RESUMO
Flippases are responsible for the asymmetric distribution of phospholipids in biological membranes. In the encapsulated fungal pathogen Cryptococcus neoformans, the putative flippase Apt1 is an important regulator of polysaccharide secretion and pathogenesis in mice by unknown mechanisms. In this study, we analyzed the role of C. neoformans Apt1 in intracellular membrane architecture and synthesis of polysaccharide and lipids. Analysis of wild type (WT), apt1Δ (mutant) and apt1Δ::APT1 (complemented) strains by transmission electron microscopy revealed that deletion of APT1 resulted in the formation of irregular vacuoles. Disorganization of vacuolar membranes in apt1Δ cells was accompanied by a significant increase in the amounts of intra-vacuolar and pigment-containing vesicles. Quantitative immunogold labeling of C. neoformans cells with a monoclonal antibody raised to a major capsular component suggested impaired polysaccharide synthesis. APT1 deletion also affected synthesis of phosphatidylserine, phosphatidylethanolamine, inositolphosphoryl ceramide, glucosylceramide and ergosterylglycoside. These results reveal novel functions of Apt1 and are in agreement with the notion that this putative flippase plays an important role in the physiology of C. neoformans.
Assuntos
Cryptococcus neoformans/genética , Proteínas Fúngicas/genética , Membranas Intracelulares/metabolismo , Lipídeos/biossíntese , Animais , Membrana Celular/genética , Membrana Celular/metabolismo , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidade , Membranas Intracelulares/química , Lipídeos/genética , Camundongos , Polissacarídeos/biossíntese , VirulênciaRESUMO
Cryptococcus neoformans is an encapsulated pathogenic fungus. The cryptococcal capsule is composed of polysaccharides and is necessary for virulence. It has been previously reported that glucuronoxylomannan (GXM), the major capsular component, is synthesized in cytoplasmic compartments and transported to the extracellular space in vesicles, but knowledge on the organelles involved in polysaccharide synthesis and traffic is extremely limited. In this paper we report the GXM distribution in C. neoformans cells sectioned by cryoultramicrotomy and visualized by transmission electron microscopy (TEM) and polysaccharide immunogold staining. Cryosections of fungal cells showed high preservation of intracellular organelles and cell wall structure. Incubation of cryosections with an antibody to GXM revealed that cytoplasmic structures associated to vesicular compartments and reticular membranes are in close proximity to the polysaccharide. GXM was generally found in association with the membrane of intracellular compartments and within different layers of the cell wall. Analysis of extracellular fractions from cryptococcal supernatants by transmission electron microscopy in combination with serologic, chromatographic and spectroscopic methods revealed fractions containing GXM and lipids. These results indicate an intimate association of GXM and lipids in both intracellular and extracellular spaces consistent with polysaccharide synthesis and transport in membrane-associated structures.
Assuntos
Cryptococcus neoformans/química , Cryptococcus neoformans/ultraestrutura , Vesículas Citoplasmáticas/química , Lipídeos de Membrana/análise , Polissacarídeos/análise , Fatores de Virulência/análise , Transporte Biológico , Fracionamento Celular , Parede Celular/química , Parede Celular/ultraestrutura , Crioultramicrotomia , Vesículas Citoplasmáticas/ultraestrutura , Espaço Extracelular/química , Imuno-Histoquímica , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Organelas/química , Organelas/ultraestruturaRESUMO
In previous studies, Mucor circinelloides YR-1 isolated from petroleum-contaminated soils grown in decane as sole carbon source, showed fatty alcohol oxidase (FAO) activities in either particulate or soluble fractions from a cell-free extract. One is associated to internal membranes (mFAO) and the other one is soluble (sFAO). Both activities appear to be located in the cells in specific compartments other than peroxisomes. Results suggested that mFAO could be located on the inner face of the membrane of these compartments, and sFAO could be in the lumen of the specific compartments. This study reports on the intracellular distribution of FAO activity and the purification of sFAOs and mFAO after several different procedures for release from the membranous fraction using the mixed membrane fraction (MMF) after cellular homogenization as enzymatic source. Results with the purified mFAO show, by molecular weight criteria, that the enzyme has only one type of subunit with molecular mass of 46 kDa, with two isoelectric point components: 6.0 and 6.3. We found that mFAO is strongly associated to the MMF, possibly in a transitory fashion. Using non-denaturating gels, we suggest that sFAO and mFAO have the same subunits in their native structures, and, due to their native molecular weight of approximately 350 kDa, each could be natively structured as an octameric complex.
