lysosomes, concomitant with increased oxidative stress. Together, we establish Drosophila Sap-r mutants as a lysosomal storage disease model suitable for studying the age-dependent progression of lysosomal dysfunction associated with lipid accumulation and the resulting pathological signaling

  12. Mucolipidosis type IV: the effect of increased lysosomal pH on the abnormal lysosomal storage.

    PubMed

    Kogot-Levin, Aviram; Zeigler, Marsha; Ornoy, Asher; Bach, Gideon

    2009-06-01

    Mucolipidosis type IV (MLIV) is a neurodegenerative channelopathy that is caused by the deficiency of TRPML1 activity, a nonselective cation channel. TRPML1 is a lysosomal membrane protein, and thus, MLIV is a lysosomal storage disorder. The basic, specific function of TRPML1 has not been yet clarified. A recent report (Soyombo AA, Tjon-Kon-Sang S, Rbaibi Y, Bashllari E, Bisceglia J, Muallem S, Kiselyov K: J Biol Chem 281:7294-7301, 2006) indicated that TRPML1 functions as an outwardly proton channel whose function is the prevention of overacidification of these organelles. Thus, in MLIV the lysosomal pH is lower than normal. Furthermore, attempts by these investigators to increase slightly the lysososmal pH with either Nigericin or Chloroquine suggested corrective effect of the abnormal storage in MLIV cells. We investigated this approach using these agents with cultured fibroblasts from severely affected and milder patients. Our data indicated that there was no reduction in the total number of storage vesicles by either agent, although Nigericin resulted in a change in the nature of the storage materials, reducing the presence of lamellated substances (lipids) so that the storage vesicles contained predominantly granulated substances. On the other hand, transfection with the normal MCOLN1 cDNA (the gene coding for TRPML1) resulted in the removal of almost all the storage materials.

  13. Size-controlled nanopores in lipid membranes with stabilizing electric fields.

    PubMed

    Fernández, M Laura; Risk, Marcelo; Reigada, Ramon; Vernier, P Thomas

    2012-06-29

    Molecular dynamics (MD) has been shown to be a useful tool for unveiling many aspects of pore formation in lipid membranes under the influence of an applied electric field. However, the study of the structure and transport properties of electropores by means of MD has been hampered by difficulties in the maintenance of a stable electropore in the typically small simulated membrane patches. We describe a new simulation scheme in which an initially larger porating field is systematically reduced after pore formation to lower stabilizing values to produce stable, size-controlled electropores, which can then be characterized at the molecular level. A new method allows the three-dimensional modeling of the irregular shape of the pores obtained as well as the quantification of its volume. The size of the pore is a function of the value of the stabilizing field. At lower fields the pore disappears and the membrane recovers its normal shape, although in some cases long-lived, fragmented pores containing unusual lipid orientations in the bilayer are observed.

  14. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity

    PubMed Central

    Pi, Huifeng; Li, Min; Tian, Li; Yang, Zhiqi; Yu, Zhengping; Zhou, Zhou

    2017-01-01

    Cadmium (Cd), a highly ubiquitous heavy metal, is a well-known inducer of neurotoxicity. However, the mechanism underlying cadmium-induced neurotoxicity remains unclear. In this study, we found that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function by reducing the levels of lysosomal-associated membrane proteins, inhibiting lysosomal proteolysis and altering lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to nerve cell death. In addition, Cd decreases transcription factor EB (TFEB) expression at both the mRNA and protein levels. Furthermore, Cd induces the nuclear translocation of TFEB and TFEB target-gene expression, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Notably, restoration of the levels of lysosomal-associated membrane protein, lysosomal proteolysis, lysosomal pH and autophagic flux through Tfeb overexpression protects against Cd-induced neurotoxicity, and this protective effect is incompletely dependent on TFEB nuclear translocation. Moreover, gene transfer of the master autophagy regulator TFEB results in the clearance of toxic proteins and the correction of Cd-induced neurotoxicity in vivo. Our study is the first to demonstrate that Cd disrupts lysosomal function and autophagic flux and manipulation of TFEB signalling may be a therapeutic approach for antagonizing Cd-induced neurotoxicity. PMID:28240313

  15. Effect of the compatible solute ectoine on the stability of the membrane proteins.

    PubMed

    Roychoudhury, Arpita; Haussinger, Dieter; Oesterhelt, Filipp

    2012-08-01

    Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.

  16. Polyacrylamide-Polydivinylbenzene Decorated Membrane for Sundry Ionic Stabilized Emulsions Separation via a Facile Solvothermal Method.

    PubMed

    Zhang, Weifeng; Liu, Na; Cao, Yingze; Chen, Yuning; Zhang, Qingdong; Lin, Xin; Qu, Ruixiang; Li, Haifang; Feng, Lin

    2016-08-24

    Aiming to solve the worldwide challenge of stabilized oil-in-water emulsion separation, a PAM-PDVB decorated nylon membrane is fabricated via a facile solvothermal route in our group. The main composition is PAM, while the PDVB plays a role as cross-linker in order to improve the interaction between the polymer and the substrate. By the combination of the superhydrophilic and underwater superoleophobic wettability of the PAM polymer with the micropore size of the substrate, the as-prepared material is able to achieve the separation of various stabilized oil-in-water emulsions including cationic type, nonionic type, and anionic type. Compared with previous works, the emulsions used in this case are more stable and can stay for several days. Besides, the solvothermal method is facile, cost saving, and relatively environmentally friendly in this experiment. Moreover, the PAM-PDVB modified membrane exhibits excellent pH stability, recyclability, and high separation efficiency (above 99%), which can be scaled up and used in the practical industrial field.

  17. Stability of spray-dried tuna oil emulsions encapsulated with two-layered interfacial membranes.

    PubMed

    Klinkesorn, Utai; Sophanodora, Pairat; Chinachoti, Pavinee; McClements, D Julian; Decker, Eric A

    2005-10-19

    omega-3 Fatty acids have numerous health benefits, but their addition to foods is limited by oxidative rancidity. Spray-drying tuna oil-in-water emulsion droplets with a coating of lecithin and chitosan multilayer system could produce emulsion droplet interfacial membranes that are cationic and thick, both factors that can help control lipid oxidation. Physicochemical and oxidative stability of the spray-dried emulsions were determined as a function of storage temperature and relative humidity (RH). The combination of ethylenediaminetetraacetic acid (EDTA) and mixed tocopherols was able to increase the oxidative stability of dried emulsions. Lipid oxidation was more rapid during storage at low relative humidity (11% and 33% compared to 52% RH). At high moisture, physical modifications in the sample were observed, including reduced dispersibility and formation of brown pigments. Sugar crystallization or Maillard products produced at the higher humidities may have inhibited oxidation. Overall, spray-dried tuna oil-in-water emulsions stabilized by lecithin-chitosan membranes were more oxidatively stable than bulk oils and thus have excellent potential as an omega-3 fatty acid ingredient for functional foods.

  18. Binding of protofibrillar Aβ trimers to lipid bilayer surface enhances Aβ structural stability and causes membrane thinning.

    PubMed

    Dong, Xuewei; Sun, Yunxiang; Wei, Guanghong; Nussinov, Ruth; Ma, Buyong

    2017-10-05

    Alzheimer's disease, a common neurodegenerative disease, is characterized by the aggregation of amyloid-β (Aβ) peptides. The interactions of Aβ with membranes cause changes in membrane morphology and ion permeation, which are responsible for its neurotoxicity and can accelerate fibril growth. However, the Aβ-lipid interactions and how these induce membrane perturbation and disruption at the atomic level and the consequences for the Aβ organization are not entirely understood. Here, we perform multiple atomistic molecular dynamics simulations on three protofibrillar Aβ9-40 trimers. Our simulations show that, regardless of the morphologies and the initial orientations of the three different protofibrillar Aβ9-40 trimers, the N-terminal β-sheet of all trimers preferentially binds to the membrane surface. The POPG lipid bilayers enhance the structural stability of protofibrillar Aβ trimers by stabilizing inter-peptide β-sheets and D23-K28 salt-bridges. The interaction causes local membrane thinning. We found that the trimer structure related to Alzheimer's disease brain tissue () is the most stable both in water solution and at membrane surface, and displays slightly stronger membrane perturbation capability. These results provide mechanistic insights into the membrane-enhanced structural stability of protofibrillar Aβ oligomers and the first step of Aβ-induced membrane disruption at the atomic level.

  19. Stability in alkaline aqueous electrolyte of air electrode protected with fluorinated interpenetrating polymer network membrane

    NASA Astrophysics Data System (ADS)

    Bertolotti, Bruno; Messaoudi, Houssam; Chikh, Linda; Vancaeyzeele, Cédric; Alfonsi, Séverine; Fichet, Odile

    2015-01-01

    We developed original anion exchange membranes to protect air electrodes operating in aqueous lithium-air battery configuration, i.e. supplied with atmospheric air and in concentrated aqueous lithium hydroxide. These protective membranes have an interpenetrating polymer network (IPN) architecture combining a hydrogenated cationic polyelectrolyte network based on poly(epichlorohydrin) (PECH) and a fluorinated neutral network based on perfluoropolyether (Fluorolink® MD700). Two phases, each one rich in one of the polymer, are co-continuous in the materials. This morphology allows combining their properties according to the weight proportions of each polymer. Thus, PECH/Fluorolink IPNs show ionic conductivity varying from 1 to 2 mS cm-1, water uptake from 30 to 90 wt.% and anionic transport number from 0.65 to 0.80 when the PECH proportion varies from 40 to 90 wt.%. These membranes have been systematically assembled on air electrodes. Air electrode protected with PECH/Fluorolink 70/30 IPN shows outstanding stability higher than 1000 h, i.e. a 20-fold increase in the lifetime of the non-modified electrode. This efficient membrane/air electrode assembly is promising for development of alkaline electrolyte based storage or production energy systems, such as metal air batteries or alkaline fuel cells.

