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Sample records for lysosomal membrane stability

  1. Stability of lysosomal membrane in Carcinus maenas acts as a biomarker of exposure to pharmaceuticals.

    PubMed

    Aguirre-Martínez, G V; Buratti, S; Fabbri, E; Del Valls, T A; Martín-Díaz, M L

    2013-05-01

    The presence of pharmaceuticals in the environment is now a major concern given their potential adverse effects on organisms, particularly human beings. Because the feeding style and habitat of the crab Carcinus maenas make this species vulnerable to organic contaminants, it has been used previously in ecotoxicological studies. Lysosomal membrane stability (LMS) in crabs is a general indicator of cellular well-being and can be visualized by the neutral red retention (NRR) assay. LMS in crab hemolymph has been evaluated as a cellular biomarker of adverse effects produced by exposure to pharmaceutical compounds. Crabs were exposed in the laboratory to four different pharmaceuticals for 28 days in a semistatic 24-h renewal assay. Filtered seawater was spiked every 2 days with various concentrations (from 0.1 to 50 μg · L(-1)) of caffeine, ibuprofen, carbamazepine, and novobiocin. Results showed that NRR time, measured at day 28, was significantly reduced (p < 0.05) after exposure to environmental concentrations of each pharmaceutical (caffeine = 15 μg · L(-1); carbamazepine = 1 μg · L(-1); ibuprofen = 5 μg · L(-1); and novobiocin = 0.1 μg · L(-1)) when compared with control organisms. The predicted "no environmental effect" concentration/measured environmental concentration results showed that the selected pharmaceuticals are toxic at environmental concentrations and need further assessment. LMS monitoring in crabs is a sensitive tool for evaluating exposure to concentrations of selected drugs under laboratory conditions and provides a robust tier 1 testing approach (screening biomarker) for rapid assessment of marine pollution and environmental impact assessments for analyzing pharmaceutical contamination in aquatic environments. PMID:23132752

  2. Lysosomes and the plasma membrane

    PubMed Central

    Andrews, Norma W.

    2002-01-01

    Studies of the cell invasion mechanism of the parasite Trypanosoma cruzi led to a series of novel findings, which revealed a previously unsuspected ability of conventional lysosomes to fuse with the plasma membrane. This regulated exocytic process, previously regarded mostly as a specialization of certain cell types, was recently shown to play an important role in the mechanism by which cells reseal their plasma membrane after injury. PMID:12147679

  3. Effect of reactive oxygen species on lysosomal membrane integrity. A study on a lysosomal fraction.

    PubMed

    Zdolsek, J M; Svensson, I

    1993-01-01

    Using a lysosome-enriched "light mitochondrial" fraction of a rat liver homogenate, the effects of the reactive oxygen species hydrogen peroxide, superoxide- and hydroxyl radicals were determined. Alterations in the intralysosomal pH and the release of a lysosomal marker enzyme, N-acetyl-glucosaminidase, were used as indicators of changes in the lysosomal membrane integrity. Lipid peroxidation of the fraction was assayed by TBARS measurement. Neither superoxide radicals, generated by hypoxanthine/xanthine oxidase, nor a bolus dose of hydrogen peroxide (0.5-1.5 mM) induced any lysosomal damage. If, however, Fe(III)ADP was included in the superoxide radical-generating system, lysosomal membrane damage was detected, both as an increase in lysosomal pH and as a release of N-acetyl-glucosaminidase, but only after a lag phase of about 7 min. Lipid peroxidation, on the other hand, proceeded gradually. Lysosomes treated with hydrogen peroxide displayed similar dose-dependent alterations, albeit only if both Fe(III)ADP and the reducing amino acid cysteine were added. In the latter system, however, alterations of the lysosomal membrane stability occurred more rapidly, showing a lag phase of only 2 min. Lipid peroxidation, which proceeded faster and displayed no lag phase, levelled out within 10 min. The results indicate that neither superoxide radicals nor hydrogen peroxide are by themselves damaging to lysosomes. Available catalytically active iron in Fe(II) form, however, allows reactions yielding powerful oxidative species--probably hydroxyl radicals formed via Fenton reactions--to take place inducing peroxidation of the lysosomal membranes resulting in dissipation of the proton-gradient and leakage of their enzyme contents. PMID:8148962

  4. 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.

  5. 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

  6. 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.

  7. 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. PMID:27102538

  8. Strategies for Assaying Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Hafner Česen, Maruša; Turk, Boris

    2016-01-01

    Late endosomal organelles have an acidic pH and contain hydrolytic enzymes to degrade cargo delivered either from the extracellular environment by endocytosis or from within the cell itself by autophagy. In the event of lysosomal membrane permeabilization (LMP), the contents of late endosomes and lysosomes can be released into the cytosol and then initiate apoptosis. Compounds that can trigger LMP are therefore candidates for the induction of apoptosis, in particular in anticancer therapy. Alternatively, drug-delivery systems, such as nanoparticles, can have side effects that can include LMP, which has toxic consequences for the cells. To determine when, to what extent, and with what consequences LMP occurs is therefore of paramount importance for the evaluation of new potentially LMP-inducing compounds. In this introduction, we provide an overview of some basic assays for assessing LMP, such as staining with lysosomotropic dyes and measurement of cysteine cathepsin activity, and discuss additional strategies for the detection of the release of endogenous lysosomal molecules or preloaded exogenous tracers into the cytosol. PMID:27250949

  9. Ecocytological and toxicological responses to copper in Perna viridis (L.) (Bivalvia: Mytilidae) haemocyte lysosomal membranes.

    PubMed

    Nicholson, S

    2001-11-01

    Bivalve lysosomes are sites of intense intracellular digestion. Lysosomes accumulate many pollutants to high concentrations resulting in membrane destabilisation. Consequently, the elucidation of lysosomal membrane integrity utilising the neutral red assay has been used to good effect in pollution monitoring. Naturally occurring environmental stressors also have the potential to destabilise the membrane. Exposure to elevated copper concentrations and extremes of temperature, salinity, hypoxia, emersion and inadequate ration were investigated in haemocyte lysosomes from the tropical bivalve, Perna viridis. Elevated copper concentrations destabilised the membrane although responses were not entirely related to the exposure-concentration. Environmental stressors induced through higher thermal regimes (29 degrees C and 35 degrees C), hyposalinity (10-25/1000) and prolonged emersion elicited significant lysosomal membrane destabilisation. Hypoxia and inadequate ration did not significantly effect membrane stability. The haemocyte lysosomal membranes were generally resistant to exogenous alterations within normal ranges and only showed significant labilisation at environmental extremes. P. viridis haemocyte lysosomal membrane biomarkers should, therefore, prove robust to natural stressors when deployed in marine monitoring programmes and thus prove a valuable, rapid, cost-effective cytological marker of pollution. PMID:11680735

  10. Neuraminidase of Influenza A Virus Binds Lysosome-Associated Membrane Proteins Directly and Induces Lysosome Rupture

    PubMed Central

    Ju, Xiangwu; Yan, Yiwu; Liu, Qiang; Li, Ning; Sheng, Miaomiao; Zhang, Lifang; Li, Xiao; Liang, Zhu; Huang, Fengming; Liu, Kangtai; Zhao, Yan; Zhang, Yanxu; Zou, Zhen; Du, Jianchao; Zhong, Ying; Zhou, Huandi; Yang, Peng; Lu, Huijun; Tian, Mingyao; Li, Dangsheng; Zhang, Jianming

    2015-01-01

    ABSTRACT As a recycling center, lysosomes are filled with numerous acid hydrolase enzymes that break down waste materials and invading pathogens. Recently, lysosomal cell death has been defined as “lysosomal membrane permeabilization and the consequent leakage of lysosome contents into cytosol.” Here, we show that the neuraminidase (NA) of H5N1 influenza A virus markedly deglycosylates and degrades lysosome-associated membrane proteins (LAMPs; the most abundant membrane proteins of lysosome), which induces lysosomal rupture, and finally leads to cell death of alveolar epithelial carcinoma A549 cells and human tracheal epithelial cells. The NA inhibitors peramivir and zanamivir could effectively block the deglycosylation of LAMPs, inhibit the virus cell entry, and prevent cell death induced by the H5N1 influenza virus. The NA of seasonal H1N1 virus, however, does not share these characteristics. Our findings not only reveal a novel role of NA in the early stage of the H5N1 influenza virus life cycle but also elucidate the molecular mechanism of lysosomal rupture crucial for influenza virus induced cell death. IMPORTANCE The integrity of lysosomes is vital for maintaining cell homeostasis, cellular defense and clearance of invading pathogens. This study shows that the H5N1 influenza virus could induce lysosomal rupture through deglycosylating lysosome-associated membrane proteins (LAMPs) mediated by the neuraminidase activity of NA protein. NA inhibitors such as peramivir and zanamivir could inhibit the deglycosylation of LAMPs and protect lysosomes, which also further interferes with the H5N1 influenza virus infection at early stage of life cycle. This work is significant because it presents new concepts for NA's function, as well as for influenza inhibitors' mechanism of action, and could partially explain the high mortality and high viral load after H5N1 virus infection in human beings and why NA inhibitors have more potent therapeutic effects for lethal avian

  11. TMEPAI increases lysosome stability and promotes autophagy.

    PubMed

    Luo, Shenheng; Yang, Meng; Lv, Dan; Jing, Lei; Li, Yuyin; Liu, Zhenxing; Diao, Aipo

    2016-07-01

    Autophagy is emerging as a critical response of normal and cancer cells to environmental changes and plays an important role in cell metabolism and maintenance of damaged organelles. Transmembrane prostate androgen-induced protein (TMEPAI) is a pro-tumorigenic factor with high expression in tumor cells. In this study, we showed that depletion of TMEPAI leads to lysosomal labilization and inhibits autophagy. Further study showed that the inhibition of autophagy induced by the depletion of TMEPAI is involved in regulation of Beclin-1. Depletion of TMEPAI increases the sensitivity of cancer cells to chemotherapeutic drugs. Our study reveals the role of TMEPAI in promoting lysosome stability and autophagy, which might be used as a target for cancer chemotherapeutic treatment. PMID:27163528

  12. 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. PMID:24218854

  13. Presence of detergent-resistant microdomains in lysosomal membranes.

    PubMed

    Taute, Antje; Wätzig, Kristin; Simons, Brigitte; Lohaus, Christiane; Meyer, Helmut; Hasilik, Andrej

    2002-10-18

    We examined the association of acetyl-CoA:alpha-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 degrees C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts. PMID:12379211

  14. Calpain 1 induce lysosomal permeabilization by cleavage of lysosomal associated membrane protein 2.

    PubMed

    Villalpando Rodriguez, Gloria E; Torriglia, Alicia

    2013-10-01

    In light induced retinal degeneration (LIRD) photoreceptor cell death is mediated by caspase independent mechanisms. The activation of LEI/L-DNase II pathway in this model, is due to cathepsin D release from lysosomes, although the underlying mechanism remains poorly understood. In this paper we studied the involvement of calpains in lysosomal permeabilization. We investigated, for the first time, the calpain targets at lysosomal membrane level. We found that calpain 1 is responsible for lysosomal permeabilization by cleavage of the lysosomal associated membrane protein 2 (LAMP 2). Moreover, LAMP 2 degradation and lysosomal permeabilization were rescued by calpain inhibition and the use of MEF(-/-)lamp 2 cells indicates that the cleavage of LAMP 2A is essential for this permeabilization. Finally, we found that LAMP 2 is cleaved in LIRD, suggesting that the mechanism of calpain induced lysosomal permeabilization is not exclusive of a single cell death model. Overall, these data shed new light on understanding the mechanisms of lysosomal and caspase-independent cell death and point to the original targets for development of the new therapeutic protocols. PMID:23747342

  15. 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.

  16. 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. PMID:12820995

  17. 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.

  18. 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. PMID:23071517

  19. Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

    2016-01-01

    During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed. PMID:27140915

  20. 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

    2015-04-01

    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. PMID:25860611

  1. Lysosomal Integral Membrane Protein-2: A New Player in Lysosome-Related Pathology

    PubMed Central

    Gonzalez, Ashley; Valeiras, Mark; Sidransky, Ellen; Tayebi, Nahid

    2014-01-01

    Lysosomes require the presence of many specialized proteins to facilitate their roles in cellular maintenance. One such protein that has proven to be an important player in the lysosomal field is lysosomal integral membrane protein-2 (LIMP-2), encoded by the gene SCARB2. LIMP-2 is required for the normal biogenesis and maintenance of lysosomes and endosomes and has been identified as the specific receptor for glucocerebrosidase, the enzyme deficient in Gaucher disease. Research into LIMP-2 and the SCARB2 gene indicate that it may be a factor contributing to the clinical heterogeneity seen among patients with Gaucher disease. Mutations in SCARB2 have also been identified as the cause of action myoclonus renal failure (AMRF), and in some cases progressive myoclonic epilepsy. A total of 14 disease-causing SCARB2 mutations have been identified to date. The role of LIMP-2 in human pathology has expanded with its identification as a component of the intercalated disc in cardiac muscle and as a receptor for specific enteroviruses, two unanticipated findings that reaffirm the myriad roles of lysosomal proteins. Studies into the full impact of LIMP-2 deficiency and the LIMP2/glucocerebrosidase molecular pathway will lead to a better understanding of disease pathogenesis in Gaucher disease and AMRF, and to new insights into lysosomal processing, trafficking and function. PMID:24389070

  2. Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

    SciTech Connect

    Haylett, T.; Thilo, L.

    1986-10-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D/sub 1/, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from <0.1% to a steady-state level of approx.2.5% of the total label. As analyzed by NaDodSO/sub 4/ PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only approx.1% of internalized membrane is recycled via a membrane pool of secondary lysosomes.

  3. (-)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization

    PubMed Central

    LeGendre, Onica; Breslin, Paul AS; Foster, David A

    2015-01-01

    (-)-Oleocanthal (OC), a phenolic compound present in extra-virgin olive oil (EVOO), has been implicated in the health benefits associated with diets rich in EVOO. We investigated the effect of OC on human cancer cell lines in culture and found that OC induced cell death in all cancer cells examined as rapidly as 30 minutes after treatment in the absence of serum. OC treatment of non-transformed cells suppressed their proliferation but did not cause cell death. OC induced both primary necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP). We provide evidence that OC promotes LMP by inhibiting acid sphingomyelinase (ASM) activity, which destabilizes the interaction between proteins required for lysosomal membrane stability. The data presented here indicate that cancer cells, which tend to have fragile lysosomal membranes compared to non-cancerous cells, are susceptible to cell death induced by lysosomotropic agents. Therefore, targeting lysosomal membrane stability represents a novel approach for the induction of cancer-specific cell death. PMID:26380379

  4. 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

  5. ESeroS-GS Protects Neuronal Cells from Oxidative Stress by Stabilizing Lysosomes.

    PubMed

    Yang, Na; Chen, Qianqian; He, Xiaolong; Zhao, Xingyu; Wei, Taotao

    2016-01-01

    γ-l-glutamyl-S-[2-[[[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl]oxy]carbonyl]-3-[[2-(1H-indol-3-yl)ethyl]amino]-3-oxopropyl]-l-cysteinylglycine sodium salt (ESeroS-GS) is a water-soluble derivative of α-tocopherol (vitamin E). We reported previously that ESeroS-GS can act as an anti-inflammatory agent and can induce cell death in breast cancer cells. However, the potential antioxidant capacities of ESeroS-GS remain elusive. Here, we measured its scavenging effects on free radicals and evaluated its protective effects on neuronal cells against oxidative stress. The results indicated that ESeroS-GS effectively scavenged both 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonate free radicals (ABTS(•+)) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals, and attenuated H₂O₂-induced neuronal cell death. H₂O₂ treatment induced lysosomal membrane permeabilization rapidly, and caused the redistribution of lysosomal proteases, which were responsible for the neuronal cell death. ESeroS-GS abolished the interaction between tBid and the lysosomal membranes, blocked the translocation of tBid to the lysosomal membranes, decreased its oligomerization within the membrane circumstances, prevented the lysosomal membrane permeabilization, and thus attenuated the neuronal cell death. These data suggest that ESeroS-GS protected the neuronal cells from oxidative stress by stabilizing lysosomal membranes, and thus might act as a novel neuroprotector for neuronal diseases associated with oxidative stress. PMID:27231890

  6. The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization.

    PubMed

    Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

    2016-01-01

    Progressive lowering of pH is characteristic for the endocytic pathway and enables efficient degradation of molecules by hydrolytic enzymes at its distal end. The existence of the proton gradient over the endosomal/lysosomal membranes depends on the action of the vacuolar ATPase (v-ATPase). During lysosomal membrane permeabilization (LMP), protons leak through the destabilized membrane, resulting in loss of the pH gradient. Here, we present a protocol showing how this effect can be detected by staining cells with lysosomotropic dyes, which accumulate in acidic organelles after protonation. During LMP, cells lose the ability to retain these dyes and therefore appear pale. Among the most commonly used lysosomotropic dyes are LysoTracker reagents and acridine orange. Cells can be analyzed with a fluorescence microscope; however, flow-cytometric analysis enables fast, objective, and reliable evaluation of differences between samples. Advantages of the technique include the fact that sample preparation is relatively simple and can be scaled-up to test several different compounds or conditions. However, as we will discuss, cells treated with v-ATPase inhibitors also lose the pH gradient across lysosomal membranes and cannot be stained with lysosomotropic dyes, although this is not accompanied by LMP. Therefore, merely observing loss of staining is not in itself a proof of LMP. PMID:27140914