Assuntos
Álcoois Graxos/metabolismo , Mucor/química , Mucor/enzimologia , Oxirredutases/análise , Microbiologia do Solo , Membrana Celular/química , Eletroforese em Gel de Poliacrilamida/métodos , Immunoblotting , Membranas Intracelulares/química , Ponto Isoelétrico , Peso Molecular , Mucor/isolamento & purificação , Oxirredutases/química , Oxirredutases/isolamento & purificação , Petróleo , Multimerização Proteica , Subunidades Proteicas , Poluentes do SoloRESUMO
Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.
Assuntos
Encéfalo/citologia , Proliferação de Células , Retículo Endoplasmático/química , Complexo de Golgi/química , Membranas Intracelulares/química , Animais , Western Blotting , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Feminino , Glioblastoma/química , Glioblastoma/metabolismo , Glioblastoma/patologia , Complexo de Golgi/ultraestrutura , Humanos , Imuno-Histoquímica , Membranas Intracelulares/ultraestrutura , Lipídeos de Membrana/análise , Proteínas de Membrana/análise , Fósforo/análise , Ratos , Ratos WistarRESUMO
Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional glycophosphoprotein, secreted by different cell types, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated in this work by immunofluorescence and immunoelectron microscopy approaches the localization of an OPN-like protein in dense granules of extracellular T. gondii tachyzoites. Western blotting and RT-PCR confirmed this protein expression by the parasites. Our results also showed, after macrophage invasion, an intense positive labeling for OPN-like protein at the sub-apical portion of tachyzoites, the site of dense granules secretion, and the localization of this protein at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we speculate that this protein participates during the parasite interaction process with host cells and parasitophorous vacuole formation.
Assuntos
Grânulos Citoplasmáticos/química , Osteopontina/análise , Proteínas de Protozoários/análise , Toxoplasma/química , Vacúolos/química , Vacúolos/parasitologia , Animais , Western Blotting , Expressão Gênica , Membranas Intracelulares/química , Macrófagos Peritoneais/parasitologia , Camundongos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Mammalian seminal plasma contains membranous vesicles (MV), which differ in composition and origin. Among these particles, human prostasomes and equine prostasome-like MV have been the most studied. The aim of the present work is to characterize the biochemical composition and membrane fluidity of MV isolated from boar seminal plasma. The MV from boar seminal plasma were isolated by ultracentrifugation and further purification by gel filtration on Sephadex G-200. The MV were examined by electron microscopy (EM), amount of cholesterol, total phospholipid, protein content, and phospholipid composition were analyzed. Membrane fluidity of MV and spermatozoa were estimated from the electron spin resonance (ESR) spectra of the 5-doxilstearic acid incorporated into the vesicle membranes by the order parameter (S). The S parameter gives a measure of degree of structural order in the membrane and is defined as the ratio of the spectral anisotropy in the membranes to the maximum anisotropy obtained in a rigidly oriented system. The S parameter takes into consideration that S = 1 for a rapid spin-label motion of about only one axis and S = 0 for a rapid isotropic motion. Intermediate S values between S = 0 and S = 1 represents the consequence of decreased membrane fluidity. The EM revealed the presence of bilaminar and multilaminar electron-dense vesicles. Cholesterol to phospholipid molar ratio from the isolated MV was 1.8. Phospholipid composition showed a predominance of sphingomyelin. The S parameter for porcine MV and for boar spermatozoa was 0.73 +/- 0.02 and 0.644 +/- 0.008, respectively, with the S for MV being greater (p < 0.001) than the S for spermatozoa. The high order for S found for boar MV was in agreement with the greater cholesterol/phospholipids ratio and the lesser ratio for phosphatidylcholine/sphingomyelin. Results obtained in the present work indicate that MV isolated from boar semen share many biochemical and morphological characteristics with equine prostasome-like MV and human prostasomes. The characteristics of the porcine MV of the seminal plasma, however, differed from those of boar sperm plasma membranes.