  20. Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia

    NASA Astrophysics Data System (ADS)

    Machta, Benjamin B.; Gray, Ellyn; Nouri, Mariam; McCarthy, Nicola L. C.; Gray, Erin M.; Miller, Ann L.; Brooks, Nicholas J.; Veatch, Sarah L.

    2016-08-01

    Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature ($T_c$) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on $T_c$. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described `intoxication reversers' raise $T_c$ and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises $T_c$ in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that $\\Delta T_c$ predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

  1. Preparation of uniform particle-stabilized emulsions using SPG membrane emulsification.

    PubMed

    Sun, Guanqing; Qi, Feng; Wu, Jie; Ma, Guanghui; Ngai, To

    2014-06-24

    Various aspects of particle-stabilized emulsions (or so-called Pickering emulsions) have been extensively investigated during the last two decades, but the preparation of uniform Pickering emulsion droplets via a simple and scalable method has been sparingly realized. We report the preparation of uniform Pickering emulsions by Shirasu porous glass (SPG) membrane emulsification. The size of the emulsion droplets ranging from 10-50 μm can be precisely controlled by the size of the membrane pore. The emulsion droplets have a high monodispersity with coefficients of variation (CV) lower than 15% in all of the investigated systems. We further demonstrate the feasibility of locking the assembled particles at the interface, and emulsion droplets have been shown to be excellent templates for the preparation of monodisperse colloidosomes that are necessary in drug-delivery systems.

  2. Sphingosine-induced apoptosis is dependent on lysosomal proteases.

    PubMed Central

    Kågedal, K; Zhao, M; Svensson, I; Brunk, U T

    2001-01-01

    We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10 mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes. PMID:11583579

  3. Lysosome-mediated processing of chromatin in senescence.

    PubMed

    Ivanov, Andre; Pawlikowski, Jeff; Manoharan, Indrani; van Tuyn, John; Nelson, David M; Rai, Taranjit Singh; Shah, Parisha P; Hewitt, Graeme; Korolchuk, Viktor I; Passos, Joao F; Wu, Hong; Berger, Shelley L; Adams, Peter D

    2013-07-08

    Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C-negative, but strongly γ-H2AX-positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression.

  4. Haematopoietic, Antioxidant and Membrane Stabilizing Property of Diallyl Disulphide in Irradiated Mice

    PubMed Central

    Tenkanidiyoor, Yogish Somayaji; Vasudeva, Vidya; Rao, Shama; Gowda, Damodara; Rao, Chandrika; Sanjeev, Ganesh

    2016-01-01

    Introduction Diallyl disulphide is an organo-sulphur compound which is present in garlic and responsible for the characteristic odor of garlic. It is known for its anticancer and invitro membrane stabilizing properties. Aim The main aim was to evaluate the haematopoietic, antioxidant and membrane stabilizing property of diallyl disulfide in irradiated mice. Materials and Methods Mice were grouped into 6 groups as control, drug control, radiation control and drug pre-treatment groups (i.e. drug administration + radiation group) The mice were fed orally for 15 consecutive days and on the 15th day, one hour after drug administration, the mice were irradiated with 6Gy electron beam radiation. The changes in blood cell count, total antioxidant levels, malondialdehyde and reduced glutathione levels were determined. The immunomodulatory response of DADS to the radiological effects was determined by the estimation of IL-6 levels. Results A significant improvement in pre-drug treatment group when compared to control groups in the haemoglobin, red blood cell count, white blood cell count, haematocrit and platelet counts was observed. There is an increased level of interleukin-6 in the drug treated groups compared to the radiation control. An increase in the malondialdehyde levels and decrease in the glutathione levels in the irradiated group indicate increased lipid peroxidation and oxidative stress, whereas, there is a significant reduction in the malondialdehyde levels and increased glutathione levels in the drug pre-treatment groups showing membrane stabilization. Conclusion Thus DADS proves to be an effective haematopoietic and antioxidative agent to counter radiation induced haematopoietic suppression and oxidative stress. PMID:27042448

  5. Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins.

    PubMed

    Dierks, Thomas; Schlotawa, Lars; Frese, Marc-André; Radhakrishnan, Karthikeyan; von Figura, Kurt; Schmidt, Bernhard

    2009-04-01

    Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been

  6. Insertion stability of poly(ethylene glycol)-cholesteryl-based lipid anchors in liposome membranes.

    PubMed

    Molnar, Daniel; Linders, Jürgen; Mayer, Christian; Schubert, Rolf

    2016-06-01

    Liposomes consist of a hydrophilic core surrounded by a phospholipid (PL) bilayer. In human blood, the half-life of such artificial vesicles is limited. To prolong their stability in the circulation, liposomal bilayers can be modified by inserting poly(ethylene glycol) (PEG) molecules using either PL or sterols as membrane anchors. This establishes a hydrophilic steric barrier, reducing the adsorption of serum proteins, recognition and elimination by cells of the immune system. In addition, targeting ligands (such as antibodies) are frequently coupled to the distal end of the PEG chains to direct the vesicles (then called 'immuno-liposomes') to specific cell types, such as tumor cells. To our knowledge, experiments on the stability of ligand anchoring have so far only been conducted with PL-based PEGs and not with sterol-based PEGs after insertion via the sterol-based post-insertion technique (SPIT). Therefore, our study examines the insertion stability of PEG-cholesteryl ester (Chol-PEG) molecules with PEG chains of 1000, 1500 and 2000Da molecular mass which have been inserted into the membranes of liposomes using SPIT. For this study we used different acceptor media and multiple analytical techniques, including pulsed-field-gradient nuclear magnetic resonance (PFG-NMR), free-flow electrophoresis, size exclusion chromatography and ultracentrifugation. The obtained data consistently showed that a higher molar mass of PEG chains positively correlates with higher release from the liposome membranes. Furthermore, we could detect and quantify the migration of Chol-PEG molecules from radioactively double-labeled surface-modified liposomes to negatively charged acceptor liposomes via free-flow electrophoresis. Insertion of Chol-PEG molecules into the membrane of preformed liposomes using SPIT is an essential step for the functionalization of liposomes with the aim of specific targeting. For the first time, we present a kinetic analysis of this insertion process using PFG

  7. Mechanisms and stability of oxide-ion transport in homogenous and heterogeneous ceramic membranes

    NASA Astrophysics Data System (ADS)

    Tichy, Robin Sarah

    Solid oxide-ion conductors are basic components of several modern technologies. Oxide-ion electrolytes are oxide-ion conductors and electronic insulators; they are used in oxygen sensors and solid oxide fuel cells. The required oxide-ion conductivity is only achieved at higher temperatures. Commercialization of this technology demands the development of a better oxide-ion electrolyte and/or the ability to fabricate a large area ceramic membrane with a thickness of L < 10 mum. This thesis addresses the development of an improved oxide-ion electrolyte and of mixed ionic-electronic conductors that can be used as the electrodes of a fuel cell. The perovskite LaGaO3 doped with Sr and Mg was known to be a superior oxide-ion electrolyte. Optimization of the composition in a narrow single-phase field was accomplished, and its stability against mixed-conductor electrodes was engineered and demonstrated in tests on a single fuel cell. In addition to fuel-cell electrodes, mixed ionic-electronic conductors are used as gas separation membranes and methane conversion reactors that produce syn-gas. Structural and chemical stability of mixed conductors are a major problem for ceramic-membrane reactors because the material must exhibit good mixed conduction in both high and very low oxygen partial pressures and at operating temperatures, 600°C ≤ Top. ≤ 900°C. The material SrMnO3 is a high-temperature, oxygen-deficient, perovskite that may be preserved at room temperature. Although this material exhibits good mixed conduction, it reverts to its stable stoichiometric phase under oxidizing operating conditions. La2NiO4+delta has a tetragonal crystal structure that is closely related to the cubic perovskite structure. The ionic conduction occurs via the migration of interstitial oxygen, which is lost in reducing atmospheres. The stability of mixed conduction within one material proved difficult to achieve in both reducing and oxidizing conditions at high temperatures. Several

  8. Graphene Oxide Nanofiltration Membranes Stabilized by Cationic Porphyrin for High Salt Rejection.

    PubMed

    Xu, Xiao-Ling; Lin, Fu-Wen; Du, Yong; Zhang, Xi; Wu, Jian; Xu, Zhi-Kang

    2016-05-25

    Swelling has great influences on the structure stability and separation performance of graphene oxide laminate membranes (GOLMs) for water desalination and purification. Herein, we report cross-linked GOLMs from GO assembled with cationic tetrakis(1-methyl-pyridinium-4-yl)porphyrin (TMPyP) by a vacuum-assisted strategy. The concave nonoxide regions (G regions) of GO are used as cross-linking sites for the first time to precisely control the channel size for water permeation and salt ion retention. Channels around 1 nm are constructed by modulating the assembly ratio of TMPyP/GO, and these cross-linked GOLMs show high salt rejection.