  7. Regulation of membrane trafficking by signalling on endosomal and lysosomal membranes

    PubMed Central

    Li, Xinran; Garrity, Abigail G; Xu, Haoxing

    2013-01-01

    Endosomal and lysosomal membrane trafficking requires the coordination of multiple signalling events to control cargo sorting and processing, and endosome maturation. The initiation and termination of signalling events in endosomes and lysosomes is not well understood, but several key regulators have been identified, which include small GTPases, phosphoinositides, and Ca2+. Small GTPases act as master regulators and molecular switches in a GTP-dependent manner, initiating signalling cascades to regulate the direction and specificity of endosomal trafficking. Phosphoinositides are membrane-bound lipids that indicate vesicular identities for recruiting specific cytoplasmic proteins to endosomal membranes, thus allowing specificity of membrane fusion, fission, and cargo sorting to occur within and between specific vesicle compartments. In addition, phosphoinositides regulate the function of membrane proteins such as ion channels and transporters in a compartment-specific manner to mediate transport and signalling. Finally, Ca2+, a locally acting second messenger released from intracellular ion channels, may provide precise spatiotemporal regulation of endosomal signalling and trafficking events. Small GTPase signalling can regulate phosphoinositide conversion during endosome maturation, and electrophysiological studies on isolated endosomes have shown that endosomal and lysosomal Ca2+ channels are directly modulated by endosomal lipids. Thus trafficking and maturation of endosomes and lysosomes can be precisely regulated by dynamic changes in GTPases and membrane lipids, as well as Ca2+ signalling. Importantly, impaired phosphoinositide and Ca2+ signalling can cause endosomal and lysosomal trafficking defects at the cellular level, and a spectrum of lysosome storage diseases. PMID:23878375

  8. 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. PMID:26302210

  9. 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. PMID:22729905

  10. Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons

    PubMed Central

    YAN, MIN; ZHU, WENBO; ZHENG, XIAOKE; LI, YUAN; TANG, LIPENG; LU, BINGZHENG; CHEN, WENLI; QIU, PENGXIN; LENG, TIANDONG; LIN, SUIZHEN; YAN, GUANGMEI; YIN, WEI

    2016-01-01

    Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death in non-human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal membranes as assessed by acridine orange redistribution and immunofluorescence of cathepsin B in the cytoplasm. Inhibition of glutamate excitotoxicity by the NMDA receptor antagonist MK-801 and the calcium chelator ethylene glycolbis (2-aminoethylether)-N, N, N′, N′-tetraacetic acid, rescued lysosomes from permeabilization. The role of calpain and reactive oxygen species (ROS) in inducing LMP was also investigated. Ca2+ overload following glutamate treatment induced the activation of calpain and the production of ROS, which are two major contributors to neuronal death. It has been reported that lysosomal-associated membrane protein 2 (LAMP2) and heat shock protein (HSP)70 are two calpain substrates that promote LMP in cancer cells; however, it was found that calpains were activated by glutamate, but only LAMP2 was subsequently degraded. Furthermore, LMP was not alleviated by treatment with the calpain inhibitors calpeptin and SJA6017, which blocked the cleavage of the calpain substrate α-fodrin. It was demonstrated that LMP was significantly alleviated by treatment with the antioxidant N-Acetyl-L-cysteine, indicating that LMP involvement in early glutamate excitotoxicity may be mediated partly by ROS rather than calpain activation. Overall, these data shed light on the role of ROS-mediated LMP in early glutamate excitotoxicity. PMID:26821268

  11. Lysosomal involvement in cellular turnover of plasma membrane sphingomyelin.

    PubMed

    Sutrina, S L; Chen, W W

    1984-04-18

    At least two isoenzymes of sphingomyelinase (sphingomyelin cholinephosphohydrolase, EC 3.1.4.12), including lysosomal acid sphingomyelinase and nonlysosomal magnesium-dependent neutral sphingomyelinase, catalyse the degradation of sphingomyelin in cultured human skin fibroblasts. A genetically determined disorder of sphingomyelin metabolism, type A Niemann-Pick disease, is characterized by a deficiency of lysosomal acid sphingomyelinase. To investigate the involvement of lysosomes in the degradation of cellular membrane sphingomyelin, we have undertaken studies to compare the turnover of plasma membrane sphingomyelin in fibroblasts from a patient with type A Niemann-Pick disease, which completely lack acid sphingomyelinase activity but retain nonlysosomal neutral sphingomyelinase activity, with turnover in fibroblasts from normal individuals. Plasma membrane sphingomyelin was labeled by incubating cells at low temperature with phosphatidylcholine vesicles containing radioactive sphingomyelin. A fluorescent analog of sphingomyelin, N-4-nitrobenzo-2-oxa-1,3-diazoleaminocaproyl sphingosylphosphorylcholine (NBD-sphingomyelin) is seen to be readily transferred at low temperature from phosphatidylcholine liposomes to the plasma membranes of cultured human fibroblasts. Moreover, when kinetic studies were done in parallel, a constant ratio of [14C]oleoylsphingosylphosphorylcholine ( [14C]sphingomyelin) to NBD-sphingomyelin was taken up at low temperature by the fibroblast cells, suggesting that [14C]sphingomyelin undergoes a similar transfer. The comparison of sphingomyelin turnover at 37 degrees C in normal fibroblasts compared to Niemann-Pick diseased fibroblasts shows that a rapid turnover of plasma membrane-associated sphingomyelin within the first 30 min appears to be similar in both normal and Niemann-Pick diseased cells. This rapid turnover appears to be primarily due to rapid removal of the [14C]sphingomyelin from the cell surface into the incubation medium. During

  12. Apolipoprotein L-I Promotes Trypanosome Lysis by Forming Pores in Lysosomal Membranes

    NASA Astrophysics Data System (ADS)

    Pérez-Morga, David; Vanhollebeke, Benoit; Paturiaux-Hanocq, Françoise; Nolan, Derek P.; Lins, Laurence; Homblé, Fabrice; Vanhamme, Luc; Tebabi, Patricia; Pays, Annette; Poelvoorde, Philippe; Jacquet, Alain; Brasseur, Robert; Pays, Etienne

    2005-07-01

    Apolipoprotein L-I is the trypanolytic factor of human serum. Here we show that this protein contains a membrane pore-forming domain functionally similar to that of bacterial colicins, flanked by a membrane-addressing domain. In lipid bilayer membranes, apolipoprotein L-I formed anion channels. In Trypanosoma brucei, apolipoprotein L-I was targeted to the lysosomal membrane and triggered depolarization of this membrane, continuous influx of chloride, and subsequent osmotic swelling of the lysosome until the trypanosome lysed.

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

    PubMed

    Encarnação, Marisa; Espada, Lília; Escrevente, Cristina; Mateus, Denisa; Ramalho, José; Michelet, Xavier; Santarino, Inês; Hsu, Victor W; Brenner, Michael B; Barral, Duarte; Vieira, Otília V

    2016-06-20

    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

  14. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

    PubMed

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+)-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D. PMID:27028538

  15. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

    PubMed Central

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W.

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D. PMID:27028538

  16. Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.

    PubMed

    Kolter, Thomas; Sandhoff, Konrad

    2005-01-01

    Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family. PMID:16212488

  17. Lysosomal Physiology

    PubMed Central

    Xu, Haoxing; Ren, Dejian

    2015-01-01

    Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases. PMID:25668017

  18. Lysosomal physiology.

    PubMed

    Xu, Haoxing; Ren, Dejian

    2015-01-01

    Lysosomes are acidic compartments filled with more than 60 different types of hydrolases. They mediate the degradation of extracellular particles from endocytosis and of intracellular components from autophagy. The digested products are transported out of the lysosome via specific catabolite exporters or via vesicular membrane trafficking. Lysosomes also contain more than 50 membrane proteins and are equipped with the machinery to sense nutrient availability, which determines the distribution, number, size, and activity of lysosomes to control the specificity of cargo flux and timing (the initiation and termination) of degradation. Defects in degradation, export, or trafficking result in lysosomal dysfunction and lysosomal storage diseases (LSDs). Lysosomal channels and transporters mediate ion flux across perimeter membranes to regulate lysosomal ion homeostasis, membrane potential, catabolite export, membrane trafficking, and nutrient sensing. Dysregulation of lysosomal channels underlies the pathogenesis of many LSDs and possibly that of metabolic and common neurodegenerative diseases. PMID:25668017

  19. Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

    SciTech Connect

    Mithieux, G.; Rousset, B.

    1989-03-15

    We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells.

  20. 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

  1. 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.

  2. β2-Microglobulin Amyloid Fibrils Are Nanoparticles That Disrupt Lysosomal Membrane Protein Trafficking and Inhibit Protein Degradation by Lysosomes*

    PubMed Central

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

    2014-01-01

    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. PMID:25378395

  3. 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

  4. Activation of Membrane NADPH Oxidase Associated with Lysosome-Targeted Acid Sphingomyelinase in Coronary Endothelial Cells

    PubMed Central

    Bao, Jun-Xiang; Jin, Si; Zhang, Fan; Wang, Zheng-Chao; Li, Ningjun

    2010-01-01

    Abstract This study explored the mechanism mediating the aggregation of membrane NADPH oxidase (NOX) subunits and subsequent activation of this enzyme in bovine coronary arterial endothelial cells (CAECs). With confocal microscopy, we found that FasL stimulated lipid rafts (LRs) clustering with NOX subunit aggregation and acid sphingomyelinase (ASM) gathering, which was blocked by the siRNA of sortilin, an intracellular protein responsible for the binding and targeting of ASM to lysosomes. Correspondingly, FasL-induced O2·− production through NOX in LRs fractions was abolished by sortilin siRNA. Further, with flow-cytometry and fluorescence resonance energy transfer (FRET) analysis, we surprisingly demonstrated that after FasL stimulation, sortilin was exposed to cell membranes from lysosomes together with Lamp-1 and ASM, and these lysosomal components were aggregated and form a signaling complex in cell membranes. With co-immunoprecipitation, lysosomal sortilin and ASM were found to interact more strongly when CAECs were stimulated by FasL. Functionally, inhibition of either sortilin expression, lysosome function, LRs clustering, or NOX activity significantly attenuated FasL-induced decrease in nitric oxide (NO) levels. It is concluded that lysosome-targeted ASM, through sortilin, is able to traffic to and expose to cell-membrane surface, which may lead to LRs clustering and NOX activation in CAECs. Antioxid. Redox Signal. 12, 703–712. PMID:19761405

  5. 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

    2016-01-01

    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. PMID:27120618

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

    PubMed Central

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

    2016-01-01

    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. PMID:27120618

  7. SID1 transmembrane family, member 2 (Sidt2): a novel lysosomal membrane protein.

    PubMed

    Jialin, Gao; Xuefan, Gu; Huiwen, Zhang

    2010-11-26

    In a recent proteomic study of lysosomal proteins [10], we identified SID1 transmembrane family, member 2 (Sidt2) as a novel lysosomal membrane protein candidate. The Sidt2 gene encodes an 832-amino acid residues protein with a calculated molecular mass of 94.5kDa. Bioinformatic analysis showed that Sidt2 is a multipass transmembrane protein that contains 10 putative N-glycosylation sites (NxS/T) and two potential tyrosine-based sorting signals (YGSF and YDTL). Using specific anti-Sidt2 antibody and lysosomal markers, the lysosomal localization of Sidt2 was determined by immunofluorescence. Furthermore, using subcellular fractionation techniques, we demonstrated that Sidt2 is a lysosomal integral membrane protein. Endogenous Sidt2 was detected in multiple tissues of mouse and rat with approximately 120-130kDa molecular weights due to extensive glycosylation. After digestion with PNGase F, the apparent molecular mass of Sidt2 decreased to the predicted value of 95kDa. In rats, Sidt2 was highly expressed in the liver, brain, and kidney, whereas no or little expression was found in the skeletal muscles, heart, and other tissues. In summary, Sidt2 is a highly glycosylated lysosomal integral membrane protein that shows tissue-specific expression. PMID:20965152

  8. Lysosome stabilization in slices of rat liver when incubated with vitamin A excess

    SciTech Connect

    Morre, D.M.; Morre, D.J.; Bowen, S.; Reutter, W.

    1986-05-01

    An organ culture of slices of livers from adult rats was used to study effect of vitamin A (all-trans retinol) on lysosome stability. Lysosomes were purified by centrifugation in Percoll gradients. Preparations were monitored by electron microscopy and evaluated by morphometry and assays of marker enzymes. Enrichments relative to homogenates and crude pellets were estimated from latent (triton X-100) acid p-nitrophenylphosphatase specific activities. Lysosomes prepared from unincubated slices were enriched 50-fold in latent acid phosphatase relative to homogenates. In contrast, lysosomes prepared from slices incubated for 30 min in PBS alone were enriched only 20-fold. When 25 ..mu..g/ml retinol was included in the incubation medium, enrichments of 40-fold were obtained. The integrity of the slices was monitored by electron microscopy and their viability was confirmed by a sustained uptake and incorporation of (/sup 3/H)leucine into protein (up to 2 h in culture). The loss of lysosomes from homogenates of slices incubated in the absence of retinol was accompanied by a loss of acid phosphatase from the lysosomal pellet to the supernatant during purification. Addition of retinol to slices just prior to homogenization was without effect. The results demonstrate a stabilizing influence of vitamin A on lysosomes during incubation of licer slices. The findings contrast earlier reports of retinol-induced lysosome fragility in other in vitro systems.

  9. 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. PMID:26818531

  10. 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.

  11. Membrane-anchored ubiquitin ligase complex is required for the turnover of lysosomal membrane proteins

    PubMed Central

    Li, Ming; Koshi, Tatsuhiro

    2015-01-01

    Cells must regulate the abundance and activity of numerous nutrient transporters in different organelle membranes to achieve nutrient homeostasis. As the recycling center and major storage organelle, lysosomes are essential for maintaining nutrient homeostasis. However, very little is known about mechanisms that govern the regulation of its membrane proteins. In this study, we demonstrated that changes of Zn2+ levels trigger the downregulation of vacuolar Zn2+ transporters. Low Zn2+ levels cause the degradation of the influx transporter Cot1, whereas high Zn2+ levels trigger the degradation of the efflux channel Zrt3. The degradation process depends on the vacuole membrane recycling and degradation pathway. Unexpectedly, we identified a RING domain–containing E3 ligase Tul1 and its interacting proteins in the Dsc complex that are important for the ubiquitination of Cot1 and partial ubiquitination of Zrt3. Our study demonstrated that the Dsc complex can function at the vacuole to regulate the composition and lifetime of vacuolar membrane proteins. PMID:26527740

  12. 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. PMID:26815913

  13. 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.

  14. Protein Networks Supporting AP-3 Function in Targeting Lysosomal Membrane Proteins

    PubMed Central

    Baust, Thorsten; Anitei, Mihaela; Czupalla, Cornelia; Parshyna, Iryna; Bourel, Line; Thiele, Christoph; Krause, Eberhard

    2008-01-01

    The AP-3 adaptor complex targets selected transmembrane proteins to lysosomes and lysosome-related organelles. We reconstituted its preferred interaction with liposomes containing the ADP ribosylation factor (ARF)-1 guanosine triphosphatase (GTPase), specific cargo tails, and phosphatidylinositol-3 phosphate, and then we performed a proteomic screen to identify new proteins supporting its sorting function. We identified ≈30 proteins belonging to three networks regulating either AP-3 coat assembly or septin polymerization or Rab7-dependent lysosomal transport. RNA interference shows that, among these proteins, the ARF-1 exchange factor brefeldin A-inhibited exchange factor 1, the ARF-1 GTPase-activating protein 1, the Cdc42-interacting Cdc42 effector protein 4, an effector of septin-polymerizing GTPases, and the phosphatidylinositol-3 kinase IIIC3 are key components regulating the targeting of lysosomal membrane proteins to lysosomes in vivo. This analysis reveals that these proteins, together with AP-3, play an essential role in protein sorting at early endosomes, thereby regulating the integrity of these organelles. PMID:18287518

  15. 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.

  16. Sensitivity to Lysosome-Dependent Cell Death Is Directly Regulated by Lysosomal Cholesterol Content

    PubMed Central

    Appelqvist, Hanna; Sandin, Linnea; Björnström, Karin; Saftig, Paul; Garner, Brett; Öllinger, Karin; Kågedal, Katarina

    2012-01-01

    Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis. PMID:23166840

  17. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells

    PubMed Central

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M.; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrPSc accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  18. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells.

    PubMed

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrP(Sc)) of the cellular prion protein (PrP(c)). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrP(c) into PrP(Sc). Within neurons, PrP(Sc) accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrP(Sc) accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  19. Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion.

    PubMed

    Dolat, Lee; Spiliotis, Elias T

    2016-08-29

    Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate-dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes. PMID:27551056

  20. 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

  1. Pneumolysin Activates Macrophage Lysosomal Membrane Permeabilization and Executes Apoptosis by Distinct Mechanisms without Membrane Pore Formation

    PubMed Central

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

    2014-01-01

    ABSTRACT 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. PMID:25293758

  2. Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

    PubMed Central

    Han, Wei-Qing; Xia, Min; Xu, Ming; Boini, Krishna M.; Ritter, Joseph K.; Li, Ning-Jun; Li, Pin-Lan

    2012-01-01

    Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production – an important step that mediates membrane raft clustering – was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation. PMID:22349696

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

    PubMed Central

    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

    2015-01-01

    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 cytotoxiс activity by mechanisms different from “classical” apoptosis, and could be a promising new compound to overcome resistance to standard therapies in genitourinary malignancies. PMID:26093146

  4. [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. PMID:24564165

  5. Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1

    PubMed Central

    Vanwalleghem, Gilles; Fontaine, Frédéric; Lecordier, Laurence; Tebabi, Patricia; Klewe, Kristoffer; Nolan, Derek P.; Yamaryo-Botté, Yoshiki; Botté, Cyrille; Kremer, Anneke; Burkard, Gabriela Schumann; Rassow, Joachim; Roditi, Isabel; Pérez-Morga, David; Pays, Etienne

    2015-01-01

    Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion. PMID:26307671

  6. Coupling of lysosomal and mitochondrial membrane permeabilization in trypanolysis by APOL1.

    PubMed

    Vanwalleghem, Gilles; Fontaine, Frédéric; Lecordier, Laurence; Tebabi, Patricia; Klewe, Kristoffer; Nolan, Derek P; Yamaryo-Botté, Yoshiki; Botté, Cyrille; Kremer, Anneke; Burkard, Gabriela Schumann; Rassow, Joachim; Roditi, Isabel; Pérez-Morga, David; Pays, Etienne

    2015-01-01

    Humans resist infection by the African parasite Trypanosoma brucei owing to the trypanolytic activity of the serum apolipoprotein L1 (APOL1). Following uptake by endocytosis in the parasite, APOL1 forms pores in endolysosomal membranes and triggers lysosome swelling. Here we show that APOL1 induces both lysosomal and mitochondrial membrane permeabilization (LMP and MMP). Trypanolysis coincides with MMP and consecutive release of the mitochondrial TbEndoG endonuclease to the nucleus. APOL1 is associated with the kinesin TbKIFC1, of which both the motor and vesicular trafficking VHS domains are required for MMP, but not for LMP. The presence of APOL1 in the mitochondrion is accompanied by mitochondrial membrane fenestration, which can be mimicked by knockdown of a mitochondrial mitofusin-like protein (TbMFNL). The BH3-like peptide of APOL1 is required for LMP, MMP and trypanolysis. Thus, trypanolysis by APOL1 is linked to apoptosis-like MMP occurring together with TbKIFC1-mediated transport of APOL1 from endolysosomal membranes to the mitochondrion. PMID:26307671

  7. 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. PMID:25246127

  8. 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

  9. Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver.