Assuntos
Membrana Celular/química , Fluidez de Membrana , Sêmen/fisiologia , Espermatozoides/citologia , Suínos , Animais , Membrana Celular/ultraestrutura , Colesterol/análise , Espectroscopia de Ressonância de Spin Eletrônica/veterinária , Membranas Intracelulares/química , Masculino , Lipídeos de Membrana/análise , Lipídeos de Membrana/química , Fosfolipídeos/análise , Sêmen/químicaRESUMO
Highly purified glycosomes from Trypanosoma cruzi epimastigotes were obtained by differential centrifugation and isopycnic ultracentrifugation. Glycosomal membranes, produced by carbonate treatment of purified glycosomes, exhibited about eight main protein bands and eight minor ones. Essentially the same protein pattern was observed in the detergent-rich fraction of a Triton X-114 fractionation of whole glycosomes, indicating that most of the membrane-bound polypeptides were highly hydrophobic. The orientation of these proteins was studied by in situ labelling followed by limited pronase hydrolysis of intact glycosomes. Three glycosome membrane proteins were characterized as peripheral by comparing the protein bands patterns of membrane fractions obtained by different treatments. Noteworthy membrane polypeptides were: (1) a peripheral 75k Da membrane protein, oriented towards the cytosol, which was the most abundant glycosomal membrane protein in exponentially growing epimastigotes but was essentially absent in stationary phase cells; (2) a pair of integral membrane proteins with molecular masses in the range of 85-100 kDa, which were only present in stationary phase cells; (3) a heme-containing 36k Da protein, strongly associated to the membrane, present in both growth phases; (4) a very immunogenic 41k Da integral membrane polypeptide, oriented towards the cytosol. The lipid composition of the glycosomal membranes was also investigated. The distribution of phospholipid species in glycosomes and glycosomal membranes was very similar to that of whole cells, with phosphatidyl-ethanolamine, phosphatidyl-choline, and phosphatidyl-serine as main components and smaller proportions of sphingomyelin and with phosphatidyl-inositol. On the other hand, glycosomes were enriched in endogenous sterols (ergosterol, 24-ethyl-5,7,22-cholesta-trien-3beta-ol), and precursors, when compared with whole cells, a finding consistent with the proposal that these organelles are involved in the de novo biosynthesis of sterols in trypanosomatids.
Assuntos
Lipídeos de Membrana/análise , Proteínas de Membrana/análise , Microcorpos/química , Proteínas de Protozoários/análise , Trypanosoma cruzi/química , Animais , Antígenos de Protozoários/análise , Centrifugação com Gradiente de Concentração , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Microcorpos/ultraestrutura , Trypanosoma cruzi/imunologia , Trypanosoma cruzi/ultraestrutura , UltracentrifugaçãoRESUMO
GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.
Assuntos
Detergentes/química , Gangliosídeos/biossíntese , Gangliosídeos/metabolismo , Glicosiltransferases/metabolismo , Complexo de Golgi/química , Membranas Intracelulares/química , Animais , Células CHO/química , Células CHO/enzimologia , Células CHO/metabolismo , Extratos Celulares/química , Linhagem Celular , Membrana Celular/química , Cricetinae , Complexo de Golgi/enzimologia , Humanos , Membranas Intracelulares/enzimologia , Microdomínios da Membrana/química , Octoxinol/metabolismo , Sialiltransferases/biossínteseRESUMO
Spironolactone (SL) increases the glucuronidation rate of several compounds. We analyzed the molecular basis of changes occurring in major rat liver UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform of family 2, in response to SL. UGT activity toward bilirubin, ethynylestradiol and p-nitrophenol was assayed in native and activated microsomes. Protein and mRNA levels were determined by Western and Northern blotting. The lipid composition and physicochemical properties of the microsomal membrane were also analyzed. Glucuronidation rates of bilirubin and ethynylestradiol (at both 3-OH and 17 beta-OH positions), determined in UDP-N-acetylglucosamine-activated membranes, were increased in SL group. Western blot analysis revealed increased levels of UGT1A1 and 1A5 (bilirubin and 3-OH ethynylestradiol conjugation), and 2B1 (17 beta-OH ethynylestradiol conjugation). Northern blot studies suggested transcriptional regulation by the steroid. Analysis of UGT activity in native vs. alamethicin-activated microsomes indicated increased latency, which was not associated to changes in physicochemical properties of the microsomal membrane. p-Nitrophenol glucuronidation rate and mRNA and protein levels of UGT1A6, the main isoform conjugating planar phenols, were not affected by the inducer. The data suggest transcriptional regulation of specific isoforms of hepatic UGT by SL, thus explaining previously reported increases in UGT activity toward selective substrates.