  9. The lysosome as a command-and-control center for cellular metabolism

    PubMed Central

    2016-01-01

    Lysosomes are membrane-bound organelles found in every eukaryotic cell. They are widely known as terminal catabolic stations that rid cells of waste products and scavenge metabolic building blocks that sustain essential biosynthetic reactions during starvation. In recent years, this classical view has been dramatically expanded by the discovery of new roles of the lysosome in nutrient sensing, transcriptional regulation, and metabolic homeostasis. These discoveries have elevated the lysosome to a decision-making center involved in the control of cellular growth and survival. Here we review these recently discovered properties of the lysosome, with a focus on how lysosomal signaling pathways respond to external and internal cues and how they ultimately enable metabolic homeostasis and cellular adaptation. PMID:27621362

  10. High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A

    PubMed Central

    Gabandé-Rodríguez, E; Boya, P; Labrador, V; Dotti, C G; Ledesma, M D

    2014-01-01

    Niemann Pick disease type A (NPA), which is caused by loss of function mutations in the acid sphingomyelinase (ASM) gene, is a lysosomal storage disorder leading to neurodegeneration. Yet, lysosomal dysfunction and its consequences in the disease are poorly characterized. Here we show that undegraded molecules build up in neurons of acid sphingomyelinase knockout mice and in fibroblasts from NPA patients in which autophagolysosomes accumulate. The latter is not due to alterations in autophagy initiation or autophagosome–lysosome fusion but because of inefficient autophago–lysosomal clearance. This, in turn, can be explained by lysosomal membrane permeabilization leading to cytosolic release of Cathepsin B. High sphingomyelin (SM) levels account for these effects as they can be induced in control cells on addition of the lipid and reverted on SM-lowering strategies in ASM-deficient cells. These results unveil a relevant role for SM in autophagy modulation and characterize autophagy anomalies in NPA, opening new perspectives for therapeutic interventions. PMID:24488099

  11. Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol‐dependent cytolysins

    PubMed Central

    Malet, Julien Karim

    2016-01-01

    Abstract Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum‐Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore‐forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore‐forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol‐dependent cytolysins. PMID:27739224

  12. Electrochemical enzymatic biosensor with long-term stability using hybrid mesoporous membrane.

    PubMed

    Itoh, Tetsuji; Shimomura, Takeshi; Hayashi, Akari; Yamaguchi, Akira; Teramae, Norio; Ono, Masatoshi; Tsunoda, Tatsuo; Mizukami, Fujio; Stucky, Galen D; Hanaoka, Taka-aki

    2014-09-21

    An acetylcholinesterase-immobilized sensor unit was successfully prepared by encapsulating the enzyme within hybrid mesoporous silica membranes (F127-MST). Through a novel combination with tetracyanoquinodimethane, both acetylcholine and organophosphorus pesticides were successfully detected with high sensitivity. Furthermore, we manufactured the working prototype of an enzyme sensor with this sensor unit for detecting dichlorvos, aldicarb and parathion. At present, the detection limit in this working prototype either equaled or surpassed that of others. Also, we have the advantage of increased stability of the enzyme against the outer environment by encapsulation of the enzymes into a silica nanospace. Consequently, acetylcholinesterase immobilized in F127-MST is a practical sensor with high sensitivity, reusability, and storage stability.

  13. Simultaneous Enhancements of Conductivity and Stability for Anion Exchange Membranes (AEMs) through Precise Structure Design

    PubMed Central

    Ran, Jin; Wu, Liang; Wei, Bing; Chen, Yaoyao; Xu, Tongwen

    2014-01-01

    Polymeric materials as anion exchange membranes (AEMs) play an essential role in the field of energy and environment. The achievement of high performance AEMs by the precise manipulation of macromolecular architecture remains a daunting challenge. Herein, we firstly report a novel rod-coil graft copolymer AEM, possessing rigid hydrophobic main chains and soft hydrophilic graft chains. The low graft density, which can alleviate the adverse influences of ioinc graft chains on the main chains, was obtained by using the living polymerization technique. Consequently, the grafted ionic groups which result in the degradation of polymer backbone was decreased to a small degree. Moreover, the relatively long graft chains induced the nanophase separation between the hydrophobic polymer chains and hydrophilic graft chains, which creates a convinient pathway for high hydroxide ion mobility. Such an accurate molecular design simultaneously improves the hydroxide ion conductivity and alkaline stability as well as dimensional stability. PMID:25255843

  14. The SM protein Car/Vps33A regulates SNARE-mediated trafficking to lysosomes and lysosome-related organelles.

    PubMed

    Akbar, Mohammed A; Ray, Sanchali; Krämer, Helmut

    2009-03-01

    The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes.

  15. The SM Protein Car/Vps33A Regulates SNARE-mediated Trafficking to Lysosomes and Lysosome-related Organelles

    PubMed Central

    Akbar, Mohammed A.; Ray, Sanchali

    2009-01-01

    The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes. PMID:19158398

  16. Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions

    PubMed Central

    Ferenczi, Emily A; Fraser, James A; Chawla, Sangeeta; Skepper, Jeremy N; Schwiening, Christof J; Huang, Christopher L-H

    2004-01-01

    This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (Em) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of Em despite cell shrinkage, which was attributable to elevation of intracellular [Cl−] above electrochemical equilibrium through Na+–Cl− and Na+−K+−2Cl− cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope xz-scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl−-free, normal and Na+-free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady-state resting potentials (Em). Fibres in isotonic Cl−-free, normal and Na+-free Ringer solutions showed similar Em values consistent with previously reported permeability ratios PNa/PK(0.03–0.05) and PCl/PK (∼2.0) and intracellular [Na+], [K+] and [Cl−]. Increased extracellular osmolarities produced hyperpolarizing shifts in Em in fibres studied in Cl−-free Ringer solution consistent with the Goldman-Hodgkin-Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such Em shifts, suggesting a Cl−-dependent stabilization of membrane potential. This stabilization of Em was abolished by withdrawing extracellular Na+ or by the combined presence of the Na+–Cl− cotransporter (NCC) inhibitor chlorothiazide (10 μm) and the Na+−K+−2Cl− cotransporter (NKCC) inhibitor bumetanide (10 μm), or the Na+−K+-ATPase inhibitor ouabain (1 or 10 μm) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a

  17. Effects of acute and chronic exercise on the osmotic stability of erythrocyte membrane of competitive swimmers

    PubMed Central

    2017-01-01

    This study aimed to evaluate the influence of acute and chronic exercise on erythrocyte membrane stability and various blood indices in a population consisting of five national-level male swimmers, over 18 weeks of training. The evaluations were made at the beginning and end of the 1st, 7th, 13th and 18th weeks, when volume and training intensity have changed. The effects manifested at the beginning of those weeks were considered due to chronic adaptations, while the effects observed at the end of the weeks were considered due to acute manifestations of the exercise load of that week. Acute changes resulting from the exercise comprised increases in creatine kinase activity (CK) and leukocyte count (Leu), and decrease in hematocrit (Ht) and mean corpuscular volume (MCV), at the end of the first week; increase in the activities of CK and lactate dehydrogenase (LDH), in the uric acid (UA) concentration and Leu count, at the end of the seventh week; increases in CK and LDH activities and in the mean corpuscular hemoglobin concentration (MCHC), at the end of the 13th week; and decrease in the value of the osmotic stability index 1/H50 and increases in the CK activity and platelets (Plt) count, at the end of the 18th week. Chronic changes due to training comprised increase in the values of 1/H50, CK, LDH, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum iron (Fe), MCV and Plt. Although acute training has resulted in decrease in the osmotic stability of erythrocytes, possibly associated with exacerbation of the oxidative processes during intense exercise, chronic training over 18 weeks resulted in increased osmotic stability of erythrocytes, possibly by modulation in the membrane cholesterol content by low and high density lipoproteins. PMID:28151958

  18. Effects of acute and chronic exercise on the osmotic stability of erythrocyte membrane of competitive swimmers.

    PubMed

    Paraiso, Lara Ferreira; Gonçalves-E-Oliveira, Ana Flávia Mayrink; Cunha, Lucas Moreira; de Almeida Neto, Omar Pereira; Pacheco, Adriana Garcia; Araújo, Karinne Beatriz Gonçalves; Garrote-Filho, Mário da Silva; Bernardino Neto, Morun; Penha-Silva, Nilson

    2017-01-01

    This study aimed to evaluate the influence of acute and chronic exercise on erythrocyte membrane stability and various blood indices in a population consisting of five national-level male swimmers, over 18 weeks of training. The evaluations were made at the beginning and end of the 1st, 7th, 13th and 18th weeks, when volume and training intensity have changed. The effects manifested at the beginning of those weeks were considered due to chronic adaptations, while the effects observed at the end of the weeks were considered due to acute manifestations of the exercise load of that week. Acute changes resulting from the exercise comprised increases in creatine kinase activity (CK) and leukocyte count (Leu), and decrease in hematocrit (Ht) and mean corpuscular volume (MCV), at the end of the first week; increase in the activities of CK and lactate dehydrogenase (LDH), in the uric acid (UA) concentration and Leu count, at the end of the seventh week; increases in CK and LDH activities and in the mean corpuscular hemoglobin concentration (MCHC), at the end of the 13th week; and decrease in the value of the osmotic stability index 1/H50 and increases in the CK activity and platelets (Plt) count, at the end of the 18th week. Chronic changes due to training comprised increase in the values of 1/H50, CK, LDH, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum iron (Fe), MCV and Plt. Although acute training has resulted in decrease in the osmotic stability of erythrocytes, possibly associated with exacerbation of the oxidative processes during intense exercise, chronic training over 18 weeks resulted in increased osmotic stability of erythrocytes, possibly by modulation in the membrane cholesterol content by low and high density lipoproteins.

  19. UV-visible spectroscopy method for screening the chemical stability of potential antioxidants for proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Banham, Dustin; Ye, Siyu; Knights, Shanna; Stewart, S. Michael; Wilson, Mahlon; Garzon, Fernando

    2015-05-01

    A novel method based on UV-visible spectroscopy is reported for screening the chemical stability of potential antioxidant additives for proton exchange membrane fuel cells, and the chemical stabilities of three CeOx samples of varying crystallite sizes (6, 13, or 25 nm) are examined. The chemical stabilities predicted by this new screening method are compared to in-situ membrane electrode assembly (MEA) accelerated stress testing, with the results confirming that this rapid and inexpensive method can be used to accurately predict performance impacts of antioxidants.

  20. Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity.