    PubMed

    Kong, Xiang Yi; Kase, Eili Tranheim; Herskedal, Anette; Schjalm, Camilla; Damme, Markus; Nesset, Cecilie Kasi; Thoresen, G Hege; Rustan, Arild C; Eskild, Winnie

    2015-01-01

    Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp gt/gt mice (formerly known as Ncu-g1gt/gt mice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury. PMID:26047317

  10. Biphosphinic palladacycle complex mediates lysosomal-membrane permeabilization and cell death in K562 leukaemia cells.

    PubMed

    Barbosa, Christiano M V; Oliveira, Carlos R; Nascimento, Fábio D; Smith, Mickaela C M; Fausto, Daniela M; Soufen, Marco Antonio; Sena, Eliana; Araújo, Ronaldo C; Tersariol, Ivarne L S; Bincoletto, Claudia; Caires, Antonio C F

    2006-08-01

    The cell death mechanism of cytotoxicity induced by the Biphosphinic Palladacycle Complex (BPC) was studied using a K562 leukaemia cell line. The IC50 values obtained for K562 cells post-72 h of BPC were less than 5.0 microM by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue assays. Using the Acridine Orange vital staining combining fluorescence microscopy it was observed that the complex triggers apoptosis in K562 cells, inducing DNA fragmentation, as analysed through electrophoresis. Lysosomal-membrane permeabilization was also observed in K562 cells post-5 h of BPC, which suggests intralysosomal accumulation by proton-trapping, since its pKa value ranged from 5.1 to 6.5. Caspase-3, and -6 activity induced by BPC in K562 cells was prevented by the cathepsin-B inhibitor [N-(L-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-L-isoleucyl-L-proline] (CA074). These events occurred in the presence of endogenous bcl-2 and bax expression. Acute toxicological studies demonstrated that BPC produces no lesions for liver and kidney fourteen-days after drug administration (100 mg/kg--i.p.). White and red blood cells of BPC-treated mice presented normal morphological characteristics. Taken together, these data suggest a novel lysosomal pathway for BPC-induced apoptosis, in which lysosomes are the primary target and cathepsin B acts as death mediator. PMID:16831419

  11. Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver

    PubMed Central

    Kong, Xiang Yi; Kase, Eili Tranheim; Herskedal, Anette; Schjalm, Camilla; Damme, Markus; Nesset, Cecilie Kasi; Thoresen, G. Hege; Rustan, Arild C.; Eskild, Winnie

    2015-01-01

    Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmpgt/gt mice (formerly known as Ncu-g1gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmpgt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmpgt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmpgt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmpgt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmpgt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmpgt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmpgt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury. PMID:26047317

  12. 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

  13. Growth of Rickettsia typhi in irradiated L cells enhanced by lysosomal stabilization.

    PubMed Central

    Woodman, D R; Schultz, W W; Woodman, K L; Weiss, E

    1979-01-01

    The growth of some obligate intracellular parasites is contingent upon avoidance of lysosomal activation during growth in their host cells. This is accomplished by the various parasites by different mechanisms and with different degrees of efficiency. The possibility was tested that the lysosomal stabilizer cortisone acetate might protect and thus enhance the growth of Rickettsia typhi in mouse L cells irradiated 6 days earlier. Beginning 2 days before infection of the L cells with a multiplicity of 10 rickettsiae, 20 microgram of cortisone per ml was added in medium 199 containing 5% fetal calf serum. This concentration of cortisone was below the cytotoxic level, as determined by viability staining, but was sufficient to significantly alter the ratios of cellular and released acid phosphatase and beta-glucuronidase in uninfected and infected cells, as shown by spectrophotometric analysis. Rickettsial replication, measured by hemolytic activity at 96 h and confirmed by microscopic observations at earlier stages of infection, was increased by the cortisone. Cortisone concentrations of 10 or 40 microgram/ml were less effective, and cortisone was ineffective when the rickettsial multiplicity per L cell was 2 or lower. These results indicate that amounts of cortisone that increase lysosomal stabilization in L cells favor rickettsial multiplication when the multiplicity of infection is relatively high. PMID:106007

  14. 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

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

    PubMed

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

    2014-08-01

    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

  16. SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium

    PubMed Central

    Han, Wei-Qing; Xia, Min; Zhang, Chun; Zhang, Fan; Xu, Ming; Li, Ning-Jun

    2011-01-01

    The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91phox, and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function. PMID:21926345

  17. The mouse lysosomal membrane protein 1 gene as a candidate for the motorneuron degeneration (mnd) locus

    SciTech Connect

    Bermingham, N.A.; Martin, J.E.; Fisher, E.M.C.

    1996-03-01

    The motorneuron degeneration (mnd) mutation causes one of the few late-onset progressive neurodegenerations in mice; therefore, the mnd mouse is a valuable paradigm for studying neurodegenerative biology. The mnd mutation may also model human neuronal ceroid lipofuscinosis (NCL) or Batten disease. Mnd maps to the centromeric region of mouse chromosome 8, which likely corresponds to portions of human chromosomes 13,8, or 19; we note that the chromosome 13 portion maps close to a region thought to contain the human Type V NCL locus. We have identified candidate genes for the mnd locus from human chromosomes 13, 8, and 19, and we are mapping these genes in the mouse to determine their proximity to the mutated locus and to refine the comparative human-mouse map in this area. A candidate gene from human chromosome 13 is LAMP1, which encodes lysosomal membrane protein 1. We found that Lamp1 in the mouse lies within the region of the mnd mutation. Therefore, we sequenced Lamp1 cDNAs from homozygous mnd mice and unrelated wildtype C57BL/6 mice. We find no differences between the two cDNA species in the regions examined, and expression analysis shows a similar LAMP1 protein distribution in wildtype and mutant mice, suggesting that an abnormal accumulation of material within normal lysosome structures is unlikely to be the pathogenetic mechanism in the mnd mouse. 19 refs., 3 figs.

  18. Stat3 controls cell death during mammary gland involution by regulating uptake of milk fat globules and lysosomal membrane permeabilization

    PubMed Central

    Resemann, Henrike K.; Ramos-Montoya, Antonio; Skepper, Jeremy; Watson, Christine J.

    2014-01-01

    We have previously demonstrated that Stat3 regulates lysosomal mediated-programmed cell death (LM-PCD) during mouse mammary gland involution in vivo. However, the mechanism that controls the release of lysosomal cathepsins to initiate cell death in this context has not been elucidated. We show here that Stat3 regulates the formation of large lysosomal vacuoles that contain triglyceride. Furthermore, we demonstrate that milk fat globules (MFGs) are toxic to epithelial cells and that, when applied to purified lysosomes, the MFG hydrolysate oleic acid potently induces lysosomal leakiness. Additionally, uptake of secreted MFGs coated in butyrophilin 1A1 is diminished in Stat3 ablated mammary glands while loss of the phagocytosis bridging molecule MFG-E8 results in reduced leakage of cathepsins in vivo. We propose that Stat3 regulates LM-PCD in mouse mammary gland by switching cellular function from secretion to uptake of MFGs. Thereafter, perturbation of lysosomal vesicle membranes by high levels of free fatty acids results in controlled leakage of cathepsins culminating in cell death. PMID:25283994

  19. 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. PMID:25849458

  20. 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

  1. 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. PMID:12069313

  2. 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

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

    PubMed

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

    2016-04-01

    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

  4. LAPTM5: A novel lysosomal-associated multispanning membrane protein preferentially expressed in hematopoietic cells

    SciTech Connect

    Adra, C.N.; Zhu, Shaochun; Ko, Jone-Long

    1996-07-15

    While a large body of knowledge about cell membrane proteins exists, much less is known about the repertoire and function of integral membrane proteins of intracellular organelles. In looking for novel classes of genes that are functionally important to hematopoietic cells, we have cloned the cDNA for a gene preferentially expressed in adult hematopoietic tissues. During embryonic development the gene is expressed in both hematopoietic and nonhematopoietic tissues. In cell lines the gene is expressed specifically in hematopoietic lineages, whereas in normal adult tissues the mRNA is preferentially detected at high levels in lymphoid and myeloid tissues. The predicted protein is a pentaspanner with no homology to known genes and conserved across evolution. Immunocytological and cell fractionation studies with a specific antibody revealed a protein localizing in lysosomes. The gene, provisionally named LAPTM5, maps to chromosome 1p34. The expression pattern of the gene together with preliminary evidence that the protein interacts with ubiquitin indicates that the protein may have a special functional role during embryogenesis and in adult hematopoietic cells. 53 refs., 9 figs.

  5. 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

  6. Studying Lysosomal Membrane Permeabilization by Analyzing the Release of Preloaded BSA-Gold Particles into the Cytosol.

    PubMed

    Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

    2016-01-01

    In addition to techniques involving assaying the release of endogenous lysosomal molecules into the cytosol, the endocytic system can be preloaded with exogenous fluorescent or electron-dense tracers. These tracers will translocate into the cytosol upon lysosomal membrane permeabilization and have the advantage of being detectable directly without additional labeling. Another benefit is that the tracers can be made more abundant than most endogenous lysosomal molecules, which facilitates their detection. Tracers that can be analyzed with fluorescence microscopy include low-molecular-mass molecules such as sulforhodamine B and also fluorescent polymers of dextran that are available in a wide range of molecular masses. This protocol shows how, for electron-microscopic analysis, cells can be fed with colloidal gold or ferrofluid particles complexed to bovine serum albumin. Although electron microscopy entails a high-resolution analysis, which can be advantageous, we caution how it is important to note that particulate tracers are larger than many endogenous lysosomal molecules and might be released only upon extensive membrane permeabilization. PMID:27250941

  7. Poor lysosomal membrane integrity in proximal tubule cells of haptoglobin 2-2 genotype mice with diabetes mellitus

    PubMed Central

    Asleh, Rabea; Nakhoul, Farid M.; Miller-Lotan, Rachel; Awad, Hoda; Farbstein, Dan; Levy, Nina S.; Nakhoul, Nakhoul; Iancu, Theodore C.; Manov, Irena; Laue, Michael; Traber, Maret G.; Lebold, Katie M.; Levy, Andrew P.

    2013-01-01

    The haptoglobin (Hp) genotype is a major determinant of progression of nephropathy in individuals with diabetes mellitus (DM). The major function of the Hp protein is to bind and modulate the fate of extracorpuscular hemoglobin and its iron cargo. We have previously demonstrated an interaction between the Hp genotype and the DM on the accumulation of iron in renal proximal tubule cells. The primary objective of this study was to determine the intracellular localization of this iron in the proximal tubule cell and to assess its potential toxicity. Transmission electron microscopy demonstrated a marked accumulation of electron-dense deposits in the lysosomes of proximal tubules cells in Hp 2-2 DM mice. Energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to perform elemental analysis of these deposits and demonstrated that these deposits were iron rich. These deposits were associated with lysosomal membrane lipid peroxidation and loss of lysosomal membrane integrity. Vitamin E administration to Hp 2-2 DM mice resulted in a significant decrease in both intralysosomal iron-induced oxidation and lysosomal destabilization. Iron-induced renal tubular injury may play a major role in the development of diabetic nephropathy and may be a target for slowing the progression of renal disease. PMID:22749805

  8. Lysosomes and autophagy in aquatic animals.

    PubMed

    Moore, Michael N; Kohler, Angela; Lowe, David; Viarengo, Aldo

    2008-01-01

    The lysosomal-autophagic system appears to be a common target for many environmental pollutants, as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, autophagic reactions frequently involving reduced lysosomal membrane integrity or stability appear to be effective generic indicators of cellular well-being in eukaryotes: in social amoebae (slime mold), mollusks and fish, autophagy/membrane destabilization is correlated with many stress and toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants can be used for predicting cellular dysfunction and health in aquatic animals, such as shellfish and fish, which are extensively used as sensitive bioindicators in monitoring ecosystem health; and also represent a significant food resource for at least 20% of the global human population. Explanatory frameworks for prediction of pollutant impact on health have been derived encompassing a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. Methods are described for tracking in vivo autophagy of fluorescently labeled cytoplasmic proteins, measuring degradation of radiolabeled intracellular proteins and morphometric measurement of lysosomal/cytoplasmic volume ratio. Additional methods for the determination of lysosomal membrane stability in lower animals are also described, which can be applied to frozen tissue sections, protozoans and isolated cells in vivo. Experimental and simulated results have also indicated that nutritional deprivation (analogous in marine mussels to caloric restriction)-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and simulation modelling is proposed as a practical toolbox for predicting toxic

  9. 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. PMID:26232047

  10. 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.

  11. 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

  12. Targeting the lysosome in cancer

    PubMed Central

    Piao, Shengfu; Amaravadi, Ravi K.

    2016-01-01

    Lysosomes are membrane-bound intracellular organelles that receive macromolecules delivered by endocytosis, phagocytosis, and autophagy for degradation and recycling. Over the last decade, advances in lysosome research have established a broad role for the lysosome in the pathophysiology of disease. In this review, we highlight the recent discoveries in lysosome biology, with an emphasis on their implications for cancer therapy. We focus on targeting the lysosome in cancer by exploring lysosomal biogenesis and its role in the crosstalk between apoptosis and autophagy. We also discuss how lysosomal inhibition could emerge as a new therapeutic strategy to overcome drug resistance in cancer. PMID:26599426

  13. 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

    2016-04-01

    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

  14. Requirement of translocated lysosomal V1 H+-ATPase for activation of membrane acid sphingomyelinase and raft clustering in coronary endothelial cells

    PubMed Central

    Xu, Ming; Xia, Min; Li, Xiao-Xue; Han, Wei-Qing; Boini, Krishna M.; Zhang, Fan; Zhang, Yang; Ritter, Joseph K; Li, Pin-Lan

    2012-01-01

    Acid sphingomyelinase (ASM) mediates the formation of membrane raft (MR) redox signalosomes in a process that depends on a local acid microenvironment in coronary arterial endothelial cells (CAECs). However, it is not known how this local acid microenvironment is formed and maintained. The present study hypothesized that lysosomal V1 H+-ATPase provides a hospitable acid microenvironment for activation of ASM when lysosomes traffic and fuse into the cell membrane. Confocal microscopy showed that local pH change significantly affected MRs, with more fluorescent patches under low pH. Correspondingly, the ASM product, ceramide, increased locally in the cell membrane. Electron spin resonance assay showed that local pH increase significantly inhibited NADPH oxidase–mediated production of O2−. in CAECs. Direct confocal microscopy demonstrated that Fas ligand resulted in localized areas of decreased pH around CAEC membranes. The inhibitors of both lysosomal fusion and H+-ATPase apparently attenuated FasL-caused pH decrease. V1 H+-ATPase accumulation and activity on cell membranes were substantially suppressed by the inhibitors of lysosomal fusion or H+-ATPase. These results provide the first direct evidence that translocated lysosomal V1 H+-ATPase critically contributes to the formation of local acid microenvironment to facilitate activation of ASM and consequent MR aggregation, forming MR redox signalosomes and mediating redox signaling in CAECs. PMID:22357614

  15. 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. PMID:26313128

  16. Lysosomal storage disorders: The cellular impact of lysosomal dysfunction

    PubMed Central

    2012-01-01

    Lysosomal storage diseases (LSDs) are a family of disorders that result from inherited gene mutations that perturb lysosomal homeostasis. LSDs mainly stem from deficiencies in lysosomal enzymes, but also in some non-enzymatic lysosomal proteins, which lead to abnormal storage of macromolecular substrates. Valuable insights into lysosome functions have emerged from research into these diseases. In addition to primary lysosomal dysfunction, cellular pathways associated with other membrane-bound organelles are perturbed in these disorders. Through selective examples, we illustrate why the term “cellular storage disorders” may be a more appropriate description of these diseases and discuss therapies that can alleviate storage and restore normal cellular function. PMID:23185029

  17. 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

  18. Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death.

    PubMed

    Shang, Tiesong; Joseph, Joy; Hillard, Cecilia J; Kalyanaraman, B

    2005-10-14

    We have investigated here the mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells (SH-SY5Y) treated with mitochondrial toxins, such as MPP(+) and rotenone. Mitochondrial respiratory chain inhibitors and uncouplers decreased mitochondrial membrane potential leading to DAPK dephosphorylation and activation. The class III phosphoinositide 3-kinase inhibitors attenuated DAPK dephosphorylation induced by mitochondrial toxins. Complex I inhibition by mitochondrial toxins (e.g. MPP(+)) resulted in mitochondrial swelling and lysosome reduction. Inhibition of class III phosphoinositide 3-kinase attenuated MPP(+)-induced lysosome reduction and cell death. The role of DAPK as a sensor of mitochondrial membrane potential in mitochondrial diseases was addressed. PMID:16085644

  19. 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

  20. 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.