Assuntos
Expressão Gênica/efeitos dos fármacos , Glucuronosiltransferase/metabolismo , Isoenzimas/metabolismo , Fígado/efeitos dos fármacos , Espironolactona/farmacologia , Animais , Bilirrubina/farmacologia , Northern Blotting , Diuréticos/farmacologia , Glucuronosiltransferase/genética , Immunoblotting , Membranas Intracelulares/química , Fígado/enzimologia , Masculino , Fluidez de Membrana/efeitos dos fármacos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Nitrofenóis/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos WistarRESUMO
The structural and functional characterization of membrane proteins includes assessment of their topology in the bilayer. In the present work, we successfully used an approach based on comparative epitope accessibility. The classical method of detergent permeabilization of fixed cells allowed antibodies to detect epitopes distributed at either side of each cellular membrane by immunofluorescent staining. Instead, freeze-thawing followed by fixation allowed antibodies to cross only the plasma membrane whereas all intracellular membranes remained impermeable. By combining the immunofluorescence results achieved with these two methods for a variety of known membrane proteins, we showed that epitope accessibility could be accurately determined in proteins residing in the plasma membrane or in intracellular compartments, including the endoplasmic reticulum, lysosomes, peroxisomes, different Golgi regions and the nucleus. Freeze-thawing neither changed the expected distribution of each tested protein nor permeabilized intracellular membranes to antibodies. It only permeabilized the plasma membrane. Furthermore, the protocol proved to be efficient in different kinds of cells, which include MDCK and FRT polarized epithelial cells, HeLa cells and fibroblasts. If the complete topology of an integral membrane protein is known, this method would allow to assign an orientation to epitopes recognized by a panel of monoclonal antibodies. It also avoids the use of toxic reagents for permeabilization. Thus, selective permeabilization of the plasma membrane by freeze-thawing provides an inexpensive and reliable method to investigate the topology of membrane proteins as well as the distribution of soluble proteins.
Assuntos
Imunofluorescência/métodos , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Animais , Linhagem Celular , Membrana Celular/química , Membrana Celular/imunologia , Permeabilidade da Membrana Celular , Detergentes , Cães , Epitopos/química , Congelamento , Células HeLa , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/imunologia , RatosRESUMO
Mitochondrial ATP-sensitive K+ channels (mitoK(ATP)) have been proposed to mediate protection against ischemic injury by increasing high-energy intermediate levels. This study was designed to verify if mitochondria are an important factor in the loss of cardiac ATP associated to ischemia, and determine the possible role of mitoK(ATP) in the control of ischemic ATP loss. Langendorff-perfused rat hearts subjected to ischemia were found to have significantly higher ATP contents when pretreated with oligomycin or atractyloside, indicating that mitochondrial ATP hydrolysis contributes toward ischemic ATP depletion. MitoK(ATP) opening induced by diazoxide promoted a similar protection against ATP loss. Diazoxide also inhibited ATP hydrolysis in isolated, nonrespiring mitochondria, an effect accompanied by a drop in the membrane potential and Ca2+ uptake. In hearts subjected to ischemia followed by reperfusion, myocardial injury was prevented by diazoxide, but not atractyloside or oligomycin, which, unlike diazoxide, decreased reperfusion ATP levels. Our results suggest that mitoK(ATP)-mediated protection occurs due to selective inhibition of mitochondrial ATP hydrolysis during ischemia, without affecting ATP synthesis after reperfusion.