    PubMed

    Chalbi, Najla; Martínez-Ballesta, Ma Carmen; Youssef, Nabil Ben; Carvajal, Micaela

    2015-03-01

    Changes in plasma membrane lipids, such as sterols and fatty acids, have been observed as a result of salt stress. These alterations, together with modification of the plasma membrane protein profile, confer changes in the physical properties of the membrane to be taken into account for biotechnological uses. In our experiments, the relationship between lipids and proteins in three different Brassicaceae species differing in salinity tolerance (Brassica oleracea, B. napus and Cakile maritima) and the final plasma membrane stability were studied. The observed changes in the sterol (mainly an increase in sitosterol) and fatty acid composition (increase in RUFA) in each species led to physical adaptation of the plasma membrane to salt stress. The in vitro vesicles stability was higher in the less tolerant (B. oleracea) plants together with low lipoxygenase activity. These results indicate that the proteins/lipids ratio and lipid composition is an important aspect to take into account for the use of natural vesicles in plant biotechnology.

  1. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    PubMed

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  2. Reconstruction of the Distal Oblique Bundle of the Interosseous Membrane: A Technique to Restore Distal Radioulnar Joint Stability.

    PubMed

    Riggenbach, Michael D; Wright, Thomas W; Dell, Paul C

    2015-11-01

    The distal radioulnar ligament reconstruction is a technique that may be used for distal radioulnar joint instability without arthritis and failed nonsurgical management; clinical results demonstrate resolved or improved stability. Recent literature has focused on the distal oblique bundle of the interosseous membrane and its contributions to stability. This article describes a technically simple surgical technique to reconstruct the distal oblique bundle and restore distal radioulnar joint stability.

  3. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein-induced lysosomal translocation of proapoptotic effectors is mediated by phosphofurin acidic cluster sorting protein-2 (PACS-2).

    PubMed

    Werneburg, Nathan W; Bronk, Steve F; Guicciardi, Maria Eugenia; Thomas, Laurel; Dikeakos, Jimmy D; Thomas, Gary; Gores, Gregory J

    2012-07-13

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis of liver cancer cell lines requires death receptor-5 (DR5)-dependent permeabilization of lysosomal membranes. Ligated DR5 triggers recruitment of the proapoptotic proteins Bim and Bax to lysosomes, releasing cathepsin B into the cytosol where it mediates mitochondria membrane permeabilization and activation of executioner caspases. Despite the requirement for lysosome membrane permeabilization during TRAIL-induced apoptosis, little is known about the mechanism that controls recruitment of Bim and Bax to lysosomal membranes. Here we report that TRAIL induces recruitment of the multifunctional sorting protein phosphofurin acidic cluster sorting protein-2 (PACS-2) to DR5-positive endosomes in Huh-7 cells where it forms an immunoprecipitatable complex with Bim and Bax on lysosomal membranes. shRNA-targeted knockdown of PACS-2 prevents recruitment of Bim or Bax to lysosomes, blunting the TRAIL-induced lysosome membrane permeabilization. Consistent with the reduced lysosome membrane permeabilization, shRNA knockdown of PACS-2 in Huh-7 cells reduced TRAIL-induced apoptosis and increased clonogenic cell survival. The determination that recombinant PACS-2 bound Bim but not Bax in vitro and that shRNA knockdown of Bim blocked Bax recruitment to lysosomes suggests that TRAIL/DR5 triggers endosomal PACS-2 to recruit Bim and Bax to lysosomes to release cathepsin B and induce apoptosis. Together, these findings provide insight into the lysosomal pathway of apoptosis.

  4. Impulse control disorder, lysosomal malfunction and ATP13A2 insufficiency in Parkinsonism.

    PubMed

    Liu, Jun-Ping; Li, Jianfeng; Lu, Yanhua; Wang, Lihui; Chen, Gang

    2017-02-01

    Lysosomal transport of cargos in neurons is essential for neuronal proteostasis, transmission and functional motors and behaviours. Lysosomal malfunction including storage disorders is involved in the pathogenesis of Parkinson's disease (PD). Given the unclear molecular mechanisms of diverse defects in PD phenotypes, especially behavioural deficits, this mini review explores the cellular contexts of PD impulse control disorders and the molecular aspects of lysosomal cross-membrane transports. Focuses are paid to trace metal involvements in α-synuclein assembly in Lewy bodies, the functions and molecular interactions of ATP13A2 as ATPase transporters in lysosomal membranes for cross-membrane trafficking and lysosomal homeostasis, and our current understandings of the neural circuits in ICD. Erroneously polarized distributions of cargos such as metals and lipids on each side of lysosomal membranes triggered by gene mutations and deregulated expression of ATP13A2 may thus instigate sensing protein structural changes such as aggregations, organelle degeneration, and specific neuronal ageing and death in Parkinsonism. © 2016 John Wiley & Sons Australia, Ltd.

  5. Poly(vinylbenzylchloride) Based Anion-Exchange Blend Membranes (AEBMs): Influence of PEG Additive on Conductivity and Stability

    PubMed Central

    Kerres, Jochen A.; Krieg, Henning M.

    2017-01-01

    In view of the many possible applications such as fuel cells and electrolysers, recent interest in novel anion exchange membranes (AEMs) has increased significantly. However, their low conductivity and chemical stability limits their current suitability. In this study, the synthesis and characterization of several three- and four-component anion exchange blend membranes (AEBMs) is described, where the compositions have been systematically varied to study the influence of the AEBM’s composition on the anion conductivities as well as chemical and thermal stabilities under strongly alkaline conditions. It was shown that the epoxide-functionalized poly(ethylene glycol)s that were introduced into the four-component AEBMs resulted in increased conductivity as well as a marked improvement in the stability of the AEBMs in an alkaline environment. In addition, the thermal stability of the novel AEBMs was excellent showing the suitability of these membranes for several electrochemical applications. PMID:28621717

  6. Poly(vinylbenzylchloride) Based Anion-Exchange Blend Membranes (AEBMs): Influence of PEG Additive on Conductivity and Stability.

    PubMed

    Kerres, Jochen A; Krieg, Henning M

    2017-06-16

    In view of the many possible applications such as fuel cells and electrolysers, recent interest in novel anion exchange membranes (AEMs) has increased significantly. However, their low conductivity and chemical stability limits their current suitability. In this study, the synthesis and characterization of several three- and four-component anion exchange blend membranes (AEBMs) is described, where the compositions have been systematically varied to study the influence of the AEBM's composition on the anion conductivities as well as chemical and thermal stabilities under strongly alkaline conditions. It was shown that the epoxide-functionalized poly(ethylene glycol)s that were introduced into the four-component AEBMs resulted in increased conductivity as well as a marked improvement in the stability of the AEBMs in an alkaline environment. In addition, the thermal stability of the novel AEBMs was excellent showing the suitability of these membranes for several electrochemical applications.

  7. Muscle intermediate filaments and their links to membranes and membranous organelles.

    PubMed

    Capetanaki, Yassemi; Bloch, Robert J; Kouloumenta, Asimina; Mavroidis, Manolis; Psarras, Stelios

    2007-06-10

    Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.

  8. Muscle intermediate filaments and their links to membranes and membranous organelles

    SciTech Connect

    Capetanaki, Yassemi . E-mail: ycapetanaki@bioacademy.gr; Bloch, Robert J.; Kouloumenta, Asimina; Mavroidis, Manolis; Psarras, Stelios

    2007-06-10

    Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.

  9. Specific lysosomal transport of small neutral amino acids

    SciTech Connect

    Pisoni, R.L.; Flickinger, K.S.; Thoene, J.G.; Christensen, H.N.

    1986-05-01

    Studies of amino acid exodus from lysosomes have allowed us previously to describe transport systems specific for cystine and another for cationic amino acids in fibroblast lysosomes. They are now able to study amino acid uptake into highly purified fibroblast lysosomes obtained by separating crude granular fraction on gradients formed by centrifugation in 35% isoosmotic Percoll solutions. Analog inhibition and saturation studies indicate that L-(/sup 14/C)proline (50 ..mu..M) uptake by fibroblast lysosomes at 37/sup 0/C in 50 mM citrate/tris pH 7.0 buffer containing 0.25 M sucrose is mediated by two transport systems, one largely specific for L-proline and the other for which transport is shared with small neutral amino acids such as alanine, serine and threonine. At 7 mM, L-proline inhibits L-(/sup 14/C)proline uptake almost completely, whereas ala, ser, val, thr, gly, N-methylalanine and sarcosine inhibit proline uptake by 50-65%. The system shared by alanine, serine and threonine is further characterized by these amino acids strongly inhibiting the uptakes of each other. Lysosomal proline transport is selective for the L-isomer of the amino acid, and is scarcely inhibited by 7 mM arg, glu, asp, leu, phe, his, met, (methylamino) isobutyrate, betaine or N,N-dimethylglycine. Cis or trans-4-hydroxy-L-proline inhibit proline uptake only slightly. In sharp contrast to the fibroblast plasma membrane in which Na/sup +/ is required for most proline and alanine transport, lysosomal uptake of these amino acids occurs independently of Na/sup +/.

  10. Isolation of Spheroplast Membranes and Stability of Spheroplasts of Bacillus stearothermophilus

    PubMed Central

    Bodman, Hollis; Welker, N. E.