  1. TRAIL-Death Receptor 4 Signaling via Lysosome Fusion and Membrane Raft Clustering In Coronary Arterial Endothelial Cells: Evidence from ASM Knockout Mice

    PubMed Central

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M.; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2012-01-01

    Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptor death receptor 4 (DR4) have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation and leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MRs) clustering and formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and its co-localization with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1+/+) mice. Further, TRAIL triggered ASM translocation, ceramide production and NADPH oxidase aggregation in MR clusters in Smpd1+/+ CAECs, whereas these observations were not found in Smpd1−/− CAECs. Moreover, ASM deficiency reduced TRAIL-induced O2−· production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer (FRET), we found that Lamp-1 (lysosome membrane marker protein) and ganglioside GM1 (MR marker) were trafficking together in Smpd1+/+ CAECs, which was absent in Smpd1−/− CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1−/− CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking and fusion with membrane and formation of MR redox signaling platforms, which may

  2. Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent M sub r approx 120,000

    SciTech Connect

    Viitala, J.; Carlsson, S.R.; Siebert, P.D.; Fukuda, M. )

    1988-06-01

    Although several lysosomal membrane glycoproteins have been characterized by using specific antibodies, none of the studies so far elucidated the amino acid sequence of a lysosomal membrane glycoprotein. Here we describe cDNA clones encoding for one of the lysosome-associated membrane proteins with apparent M{sub r} {approx} 120,000, lamp A. The amino acid sequence based on the fully coded cDNA shows that as many as 18 potential N-glycosylation sites can be found in the total of 385 amino acid residues. The results obtained by endoglycosidase F digestion support the conclusion that this glycoprotein contains 18 N-glycans. These N-glycosylation sites are clustered in two domains; one contains 10 and the other contains 8 N-glycosylation sites. These domains are separated by a (proline-serine)-rich region that has a distinct homology to the IgA hinge structure. The first N-glycosylated domain is elongated to a potential leader peptide toward the NH{sub 2}-terminal end. The second N-glycosylated domain, on the other hand, is connected to a putative transmembrane portion consisting of hydrophobic amino acids. This segment, in turn, is elongated to a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end.

  3. Lysosomal and autophagic reactions as predictive indicators of environmental impact in aquatic animals.

    PubMed

    Moore, Michael N; Allen, J Icarus; McVeigh, Allan; Shaw, Jenny

    2006-01-01

    The lysosomal-autophagic system appears to be a common target for many environmental pollutants as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, lysosomal membrane integrity or stability appears to be an effective generic indicator of cellular well-being in eukaryotes: in bivalve molluscs and fish, stability is correlated with many toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants has been explored in relation to predicting cellular dysfunction and health in marine mussels, which are extensively used as sensitive bioindicators in monitoring ecosystem health. Derivation of explanatory frameworks for prediction of pollutant impact on health is a major goal; and we have developed a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. This model has also complemented the creation of a cell-based computational model for molluscan hepatopancreatic cells that simulates lysosomal, autophagic and other cellular reactions to pollutants. Experimental and simulated results have also indicated that nutritional deprivation-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and modelling is proposed as a practical toolbox for predicting toxic environmental risk. PMID:16874099

  4. Anthrax Lethal Toxin Induced Lysosomal Membrane Permeabilization and Cytosolic Cathepsin Release Is Nlrp1b/Nalp1b-Dependent

    PubMed Central

    Averette, Kathleen M.; Pratt, Matthew R.; Yang, Yanan; Bassilian, Sara; Whitelegge, Julian P.; Loo, Joseph A.; Muir, Tom W.; Bradley, Kenneth A.

    2009-01-01

    NOD-like receptors (NLRs) are a group of cytoplasmic molecules that recognize microbial invasion or ‘danger signals’. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT), is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP). The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis. PMID:19924255

  5. Heat Shock Protein 70.1 (Hsp70.1) Affects Neuronal Cell Fate by Regulating Lysosomal Acid Sphingomyelinase*

    PubMed Central

    Zhu, Hong; Yoshimoto, Tanihiro; Yamashima, Tetsumori

    2014-01-01

    The inducible expression of heat shock protein 70.1 (Hsp70.1) plays cytoprotective roles in its molecular chaperone function. Binding of Hsp70 to an endolysosomal phospholipid, bis(monoacylglycero)phosphate (BMP), has been recently shown to stabilize lysosomal membranes by enhancing acid sphingomyelinase (ASM) activity in cancer cells. Using the monkey experimental paradigm, we have reported that calpain-mediated cleavage of oxidized Hsp70.1 causes neurodegeneration in the hippocampal cornu ammonis 1 (CA1), whereas expression of Hsp70.1 in the motor cortex without calpain activation contributes to neuroprotection. However, the molecular mechanisms of the lysosomal destabilization/stabilization determining neuronal cell fate have not been elucidated. To elucidate whether regulation of lysosomal ASM could affect the neuronal fate, we analyzed Hsp70.1-BMP binding and ASM activity by comparing the motor cortex and the CA1. We show that Hsp70.1 being localized at the lysosomal membrane, lysosomal lipid BMP levels, and the lipid binding domain of Hsp70.1 are crucial for Hsp70.1-BMP binding. In the postischemic motor cortex, Hsp70.1 being localized at the lysosomal membrane could bind to BMP without calpain activation and decreased BMP levels, resulting in increasing ASM activity and lysosomal stability. However, in the postischemic CA1, calpain activation and a concomitant decrease in the lysosomal membrane localization of Hsp70.1 and BMP levels may diminish Hsp70.1-BMP binding, resulting in decreased ASM activity and lysosomal rupture with leakage of cathepsin B into the cytosol. A TUNEL assay revealed the differential neuronal vulnerability between the CA1 and the motor cortex. These results suggest that regulation of ASM activation in vivo by Hsp70.1-BMP affects lysosomal stability and neuronal survival or death after ischemia/reperfusion. PMID:25074941

  6. Vitamin A-deficiency and its effects on the lysosomal enzymes of fish.

    PubMed

    Harikumar, P; Kakati, R; Goswami, U C

    1996-01-01

    The effect of vitamin A-deficiency on the structural integrity of lysosomes in the skeletal muscle and skin of Heteropneustes fossilis, a dehydroretinol-rich freshwater siluroid used in pisciculture, has been evaluated. Dietary stress was found to cause enhanced release of acid hydrolases from both skeletal muscle and skin tissues. The results indicate that the regulation of lysosomal membrane stability in these tissues is a function of vitamin A. PMID:8843982

  7. Transporter associated with antigen processing-like (ABCB9) stably expressed in Chinese hamster ovary-K1 cells is sorted to the microdomains of lysosomal membranes.

    PubMed

    Fujimoto, Yasuyuki; Kamakura, Aya; Motohashi, Yu; Ohashi-Kobayashi, Ayako; Maeda, Masatomo

    2011-01-01

    The carboxyl terminus of a human ATP-binding cassette (ABC) transporter, transporter associated with antigen processing (TAP)-like (TAPL), was tagged with green fluorescence protein (GFP), and the resulting fusion protein (TAPL-GFP) was stably expressed in Chinese hamster ovary (CHO)-K1 cells. The GFP signal was co-localized with that of LysoTracker but not that of MitoTracker, as visualized under a microscope. TAPL-GFP was co-sedimented with lysosomal marker cathepsin D on Percoll density gradient centrifugation. These results indicated that TAPL is a lysosomal ABC transporter but not a mitochondrial one. It was not solubilized completely with a non-ionic detergent under ice-cold conditions, and was co-sedimented with flotillin-1 on sucrose density gradient centrifugation. A similar result was obtained with high pH-treatment. Furthermore, treatment with methyl-β-cyclodextrin resulted in an altered distribution of TAPL-GFP. These results suggest that TAPL may be localized to the microdomains (lipid rafts) of lysosomal membranes enriched in cholesterol. PMID:21212514

  8. S-layer stabilized lipid membranes (Review)

    PubMed Central

    Schuster, Bernhard; Pum, Dietmar; Sleytr, Uwe B.

    2010-01-01

    The present review focuses on a unique bio-molecular construction kit based on surface-layer (S-layer) proteins as building blocks and patterning elements, but also major classes of biological molecules such as lipids, membrane-active peptides and membrane proteins, and glycans for the design of functional supported lipid membranes. The biomimetic approach copying the supramolecular building principle of most archaeal cell envelopes merely composed of a plasma membrane and a closely associated S-layer lattice has resulted in robust and fluid lipid membranes. Most importantly, S-layer supported lipid membranes spanning an aperture or generated on solid and porous substrates constitute highly interesting model membranes for the reconstitution of responsive transmembrane proteins and membrane-active peptides. This is of particular challenge as one-third of all proteins are membrane proteins such as pore-forming proteins, ion channels, and receptors. S-layer supported lipid membranes are seen as one of the most innovative strategies in membrane protein-based nanobiotechnology with potential applications that range from pharmaceutical (high-throughput) drug screening over lipid chips to the detection of biological warfare agents. PMID:20408666

  9. Lysosomal Lipid Storage Diseases

    PubMed Central

    Schulze, Heike; Sandhoff, Konrad

    2011-01-01

    Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a “traffic jam.” This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement. PMID:21502308

  10. 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. PMID:26907692

  11. TOPICAL REVIEW: Stability of macroion-decorated lipid membranes

    NASA Astrophysics Data System (ADS)

    May, Sylvio

    2005-08-01

    Adsorption of macroions such as colloidal particles, proteins, or other rigid biopolymers onto oppositely charged, mixed lipid membranes is a ubiquitous phenomenon encountered in biotechnology, drug delivery, and cellular biology. The softness and self-assembled nature of the membrane enable the macroion-membrane complex to laterally reorganize via forming macroion clusters, lipid domains, or separate phases, and to exhibit curvature modulations or even morphological transitions. Almost always, the lateral organization of the membrane and associated macroion layer mutually depend on each other so that neither of the two extreme views—macroion-induced membrane domain formation or membrane-mediated macroion clustering—strictly accounts for the underlying energetics. We review and discuss some recent efforts to describe the lateral organization and stability of macroion-decorated lipid membranes using different levels of mean-field electrostatics, thereby focusing on binary membranes and the destabilizing role of compositional gradients.

  12. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    PubMed Central

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer; Miercke, Larry J. W.; Rasmussen, Søren G. F.; Robbins, Rebecca A.; Rana, Rohini R.; Loland, Claus J.; Kobilka, Brian; Stroud, Robert; Byrne, Bernadette; Gether, Ulrik; Gellman, Samuel H.

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles. PMID:21049926

  13. Quantification of age-related changes of α-tocopherol in lysosomal membranes in murine tissues and human fibroblasts.

    PubMed

    König, Jeannette; Besoke, Fabian; Stuetz, Wolfgang; Malarski, Angelika; Jahreis, Gerhard; Grune, Tilman; Höhn, Annika

    2016-05-01

    Considering the biological function of α-tocopherol (α-Toc) as a potent protective factor against oxidative stress, this antioxidant is in the focus of aging research. To understand the role of α-Toc during aging we investigated α-Toc concentrations in young and aged primary human fibroblasts after supplementation with RRR-α-Toc. Additionally, α-Toc contents were determined in brain, kidney, and liver tissue of 10 week-, 18 month-, and 24 month-old mice, which were fed a standard diet containing 100 mg/kg dl-α-tocopheryl acetate. α-Toc concentrations in isolated lysosomes and the expression of the α-Toc transport proteins Niemann Pick C1 (NPC1), Niemann Pick C2 (NPC2), and lipoprotein lipase were also analyzed. Obtained data show a significant age-related increase of α-Toc in murine liver, kidney, and brain tissue as well as in human dermal fibroblasts. Also liver and kidney lysosomes are marked by elevated α-Toc contents with aging. NPC1 and NPC2 protein amounts are significantly decreased in adult and aged murine kidney tissue. Also aged human dermal fibroblasts show decreased NPC1 amounts. Supplementation of young and aged fibroblasts led also to decreased NPC1 amounts, suggesting a direct role of this protein in α-Toc distribution. Our results indicate an age-dependent increase of α-Toc in different murine tissues as well as in human fibroblasts. Furthermore saturation and intracellular distribution of α-Toc seem to be strongly dependent on the availability of this vitamin as well as on the presence of the lysosomal protein NPC1. © 2016 BioFactors, 42(3):307-315, 2016. PMID:27095633

  14. Novel Mechanism of Cytotoxicity for the Selective Selenosemicarbazone, 2-Acetylpyridine 4,4-Dimethyl-3-selenosemicarbazone (Ap44mSe): Lysosomal Membrane Permeabilization.

    PubMed

    Al-Eisawi, Zaynab; Stefani, Christian; Jansson, Patric J; Arvind, Akanksha; Sharpe, Philip C; Basha, Maram T; Iskander, George M; Kumar, Naresh; Kovacevic, Zaklina; Lane, Darius J R; Sahni, Sumit; Bernhardt, Paul V; Richardson, Des R; Kalinowski, Danuta S

    2016-01-14

    Selenosemicarbazones show marked antitumor activity. However, their mechanism of action remains unknown. We examined the medicinal chemistry of the selenosemicarbazone, 2-acetylpyridine 4,4-dimethyl-3-selenosemicarbazone (Ap44mSe), and its iron and copper complexes to elucidate its mechanisms of action. Ap44mSe demonstrated a pronounced improvement in selectivity toward neoplastic relative to normal cells compared to its parent thiosemicarbazone. It also effectively depleted cellular Fe, resulting in transferrin receptor-1 up-regulation, ferritin down-regulation, and increased expression of the potent metastasis suppressor, N-myc downstream regulated gene-1. Significantly, Ap44mSe limited deleterious methemoglobin formation, highlighting its usefulness in overcoming toxicities of clinically relevant thiosemicarbazones. Furthermore, Cu-Ap44mSe mediated intracellular reactive oxygen species generation, which was attenuated by the antioxidant, N-acetyl-L-cysteine, or Cu sequestration. Notably, Ap44mSe forms redox active Cu complexes that target the lysosome to induce lysosomal membrane permeabilization. This investigation highlights novel structure-activity relationships for future chemotherapeutic design and underlines the potential of Ap44mSe as a selective anticancer/antimetastatic agent. PMID:26645570

  15. Autophagy in lysosomal storage disorders

    PubMed Central

    Lieberman, Andrew P.; Puertollano, Rosa; Raben, Nina; Slaugenhaupt, Susan; Walkley, Steven U.; Ballabio, Andrea

    2012-01-01

    Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as “autophagy disorders.” PMID:22647656

  16. Human recombinant lysosomal enzymes produced in microorganisms.

    PubMed

    Espejo-Mojica, Ángela J; Alméciga-Díaz, Carlos J; Rodríguez, Alexander; Mosquera, Ángela; Díaz, Dennis; Beltrán, Laura; Díaz, Sergio; Pimentel, Natalia; Moreno, Jefferson; Sánchez, Jhonnathan; Sánchez, Oscar F; Córdoba, Henry; Poutou-Piñales, Raúl A; Barrera, Luis A

    2015-01-01

    Lysosomal storage diseases (LSDs) are caused by accumulation of partially degraded substrates within the lysosome, as a result of a function loss of a lysosomal protein. Recombinant lysosomal proteins are usually produced in mammalian cells, based on their capacity to carry out post-translational modifications similar to those observed in human native proteins. However, during the last years, a growing number of studies have shown the possibility to produce active forms of lysosomal proteins in other expression systems, such as plants and microorganisms. In this paper, we review the production and characterization of human lysosomal proteins, deficient in several LSDs, which have been produced in microorganisms. For this purpose, Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, and Ogataea minuta have been used as expression systems. The recombinant lysosomal proteins expressed in these hosts have shown similar substrate specificities, and temperature and pH stability profiles to those produced in mammalian cells. In addition, pre-clinical results have shown that recombinant lysosomal enzymes produced in microorganisms can be taken-up by cells and reduce the substrate accumulated within the lysosome. Recently, metabolic engineering in yeasts has allowed the production of lysosomal enzymes with tailored N-glycosylations, while progresses in E. coli N-glycosylations offer a potential platform to improve the production of these recombinant lysosomal enzymes. In summary, microorganisms represent convenient platform for the production of recombinant lysosomal proteins for biochemical and physicochemical characterization, as well as for the development of ERT for LSD. PMID:26071627

  17. 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.

  18. 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.

  19. 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.

  20. A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.

    PubMed

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

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling 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 to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation. PMID:26950892

  1. 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

  2. Lysosomal Acid Phosphatase Biosynthesis and Dysfunction: A Mini Review Focused on Lysosomal Enzyme Dysfunction in Brain.