Assuntos
Trifosfato de Adenosina/metabolismo , Membranas Intracelulares/fisiologia , Mitocôndrias/fisiologia , Isquemia Miocárdica , Miocárdio/metabolismo , Canais de Potássio/fisiologia , Animais , Cálcio/metabolismo , Respiração Celular , Hidrólise , Técnicas In Vitro , Membranas Intracelulares/química , Masculino , Potenciais da Membrana , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/fisiologia , Miocárdio/patologia , Miocárdio/ultraestrutura , Permeabilidade , Ratos , Ratos Sprague-DawleyRESUMO
MI-D (4-phenyl-5-(4-nitrocinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride), a new mesoionic compound, decreased the rate of swelling induced by valinomycin-K+, as well as induced swelling in the presence of nigericin-K+. Shrinkage was also affected, suggesting interference with the inner mitochondrial membrane, which would affect both fluidity and elasticity. Fluorescence polarization of DPH and DPH-PA, probing the core and outer regions respectively, of the DMPC and native membranes, indicated that MI-D shifts the midpoint of phase transition to higher values and orders of the fluid phase. These alterations in membrane fluidity are thus related to MI-D effects on the energy-linked functions of mitochondria.
Assuntos
Cinamatos/farmacologia , Membranas Artificiais , Mitocôndrias Hepáticas/efeitos dos fármacos , Tiazóis/farmacologia , Desacopladores/farmacologia , Animais , Difenilexatrieno/análogos & derivados , Difenilexatrieno/metabolismo , Polarização de Fluorescência , Membranas Intracelulares/química , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Ionóforos/antagonistas & inibidores , Cinética , Masculino , Fluidez de Membrana/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Nigericina/antagonistas & inibidores , Concentração Osmolar , Permeabilidade/efeitos dos fármacos , Potássio/metabolismo , Ratos , Ratos Wistar , Tiadiazóis , Valinomicina/antagonistas & inibidoresRESUMO
The phase behavior of plasma membrane (PM), endoplasmic reticulum (ER), and nuclear membranes (NM) isolated from adult rat papillary cells was studied using the molecular probe Laurdan. The steady-state fluorescence data analysis was correlated with the lipid composition obtained by biochemical assays. The comparison between intact membranes and protein-free reconstituted vesicles using the whole lipid extract shows the essential role of proteins on the temperature response of natural membranes. The phospholipid (PL) and cholesterol (Cho) content was measured in the three membrane fractions, the PL/Cho molar ratio being between 1.5 and 1.9. However, Laurdan's parameters in NM show a fluid phase state pattern even at low temperature (5 degrees C), with a restricted dipole relaxation in comparison with that displayed in liquid crystalline phase state lipid model membranes. PM and ER are in a gel-like state at temperatures below 20 degrees C, showing increasing dipole relaxation with temperature. The curved fits obtained are characteristic of cholesterol-enriched membranes. The distinctive phase behavior of nuclear membranes vanishes when proteins are extracted. However, relaxation is still faster in this fraction, which correlates with the native lipid composition. NM has the lowest percentage of phosphatidylinositol and sphingomyelin-the latter being a highly saturated phospholipid- and the highest percentage of phosphatidylcholine and phosphatidylethanolamine (PE), nuclear PE being enriched in arachidonic acid. All these changes agree with the higher fluidity of NM compared with ER or PM in the conditions assayed.
Assuntos
2-Naftilamina/análogos & derivados , Membrana Celular/química , Membranas Intracelulares/química , Medula Renal/química , Medula Renal/metabolismo , Lipídeos/química , Proteínas/química , 2-Naftilamina/farmacologia , Animais , Núcleo Celular/química , Colesterol/química , Cromatografia em Camada Fina , Retículo Endoplasmático/química , Ácidos Graxos/química , Corantes Fluorescentes/farmacologia , Lauratos/farmacologia , Bicamadas Lipídicas/química , Metabolismo dos Lipídeos , Lipossomos/química , Masculino , Fosfolipídeos/química , Proteínas/metabolismo , Ratos , Ratos Wistar , Espectrometria de Fluorescência , Frações Subcelulares/química , TemperaturaRESUMO
In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.