    1969-01-01

    Spheroplasts were prepared by lysozyme digestion of the cell wall and ruptured by suspension in 0.15 m NaCl, followed by centrifugation at 30,900 × g for 35 min, and by a final suspension in 0.05 m NaCl for 12 to 16 hr at 5 C. The membrane ghosts were washed four times in tris(hydroxylmethyl)aminomethane (Tris) magnesium buffer and once in distilled water. The intact membranes resembled empty sacs with narrow slits in which the cytoplasm was extruded. A 92% recovery of cell membrane was obtained with all membrane preparations. The spheroplasts do not require a stabilizing medium to keep them from rupturing, and they are stable for 2 to 3 hr when exposed to a temperature of 65 C. The membrane content of the cell increases with age of culture (mid-log, 16.5%; late-log, 17.0%; and stationary, 17.6%) and temperature of growth (55 C, 16.5%; and 65 C, 17.8%), and it is unaffected by composition of the growth medium. The ratio of the protein to lipid content of the membrane increases with the complexity of the medium, age of culture (mid-log, 3.65; late-log, 3.91; and stationary, 4.15), and temperature of growth (55 C, 3.65; and 65 C, 5.22). The ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) content of the membranes was 9.0 to 13.7% and 0.3 to 0.8%, respectively. Reducing sugar (determined as glucose) amounts to 0.9 to 1.0% of the membrane weight and did not significantly vary for the different membrane preparations. Medium composition, age of culture, and temperature of growth have no significant effect on the amount of each amino acid in the membrane. Aspartic acid, glutamic acid, alanine, leucine, and lysine are present in the greatest amount and represent 12.9 to 14.1%, 10.4 to 11.3%, 9.6 to 10.3%, 7.7 to 8.8%, and 7.6 to 8.5% of the membrane peptide, respectively. Prior to the rupture of the spheroplasts, 25.0, 15.7, and 50.0% of the protein, RNA, and DNA, respectively, is lost. In potassium phosphate-magnesium buffer without sucrose, 90% of the protein and

  11. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    PubMed

    Burkard, Christine; Verheije, Monique H; Wicht, Oliver; van Kasteren, Sander I; van Kuppeveld, Frank J; Haagmans, Bart L; Pelkmans, Lucas; Rottier, Peter J M; Bosch, Berend Jan; de Haan, Cornelis A M

    2014-11-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  12. Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

    PubMed Central

    Burkard, Christine; Verheije, Monique H.; Wicht, Oliver; van Kasteren, Sander I.; van Kuppeveld, Frank J.; Haagmans, Bart L.; Pelkmans, Lucas; Rottier, Peter J. M.; Bosch, Berend Jan; de Haan, Cornelis A. M.

    2014-01-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion. PMID:25375324

  13. The Bilayer Enhances Rhodopsin Kinetic Stability in Bovine Rod Outer Segment Disk Membranes

    PubMed Central

    Corley, Scott C.; Sprangers, Peter; Albert, Arlene D.

    2011-01-01

    Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As determined by DSC, rhodopsin exhibited a scan-rate-dependent irreversible endothermic transition at all stages of solubilization. The transition temperature (Tm) decreased in the subsolubilizing stage. However, once the rhodopsin was in a micelle environment there was little change of the Tm as the phospholipid/rhodopsin ratio in the mixed micelles decreased during the fully solubilized stage. Rhodopsin thermal denaturation is consistent with the two-state irreversible model at all stages of solubilization. The activation energy of denaturation (Eact) was calculated from the scan rate dependence of the Tm and from the rate of rhodopsin thermal bleaching at all stages of solubilization. The Eact as determined by both techniques decreased in the subsolubilizing stage, but remained constant once fully solubilized. These results indicate the bilayer structure increases the Eact to rhodopsin denaturation. PMID:21689528

  14. Structural changes in bacteriorhodopsin in purple membranes induced by irreversible photobleaching with heterogeneous and homogeneous stability.

    PubMed

    Yokoyama, Yasunori; Sonoyama, Masashi; Mitaku, Shigeki

    2010-01-01

    Kinetic studies of irreversible photobleaching of bacteriorhodopsin (bR) in purple membrane (PM) at neutral pH have previously indicated the existence of two kinds of species which differ in their structural stability. bR was shown to have kinetically slow- and fast-decayed components with the faster one increasing with changes in intra- and intermolecular structures in darkness. However, our recent work reported that photobleaching kinetics above pH 10 were characterized by a single-decay component. In order to elucidate the factors responsible for the heterogeneous or homogeneous stability of photobleaching, we conducted investigations into the structural changes in bR in PM induced by photobleaching at pH 7 and 11 by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. ATR-FTIR spectra of bR photobleached at pH 7 and 11 showed that an increase in IR peak intensity at 1656 cm(-1) occurred simultaneously with decreases at 1666 cm(-1), indicating an alpha(II)-to-alpha(I) transition in transmembrane helices during photobleaching. The most significant change in IR spectra occurred at 1626 cm(-1) for samples photobleached at pH 7, and was attributed to structures formed between adjacent molecules. The origin of the heterogeneity of photobleaching is discussed on the basis of structural characteristics found in the bleached membranes.

  15. Phosphoinositides in the mammalian endo-lysosomal network

    PubMed Central

    Cullen, Peter J.; Carlton, Jeremy G.

    2014-01-01

    The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform. PMID:22374088

  16. Imidazoacridinone-dependent lysosomal photodestruction: a pharmacological Trojan horse approach to eradicate multidrug-resistant cancers

    PubMed Central

    Adar, Y; Stark, M; Bram, E E; Nowak-Sliwinska, P; van den Bergh, H; Szewczyk, G; Sarna, T; Skladanowski, A; Griffioen, A W; Assaraf, Y G

    2012-01-01

    Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC50 values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction. PMID:22476101

  17. Activation of Peroxisome Proliferator-activated Receptor α Induces Lysosomal Biogenesis in Brain Cells

    PubMed Central

    Ghosh, Arunava; Jana, Malabendu; Modi, Khushbu; Gonzalez, Frank J.; Sims, Katherine B.; Berry-Kravis, Elizabeth; Pahan, Kalipada

    2015-01-01

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174

  18. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture.

    PubMed

    Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2017-07-27

    TMEM175 is a lysosomal K(+) channel that is important for maintaining the membrane potential and pH stability in lysosomes. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K(+) channels and lacks the TVGYG selectivity filter motif found in these channels. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K(+) channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K(+) channel family.

  19. Differential stability of photosynthetic membranes and fatty acid composition at elevated temperature in Symbiodinium

    NASA Astrophysics Data System (ADS)

    Díaz-Almeyda, E.; Thomé, P. E.; El Hafidi, M.; Iglesias-Prieto, R.

    2011-03-01

    Coral reefs are threatened by increasing surface seawater temperatures resulting from climate change. Reef-building corals symbiotic with dinoflagellates in the genus Symbiodinium experience dramatic reductions in algal densities when exposed to temperatures above the long-term local summer average, leading to a phenomenon called coral bleaching. Although the temperature-dependent loss in photosynthetic function of the algal symbionts has been widely recognized as one of the early events leading to coral bleaching, there is considerable debate regarding the actual damage site. We have tested the relative thermal stability and composition of membranes in Symbiodinium exposed to high temperature. Our results show that melting curves of photosynthetic membranes from different symbiotic dinoflagellates substantiate a species-specific sensitivity to high temperature, while variations in fatty acid composition under high temperature rather suggest a complex process in which various modifications in lipid composition may be involved. Our results do not support the role of unsaturation of fatty acids of the thylakoid membrane as being mechanistically involved in bleaching nor as being a dependable tool for the diagnosis of thermal susceptibility of symbiotic reef corals.

  20. Membrane-anchoring stabilizes and favors secretion of New Delhi Metallo-β-lactamase

    PubMed Central

    González, Lisandro J.; Bahr, Guillermo; Nakashige, Toshiki G.; Nolan, Elizabeth M.; Bonomo, Robert A.; Vila, Alejandro J.

    2016-01-01

    Carbapenems, “last resort” β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, since these enzymes are readily degraded in their non-metallated form. However, the New Delhi Metallo-β-lactamase (NDM-1) is able to persist under conditions of metal depletion. NDM-1 is a lipidated protein anchored to the outer membrane of Gram-negative bacteria. Membrane-anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the blaNDM gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies. PMID:27182662

  1. Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery.

    PubMed

    Thibault, Marc; Lavertu, Marc; Astolfi, Mélina; Buschmann, Michael D

    2016-10-01

    Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.

  2. Activity-dependent trafficking of lysosomes in dendrites and dendritic spines.

    PubMed

    Goo, Marisa S; Sancho, Laura; Slepak, Natalia; Boassa, Daniela; Deerinck, Thomas J; Ellisman, Mark H; Bloodgood, Brenda L; Patrick, Gentry N

    2017-08-07

    In neurons, lysosomes, which degrade membrane and cytoplasmic components, are thought to primarily reside in somatic and axonal compartments, but there is little understanding of their distribution and function in dendrites. Here, we used conventional and two-photon imaging and electron microscopy to show that lysosomes traffic bidirectionally in dendrites and are present in dendritic spines. We find that lysosome inhibition alters their mobility and also decreases dendritic spine number. Furthermore, perturbing microtubule and actin cytoskeletal dynamics has an inverse relationship on the distribution and motility of lysosomes in dendrites. We also find trafficking of lysosomes is correlated with synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors. Strikingly, lysosomes traffic to dendritic spines in an activity-dependent manner and can be recruited to individual spines in response to local activation. These data indicate the position of lysosomes is regulated by synaptic activity and thus plays an instructive role in the turnover of synaptic membrane proteins. © 2017 Goo et al.

  3. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes

    PubMed Central

    1996-01-01

    We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRP-loaded lysosomes are lysosomal- associated membrane protein-1 (LAMP-1) positive, mannose-6-phosphate receptor (M6PR) negative. and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded. Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact. Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step. PMID:8601581

  4. The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

    PubMed Central

    Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

    2015-01-01

    The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

  5. Ferritin-stimulated lipid peroxidation, lysosomal leak, and macroautophagy promote lysosomal "metastability" in primary hepatocytes determining in vitro cell survival.

    PubMed

    Krenn, Margit A; Schürz, Melanie; Teufl, Bernhard; Uchida, Koji; Eckl, Peter M; Bresgen, Nikolaus

    2015-03-01

    Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.

  6. Stabilizing effects of coenzyme Q10 on potassium ion release, membrane potential and fluidity of rabbit red blood cells.