    PubMed

    Ashtari, N; Jiao, X; Rahimi-Balaei, M; Amiri, S; Mehr, S E; Yeganeh, B; Marzban, H

    2016-01-01

    Lysosomes are membrane-bound organelles that are responsible for degrading and recycling macromolecules. Lysosomal dysfunction occurs in enzymatic and non-enzymatic deficiencies, which result in abnormal accumulation of materials. Although lysosomal storage disorders affect different organs, the central nervous system is the most vulnerable. Evidence shows the role of lysosomal dysfunction in different neurodegenerative diseases, such as Niemann-Pick Type C disease, juvenile neuronal ceroid lipofuscinosis, Alzheimer's disease and Parkinson's disease. Lysosomal enzymes such as lysosomal acid phosphatase 2 (Acp2) play a critical role in mannose-6-phosphate removal and Acp2 controls molecular and cellular functions in the brain during development and adulthood. Acp2 is essential in cerebellar development, and mutations in this gene cause severe cerebellar neurodevelopmental and neurodegenerative disorders. In this mini-review, we highlight lysosomal dysfunctions in the pathogenesis of neurodevelopmental and/or neurodegenerative diseases with special attention to Acp2 dysfunction. PMID:27132795

  3. A topological and conformational stability alphabet for multipass membrane proteins.

    PubMed

    Feng, Xiang; Barth, Patrick

    2016-03-01

    Multipass membrane proteins perform critical signal transduction and transport across membranes. How transmembrane helix (TMH) sequences encode the topology and conformational flexibility regulating these functions remains poorly understood. Here we describe a comprehensive analysis of the sequence-structure relationships at multiple interacting TMHs from all membrane proteins with structures in the Protein Data Bank (PDB). We found that membrane proteins can be deconstructed in interacting TMH trimer units, which mostly fold into six distinct structural classes of topologies and conformations. Each class is enriched in recurrent sequence motifs from functionally unrelated proteins, revealing unforeseen consensus and evolutionary conserved networks of stabilizing interhelical contacts. Interacting TMHs' topology and local protein conformational flexibility were remarkably well predicted in a blinded fashion from the identified binding-hotspot motifs. Our results reveal universal sequence-structure principles governing the complex anatomy and plasticity of multipass membrane proteins that may guide de novo structure prediction, design, and studies of folding and dynamics. PMID:26780406

  4. The effects of aspirin and dimethyl sulphoxide on the latency of lysosomes in a cell-free system.

    PubMed

    Zodrow, J; Rogers, S H

    1984-05-14

    Lysosomal preparations were exposed to various concentrations of dimethylsulphoxide (DMSO) and aspirin singularly and in combination. Acid phosphatase and beta-glucuronidase activities were measured and utilized as an indication of lysosomal membrane stability under experimental conditions in the presence and absence of these drugs. Extremely low concentrations of each drug were employed in an attempt to mimic the levels which might be feasible in vivo. There was a significant decrease of enzyme activity (increased structure-linked latency) in the presence of DMSO. Aspirin had no significant effect on the latency of the lysosomes. There was no indication of synergism between DMSO and aspirin. It was concluded that some of the therapeutic advantages attributed to DMSO in the treatment of arthritis and other musculoskeletal diseases may come from the stabilization of lysosomes in cells that contribute to the pathological condition. Aspirin did not seem to exert a therapeutic effect through this mechanism. PMID:6727549

  5. Stabilized composite membranes and membrane electrode assemblies for elevated temperature/low relative humidity PEFC operation

    NASA Astrophysics Data System (ADS)

    Ramani, Vijay; Kunz, H. R.; Fenton, J. M.

    An approach is presented to combine existing heteropolyacid (HPA) additive and membrane electrode assembly (MEA) stabilization techniques to yield a stabilized MEA for operation at 120 °C and 35% relative humidity (RH). MEAs were prepared using Nafion ®/phosphotungstic acid composite membranes with a phosphotungstic acid (PTA) particle size of 30-50 nm. The PTA additive was stabilized by substituting its protons with cesium counter ions. The Nafion ® in the membrane and electrodes was simultaneously converted to the Cs + form by an ion-exchange process. The melt processability of the Nafion ® in the Cs + form permitted the MEA to be heat treated at 200 °C and 30 atm, promoting the development of a durable membrane/electrode interface. The prior stabilization of the PTA permitted MEA re-protonation with minimal additive loss. FTIR spectroscopy and thermogravimetric analysis (TGA) were employed to present evidence of ion-exchange and protonation. In situ electrochemical impedance measurements (EIS) and cyclic voltammetry (CV) measurements confirmed ion-exchange and protonation within the active portion of the stabilized MEA. The stabilization process did not affect the integrity of the MEA, with the hydrogen crossover currents through the membrane remaining unchanged at 2 mA cm -2. The MEA was evaluated at 120 °C and 35% relative humidity in an operating fuel cell environment and yielded respectable performance under these conditions.

  6. 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.

  7. 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.

  8. Identification of differential anti-neoplastic activity of copper bis(thiosemicarbazones) that is mediated by intracellular reactive oxygen species generation and lysosomal membrane permeabilization.

    PubMed

    Stefani, Christian; Al-Eisawi, Zaynab; Jansson, Patric J; Kalinowski, Danuta S; Richardson, Des R

    2015-11-01

    Bis(thiosemicarbazones) and their copper (Cu) complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined the structure-activity relationships of twelve bis(thiosemicarbazones) to elucidate factors regarding their anti-cancer efficacy. Importantly, the alkyl substitutions at the diimine position of the ligand backbone resulted in two distinct groups, namely, unsubstituted/monosubstituted and disubstituted bis(thiosemicarbazones). This alkyl substitution pattern governed their: (1) Cu(II/I) redox potentials; (2) ability to induce cellular (64)Cu release; (3) lipophilicity; and (4) anti-proliferative activity. The potent anti-cancer Cu complex of the unsubstituted bis(thiosemicarbazone) analog, glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), generated intracellular reactive oxygen species (ROS), which was attenuated by Cu sequestration by a non-toxic Cu chelator, tetrathiomolybdate, and the anti-oxidant, N-acetyl-l-cysteine. Fluorescence microscopy suggested that the anti-cancer activity of Cu(GTSM) was due, in part, to lysosomal membrane permeabilization (LMP). For the first time, this investigation highlights the role of ROS and LMP in the anti-cancer activity of bis(thiosemicarbazones). PMID:26335599

  9. 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. PMID:16917129

  10. 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

  11. Lysosome-associated membrane glycoprotein (LAMP) – preliminary study on a hidden antigen target for vaccination against schistosomiasis

    PubMed Central

    Nawaratna, Sujeevi S. K.; Gobert, Geoffrey N.; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P.; Jones, Malcolm K.

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16–25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  12. Lysosome-associated membrane glycoprotein (LAMP)--preliminary study on a hidden antigen target for vaccination against schistosomiasis.

    PubMed

    Nawaratna, Sujeevi S K; Gobert, Geoffrey N; Willis, Charlene; Mulvenna, Jason; Hofmann, Andreas; McManus, Donald P; Jones, Malcolm K

    2015-01-01

    Our previously reported gene atlasing of schistosome tissues revealed transcripts that were highly enriched in the digestive tract of Schistosoma mansoni. From these, we selected two candidates, Sm-LAMP and Sm-NPC2 for testing as vaccine targets. The two molecules were selected on the basis of relatively high expression in the gastrodermis, their potentially important biological function, divergence from homologous molecules of the host and possible apical membrane expression in the gastrodermis. Bacterially expressed recombinant peptides corresponding to regions excluding trans-membrane domains of the selected vaccine targets were used in blinded vaccine trials in CBA mice using alum-CpG as adjuvant. Vaccine trials using the recombinant insoluble Sm-LAMP protein showed 16-25% significant reduction in total worm burden. Faecal egg count reduction was 52% and 60% in two trials, respectively, with similar results for the solubly expressed protein. Liver egg burden was reduced significantly (20% and 38%) with an insoluble recombinant Sm-LAMP in two trials, but not with the soluble recombinant form. Parasite fecundity was not affected by either Sm-LAMP protein preparations in the trials. It is concluded that Sm-LAMP may provide limited protection towards S. mansoni infections but could be used in combination with other vaccine candidates, to provide more comprehensive protection. PMID:26472258

  13. Chelation of lysosomal iron protects against ionizing radiation.

    PubMed

    Berndt, Carsten; Kurz, Tino; Selenius, Markus; Fernandes, Aristi P; Edgren, Margareta R; Brunk, Ulf T

    2010-12-01

    Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH. PMID:20846118

  14. BORC, a Multisubunit Complex that Regulates Lysosome Positioning

    PubMed Central

    Pu, Jing; Schindler, Christina; Jia, Rui; Jarnik, Michal; Backlund, Peter; Bonifacino, Juan S.

    2016-01-01

    SUMMARY The positioning of lysosomes within the cytoplasm is emerging as a critical determinant of many lysosomal functions. Here we report the identification of a multi-subunit complex named BORC that regulates lysosome positioning. BORC comprises eight subunits, some of which are shared with the BLOC-1 complex involved in the biogenesis of lysosome-related organelles, and the others of which are products of previously uncharacterized open reading frames. BORC associates peripherally with the lysosomal membrane, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that promotes the Kinesin-1-dependent movement of lysosomes toward the plus ends of microtubules in the peripheral cytoplasm. Interference with BORC or other components of this pathway results in collapse of the lysosomal population into the pericentriolar region. In turn, this causes reduced cell spreading and migration, highlighting the importance of BORC-dependent centrifugal transport for non-degradative functions of lysosomes. PMID:25898167

  15. 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.

  16. Involvement of Lysosome Membrane Permeabilization and Reactive Oxygen Species Production in the Necrosis Induced by Chlamydia muridarum Infection in L929 Cells.

    PubMed

    Chen, Lixiang; Wang, Cong; Li, Shun; Yu, Xin; Liu, Xue; Ren, Rongrong; Liu, Wenwen; Zhou, Xiaojing; Zhang, Xiaonan; Zhou, Xiaohui

    2016-04-28

    Chlamydiae, obligate intracellular bacteria, are associated with a variety of human diseases. The chlamydial life cycle undergoes a biphasic development: replicative reticulate bodies (RBs) phase and infectious elementary bodies (EBs) phase. At the end of the chlamydial intracellular life cycle, EBs have to be released to the surrounded cells. Therefore, the interactions between Chlamydiae and cell death pathways could greatly influence the outcomes of Chlamydia infection. However, the underlying molecular mechanisms remain elusive. Here, we investigated host cell death after Chlamydia infection in vitro, in L929 cells, and showed that Chlamydia infection induces cell necrosis, as detected by the propidium iodide (PI)-Annexin V double-staining flow-cytometric assay and Lactate dehydrogenase (LDH) release assay. The production of reactive oxygen species (ROS), an important factor in induction of necrosis, was increased after Chlamydia infection, and inhibition of ROS with specific pharmacological inhibitors, diphenylene iodonium (DPI) or butylated hydroxyanisole (BHA), led to significant suppression of necrosis. Interestingly, live-cell imaging revealed that Chlamydia infection induced lysosome membrane permeabilization (LMP). When an inhibitor upstream of LMP, CA-074-Me, was added to cells, the production of ROS was reduced with concomitant inhibition of necrosis. Taken together, our results indicate that Chlamydia infection elicits the production of ROS, which is dependent on LMP at least partially, followed by induction of host-cell necrosis. To our best knowledge, this is the first live-cell-imaging observation of LMP post Chlamydia infection and report on the link of LMP to ROS to necrosis during Chlamydia infection. PMID:26838343

  17. PPARα in lysosomal biogenesis: A perspective

    PubMed Central

    Ghosh, Arunava; Pahan, Kalipada

    2016-01-01

    Lysosomes are membrane-bound vesicles containing hydrolytic enzymes, ubiquitously present in all eukaryotic cells. Classically considered to be central to the cellular waste management machinery, recent studies revealed the role of lysosomes in a wide array of cellular processes like, degradation, cellular development, programmed cell death, secretion, plasma membrane repair, nutritional responses, and lipid metabolism. We recently studied the regulation of TFEB, considered to be the master regulator of lysosomal biogenesis, by activation of peroxisomal proliferator activated receptor α (PPARα), one of the key regulators of lipid metabolism. In this article, we discuss how the recent finding could be put in to perspective with the previous findings that relate lysosomal biogenesis to lipid metabolism, and comment on the possibility of a bi-directional interplay between these two distinct cellular processes upon activation of PPARα. PMID:26621249

  18. 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

  19. Lysosomal destabilization in p53-induced apoptosis

    PubMed Central

    Yuan, Xi-Ming; Li, Wei; Dalen, Helge; Lotem, Joseph; Kama, Rachel; Sachs, Leo; Brunk, Ulf T.

    2002-01-01

    The tumor suppressor wild-type p53 can induce apoptosis. M1-t-p53 myeloid leukemic cells have a temperature-sensitive p53 protein that changes its conformation to wild-type p53 after transfer from 37°C to 32°C. We have now found that these cells showed an early lysosomal rupture after transfer to 32°C. Mitochondrial damage, including decreased membrane potential and release of cytochrome c, and the appearance of apoptotic cells occurred later. Lysosomal rupture, mitochondrial damage, and apoptosis were all inhibited by the cytokine IL-6. Some other compounds can also inhibit apoptosis induced by p53. The protease inhibitor N-tosyl-l-phenylalanine chloromethyl ketone inhibited the decrease in mitochondrial membrane potential and cytochrome c release, the Ca2+-ATPase inhibitor thapsigargin inhibited only cytochrome c release, and the antioxidant butylated hydroxyanisole inhibited only the decrease in mitochondrial membrane potential. In contrast to IL-6, these other compounds that inhibited some of the later occurring mitochondrial damage did not inhibit the earlier p53-induced lysosomal damage. The results indicate that apoptosis is induced by p53 through a lysosomal-mitochondrial pathway that is initiated by lysosomal destabilization, and that this pathway can be dissected by using different apoptosis inhibitors. These findings on the induction of p53-induced lysosomal destabilization can also help to formulate new therapies for diseases with apoptotic disorders. PMID:11959917

  20. Podocytes degrade endocytosed albumin primarily in lysosomes.

    PubMed

    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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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. PMID:25201349

  6. Enzyme stabilization by linear chain polymers in ultrafiltration membrane reactors

    SciTech Connect

    Greco, G.; Gianfreda, L.

    1981-10-01

    The experimental results discussed in this article concern pi-nitrophenylphosphate hydrolysis by acid phosphatase in an ultrafiltration membrane reactor. The basic conclusions drawn are : 1) Linking the enzyme to a soluble support does not give rise to an increase in its stability while the chemical manipulations involved result in marked reductions in enzymic activity. 2) Enzyme entrapment within a proteic gel produces a considerable increase in its thermal stability as compared to the diluted native enzyme; this presumably stems from drastic reductions in enzyme mobility. 3) Correspondingly, considerable reductions occur in enzyme activity that depend on substrate mass transfer resistances within the gel layer. 4) Small amounts of linear chain water-soluble synthetic polymers (polyacrylamides) give rise to high macromolecular concentration levels in the reactor region where the enzyme is dynamically immobilized and produce the same enzyme stabilization as gel entrapment. 5) Only minor substrate mass transfer limitations take place in this region and hence enzyme activity is virtually unaffected. 6) Both effects (stabilization and slight activity reduction) seem not to depend strongly on the characteristics of the soluble polymer (molecular weight and ionic character). (Refs. 16).

  7. 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.

  8. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    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 cm2 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.Nanopore size, shape, and surface charge all play

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

    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 cm2 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.Nanopore size, shape, and surface charge all play

  11. The late endosome/lysosome-anchored p18-mTORC1 pathway controls terminal maturation of lysosomes

    SciTech Connect

    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose; Okada, Masato

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. Black-Right-Pointing-Pointer We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. Black-Right-Pointing-Pointer The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. Black-Right-Pointing-Pointer Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. Black-Right-Pointing-Pointer The p18-mTORC1 pathway plays crucial roles in the terminal maturation of lysosomes. -- Abstract: The late endosome/lysosome membrane adaptor p18 (or LAMTOR1) serves as an anchor for the mammalian target of rapamycin complex 1 (mTORC1) and is required for its activation on lysosomes. The loss of p18 causes severe defects in cell growth as well as endosome dynamics, including membrane protein transport and lysosome biogenesis. However, the mechanisms underlying these effects on lysosome biogenesis remain unknown. Here, we show that the p18-mTORC1 pathway is crucial for terminal maturation of lysosomes. The loss of p18 causes aberrant intracellular distribution and abnormal sizes of late endosomes/lysosomes and an accumulation of late endosome specific components, including Rab7, RagC, and LAMP1; this suggests that intact late endosomes accumulate in the absence of p18. These defects are phenocopied by inhibiting mTORC1 activity with rapamycin. Loss of p18 also suppresses the integration of late endosomes and lysosomes, resulting in the defective degradation of tracer proteins. These results suggest that the p18-mTORC1 pathway plays crucial roles in the late stages of lysosomal maturation, potentially in late endosome-lysosome fusion, which is required for processing of various macromolecules.

  12. Supported liquid membrane stability in chiral resolution by chemically and physically modified membranes.

    PubMed

    Molinari, R; Argurio, P

    2001-01-01

    In the present work some stability studies on Supported Liquid Membranes (SLMs) to be used for chiral separations were realized. In particular, primary aim was to determine how a modification of the support surface influences the SLM stability. First, the procedure for support modification was optimised, making a screening of various compounds (sulphuric acid, nitric acid, chromic acid, sodium dodecyl sulphate (SDS), glycerol, oleic alcohol, propylene glycol (PPG), bovine serum albumin (BSA)) and testing their performance by means of contact angle measurements. Next, a second screening was realized by permeation tests in a stirred cell. Finally, to compare the stability of modified with unmodified support in a process of interest for chemical and/or biochemical industries, some permeation tests for resolution of DNB-DL-Leucine were realized in a re-circulation system. Results showed a better surface hydrophilization of chemically modified support and better stability of the sulphonated support. However, in operating conditions a little high stability of the unmodified support was obtained. PMID:11381544

  13. 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. PMID:27430889

  14. 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. PMID:25450976

  15. Isolation of Lysosomes from Mammalian Tissues and Cultured Cells.

    PubMed

    Aguado, Carmen; Pérez-Jiménez, Eva; Lahuerta, Marcos; Knecht, Erwin

    2016-01-01

    Lysosomes participate within the cells in the degradation of organelles, macromolecules, and a wide variety of substrates. In any study on specific roles of lysosomes, both under physiological and pathological conditions, it is advisable to include methods that allow their reproducible and reliable isolation. However, purification of lysosomes is a difficult task, particularly in the case of cultured cells. This is mainly because of the heterogeneity of these organelles, along with their low number and high fragility. Also, isolation methods, while disrupting plasma membranes, have to preserve the integrity of lysosomes, as the breakdown of their membranes releases enzymes that could damage all cell organelles, including themselves. The protocols described below have been routinely used in our laboratory for the specific isolation of lysosomes from rat liver, NIH/3T3, and other cultured cells, but can be adapted to other mammalian tissues or cell lines. PMID:27613045

  16. 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.