Assuntos
Transporte Axonal , Cinesinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso , Moléculas de Adesão de Célula Nervosa/metabolismo , Vacúolos/química , Vacúolos/metabolismo , Animais , Especificidade de Anticorpos , Axônios/metabolismo , Biomarcadores/análise , Células Cultivadas , Córtex Cerebral/citologia , Imunofluorescência , Cones de Crescimento/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Cinesinas/química , Cinesinas/genética , Cinesinas/imunologia , Complexo Antígeno L1 Leucocitário , Camundongos , Microssomos/química , Microssomos/metabolismo , Peso Molecular , Oligonucleotídeos Antissenso/genética , Células Piramidais/citologia , Células Piramidais/embriologia , Células Piramidais/metabolismo , Ratos , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismoRESUMO
Glycosylphosphatidylinositols (GPI) are essential components in the plasma membrane of the protozoan parasite Leishmania mexicana, both as membrane anchors for the major surface macromolecules and as the sole class of free glycolipids. We provide evidence that L.mexicana dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis, is localized to a distinct tubular subdomain of the endoplasmic reticulum (ER), based on the localization of a green fluorescent protein (GFP)-DPMS chimera and subcellular fractionation experiments. This tubular membrane (termed the DPMS tubule) is also enriched in other enzymes involved in GPI biosynthesis, can be specifically stained with the fluorescent lipid, BODIPY-C5-ceramide, and appears to be connected to specific subpellicular microtubules that underlie the plasma membrane. Perturbation of microtubules and DPMS tubule structure in vivo results in the selective accumulation of GPI anchor precursors, but not free GPIs. The DPMS tubule is closely associated morphologically with the single Golgi apparatus in non-dividing and dividing cells, appears to exclude luminal ER resident proteins and is labeled, together with the Golgi apparatus, with another GFP chimera containing the heterologous human Golgi marker beta1,2-N-acetylglucosaminyltransferase-I. The possibility that the DPMS-tubule is a stable transitional ER is discussed.
Assuntos
Retículo Endoplasmático/enzimologia , Glicosilfosfatidilinositóis/biossíntese , Membranas Intracelulares/enzimologia , Leishmania mexicana/enzimologia , Manosiltransferases/metabolismo , Animais , Biomarcadores/análise , Divisão Celular , Fracionamento Celular , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Fluorescência , Glicosilfosfatidilinositóis/metabolismo , Complexo de Golgi/metabolismo , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Leishmania mexicana/citologia , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Mitose , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.
Assuntos
2-Naftilamina/análogos & derivados , Corantes Fluorescentes/metabolismo , Membranas Intracelulares/metabolismo , Lauratos/metabolismo , Fosfolipídeos/metabolismo , Receptores Nicotínicos/metabolismo , Esteróis/metabolismo , 2-Naftilamina/química , 2-Naftilamina/metabolismo , Animais , Sítios de Ligação , Colesterol/química , Colesterol/metabolismo , Ésteres do Colesterol/química , Ésteres do Colesterol/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Polarização de Fluorescência , Corantes Fluorescentes/química , Membranas Intracelulares/química , Membranas Intracelulares/efeitos dos fármacos , Lauratos/química , Metabolismo dos Lipídeos , Lipídeos/farmacologia , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolipídeos/química , Receptores Nicotínicos/química , Solventes , Espectrometria de Fluorescência , Esteróis/química , TorpedoRESUMO
In this work, the mitochondrial transmembrane electric potential (delta psi) of isolated mitochondria was used to evaluate the toxicity of some chemicals (endosulfan, 3,4-dichloroaniline, parathion, tributyltin and cadmium) and wastewater. Mitochondria were isolated from rat liver, and the delta psi measured in a suitable assay medium, using a sensitive tetraphenylphosphonium (TPP+) electrode. The test substance was pre-incubated in a rotenone-containing medium during 3 min with 1.0 mg of mitochondrial protein. Mitochondria were energised with succinate and after the establishment of a constant maximal potential ADP was added to induce the phosphorylative cycle. Chosen endpoints were the membrane potential from mitochondria oxidising succinate and the depolarisation induced by ADP. After the appropriate transformations the EC50 (effective concentration) was calculated for each toxicant. Even very low concentrations of a toxicant were able to affect the delta psi, thus showing its suitability as a biosensor in ecotoxicology and results were reproducible between tests. The utilisation of delta psi in screening tests of pure substances and wastewater seems to be very effective and can be carried out routinely.