    PubMed

    Shinozawa, S; Araki, Y; Oda, T

    1980-09-01

    The effects of coenzyme Q10 (Co Q10) on potassium ion release, membrane potential and fluidity of rabbit red blood cells were studied. Co Q10 inhibited the increased potassium ion release induced by cetylamine or lysolecithin from the cells. Co Q10 slightly decreased the membrane potential monitored by changes in fluorescence intensity of cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide [diS-C3-(5)], and also slightly decreased the membrane fluidity measured by using 1,6-diphenyl-1,3,5-hexatriene (DPH). These effects of Co Q10 on the membrane are considered to be due to its membrane stabilizing activity by interaction with lipid bilayers of the membrane.

  7. Stability study and lyophilization of vitamin E-loaded nanocapsules prepared by membrane contactor.

    PubMed

    Khayata, N; Abdelwahed, W; Chehna, M F; Charcosset, C; Fessi, H

    2012-12-15

    In this research, we studied the accelerated stability of vitamin E-loaded nanocapsules (NCs) prepared by the nanoprecipitation method. Vitamin E-loaded NCs were optimized firstly at the laboratory scale and then scaled up using the membrane contactor technique. The optimum conditions of the membrane contactor preparation (pilot scale) produced vitamin E-loaded NCs with an average size of 253 nm, polydispersity index 0.19 and a zeta potential -16 mV. The average size, polydispersity index and zeta potential values were 185 nm, 0.12 and -15 mV, respectively for the NCs prepared at laboratory scale. No significant changes were noticed in these values after 3 and 6 months of storage at high temperature (40±2 °C) and relative humidity (75±5%) in spite of vitamin E sensitivity to light, heat and oxygen. The entrapment efficiency of NCs prepared at pilot scale was 97% at the beginning of the stability study, and became (95%, 59%) after 3 and 6 months of storage, respectively. These values at lab-scale were (98%, 96%, and 89%) at time zero and after 3 and 6 months of storage, respectively. This confirms the ability of vitamin E encapsulation to preserve its stability, which is one major goal of our work. Lyophilization of the optimized formula at lab-scale was also performed. Four types of cryoprotectants were tested (poly(vinyl pyrrolidone), sucrose, mannitol, and glucose). Freeze-dried NCs prepared with sucrose were found acceptable. The other lyophilized NCs obtained at different conditions presented large aggregates. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes

    PubMed Central

    Clafshenkel, William P.; Murata, Hironobu; Andersen, Jill; Creeger, Yehuda; Russell, Alan J.

    2016-01-01

    Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system. PMID:27331401

  9. The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes.

    PubMed

    Clafshenkel, William P; Murata, Hironobu; Andersen, Jill; Creeger, Yehuda; Koepsel, Richard R; Russell, Alan J

    2016-01-01

    Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system.

  10. Performance and long term stability of mesoporous silica membranes for desalination.

    PubMed

    Elma, Muthia; Yacou, Christelle; Diniz da Costa, João C; Wang, David K

    2013-07-12

    This work shows the preparation of silica membranes by a two-step sol-gel method using tetraethyl orthosilicate in ethanolic solution by employing nitric acid and ammonia as co-catalysts. The sols prepared in pH 6 resulted in the lowest concentration of silanol (Si-OH) species to improve hydrostability and the optimized conditions for film coating. The membrane was tested to desalinate 0.3-15 wt % synthetic sodium chloride (NaCl) solutions at a feed temperature of 22 °C followed by long term membrane performance of up to 250 h in 3.5 wt % NaCl solution. Results show that the water flux (and salt rejection) decrease with increasing salt concentration delivering an average value of 9.5 kg m-2 h-1 (99.6%) and 1.55 kg m-2 h-1 (89.2%) from the 0.3 and 15 wt % saline feed solutions, respectively. Furthermore, the permeate salt concentration was measured to be less than 600 ppm for testing conditions up to 5 wt % saline feed solutions, achieving below the recommended standard for potable water. Long term stability shows that the membrane performance in water flux was stable for up to 150 h, and slightly reduced from thereon, possibly due to the blockage of large hydrated ions in the micropore constrictions of the silica matrix. However, the integrity of the silica matrix was not affected by the long term testing as excellent salt rejection of >99% was maintained for over 250 h.

  11. Performance and Long Term Stability of Mesoporous Silica Membranes for Desalination

    PubMed Central

    Elma, Muthia; Yacou, Christelle; Diniz da Costa, João C.; Wang, David K.

    2013-01-01

    This work shows the preparation of silica membranes by a two-step sol-gel method using tetraethyl orthosilicate in ethanolic solution by employing nitric acid and ammonia as co-catalysts. The sols prepared in pH 6 resulted in the lowest concentration of silanol (Si–OH) species to improve hydrostability and the optimized conditions for film coating. The membrane was tested to desalinate 0.3–15 wt % synthetic sodium chloride (NaCl) solutions at a feed temperature of 22 °C followed by long term membrane performance of up to 250 h in 3.5 wt % NaCl solution. Results show that the water flux (and salt rejection) decrease with increasing salt concentration delivering an average value of 9.5 kg m–2 h–1 (99.6%) and 1.55 kg m–2 h–1 (89.2%) from the 0.3 and 15 wt % saline feed solutions, respectively. Furthermore, the permeate salt concentration was measured to be less than 600 ppm for testing conditions up to 5 wt % saline feed solutions, achieving below the recommended standard for potable water. Long term stability shows that the membrane performance in water flux was stable for up to 150 h, and slightly reduced from thereon, possibly due to the blockage of large hydrated ions in the micropore constrictions of the silica matrix. However, the integrity of the silica matrix was not affected by the long term testing as excellent salt rejection of >99% was maintained for over 250 h. PMID:24956942

  12. A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events.

    PubMed

    Kaufmann, A M; Goldman, S D B; Krise, J P

    2009-03-01

    Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders-Niemann-Pick type C, mucolipidosis type IV, and Sandhoff's disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.

  13. Association of myosin I alpha with endosomes and lysosomes in mammalian cells.

    PubMed

    Raposo, G; Cordonnier, M N; Tenza, D; Menichi, B; Dürrbach, A; Louvard, D; Coudrier, E

    1999-05-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIalpha) is associated with endosomes and lysosomes. We show that the overproduction of MMIalpha or the production of nonfunctional truncated MMIalpha affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIalpha, promote the dissociation of MMIalpha from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIalpha might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

  14. Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells

    PubMed Central

    Raposo, Graça; Cordonnier, Marie-Neige; Tenza, Danièle; Menichi, Bernadette; Dürrbach, Antoine; Louvard, Daniel; Coudrier, Evelyne

    1999-01-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes. PMID:10233157

  15. Alkaline stability of quaternary ammonium cations for alkaline fuel cell membranes and ionic liquids.

    PubMed

    Marino, M G; Kreuer, K D

    2015-02-01

    The alkaline stability of 26 different quaternary ammonium groups (QA) is investigated for temperatures up to 160 °C and NaOH concentrations up to 10 mol L(-1) with the aim to provide a basis for the selection of functional groups for hydroxide exchange membranes in alkaline fuel cells and of ionic-liquid cations stable in basic conditions. Most QAs exhibit unexpectedly high alkaline stability with the exception of aromatic cations. β-Protons are found to be far less susceptible to nucleophilic attack than previously suggested, whereas the presence of benzyl groups, nearby hetero-atoms, or other electron-withdrawing species promote degradation reactions significantly. Cyclic QAs proved to be exceptionally stable, with the piperidine-based 6-azonia-spiro[5.5]undecane featuring the highest half-life at the chosen conditions. Absolute and relative stabilities presented herein stand in contrast to literature data, the differences being ascribed to solvent effects on degradation.

  16. Plant plasma membrane aquaporins in natural vesicles as potential stabilizers and carriers of glucosinolates.

    PubMed

    Martínez-Ballesta, Maria Del Carmen; Pérez-Sánchez, Horacio; Moreno, Diego A; Carvajal, Micaela

    2016-07-01

    Their biodegradable nature and ability to target cells make biological vesicles potential nanocarriers for bioactives delivery. In this work, the interaction between proteoliposomes enriched in aquaporins derived from broccoli plants and the glucosinolates was evaluated. The vesicles were stored at different temperatures and their integrity was studied. Determination of glucosinolates, showed that indolic glucosinolates were more sensitive to degradation in aqueous solution than aliphatic glucosinolates. Glucoraphanin was stabilized by leaf and root proteoliposomes at 25°C through their interaction with aquaporins. An extensive hydrogen bond network, including different aquaporin residues, and hydrophobic interactions, as a consequence of the interaction between the linear alkane chain of glucoraphanin and Glu31 and Leu34 protein residues, were established as the main stabilizing elements. Combined our results showed that plasma membrane vesicles from leaf and root tissues of broccoli plants may be considered as suitable carriers for glucosinolate which stabilization can be potentially attributed to aquaporins. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Model Systems of Precursor Cellular Membranes: Long-Chain Alcohols Stabilize Spontaneously Formed Oleic Acid Vesicles

    PubMed Central

    Rendón, Adela; Carton, David Gil; Sot, Jesús; García-Pacios, Marcos; Montes, Ruth; Valle, Mikel; Arrondo, José-Luis R.; Goñi, Felix M.; Ruiz-Mirazo, Kepa

    2012-01-01

    Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid. PMID:22339864

  18. Nature of forces stabilizing the transmembrane protein bacteriorhodopsin in purple membrane

    PubMed Central

    Gibson, Nicholas J.; Cassim, Joseph Y.