  17. The amino acid transporter SLC38A9 is a key component of a lysosomal membrane complex that signals arginine sufficiency to mTORC1

    PubMed Central

    Wang, Shuyu; Tsun, Zhi-Yang; Wolfson, Rachel; Shen, Kuang; Wyant, Gregory A.; Plovanich, Molly E.; Yuan, Elizabeth D.; Jones, Tony D.; Chantranupong, Lynne; Comb, William; Wang, Tim; Bar-Peled, Liron; Zoncu, Roberto; Straub, Christoph; Kim, Choah; Park, Jiwon; Sabatini, Bernardo L.; Sabatini, David M.

    2015-01-01

    The mTOR complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and v-ATPase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. Here, we identify SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag GTPases and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Km and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway. PMID:25567906

  18. 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. PMID:27050453

  19. A lysosome-centered view of nutrient homeostasis

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:27050453

  20. Haptoglobin Genotype-dependent Differences in Macrophage Lysosomal Oxidative Injury*

    PubMed Central

    Asleh, Rabea; Ward, John; Levy, Nina S.; Safuri, Shady; Aronson, Doron; Levy, Andrew P.

    2014-01-01

    The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb. PMID:24778180

  1. 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. PMID:23393155

  2. 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

  3. 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. PMID:25926456

  4. The thermomechanical stability of micro-solid oxide fuel cells fabricated on anodized aluminum oxide membranes

    NASA Astrophysics Data System (ADS)

    Kwon, Chang-Woo; Lee, Jae-Il; Kim, Ki-Bum; Lee, Hae-Weon; Lee, Jong-Ho; Son, Ji-Won

    2012-07-01

    The thermomechanical stability of micro-solid oxide fuel cells (micro-SOFCs) fabricated on an anodized aluminum oxide (AAO) membrane template is investigated. The full structure consists of the following layers: AAO membrane (600 nm)/Pt anode/YSZ electrolyte (900 nm)/porous Pt cathode. The utilization of a 600-nm-thick AAO membrane significantly improves the thermomechanical stability due to its well-known honeycomb-shaped nanopore structure. Moreover, the Pt anode layer deposited in between the AAO membrane and the YSZ electrolyte preserves its integrity in terms of maintaining the triple-phase boundary (TPB) and electrical conductivity during high-temperature operation. Both of these results guarantee thermomechanical stability of the micro-SOFC and extend the cell lifetime, which is one of the most critical issues in the fabrication of freestanding membrane-type micro-SOFCs.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Spin Stability Criteria for Axisymmetric Spinning Body with Large Flexible Membrane

    NASA Astrophysics Data System (ADS)

    Tsuda, Yuichi

    Stability of the dynamics of spinning spacecraft equipped with a large flexible membrane is discussed. The spin stability of an object having a flexible structure is deteriorated from that of a rigid object with the same shape in general, which makes a significant impact on the design of such spacecraft systems. This paper provides a fully analytical solution for the stability criteria of axisymmetric spinner spacecraft equipped with a large flexible membrane. Distinct from past studies, this paper does not impose restrictions on axial offset of the membrane attachment, and provides a stability condition using energy-angular momentum analysis when the membrane is subject only to an outer-plane first-order mode deformation. These criteria can be applied to the design of such missions as spinner solar sail spacecraft, large solar power satellite and other general spacecraft with large axisymmetric structures.

  7. 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

  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. PMID:27241615

  9. Hyper-branched anion exchange membranes with high conductivity and chemical stability.

    PubMed

    Ge, Qianqian; Liu, Yazhi; Yang, Zhengjin; Wu, Bin; Hu, Min; Liu, Xiaohe; Hou, Jianqiu; Xu, Tongwen

    2016-08-01

    In the manuscript, we report the design and preparation of hyper-branched polymer electrolytes intended for alkaline anion exchange membrane fuel cells. The resulting membrane exhibits high conductivity, lower water swelling and shows prolonged chemical stability under alkaline conditions. PMID:27456659

  10. Distinct Lysosomal Network Protein Profiles in Parkinsonian Syndrome Cerebrospinal Fluid

    PubMed Central

    Boman, Andrea; Svensson, Samuel; Boxer, Adam; Rojas, Julio C.; Seeley, William W.; Karydas, Anna; Miller, Bruce; Kågedal, Katarina; Svenningsson, Per

    2016-01-01

    Background: Clinical diagnosis of parkinsonian syndromes like Parkinson’s disease (PD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP) is hampered by overlapping symptomatology and lack of diagnostic biomarkers, and definitive diagnosis is only possible post-mortem. Objective: Since impaired protein degradation plays an important role in many neurodegenerative disorders, we hypothesized that profiles of select lysosomal network proteins in cerebrospinal fluid could be differentially expressed in these parkinsonian syndromes. Methods: Cerebrospinal fluid samples were collected from PD patients (n = 18), clinically diagnosed 4-repeat tauopathy patients; corticobasal syndrome (CBS) (n = 3) and PSP (n = 8); and pathologically diagnosed PSP (n = 8) and CBD patients (n = 7). Each patient set was compared to its appropriate control group consisting of age and gender matched individuals. Select lysosomal network protein levels were detected via Western blotting. Factor analysis was used to test the diagnostic sensitivity, specificity and accuracy of the select lysosomal network protein expression profiles. Results: PD, CBD and PSP were markedly different in their cerebrospinal fluid lysosomal network protein profiles. Lysosomal-associated membrane proteins 1 and 2 were significantly decreased in PD; early endosomal antigen 1 was decreased and lysozyme increased in PSP; and lysosomal-associated membrane proteins 1 and 2, microtubule-associated protein 1 light chain 3 and lysozyme were increased in CBD. A panel of lysosomal-associated membrane protein 2, lysozyme and microtubule-associated protein 1 light chain discriminated between controls, PD and 4-repeat tauopathies. Conclusions: This study offers proof of concept that select lysosomal network proteins are differentially expressed in cerebrospinal fluid of Parkinson’s disease, corticobasal syndrome and progressive supranuclear palsy. Lysosomal network protein analysis

  11. Stability of Beams, Plates and Membranes due to Subsonic Aerodynamic Flows and Solar Radiation Pressure

    NASA Astrophysics Data System (ADS)

    Gibbs, Samuel Chad, IV

    This dissertation explores the stability of beams, plates and membranes due to subsonic aerodynamic flows or solar radiation forces. Beams, plates and membranes are simple structures that may act as building blocks for more complex systems. In this dissertation we explore the stability of these simple structures so that one can predict instabilities in more complex structures. The theoretical models include both linear and nonlinear energy based models for the structural dynamics of the featureless rectangular structures. The structural models are coupled to a vortex lattice model for subsonic fluid flows or an optical reflection model for solar radiation forces. Combinations of these theoretical models are used to analyze the dynamics and stability of aeroelastic and solarelastic systems. The dissertation contains aeroelastic analysis of a cantilevered beam and a plate / membrane system with multiple boundary conditions. The dissertation includes analysis of the transition from flag-like to wing-like flutter for a cantilevered beam and experiments to quantify the post flutter fluid and structure response of the flapping flag. For the plate / membrane analysis, we show that the boundary conditions in the flow direction determine the type of instability for the system while the complete set of boundary conditions is required to accurately predict the flutter velocity and frequency. The dissertation also contains analysis of solarelastic stability of membranes for solar sail applications. For a fully restrained membrane we show that a flutter instability is possible, however the post flutter response amplitude is small. The dissertation also includes analysis of a membrane hanging in gravity. This systems is an analog to a spinning solar sail and is used to validate the structural dynamics of thin membranes on earth. A linear beam structural model is able to accurately capture the natural frequencies and mode shapes. Finally, the dissertation explores the stability

  12. Stability of DNA-Tethered Lipid Membranes with Mobile Tethers

    PubMed Central

    Chung, Minsub; Boxer, Steven G.

    2011-01-01

    We recently introduced two approaches for tethering planar lipid bilayers as membrane patches to either a supported lipid bilayer or DNA-functionalized surface using DNA hybridization (Chung, M., Lowe, R. D., Chan, Y-H. M., Ganesan, P. V., Boxer, S. G. J. Struct. Biol. 2009, 168, 190–9). When mobile DNA tethers are used, the tethered bilayer patches become unstable, while they are stable if the tethers are fixed on the surface. Because the mobile tethers between a patch and a supported lipid bilayer offer a particularly interesting architecture for studying the dynamics of membrane-membrane interactions, we have investigated the sources of instability, focusing on membrane composition. The most stable patches were made with a mixture of saturated lipids and cholesterol, suggesting an important role for membrane stiffness. Other factors such as the effect of tether length, lateral mobility and patch membrane edge were also investigated. Based on these results, a model for the mechanism of patch destruction is developed. PMID:21452847

  13. Lysosomal Trafficking Regulator (LYST).

    PubMed

    Ji, Xiaojie; Chang, Bo; Naggert, Jürgen K; Nishina, Patsy M

    2016-01-01

    Regulation of vesicle trafficking to lysosomes and lysosome-related organelles (LROs) as well as regulation of the size of these organelles are critical to maintain their functions. Disruption of the lysosomal trafficking regulator (LYST) results in Chediak-Higashi syndrome (CHS), a rare autosomal recessive disorder characterized by oculocutaneous albinism, prolonged bleeding, severe immunodeficiency, recurrent bacterial infection, neurologic dysfunction and hemophagocytic lympohistiocytosis (HLH). The classic diagnostic feature of the syndrome is enlarged LROs in all cell types, including lysosomes, melanosomes, cytolytic granules and platelet dense bodies. The most striking CHS ocular pathology observed is an enlargement of melanosomes in the retinal pigment epithelium (RPE), which leads to aberrant distribution of eye pigmentation, and results in photophobia and decreased visual acuity. Understanding the molecular function of LYST and identification of its interacting partners may provide therapeutic targets for CHS and other diseases associated with the regulation of LRO size and/or vesicle trafficking, such as asthma, urticaria and Leishmania amazonensis infections. PMID:26427484

  14. 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. PMID:25750174

  15. Proton-Assisted Amino Acid Transporter PAT1 Complexes with Rag GTPases and Activates TORC1 on Late Endosomal and Lysosomal Membranes

    PubMed Central

    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. PMID:22574197

  16. Structural design considerations for stretched-membrane heliostat reflector modules with stability and initial imperfection considerations

    SciTech Connect

    Murphy, L.M.; Simms, D.; Sallis, D.V.

    1986-10-01

    This report extends the work of several previous reports that present the background leading to the development of stretched-membrane modules and analysis methods to study the structural response of the stretched-membrane module. Specifically, this report presents and discusses the design implications based on our analysis of single- or double-membrane concepts, and the amplification of initial imperfections and deflections caused by loading, which results from stability considerations. In this document, we present analysis results for both single- and double-membrane concepts corresponding to a range of design and loading conditions. Further, we show that stretched-membrane/frame combinations respond quite differently to external loads than can be inferred by studying the decoupled frame and membrane independently. Thus the coupled membrane/frame problem should be considered to assure an accurate description of its response. For idealized configurations and loadings, we discuss the relative merits of various design features for both of these designs. In addition, we studied the structural stability (i.e., the tendency of structural deformation to grow with little increase in applied load) of the tensioned-membrane, compressed-frame combination. Moreover, we demonstrate how stability considerations are important in determining the amplification of both initial displacement imperfection and the deformations caused by wind and weight loading on the structure.

  17. Symmetry aspects in stability investigations for thin membranes

    NASA Astrophysics Data System (ADS)

    Eriksson, Anders; Nordmark, Arne

    2016-08-01

    Modelling of structural instability problems is considered for thin square membranes subjected to hydrostatic pressure, with a focus on the effects from symmetry conditions considered or neglected in the model. An analysis is performed through group-theoretical concepts of the symmetry aspects present in a flat membrane with one-sided pressure loading. The response of the membrane is described by its inherent differential eigensolutions, which are shown to be of five different types with respect to symmetry. A discussion is given on how boundary conditions must be introduced in order to catch all types of eigensolutions when modelling only a subdomain of the whole. Lacking symmetry in a FEM model of the whole domain is seen as a perturbation to the problem, and is shown to affect the calculated instability response, hiding or modifying instability modes. Numerical simulations verify and illustrate the analytical results, and further show the convergence with mesh fineness of different aspects of instability results.

  18. 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. PMID:23223708

  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. PMID:27214685

  20. 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

  1. Entropic forces stabilize diverse emergent structures in colloidal membranes

    NASA Astrophysics Data System (ADS)

    Kang, Louis; Gibaud, Thomas; Dogic, Zvonimir; Lubensky, T. C.

    The depletion interaction mediated by non-adsorbing polymers promotes condensation and assembly of repulsive colloidal particles into diverse higher-order structures and materials. One example, with particularly rich emergent behaviors, is the formation of two-dimensional colloidal membranes from a suspension of filamentous $\\it{fd}$ viruses, which act as rods with effective repulsive interactions, and dextran, which acts as a condensing, depletion-inducing agent. Colloidal membranes exhibit chiral twist even when the constituent virus mixture lacks macroscopic chirality, change from a circular shape to a striking starfish shape upon changing the chirality of constituent rods, and partially coalesce via domain walls through which the viruses twist by $180^\\circ$. We formulate an entropically-motivated theory that can quantitatively explain these experimental structures and measurements, both previously published and newly performed, over a wide range of experimental conditions. Our results elucidate how entropy alone, manifested through the viruses as Frank elastic energy and through the depletants as an effective surface tension, drives the formation and behavior of these diverse structures. Our generalizable principles propose the existence of analogous effects in molecular membranes and can be exploited in the design of reconfigurable colloidal structures.

  2. Entropic forces stabilize diverse emergent structures in colloidal membranes.

    PubMed

    Kang, Louis; Gibaud, Thomas; Dogic, Zvonimir; Lubensky, T C

    2016-01-14

    The depletion interaction mediated by non-adsorbing polymers promotes condensation and assembly of repulsive colloidal particles into diverse higher-order structures and materials. One example, with particularly rich emergent behaviors, is the formation of two-dimensional colloidal membranes from a suspension of filamentous fd viruses, which act as rods with effective repulsive interactions, and dextran, which acts as a condensing, depletion-inducing agent. Colloidal membranes exhibit chiral twist even when the constituent virus mixture lacks macroscopic chirality, change from a circular shape to a striking starfish shape upon changing the chirality of constituent rods, and partially coalesce via domain walls through which the viruses twist by 180°. We formulate an entropically-motivated theory that can quantitatively explain these experimental structures and measurements, both previously published and newly performed, over a wide range of experimental conditions. Our results elucidate how entropy alone, manifested through the viruses as Frank elastic energy and through the depletants as an effective surface tension, drives the formation and behavior of these diverse structures. Our generalizable principles propose the existence of analogous effects in molecular membranes and can be exploited in the design of reconfigurable colloidal structures. PMID:26472139

  3. 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. PMID:25340256

  4. 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

  5. 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

  6. Enriching membrane cholesterol improves stability and cryosurvival of buffalo spermatozoa.

    PubMed

    Rajoriya, J S; Prasad, J K; Ramteke, S S; Perumal, P; Ghosh, S K; Singh, M; Pande, Megha; Srivastava, N

    2016-01-01

    Buffalo spermatozoa are comparatively more susceptible to freezing hazards than cattle spermatozoa. In recent times incubation of spermatozoa with cholesterol-loaded-cyclodextrins (CLC) has shown improvements in semen quality in several species. Therefore, this study was undertaken to evaluate the incubation level of CLC at which maximum benefit is derived for the buffalo spermatozoa. For the study, 120 million spermatozoa were incubated in 2, 3 and 4 mg/mL of CLC (Gr II, III and IV, respectively) and cholesterol and phospholipids content, their ratio, flow cytometric evaluation of plasma membrane integrity (PMI), plasma membrane fluidity and extent of cryoinjury (Chlortetracycline, CTC assay) were compared with an untreated control (Gr I). Additionally the ability of cholesterol-loaded-spermatozoa to undergo induced acrosome reaction (IAR) using ionophore calcium (A23187) was evaluated in frozen-thaw samples. Data show a significant and linear increase (CV=0.88) in cholesterol content of spermatozoa in Gr II, III and IV and a significant decrease in phospholipids content at frozen-thaw stage in Gr IV than Gr III spermatozoa. The study revealed a significant improvement in PMI and significant reduction in plasma membrane fluidity and cryoinjury of CLC treated spermatozoa at progressive stages in three groups compared to control. Nevertheless, spermatozoa of Gr II, III and IV were significantly less responsive to ionophore calcium (A23187) than Gr I. This study shows for the first time that incubation of buffalo bull spermatozoa with CLC (3mg/120×10(6)) prior to processing permits greater numbers of sperm to survive cryopreservation while allowing spermatozoa to capacitate and the acrosome to react to AR inducer ionophore calcium (A23187). PMID:26619942

  7. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    PubMed Central

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

    2014-01-01

    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. PMID:25183830

  8. 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

  9. 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. PMID:24245310

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

    PubMed

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

    2011-08-11

    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

  11. Low pH modulates the macroorganization and thermal stability of PSII supercomplexes in grana membranes.

    PubMed

    Stoichev, Svetozar; Krumova, Sashka B; Andreeva, Tonya; Busto, Jon V; Todinova, Svetla; Balashev, Konstantin; Busheva, Mira; Goñi, Félix M; Taneva, Stefka G

    2015-02-17

    Protonation of the lumen-exposed residues of some photosynthetic complexes in the grana membranes occurs under conditions of high light intensity and triggers a major photoprotection mechanism known as energy dependent nonphotochemical quenching. We have studied the role of protonation in the structural reorganization and thermal stability of isolated grana membranes. The macroorganization of granal membrane fragments in protonated and partly deprotonated state has been mapped by means of atomic force microscopy. The protonation of the photosynthetic complexes has been found to induce large-scale structural remodeling of grana membranes-formation of extensive domains of the major light-harvesting complex of photosystem II and clustering of trimmed photosystem II supercomplexes, thinning of the membrane, and reduction of its size. These events are accompanied by pronounced thermal destabilization of the photosynthetic complexes, as evidenced by circular dichroism spectroscopy and differential scanning calorimetry. Our data reveal a detailed nanoscopic picture of the initial steps of nonphotochemical quenching. PMID:25692589

  12. Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

    PubMed

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

    2016-02-19

    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

  13. Lysosomal Storage Diseases.

    PubMed

    Kaye, Edward M.