Assuntos
Técnicas Biossensoriais , Membranas Intracelulares/química , Mitocôndrias Hepáticas/química , Poluentes Químicos da Água/análise , Compostos de Anilina/química , Compostos de Anilina/toxicidade , Animais , Cádmio/química , Cádmio/toxicidade , Endossulfano/química , Endossulfano/toxicidade , Inseticidas/química , Inseticidas/toxicidade , Masculino , Potenciais da Membrana/fisiologia , Paration/química , Paration/toxicidade , Ratos , Ratos Wistar , Esgotos/análise , Compostos de Trialquitina/química , Compostos de Trialquitina/toxicidade , Abastecimento de Água/análiseRESUMO
During intracellular development of the malarial parasite numerous membranous vesicles appear in the infected erythrocyte cytoplasm between the parasitophorous vacuolar membrane (PVM) and the erythrocyte plasma membrane. In this study we describe the characterization of a monoclonal antibody which recognizes two major parasite-encoded proteins of 50 and 41 kDa. Immunofluorescence and immunoelectron microscopy demonstrated that the monoclonal antibody reacts with cytoplasmic vesicles of Plasmodium falciparum infected erythrocyte referred to as Maurer's clefts. The antigens recognized by the monoclonal antibody were expressed very early during the erythrocytic life cycle of the parasite, and remained tightly associated within membrane vesicles even after merozoites are released from infected erythrocytes. The antigens were partially soluble in non-ionic detergents, and were released from the membrane by alkali treatment, indicating that the proteins recognized by the monoclonal antibody are peripheral membrane proteins. It is proposed that the 50 and 41 kDa antigens might be part of an underlying membrane skeletal network that provides structural support to vesicles and tubules present in the infected erythrocyte cytoplasm.
Assuntos
Antígenos de Protozoários/isolamento & purificação , Eritrócitos/parasitologia , Proteínas de Membrana/isolamento & purificação , Plasmodium falciparum/crescimento & desenvolvimento , Proteínas de Protozoários/isolamento & purificação , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/imunologia , Especificidade de Anticorpos , Antígenos de Protozoários/biossíntese , Antígenos de Protozoários/química , Imunofluorescência , Interações Hospedeiro-Parasita , Membranas Intracelulares/química , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Microscopia Imunoeletrônica , Testes de Precipitina , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , SolubilidadeRESUMO
We examined the effect of n-3 fatty acid consumption on the lipid composition and physical properties of liver microsomal membranes in normal and experimental diabetic rats. Lipid analysis showed a significant increase in the cholesterol:phospholipid ratio in membranes of normal animals fed n-3 fatty acids as well as in both groups of diabetic rats. These changes would be in part responsible for the higher fluorescent polarization of DPH (1,6-diphenyl-1,3,5 hexatriene) observed in the diabetic groups compared with the normal ones. These alterations were partially compensated by an increase in the amount of phosphatidylcholine in the diabetic rats fed on n-3 fatty acids. However, proteins also play a role in determining the physical properties of the liver microsomes because in the liposomes derived from them, the fluorescent polarization of DPH decreased in the diabetics fed n-3 fatty acids. Measurements of fluorescence anisotropy of n-AS (2-, 7 and 12 (9 anthroyloxy) stearic acid) probes revealed a restricted rotational mobility in the middle zone of the bilayer. Consistently with this finding there was an elevation in the calculated unsaturation density of the fatty acids at the carbon 8 position. These experiments confirm the lipid abnormalities that take place in experimental diabetes and they show further that n-3 fatty-acid administration causes certain compensatory, and thus beneficial, changes in these abnormalities.