    1989-01-01

    Analysis of the far-ultraviolet solution and the oriented-film circular dichroic (CD) spectra of the purple membrane (PM) has indicated that the α-helical segments of its sole protein bacteriorhodopsin (bR) can undergo a significant tilting from the normal to the membrane plane during light-dependent hydroxylamine-mediated bleaching of the bR. However, this drastic change in tertiary structure is free of any observable secondary structural changes. This phenomenon can provide an excellent means for studying the relative contributions of forces responsible for the stability of this transmembrane protein within the membrane bilayer. Perturbation of the PM by varying degrees of papain digestion (resulting in changes in the bR ranging from only an elimination of the long COOH-terminal tail to the additional eliminations of the short NH2-terminal tail and a number of linkage amino acids between the helical segments of the bR) and by chemical cross-linking with dimethyl adipimidate (resulting primarily in the formation of intramolecular cross-links) resulted in a significant increase in this bleaching-induced tilting in all cases except the one in which only the COOH-tail was eliminated. The most severe perturbation (2-wk papain digestion) increased the net tilt angle per segment from 24 to 39° with no indication of any secondary structural changes. Although these perturbations drastically reduced the structural stability of the bR to bleaching, they caused virtually no observable changes in the intramolecular structure of the bR or the supramolecular structure of the PM based on analysis of extensive absorption, linear dichroic, and CD spectra. In addition, study of the bleaching rates for the perturbed PM samples indicated that a linear correlation exists between the calculated initial bleaching rates and the net tilt angles. Considering the forces generally assumed to account for the stability of transmembrane proteins in membranes, (a) intersegmental hydrogen

  19. Artesunate Activates Mitochondrial Apoptosis in Breast Cancer Cells via Iron-catalyzed Lysosomal Reactive Oxygen Species Production*

    PubMed Central

    Hamacher-Brady, Anne; Stein, Henning A.; Turschner, Simon; Toegel, Ina; Mora, Rodrigo; Jennewein, Nina; Efferth, Thomas; Eils, Roland; Brady, Nathan R.

    2011-01-01

    The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment. PMID:21149439

  20. Genetic Regulation of Caenorhabditis elegans Lysosome Related Organelle Function

    PubMed Central

    Soukas, Alexander A.; Carr, Christopher E.; Ruvkun, Gary

    2013-01-01

    Lysosomes are membrane-bound organelles that contain acid hydrolases that degrade cellular proteins, lipids, nucleic acids, and oligosaccharides, and are important for cellular maintenance and protection against age-related decline. Lysosome related organelles (LROs) are specialized lysosomes found in organisms from humans to worms, and share many of the features of classic lysosomes. Defective LROs are associated with human immune disorders and neurological disease. Caenorhabditis elegans LROs are the site of concentration of vital dyes such as Nile red as well as age-associated autofluorescence. Even though certain short-lived mutants have high LRO Nile red and high autofluorescence, and other long-lived mutants have low LRO Nile red and low autofluorescence, these two biologies are distinct. We identified a genetic pathway that modulates aging-related LRO phenotypes via serotonin signaling and the gene kat-1, which encodes a mitochondrial ketothiolase. Regulation of LRO phenotypes by serotonin and kat-1 in turn depend on the proton-coupled, transmembrane transporter SKAT-1. skat-1 loss of function mutations strongly suppress the high LRO Nile red accumulation phenotype of kat-1 mutation. Using a systems approach, we further analyzed the role of 571 genes in LRO biology. These results highlight a gene network that modulates LRO biology in a manner dependent upon the conserved protein kinase TOR complex 2. The results implicate new genetic pathways involved in LRO biology, aging related physiology, and potentially human diseases of the LRO. PMID:24204312

  1. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter*

    PubMed Central

    Seebacher, Nicole A.; Lane, Darius J. R.; Jansson, Patric J.; Richardson, Des R.

    2016-01-01

    Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a “safe house” to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

  2. [Clinico-functional efficacy of medicinal and photon stabilization of cell membrane in patients with angina pectoris].

    PubMed

    Vasil'ev, A P; Senatorov, Iu N; Strel'tsova, N N; Gorbunova, T Iu

    2003-01-01

    Modification of erythrocytic membrane and the trend in clinicofunctional indices were studied in 90 patients with angina of effort (FC I-IV) in the course of treatment with a combination of membranoprotective drugs (group 1), magneto-laser radiation (group 2) and imitation of laser radiation (group 3). In patients of groups 1 and 2 the treatment resulted in stabilization of cell membrane accompanied with a hypotensive effect and increased exercise tolerance due to more effective cardiac performance.

  3. Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.

    PubMed

    Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A; Kappes, John C; DeLucas, Lawrence J; Riordan, John R; Urbatsch, Ina L; Hunt, John F; Brouillette, Christie G

    2014-06-01

    Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification. © 2014 The Protein Society.

  4. Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

    PubMed Central

    Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A; Kappes, John C; DeLucas, Lawrence J; Riordan, John R; Urbatsch, Ina L; Hunt, John F; Brouillette, Christie G

    2014-01-01

    Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification. PMID:24652590

  5. Stability of Mitochondrial Membrane Proteins in Terrestrial Vertebrates Predicts Aerobic Capacity and Longevity

    PubMed Central

    Kitazoe, Yasuhiro; Kishino, Hirohisa; Hasegawa, Masami; Matsui, Atsushi; Lane, Nick; Tanaka, Masashi

    2011-01-01

    The cellular energy produced by mitochondria is a fundamental currency of life. However, the extent to which mitochondrial (mt) performance (power and endurance) is adapted to habitats and life strategies of vertebrates is not well understood. A global analysis of mt genomes revealed that hydrophobicity (HYD) of mt membrane proteins (MMPs) is much lower in terrestrial vertebrates than in fishes and shows a strong negative correlation with serine/threonine composition (STC). Here, we present evidence that this systematic feature of MMPs was crucial for the evolution of large terrestrial vertebrates with high aerobic capacity. An Arrhenius-type equation gave positive correlations between STC and maximum life span (MLS) in terrestrial vertebrates (with a few exceptions relating to the lifestyle of small animals with a high resting metabolic rate [RMR]) and negative correlations in secondary marine vertebrates, such as cetaceans and alligators (which returned from land to water, utilizing buoyancy with increased body size). In particular, marked STC increases in primates (especially hominoids) among placentals were associated with very high MLS values. We connected these STC increases in MMPs with greater stability of respiratory complexes by estimating the degradation of the Arrhenius plot given by accelerating mtRMR up to mt maximum metabolic rate. Both mtRMR and HYD in terrestrial vertebrates decreased with increasing body mass. Decreases in mtRMR raise MMP stability when high mobility is not required, whereas decreased HYD may weaken this stability under the hydrophobic environment of lipid bilayer. High maximal metabolic rates (5–10 RMR), which we postulate require high MMP mobility, presumably render MMPs more unstable. A marked rise in STC may therefore be essential to stabilize MMPs, perhaps as dynamic supercomplexes, via hydrogen bonds associated with serine/threonine motifs. PMID:21824868

  6. Lysosomal Membrane Glycoproteins: Properties of LAMP-1 (Lysosome Associated Membrane Protein) and LAMP-2

    DTIC Science & Technology

    1985-01-01

    Hank’s Buffered Salt Solution " NP- 10, Nonidet - P40 - .A, Bovine Serum Albumin, FBS, Fetal Bovine Serum. *Present address: University of California... Nonidet -P-"- ( ’P- ,9,Particle Dta Inc., Elmhurst, IL), 5 mM EDTA, I mM phenyl7.eth/ls,,!f, nv fluori , . .nd 0.15 711 ,NaCl (Hughes and August, 1982...for 3 -ii n the cells were centrifuged at 100.000 x n for . e supernatants were dialyzed exhaustively auainst IK’is putter i. , 5 percent Nonidet P-40

  7. Prodigiosins uncouple lysosomal vacuolar-type ATPase through promotion of H+/Cl- symport.

    PubMed Central

    Ohkuma, S; Sato, T; Okamoto, M; Matsuya, H; Arai, K; Kataoka, T; Nagai, K; Wasserman, H H

    1998-01-01

    We reported previously [Kataoka, Muroi, Ohkuma, Waritani, Magae, Takatsuki, Kondo, Yamasaki and Nagai (1995) FEBS Lett. 359, 53-59] that prodigiosin 25-C (one of the red pigments of the prodigiosin group produced by micro-organisms like Streptomyces and Serratia) uncoupled vacuolar H+-ATPase, inhibited vacuolar acidification and affected glycoprotein processing. In the present study we show that prodigiosin, metacycloprodigiosin and prodigiosin 25-C, all raise intralysosomal pH through inhibition of lysosomal acidification driven by vacuolar-type (V-)ATPase without inhibiting ATP hydrolysis in a dose-dependent manner with IC50 values of 30-120 pmol/mg of protein. The inhibition against lysosomal acidification was quick and reversible, showing kinetics of simple non-competitive (for ATP) inhibition. However, the prodigiosins neither raised the internal pH of isolated lysosomes nor showed ionophoric activity against H+ or K+ at concentrations where they strongly inhibited lysosomal acidification. They required Cl- for their acidification inhibitory activity even when driven in the presence of K+ and valinomycin, suggesting that their target is not anion (chloride) channel(s). In fact, the prodigiosins inhibited acidification of proteoliposomes devoid of anion channels that were reconstituted from lysosomal vacuolar-type (V-)ATPase and Escherichia coli phospholipids. However, they did not inhibit the formation of an inside-positive membrane potential driven by lysosomal V-ATPase. Instead, they caused quick reversal of acidified pH driven by lysosomal V-ATPase and, in acidic buffer, produced quick acidification of lysosomal pH, both only in the presence of Cl-. In addition, they induced swelling of liposomes and erythrocytes in iso-osmotic ammonium salt of chloride but not of gluconate, suggesting the promotion of Cl- entry by prodigiosins. These results suggest that prodigiosins facilitate the symport of H+ with Cl- (or exchange of OH- with Cl-) through lysosomal

  8. Cell Division and Targeted Cell Cycle Arrest Opens and Stabilizes Basement Membrane Gaps

    PubMed Central

    Matus, David Q.; Chang, Emily; Makohon-Moore, Sasha C.; Hagedorn, Mary A.; Chi, Qiuyi; Sherwood, David R.