    2001-05-01

    Lysosomal storage disorders (LSDs), over 40 different diseases, are now considered treatable disorders. Only a few short years ago, Lysosomal storage disorders were seen as interesting neurodegenerative disorders without any potential for treatment. Effective treatment strategies such as bone marrow transplantation (BMT), enzyme replacement therapy (ERT), and glycolipid synthesis inhibition have been developed in the last 20 years and continue to be researched and evaluated. Bone marrow transplantation began approximately 15 years ago and has shown benefit for some of the lysosomal storage disorders. In order to be effective, the transplant must be performed early in the course of the disease, before the development of irreversible neurologic damage. Diseases such as Hurler appear to respond to BMT, however, improvement in bone disease is much less vigorous than responses in other organs. Krabbe disease responds if the transplant is performed before irreversible signs of neurologic damage appear. Metachromatic leukodystrophy may respond if the transplant can be performed early enough although peripheral nerve findings appear to progress. Other diseases, eg, GM1- and GM2-gangliosidoses do not appear to be altered by BMT. Despite its high cost, ERT has been very effective treatment for type I (non-neuronopathic) Gaucher disease. Enzyme replacement therapy for other LSDs, including ERT for Fabry and Pompe diseases, which are planned to be imminently introduced, and other enzymes such as for Morquio and Hunter diseases that are in the study phases, may be marketed in the very near future. Glycolipid inhibitors, such as N-butyldeoxynijirimycin (OGS-918), have been effective in reducing the liver and spleen volume in type I Gaucher disease. These oral inhibitors may prove to be important adjuncts to ERT and provide the advantage of being able to cross the blood/brain barrier, which limits enzyme access to brain. Currently, clinical studies are being conducted on patients

  14. 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

  15. 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

  16. An amphipathic polypeptide derived from poly-γ-glutamic acid for the stabilization of membrane proteins

    PubMed Central

    Han, Seong-Gu; Na, Jung-Hyun; Lee, Won-Kyu; Park, Dongkook; Oh, Jihye; Yoon, Sung-Ho; Lee, Cheng-Kang; Sung, Moon-Hee; Shin, Yeon-Kyun; Yu, Yeon Gyu

    2014-01-01

    Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-γ-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4–5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ETA) in their active conformations. Furthermore, ETA in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins. PMID:25283538

  17. Exocytotic fusion pore stability and topological defects in the membrane with orientational degree of ordering.

    PubMed

    Jesenek, Dalija; Perutková, Sárka; Kralj-Iglič, Veronika; Kralj, Samo; Iglič, Aleš

    2012-01-01

    Regulated exocytosis is a process that strongly depends on the formation and stability of the fusion pore. It was indicated experimentally and theoretically that narrow and highly curved fusion pore may be stabilized by accumulation of anisotropic membrane components possessing orientational ordering. On the other hand, narrow fusion pore may also undergo repetitive opening and closing, disruption in the so called kiss and run process or become completely opened in the process of full fusion of the vesicle with the membrane. In this paper we attempt to elucidate the subtle interplay between the stabilizing and destabilizing processes in the fusion neck. A possible physical mechanism which may lead to disruption of the stable fusion pore or complete fusion of the vesicle with the membrane is discussed. It is indicated that topologically driven defects of the in-plane orientational membrane ordering in the region of the fusion pore may disrupt the fusion. Alternatively, it may facilitate repetitive opening and closing of the fusion pore or induce full fusion of the vesicle with the target membrane. PMID:22541648

  18. Lysosomal Adaptation: How the Lysosome Responds to External Cues

    PubMed Central

    Settembre, Carmine; Ballabio, Andrea

    2014-01-01

    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. PMID:24799353

  19. Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures.

    PubMed

    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

    2011-01-12

    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

  20. 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.

  1. Effect of phosphorylation on the thermal and light stability of the thylakoid membranes.

    PubMed

    Várkonyi, Zsuzsanna; Nagy, Gergely; Lambrev, Petar; Kiss, Anett Z; Székely, Noémi; Rosta, László; Garab, Gyözö

    2009-03-01

    Higher plant thylakoid membranes contain a protein kinase that phosphorylates certain threonine residues of light-harvesting complex II (LHCII), the main light-harvesting antenna complexes of photosystem II (PSII) and some other phosphoproteins (Allen, Biochim Biophys Acta 1098:275, 1992). While it has been established that phosphorylation induces a conformational change of LHCII and also brings about changes in the lateral organization of the thylakoid membrane, it is not clear how phosphorylation affects the dynamic architecture of the thylakoid membranes. In order to contribute to the elucidation of this complex question, we have investigated the effect of duroquinol-induced phosphorylation on the membrane ultrastructure and the thermal and light stability of the chiral macrodomains and of the trimeric organization of LHCII. As shown by small angle neutron scattering on thylakoid membranes, duroquinol treatment induced a moderate (~10%) increase in the repeat distance of stroma membranes, and phosphorylation caused an additional loss of the scattering intensity, which is probably associated with the partial unstacking of the granum membranes. Circular dichroism (CD) measurements also revealed only minor changes in the chiral macro-organization of the complexes and in the oligomerization state of LHCII. However, temperature dependences of characteristic CD bands showed that phosphorylation significantly decreased the thermal stability of the chiral macrodomains in phosphorylated compared to the non-phosphorylated samples (in leaves and isolated thylakoid membranes, from 48.3 degrees C to 42.6 degrees C and from 47.5 degrees C to 44.3 degrees C, respectively). As shown by non-denaturing PAGE of thylakoid membranes and CD spectroscopy on EDTA washed membranes, phosphorylation decreased by about 5 degrees C, the trimer-to-monomer transition temperature of LHCII. It also enhanced the light-induced disassembly of the chiral macrodomains and the monomerization of the

  2. Synthesis of "group polysaccharide" by membranes from Streptococcus pyogenes and its stabilized L-form.

    PubMed Central

    Reusch, V M; Panos, C

    1977-01-01

    Rhamnose and N-acetylglucosamine (GlcNAc) are incorporated from thymidine 5'-diphosphorhamnose and uridine 5-diphospho-N-acetylglucosamine (UDPGlcNAc) into membrane fragments prepared from Streptococcus pyogenes but not into membrane fragements prepared from a stabilized L-form of this organism. Incorporation from TDPrhamnose is partially dependent upon UDPGlcNAc and vice versa. The oligomeric GlcNAc and rhamonose-containing products are easily extracted from membrane particles by sedimentation through detergent solutions. They are substantially extracted into methanol but not into chloroform-methanol (2:1). When product containing both radioactive rhamnose and GlcNAc is deacetylated and hydrolyzed briefy in acid, glucosaminyl rhamnose is obtained, byt not higher oligomers, suggesting that oligomer synthesis in vitro is terminated because unidentified wnzymatic requirements are not satisfied. The data are consistent with the assembly of group A-specific polysaccharide at the cellular membrane with participation of a lipoid anchor (acceptor) molecule. PMID:321425

  3. Chain ordering of hybrid lipids can stabilize domains in saturated/hybrid/cholesterol lipid membranes

    NASA Astrophysics Data System (ADS)

    Yamamoto, T.; Brewster, R.; Safran, S. A.

    2010-07-01

    We use a liquid-crystal model to predict that hybrid lipids (lipids that have one saturated and one unsaturated tail) can stabilize line interfaces between domains in mixed membranes of saturated lipids, hybrid lipids, and cholesterol (SHC membranes). The model predicts the phase separation of SHC membranes with both parabolic and loop binodals depending on the cholesterol concentration, modeled via an effective pressure. In some cases, the hybrid lipids can reduce the line tension to zero in SHC membranes at temperatures that approach the critical temperature as the pressure is increased. The differences in the hybrid saturated tail conformational order in bulk and at the interface are responsible for the reduction of the line tension.

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

    PubMed

    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

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

    PubMed

    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 Ca(2+)-dependent manner, and are known to play a role in the storage of Ca(2+) and membrane healing. Little is known, however, about the dynamic processes of protein-Ca(2+)-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 Ca(2+) and membrane affinities depending on the assembly location, providing a wide Ca(2+) 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 Ca(2+) in the reversible binding of annexin to the membrane surface. PMID:27271964

  6. MCOLN1 is a ROS sensor in lysosomes that regulates autophagy.

    PubMed

    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 Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) 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

  7. Mannose 6 Dephosphorylation of Lysosomal Proteins Mediated by Acid Phosphatases Acp2 and Acp5

    PubMed Central

    Makrypidi, Georgia; Damme, Markus; Müller-Loennies, Sven; Trusch, Maria; Schmidt, Bernhard; Schlüter, Hartmut; Heeren, Joerg; Lübke, Torben; Saftig, Paul

    2012-01-01

    Mannose 6-phosphate (Man6P) residues represent a recognition signal required for efficient receptor-dependent transport of soluble lysosomal proteins to lysosomes. Upon arrival, the proteins are rapidly dephosphorylated. We used mice deficient for the lysosomal acid phosphatase Acp2 or Acp5 or lacking both phosphatases (Acp2/Acp5−/−) to examine their role in dephosphorylation of Man6P-containing proteins. Two-dimensional (2D) Man6P immunoblot analyses of tyloxapol-purified lysosomal fractions revealed an important role of Acp5 acting in concert with Acp2 for complete dephosphorylation of lysosomal proteins. The most abundant lysosomal substrates of Acp2 and Acp5 were identified by Man6P affinity chromatography and mass spectrometry. Depending on the presence of Acp2 or Acp5, the isoelectric point of the lysosomal cholesterol-binding protein Npc2 ranged between 7.0 and 5.4 and may thus regulate its interaction with negatively charged lysosomal membranes at acidic pH. Correspondingly, unesterified cholesterol was found to accumulate in lysosomes of cultured hepatocytes of Acp2/Acp5−/− mice. The data demonstrate that dephosphorylation of Man6P-containing lysosomal proteins requires the concerted action of Acp2 and Acp5 and is needed for hydrolysis and removal of degradation products. PMID:22158965

  8. 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

  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. Ceramic membrane fouling during ultrafiltration of oil/water emulsions: roles played by stabilization surfactants of oil droplets.

    PubMed

    Lu, Dongwei; Zhang, Tao; Ma, Jun

    2015-04-01

    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. PMID:25730119

  11. Decreased aperture surface energy enhances electrical, mechanical, and temporal stability of suspended lipid membranes.

    PubMed

    Bright, Leonard K; Baker, Christopher A; Agasid, Mark T; Ma, Lin; Aspinwall, Craig A

    2013-11-27

    The development of next-generation transmembrane protein-based biosensors relies heavily on the use of black lipid membranes (BLMs); however, electrical, mechanical, and temporal instability of BLMs poses a limiting challenge to biosensor development. In this work, micrometer-sized glass apertures were modified with silanes of different chain length and fluorine composition, including 3-cyanopropyldimethychlorosilane (CPDCS), ethyldimethylchlorosilane (EDCS), n-octyldimethylchlorosilane (ODCS), (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)dimethylchlorosilane (PFDCS), or (heptadecafluoro-1,1,2,2-tetrahydrodecyl)dimethylchlorosilane (PFDDCS), to explore the effect of substrate surface energy on BLM stability. Low energy silane-modified surfaces promoted enhanced lipid-substrate interactions that facilitate the formation of low-leakage, stable BLMs. The surface energies of silane-modified substrates were 30 ± 3, 16 ± 1, 14 ± 2, 11 ± 1, and 7.1 ± 2 mJ m(-2) for CDCS, EDCS, ODCS, PFDCS, and PFDDCS, respectively. Decreased surface energy directly correlated to improved electrical, mechanical, and temporal BLM stability. Amphiphobic perfluorinated surface modifiers yielded superior performance compared to traditional hydrocarbon modifiers in terms of stability and BLM formation, with only marginal effects on BLM membrane permeability. Leakage currents obtained for PFDCS and PFDDCS BLMs were elevated only 10-30%, though PFDDCS modification yielded >5-fold increase in electrical stability as indicated by breakdown voltage (> 2000 mV vs 418 ± 73 mV), and >25-fold increase in mechanical stability as indicated by air-water transfers (> 50 vs 2 ± 0.2) when compared to previously reported CPDCS modification. Importantly, the dramatically improved membrane stabilities were achieved with no deleterious effects on reconstituted ion channel function, as evidenced by α-hemolysin activity. Thus, this approach provides a simple, low cost, and broadly applicable alternative for

  12. Polar interactions trump hydrophobicity in stabilizing the self-inserting membrane protein Mistic.

    PubMed

    Broecker, Jana; Fiedler, Sebastian; Gimpl, Katharina; Keller, Sandro

    2014-10-01

    Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane-interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and β-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins. PMID:25177765

  13. Stability of KcsA tetramer depends on membrane lateral pressure.

    PubMed

    van den Brink-van der Laan, Els; Chupin, Vladimir; Killian, J Antoinette; de Kruijff, Ben

    2004-04-13

    The potassium channel KcsA forms an extremely stable tetramer. Despite this high stability, it has been shown that the membrane-mimicking solvent 2,2,2-trifluoroethanol (TFE) can induce tetramer dissociation [Valiyaveetil, F. I., et al. (2002) Biochemistry 41, 10771-7, and Demmers, J. A. A., et al. (2003) FEBS Lett. 541, 69-77]. Here we have studied the effect of TFE on the structure and oligomeric state of the KcsA tetramer, reconstituted in different lipid systems. It was found that TFE changes the secondary and tertiary structure of KcsA and that it can dissociate the KcsA tetramer in all systems used. The tetramer is stabilized by a lipid bilayer as compared to detergent micelles. The extent of stabilization was found to depend on the nature of the lipids: a strong stabilizing effect of the nonbilayer lipid phosphatidylethanolamine (PE) was observed, but no effect of the charged phoshosphatidylglycerol (PG) as compared to phosphatidylcholine (PC) was found. To understand how lipids stabilize KcsA against TFE-induced tetramer dissociation, we also studied the effect of TFE on the bilayer organization in the various lipid systems, using (31)P and (2)H NMR. The observed lipid dependency was similar as was found for tetramer stabilization: PE increased the bilayer stability as compared to PC, while PG behaved similar to PC. Furthermore, it was found that TFE has a large effect on the acyl chain ordering. The results indicate that TFE inserts primarily in the membrane interface. We suggest that the lipid bilayer stabilizes the KcsA tetramer by the lateral pressure in the acyl chain region and that this stabilizing effect increases when a nonbilayer lipid like PE is present. PMID:15065868

  14. Pluronic F108 coating decreases the lung fibrosis potential of multiwall carbon nanotubes by reducing lysosomal injury.

    PubMed

    Wang, Xiang; Xia, Tian; Duch, Matthew C; Ji, Zhaoxia; Zhang, Haiyuan; Li, Ruibin; Sun, Bingbing; Lin, Sijie; Meng, Huan; Liao, Yu-Pei; Wang, Meiying; Song, Tze-Bin; Yang, Yang; Hersam, Mark C; Nel, André E

    2012-06-13

    We compared the use of bovine serum albumin (BSA) and pluronic F108 (PF108) as dispersants for multiwalled carbon nanotubes (MWCNTs) in terms of tube stability as well as profibrogenic effects in vitro and in vivo. While BSA-dispersed tubes were a potent inducer of pulmonary fibrosis, PF108 coating protected the tubes from damaging the lysosomal membrane and initiating a sequence of cooperative cellular events that play a role in the pathogenesis of pulmonary fibrosis. Our results suggest that PF108 coating could serve as a safer design approach for MWCNTs. PMID:22546002

  15. PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins.

    PubMed

    McEwan, David G; Popovic, Doris; Gubas, Andrea; Terawaki, Seigo; Suzuki, Hironori; Stadel, Daniela; Coxon, Fraser P; Miranda de Stegmann, Diana; Bhogaraju, Sagar; Maddi, Karthik; Kirchof, Anja; Gatti, Evelina; Helfrich, Miep H; Wakatsuki, Soichi; Behrends, Christian; Pierre, Philippe; Dikic, Ivan

    2015-01-01

    The lysosome is the final destination for degradation of endocytic cargo, plasma membrane constituents, and intracellular components sequestered by macroautophagy. Fusion of endosomes and autophagosomes with the lysosome depends on the GTPase Rab7 and the homotypic fusion and protein sorting (HOPS) complex, but adaptor proteins that link endocytic and autophagy pathways with lysosomes are poorly characterized. Herein, we show that Pleckstrin homology domain containing protein family member 1 (PLEKHM1) directly interacts with HOPS complex and contains a LC3-interacting region (LIR) that mediates its binding to autophagosomal membranes. Depletion of PLEKHM1 blocks lysosomal degradation of endocytic (EGFR) cargo and enhances presentation of MHC class I molecules. Moreover, genetic loss of PLEKHM1 impedes autophagy flux upon mTOR inhibition and PLEKHM1 regulates clearance of protein aggregates in an autophagy- and LIR-dependent manner. PLEKHM1 is thus a multivalent endocytic adaptor involved in the lysosome fusion events controlling selective and nonselective autophagy pathways. PMID:25498145

  16. 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

    2016-07-01

    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

  17. 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. PMID:26170381

  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. PMID:12926908

  19. The position of lysosomes within the cell determines their luminal pH.

    PubMed

    Johnson, Danielle E; Ostrowski, Philip; Jaumouillé, Valentin; Grinstein, Sergio

    2016-03-14

    We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H(+)-adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility. PMID:26975849

  20. The position of lysosomes within the cell determines their luminal pH

    PubMed Central

    Johnson, Danielle E.; Ostrowski, Philip; Jaumouillé, Valentin

    2016-01-01

    We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H+–adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility. PMID:26975849

  1. Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography

    PubMed Central

    Gallagher, Elyssia S.; Adem, Seid M.; Baker, Christopher A.; Ratnayaka, Saliya N.; Jones, Ian W.; Hall, Henry K.; Saavedra, S. Scott; Aspinwall, Craig A.

    2015-01-01

    The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2′,4′-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50mM triton X-100 or 50% acetonitrile) and physical (15 min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed > 44% increase in retention times (P < 0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance towards the development of robust membrane protein-functionalized chromatographic stationary phases. PMID:25670414

  2. Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22.

    PubMed

    Schlebach, Jonathan P; Narayan, Malathi; Alford, Catherine; Mittendorf, Kathleen F; Carter, Bruce D; Li, Jun; Sanders, Charles R

    2015-07-15

    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

  3. 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

  4. Using tryptophan fluorescence to measure the stability of membrane proteins folded in liposomes

    PubMed Central

    Moon, C. Preston; Fleming, Karen G.

    2013-01-01

    Accurate measurements of the thermodynamic stability of folded membrane proteins require methods for monitoring their conformation that are free of experimental artifacts. For tryptophan fluorescence emission experiments with membrane proteins folded into liposomes, there are two significant sources of artifacts: the first is light scattering by the liposomes; the second is the nonlinear relationship of some tryptophan spectral parameters with changes in protein conformation. Both of these sources of error can interfere with the method of determining the reversible equilibrium thermodynamic stability of proteins using titrations of chemical denaturants. Here, we present methods to manage light scattering by liposomes for tryptophan emission experiments and to properly monitor tryptophan spectra as a function of protein conformation. Our methods are tailored to the titrations of membrane proteins using common chemical denaturants. One of our recommendations is to collect and analyze the right-angle light scattering peak that occurs around the excitation wave- length in a fluorescence experiment. Another recommendation is to use only those tryptophan spectral parameters that are linearly proportional to the protein conformational population. We show that other commonly used spectral commonly used parameters lead to errors in protein stability measurements. PMID:21333792

  5. 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. PMID:27223603

  6. 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. PMID:25513883

  7. mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells

    PubMed Central

    Saric, Amra; Hipolito, Victoria E. B.; Kay, Jason G.; Canton, Johnathan; Antonescu, Costin N.; Botelho, Roberto J.

    2016-01-01

    Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase–Akt–mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes. PMID:26582390

  8. mTOR controls lysosome tubulation and antigen presentation in macrophages and dendritic cells.

    PubMed

    Saric, Amra; Hipolito, Victoria E B; Kay, Jason G; Canton, Johnathan; Antonescu, Costin N; Botelho, Roberto J

    2016-01-15

    Macrophages and dendritic cells exposed to lipopolysaccharide (LPS) convert their lysosomes from small, punctate organelles into a network of tubules. Tubular lysosomes have been implicated in phagosome maturation, retention of fluid phase, and antigen presentation. There is a growing appreciation that lysosomes act as sensors of stress and the metabolic state of the cell through the kinase mTOR. Here we show that LPS stimulates mTOR and that mTOR is required for LPS-induced lysosome tubulation and secretion of major histocompatibility complex II in macrophages and dendritic cells. Specifically, we show that the canonical phosphatidylinositol 3-kinase-Akt-mTOR signaling pathway regulates LPS-induced lysosome tubulation independently of IRAK1/4 and TBK. Of note, we find that LPS treatment augmented the levels of membrane-associated Arl8b, a lysosomal GTPase required for tubulation that promotes kinesin-dependent lysosome movement to the cell periphery, in an mTOR-dependent manner. This suggests that mTOR may interface with the Arl8b-kinesin machinery. To further support this notion, we show that mTOR antagonists can block outward movement of lysosomes in cells treated with acetate but have no effect in retrograde movement upon acetate removal. Overall our work provides tantalizing evidence that mTOR plays a role in controlling lysosome morphology and trafficking by modulating microtubule-based motor activity in leukocytes. PMID:26582390

  9. Deficiency of ATP13A2 leads to lysosomal dysfunction, α-synuclein accumulation and neurotoxicity

    PubMed Central

    Usenovic, Marija; Tresse, Emilie; Mazzulli, Joseph R.; Taylor, J. Paul; Krainc, Dimitri

    2012-01-01

    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. PMID:22442086

  10. 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.

  11. 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. PMID:27022872

  12. Use of BODIPY-Cholesterol (TF-Chol) for Visualizing Lysosomal Cholesterol Accumulation.

    PubMed

    Hölttä-Vuori, Maarit; Sezgin, Erdinc; Eggeling, Christian; Ikonen, Elina

    2016-09-01

    Dipyrromethene difluoride-cholesterol (TopFluor-Cholesterol, TF-Chol) is a widely used cholesterol analogue due to its excellent fluorescence properties and considerable similarity with natural cholesterol in terms of membrane partitioning. However, the suitability of TF-Chol for detecting lysosomal cholesterol deposition has recently been questioned. Here, we highlight the fact that the method of lipid delivery and the analysis of time-point both affect the membrane distribution and labeling pattern of TF-Chol, similarly as with radiolabeled cholesterol. Lysosomal sterol accumulation characteristic to a lysosomal storage disease is most readily detected when the probe is introduced via the physiological route, i.e. as a sterol fatty acid ester in low-density lipoprotein particles. When administered to cells from solvent, lysosomal sterol sequestration becomes evident after an overnight equilibration between membranes. PMID:27187581

  13. Ultrasonic-assisted synthesis of ZrO2 nanoparticles and their application to improve the chemical stability of Nafion membrane in proton exchange membrane (PEM) fuel cells.

    PubMed

    Taghizadeh, Mohammad Taghi; Vatanparast, Morteza

    2016-12-01

    Zirconium dioxide (ZrO2) nanoparticles were fabricated successfully via ultrasonic-assisted method using ZrO(NO3)2·H2O, ethylenediamine and hydrazine as precursors in aqueous solution. Morphology, structure and composition of the obtained products were characterized by means of X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FT-IR) and diffuse reflectance spectroscopy (DRS). Then, the synthesized nanoparticles were used to prepare Nafion/ZrO2 nanocomposite membranes. The properties of the membranes were studied by ion exchange capacity (IEC) proton conductivity (σ), thermal stability and water uptake measurements. The ex-situ Fenton's test was used to investigate the chemical stability of the membranes. From our results, compared with Nafion membrane, the nanocomposite membrane exhibited lower fluoride release and weight loss. Therefore, it can concluded that Nafion/ZrO2 nanocomposite exhibit more chemical stability than the pure Nafion membrane. ATR-FTIR spectra and SEM surface images of membranes also confirm these results. PMID:27544443

  14. Protective effect of betaine on changes in the levels of lysosomal enzyme activities in heart tissue in isoprenaline-induced myocardial infarction in Wistar rats

    PubMed Central

    Anandan, Rangasamy

    2009-01-01

    Myocardial infarction is one of the most common manifestations of cardiovascular disease. In the present study, we investigated the protective effect of betaine, a potent lipotropic molecule, on changes in the levels of lysosomal enzymes and lipid peroxidation in isoprenaline-induced myocardial infarction in Wistar rats, an animal model of myocardial infarction in man. Male albino Wistar rats were pretreated with betaine (250 mg/kg body weight) daily for a period of 30 days. After the treatment period, isoprenaline (11 mg/100 g body weight) was intraperitoneally administered to rats at intervals of 24 h for 2 days. The activities of lysosomal enzymes (β-glucuronidase, β-galactosidase, β-glucosidase, and acid phosphatase) were significantly (p < 0.05) increased in plasma with a concomitant decline in the activities of these enzymes in heart tissue of isoprenaline-administered rats. Also, the level of lipid peroxidation was higher in heart lysosomes of isoprenaline-injected rats. Pretreatment with betaine daily for a period of 30 days to isoprenaline-induced rats prevented the changes in the activities of these lysosomal enzymes. Oral treatment with betaine (250 mg/kg body weight) to normal control rats did not show any significant effect in all the biochemical parameters studied. Thus, the results of our study show that betaine protects the lysosomal membrane against isoprenaline-induced myocardial infarction. The observed effects might be due to the free radical-scavenging and membrane-stabilizing properties of betaine. PMID:19294532

  15. Neuronal lysosomal enzyme replacement using fragment C of tetanus toxin.

    PubMed

    Dobrenis, K; Joseph, A; Rattazzi, M C

    1992-03-15

    Development of a strategy for efficient delivery of exogenous enzyme to neuronal lysosomes is essential to achieve enzyme replacement in neurodegenerative lysosomal storage diseases. We tested whether effective lysosomal targeting of the human enzyme beta-N-acetylhexosaminidase A (Hex A; beta-N-acetyl-D-hexosaminide N-acetylhexosaminohydrolase, EC 3.2.1.52) can be obtained by coupling it via disulfide linkage to the atoxic fragment C of tetanus toxin (TTC) that is bound avidly by neuronal membrane. TTC-Hex A conjugation resulted in neuronal surface binding and enhanced endocytosis of enzyme as observed in immunofluorescence studies with rat brain cultures. In immunoelectrophoretic quantitative uptake studies, rat neuronal cell cultures contained 16- and 40-fold greater amounts of enzyme after incubation with TTC-Hex A than with nonderivatized Hex A. In cerebral cortex cell cultures from a feline model of human GM2 gangliosidosis (Tay-Sachs and Sandhoff diseases), binding and uptake patterns of the enzymes were similar to those in the rat brain cell cultures. After exposure to extracellular concentrations of enzyme attainable in vivo, lysosomal storage of immunodetectable GM2 ganglioside was virtually eliminated in neurons exposed to TTC-Hex A, whereas a minimal effect was observed with Hex A. These findings demonstrate the usefulness of TTC adducts for effective neuronal lysosomal enzyme replacement. PMID:1532255

  16. LAMP proteins are required for fusion of lysosomes with phagosomes.

    PubMed

    Huynh, Kassidy K; Eskelinen, Eeva-Liisa; Scott, Cameron C; Malevanets, Anatoly; Saftig, Paul; Grinstein, Sergio

    2007-01-24

    Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) are delivered to phagosomes during the maturation process. We used cells from LAMP-deficient mice to analyze the role of these proteins in phagosome maturation. Macrophages from LAMP-1- or LAMP-2-deficient mice displayed normal fusion of lysosomes with phagosomes. Because ablation of both the lamp-1 and lamp-2 genes yields an embryonic-lethal phenotype, we were unable to study macrophages from double knockouts. Instead, we reconstituted phagocytosis in murine embryonic fibroblasts (MEFs) by transfection of FcgammaIIA receptors. Phagosomes formed by FcgammaIIA-transfected MEFs obtained from LAMP-1- or LAMP-2- deficient mice acquired lysosomal markers. Remarkably, although FcgammaIIA-transfected MEFs from double-deficient mice ingested particles normally, phagosomal maturation was arrested. LAMP-1 and LAMP-2 double-deficient phagosomes acquired Rab5 and accumulated phosphatidylinositol 3-phosphate, but failed to recruit Rab7 and did not fuse with lysosomes. We attribute the deficiency to impaired organellar motility along microtubules. Time-lapse cinematography revealed that late endosomes/lysosomes as well as phagosomes lacking LAMP-1 and LAMP-2 had reduced ability to move toward the microtubule-organizing center, likely precluding their interaction with each other. PMID:17245426

  17. Non-esterified Cholesterol Content of Lysosomes Modulates Susceptibility to Oxidant-induced Permeabilization

    PubMed Central

    Reiners, John J.; Kleinman, Miriam; Kessel, David; Mathieu, Patricia A.; Caruso, Joseph A.

    2010-01-01

    Reactive oxygen species (ROS) can induce lysosomal membrane permeabilization (LMP). Photoirradiation of murine hepatoma 1c1c7 cultures preloaded with the photosensitizer NPe6 generates singlet oxygen within acidic organelles, and causes LMP and the activation of procaspases. Treatment with the cationic amphiphilic drugs (CADs) U18666A, imipramine, and clozapine stimulated the accumulation of filipin-stainable non-esterified cholesterol/sterols in late endosomes/lysosomes, but not in mitochondria. Concentration-response studies demonstrated an inverse relationship between lysosomal non-esterified cholesterol/sterol contents and susceptibility to NPe6 photoirradiation-induced intracellular membrane oxidation, LMP, and activation of procaspases-9 and -3. Similarly, the kinetics of restoration of NPe6 photoirradiation-induced LMP paralleled the losses of lysosomal cholesterol that occurred upon replating U18666A-treated cultures in CAD-free medium. Consistent with the oxidation of lysosomal cholesterol, filipin staining in U18666A-treated cultures progressively decreased with increasing photoirradiating light dose. U18666A also suppressed the inductions of LMP and procaspase activation by exogenously added hydrogen peroxide. However, neither U18666A nor imipramine suppressed the induction of apoptosis by agents that did not directly induce LMP. These studies indicate that lysosomal non-esterified cholesterol/sterol content modulates susceptibility to ROS-induced LMP, and possibly does so by being an alternative target for oxidants and lowering the probability of damage to other lysosomal membrane lipids and/or proteins. PMID:21074609

  18. 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.

  19. 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.

  20. Oxidative stability and in vitro digestibility of fish oil-in-water emulsions containing multilayered membranes.

    PubMed

    Gudipati, Venkateshwarlu; Sandra, Sandra; McClements, David Julian; Decker, Eric Andrew

    2010-07-14

    The oxidative stability and lipid digestibility of fish oil-in-water emulsions (d(43); 5.26-5.71 microm) laminated by primary, secondary, and/or tertiary layers of interfacial membranes have been investigated. The primary emulsion (5 and 0.5% wt % of fish oil and Citrem in acetate buffer) was produced through a membrane homogenizer. The second and tertiary emulsions were prepared by electrostatic deposition of chitosan and sodium alginate on the surfaces of the oil droplets, respectively. The lamination of biopolymers was measured by zeta potential. The lipid oxidative stability was assessed with peroxide value, thiobabituric acid reactive substances, and headspace aldehydes of the emulsions stored at 20 degrees C for 40 days. The positively charged secondary emulsions (+56.27 +/- 2.5 mV) were more stable to lipid oxidation compared to negatively charged primary (-45.13 +/- 1.7 mV) and tertiary emulsions (-24.8 +/- 1.2 mV). An in vitro digestion model was used to study the impact of different layers on the digestibility of oil droplets. Lipid digestion was decreased with multilayer coating, and chitosan coating further reduced the digestion. These findings have implications for the design of structured emulsions to achieve better oxidative stability with more controlled digestibility of lipids. PMID:20527781

  1. 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. PMID:27494174

  2. Stabilization of Model Membrane Systems by Disaccharides. Quasielastic Neutron Scattering Experiments and Atomistic Simulations

    NASA Astrophysics Data System (ADS)

    Doxastakis, Emmanouil; Garcia Sakai, Victoria; Ohtake, Satoshi; Maranas, Janna K.; de Pablo, Juan J.

    2006-03-01

    Trehalose, a disaccharide of glucose, is often used for the stabilization of cell membranes in the absence of water. This work studies the effects of trehalose on model membrane systems as they undergo a melting transition using a combination of experimental methods and atomistic molecular simulations. Quasielastic neutron scattering experiments on selectively deuterated samples provide the incoherent dynamic structure over a wide time range. Elastic scans probing the lipid tail dynamics display clear evidence of a main melting transition that is significantly lowered in the presence of trehalose. Lipid headgroup mobility is considerably restricted at high temperatures and directly associated with the dynamics of the sugar in the mixture. Molecular simulations provide a detailed overview of the dynamics and their spatial and time dependence. The combined simulation and experimental methodology offers a unique, molecular view of the physics of systems commonly employed in cryopreservation and lyophilization processes.

  3. 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

  4. The lysosome as a command-and-control center for cellular metabolism.

    PubMed

    Lim, Chun-Yan; Zoncu, Roberto

    2016-09-12

    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

  5. 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

  6. Amylase release from dissociated mouse pancreatic acinar cells stimulated by glucagon: effect of membrane stabilizers.

    PubMed Central

    Singh, M

    1980-01-01

    1. The effect of membrane stabilizers and cytochalasin-B on amylase secretion, basal and induced by ionophore A23187, CCK-PZ, bethanechol and glucagon, was studied in dissociated mouse pancreatic acinar cells. 2. Cytochalasin-B did not affect basal or secretagogue-stimulated amylase secretion. 3. Membrane stabilizers [thymol (10(-7)-10(-4) M), chlorpromazine (10(-7)-10(-4) M) and propranolol (10(-7)-10(-5) M) did not alter basal release of amylase. At higher concentrations of thymol (10(-3) M), chlorpromazine (10(-3) M) and propranolol (10(-4) M), dissociated acinar cells were lysed as indicated by an increase in release of lactic dehydrogenase (LDH). 4. Ionophore A23187, CCK-PZ (maximal effective concentrations, 0.01 u. ml.-1), bethanechol (maximal effective concentrations, 10(-4) M) and glucagon increased amylase secretion in a dose-dependent fashion. Concentrations of CCK-PZ and bethanechol beyond optimal levels decreased amylase secretion. Concentrations of ionophore A23187 and glucagon when tested beyond 10(-6) M and 10(-4) M respectively increased the release of LDH. In concentrations that were non-toxic, membrane stabilizers blocked the stimulating effect of cholecystokinin-pancreozymin and bethanechol on amylase secretion but did not alter the response to A23187 and glucagon. 5. Unlike bethanechol, glucagon neither increased the uptake of 45Ca nor did it alter the release of 45Ca from cells previously loaded with 45CaCl2. 6. These data provide evidence that stimulus-secretion coupling in dissociated pancreatic acinar cells is basically similar to cells in situ. The effect of glucagon is consistent with the model in which hormone-dependent mobilization of Ca2+ from intra- or extracellular sources is bypassed leading to digestive enzyme secretion. PMID:6166745

  7. 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

  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. 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. PMID:27158976

  10. 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