    2014-01-01

    Large gaps in basement membrane (BM) occur during organ remodeling and cancer cell invasion. Whether dividing cells, which temporarily reduce their attachment to BM, influence these breaches is unknown. Here we analyse uterine-vulval attachment during development across 21 species of rhabditid nematodes and find that the BM gap that forms between these organs is always bounded by a non-dividing vulval cell. Through cell cycle manipulation and live cell imaging in Caenorhabditis elegans, we show that actively dividing vulval cells facilitate enlargement of this breach by promoting BM movement. In contrast, targeted cell-cycle arrest halts BM movement and limits gap opening. Further, we demonstrate that the BM component laminin accumulates at the BM gap edge and promotes increased integrin levels in non-dividing vulval cells, stabilizing gap position. Together, these studies reveal that cell division can be used as a mechanism to regulate BM breaches, thus controlling the exchange of cells between tissues. PMID:24924309

  9. Antimicrobial Properties of Lysosomal Enzymes Immobilized on NH₂Functionalized Silica-Encapsulated Magnetite Nanoparticles.

    PubMed

    Bang, Seung Hyuck; Sekhon, Simranjeet Singh; Cho, Sung-Jin; Kim, So Jeong; Le, Thai-Hoang; Kim, Pil; Ahn, Ji-Young; Kim, Yang-Hoon; Min, Jiho

    2016-01-01

    The immobilization efficiency, antimicrobial activity and recovery of lysosomal enzymes on NH2 functionalized magnetite nanoparticles have been studied under various conditions. The immobi- lization efficiency depends upon the ratio of the amount of enzyme and magnetite and it shows an increase with magnetite concentration which is due to the presence of amine group at the magnetite surface that leads to a strong attraction. The optimized reaction time to immobilize the lysosomal enzymes on magnetite was determined by using a rolling method. The immobilization efficiency increases with reaction time and reached a plateau after 5 minutes and then remained constant for 10 minutes. However, after 30 minutes the immobilization efficiency decreased to 85%, which is due to the weaker electrostatic interactions between magnetite and detached lysosomal enzymes. The recovery and stability of immobilized lysosomal enzymes has also been studied. The antimicrobial activity was almost 100% but it decreased upon reuse and no activity was observed after its reuse for seven times. The storage stability of lysosomal enzymes as an antimicrobial agent was about 88%, which decreased to 53% after one day and all activity of immobilized lysosomal enzymes was maintained after five days. Thus, the lysosomal enzymes immobilized on magnetite nanoparticles could potentially be used as antimicrobial agents to remove bacteria.

  10. Sialic acid storage diseases. A multiple lysosomal transport defect for acidic monosaccharides.

    PubMed Central

    Mancini, G M; Beerens, C E; Aula, P P; Verheijen, F W

    1991-01-01

    A defective efflux of free sialic acid from the lysosomal compartment has been found in the clinically heterogeneous group of sialic acid storage disorders. Using radiolabeled sialic acid (NeuAc) as a substrate, we have recently detected and characterized a proton-driven carrier for sialic acid in the lysosomal membrane from rat liver. This carrier also recognizes and transports other acidic monosaccharides, among which are uronic acids. If no alternative routes of glucuronic acid transport exist, the disposal of uronic acids can be affected in the sialic acid storage disorders. In this study we excluded the existence of more than one acidic monosaccharide carrier by measuring uptake kinetics of labeled glucuronic acid [( 3H]GlcAc) in rat lysosomal membrane vesicles. [3H]GlcAc uptake was carrier-mediated with an affinity constant of transport (Kt) of 0.3 mM and the transport could be cis-inhibited or trans-stimulated to the same extent by sialic acid or glucuronic acid. Human lysosomal membrane vesicles isolated from cultured fibroblasts showed the existence of a similar proton-driven transporter with the same properties as the rat liver system (Kt of [3H]GlcAc uptake 0.28 mM). Uptake studies with [3H]NeuAc and [3H]GlcAc in resealed lysosome membrane vesicles from cultured fibroblasts of patients with different clinical presentation of sialic acid storage showed defective carrier-mediated transport for both sugars. Further evidence that the defective transport of acidic sugars represents the primary genetic defect in sialic acid storage diseases was provided by the observation of reduced, half-normal transport rates in lymphoblast-derived lysosomal membrane vesicles from five unrelated obligate heterozygotes. This study reports the first observation of a human lysosomal transport defect for multiple physiological compounds. PMID:2010546

  11. Stability of milk fat globule membrane proteins toward human enzymatic gastrointestinal digestion.

    PubMed

    Le, T T; Van de Wiele, T; Do, T N H; Debyser, G; Struijs, K; Devreese, B; Dewettinck, K; Van Camp, J

    2012-05-01

    The milk fat globule membrane (MFGM) fraction refers to the thin film of polar lipids and membrane proteins that surrounds fat globules in milk. It is its unique biochemical composition that renders MFGM with some beneficial biological activities, such as anti-adhesive effects toward pathogens. However, a prerequisite for the putative bioactivity of MFGM is its stability during gastrointestinal digestion. We, therefore, subjected MFGM material, isolated from raw milk, to an in vitro enzymatic gastrointestinal digestion. Sodium dodecyl sulfate PAGE, in combination with 2 staining methods, Coomassie Blue and periodic acid Schiff staining, was used to evaluate polypeptide patterns of the digest, whereas mass spectrometry was used to confirm the presence of specific MFGM proteins. Generally, it was observed that glycoproteins showed higher resistance to endogenous proteases compared with non-glycosylated proteins. Mucin 1 displayed the highest resistance to digestion and a considerable part of this protein was still detected at its original molecular weight after gastric and small intestine digestion. Cluster of differentiation 36 was also quite resistant to pepsin. A significant part of periodic acid Schiff 6/7 survived the gastric digestion, provided that the lipid moiety was not removed from the MFGM material. Overall, MFGM glycoproteins are generally more resistant to gastrointestinal digestion than serum milk proteins and the presence of lipids, besides glycosylation, may protect MFGM glycoproteins from gastrointestinal digestion. This gastrointestinal stability makes MFGM glycoproteins amenable to further studies in which their putative health-promoting effects can be explored. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  12. Pluronic F127 as a cell encapsulation material: utilization of membrane-stabilizing agents.

    PubMed

    Khattak, Sarwat F; Bhatia, Surita R; Roberts, Susan C

    2005-01-01

    Thermoreversible gelation of the copolymer Pluronic F127 (generic name, poloxamer 407) in water makes it a unique candidate for cell encapsulation applications, either alone or to promote cell seeding and attachment in tissue scaffolds. At concentrations of 15-20% (w/w), aqueous Pluronic F127 (F127) solutions gel at physiological temperatures. The effect of F127 on viability and proliferation of human liver carcinoma cells (HepG2) was determined for both liquid and gel formulations. Cell concentration and viability over a 5-day period were measured by the trypan blue assay via hemocytometry and results were confirmed in both the MTT and LDH assays. With 0.1-5% (w/w) F127 (liquid), cells proliferated and maintained high viability over 5 days. However, at 10% (w/w) F127 (liquid), there was a significant decrease in cell viability and no cell proliferation was evident. HepG2 cell encapsulation in F127 concentrations ranging from 15 to 20% (w/w) (gel) resulted in complete cell death by 5 days. This was also true for the HMEC-1 (endothelial) and L6 (muscle) cell lines evaluated. Cell-seeding density did not affect cell survival or proliferation. Membrane-stabilizing agents (hydrocortisone, glucose, and glycerol) were added to the F127 gel formulations to improve cell viability. The steroid hydrocortisone demonstrated the most significant improvement in viability, from <2% (in F127 alone) to >70% (with 60 nM hydrocortisone added). These results suggest that F127 formulations supplemented with membrane-stabilizing agents can serve as viable cell encapsulation materials. In addition, hydrocortisone may be generally useful in the promotion of cell viability for a wide range of encapsulation materials.

  13. Jobelyn® exhibited anti-inflammatory, antioxidant, and membrane-stabilizing activities in experimental models.

    PubMed

    Umukoro, Solomon; Oluwole, Oluwafemi Gabriel; Eduviere, Anthony T; Adrian, Omogbiya Itievere; Ajayi, Abayomi M

    2015-09-01

    Jobelyn® (JB) is an African sorghum-based food supplement claimed to be efficacious for the treatment of rheumatoid arthritis (RA). Although in vitro studies confirmed its anti-inflammatory property, no study had shown the effect of JB using in vivo animal models of inflammation. Thus, its effects on acute and chronic inflammation in rats were evaluated in this study. Its effect on rat red blood cell (RBC) lysis was also assessed. Acute inflammation was induced with intraplanter injection of carrageenan and increase in rat paw volume was measured using plethysmometer. The volume of fluid exudates, number of leukocytes, concentrations of malondialdehyde (MDA), and glutathione (GSH) in the fluid were measured on day 5 after induction of chronic inflammation with carrageenan in the granuloma air pouch model. RBC lysis induced by hypotonic medium as determined by release of hemoglobin was measured spectrophotometerically. JB (50-200 mg/kg) given orally produced a significant inhibition of acute inflammation induced by carrageenan in rats. It reduced the volume and number of leukocytes in inflammatory fluid in the granuloma air pouch model of chronic inflammation. It further decreased the levels of MDA in the fluid suggesting antioxidant property. JB elevated the concentrations of GSH in inflammatory exudates indicating free radical scavenging activity. It also significantly inhibited RBC lysis caused by hypotonic medium, suggesting membrane-stabilizing property. JB has in vivo anti-inflammatory activity, which may be related to its antioxidant and membrane-stabilizing properties, supporting its use for the treatment of arthritic disorder.

  14. The influence of a membrane environment on the structure and stability of a prokaryotic potassium channel, KcsA.

    PubMed

    Encinar, J A; Molina, M L; Poveda, J A; Barrera, F N; Renart, M L; Fernández, A M; González-Ros, J M

    2005-09-26

    The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440-446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA a