RESUMEN
BACKGROUND: ATM, the protein defective in the human genetic disorder, ataxia-telangiectasia (A-T) plays a central role in response to DNA double-strand breaks (DSBs) and in protecting the cell against oxidative stress. We showed that A-T cells are hypersensitive to metabolic stress which can be accounted for by a failure to exhibit efficient endoplasmic reticulum (ER)-mitochondrial signalling and Ca2+ transfer in response to nutrient deprivation resulting in mitochondrial dysfunction. The objective of the current study is to use an anaplerotic approach using the fatty acid, heptanoate (C7), a metabolic product of the triglyceride, triheptanoin to correct the defect in ER-mitochondrial signalling and enhance cell survival of A-T cells in response to metabolic stress. METHODS: We treated control cells and A-T cells with the anaplerotic agent, heptanoate to determine their sensitivity to metabolic stress induced by inhibition of glycolysis with 2- deoxyglucose (2DG) using live-cell imaging to monitor cell survival for 72 h using the Incucyte system. We examined ER-mitochondrial signalling in A-T cells exposed to metabolic stress using a suite of techniques including immunofluorescence staining of Grp75, ER-mitochondrial Ca2+ channel, the VAPB-PTPIP51 ER-mitochondrial tether complexes as well as proximity ligation assays between Grp75-IP3R1 and VAPB1-PTPIP51 to establish a functional interaction between ER and mitochondria. Finally, we also performed metabolomic analysis using LC-MS/MS assay to determine altered levels of TCA intermediates A-T cells compared to healthy control cells. RESULTS: We demonstrate that heptanoate corrects all aspects of the defective ER-mitochondrial signalling observed in A-T cells. Heptanoate enhances ER-mitochondrial contacts; increases the flow of calcium from the ER to the mitochondrion; restores normal mitochondrial function and mitophagy and increases the resistance of ATM-deficient cells and cells from A-T patients to metabolic stress-induced killing. The defect in mitochondrial function in ATM-deficient cells was accompanied by more reliance on aerobic glycolysis as shown by increased lactate dehydrogenase A (LDHA), accumulation of lactate, and reduced levels of both acetyl CoA and ATP which are all restored by heptanoate. CONCLUSIONS: We conclude that heptanoate corrects metabolic stress in A-T cells by restoring ER-mitochondria signalling and mitochondrial function and suggest that the parent compound, triheptanoin, has immense potential as a novel therapeutic agent for patients with A-T.
Asunto(s)
Ataxia Telangiectasia/metabolismo , Mitocondrias/metabolismo , Células Cultivadas , Retículo Endoplásmico/metabolismo , HumanosRESUMEN
The centrosome is the primary microtubule-organizing center (MTOC) of most animal cells; however, this organelle is absent during early mammalian development. Therefore, the mechanism by which the mammalian embryo organizes its microtubules (MTs) is unclear. We visualize MT bridges connecting pairs of cells and show that the cytokinetic bridge does not undergo stereotypical abscission after cell division. Instead, it serves as scaffold for the accumulation of the MT minus-end-stabilizing protein CAMSAP3 throughout interphase, thereby transforming this structure into a noncentrosomal MTOC. Transport of the cell adhesion molecule E-cadherin to the membrane is coordinated by this MTOC and is required to form the pluripotent inner mass. Our study reveals a noncentrosomal form of MT organization that directs intracellular transport and is essential for mammalian development.
Asunto(s)
Embrión de Mamíferos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Animales , Transporte Biológico , Cadherinas/metabolismo , División Celular , Centrosoma/metabolismo , Embrión de Mamíferos/citología , Desarrollo Embrionario , Interfase , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismoRESUMEN
Caveolin-1 has a complex role in prostate cancer and has been suggested to be a potential biomarker and therapeutic target. As mature caveolin-1 resides in caveolae, invaginated lipid raft domains at the plasma membrane, caveolae have been suggested as a tumor-promoting signaling platform in prostate cancer. However, caveola formation requires both caveolin-1 and cavin-1 (also known as PTRF; polymerase I and transcript release factor). Here, we examined the expression of cavin-1 in prostate epithelia and stroma using tissue microarray including normal, non-malignant and malignant prostate tissues. We found that caveolin-1 was induced without the presence of cavin-1 in advanced prostate carcinoma, an expression pattern mirrored in the PC-3 cell line. In contrast, normal prostate epithelia expressed neither caveolin-1 nor cavin-1, while prostate stroma highly expressed both caveolin-1 and cavin-1. Utilizing PC-3 cells as a suitable model for caveolin-1-positive advanced prostate cancer, we found that cavin-1 expression in PC-3 cells inhibits anchorage-independent growth, and reduces in vivo tumor growth and metastasis in an orthotopic prostate cancer xenograft mouse model. The expression of α-smooth muscle actin in stroma along with interleukin-6 (IL-6) in cancer cells was also decreased in tumors of mice bearing PC-3-cavin-1 tumor cells. To determine whether cavin-1 acts by neutralizing caveolin-1, we expressed cavin-1 in caveolin-1-negative prostate cancer LNCaP and 22Rv1 cells. Caveolin-1 but not cavin-1 expression increased anchorage-independent growth in LNCaP and 22Rv1 cells. Cavin-1 co-expression reversed caveolin-1 effects in caveolin-1-positive LNCaP cells. Taken together, these results suggest that caveolin-1 in advanced prostate cancer is present outside of caveolae, because of the lack of cavin-1 expression. Cavin-1 expression attenuates the effects of non-caveolar caveolin-1 microdomains partly via reduced IL-6 microenvironmental function. With circulating caveolin-1 as a potential biomarker for advanced prostate cancer, identification of the molecular pathways affected by cavin-1 could provide novel therapeutic targets.
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Caveolina 1/metabolismo , Microdominios de Membrana/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas de Unión al ARN/metabolismo , Actinas/metabolismo , Anciano , Animales , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-6/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Metástasis de la Neoplasia , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores Androgénicos/metabolismoRESUMEN
We have previously identified a Rho protein, RhoD, which localizes to the plasma membrane and the early endocytic compartment. Here, we show that a GTPase-deficient mutant of RhoD, RhoDG26V, causes hyperplasia and perturbed differentiation of the epidermis, when targeted to the skin of transgenic mice. In vitro, gain-of-function and loss-of-function approaches revealed that RhoD is involved in the regulation of G1/S-phase progression and causes overduplication of centrosomes. Centriole overduplication assays in aphidicolin-arrested p53-deficient U2OS cells, in which the cell and the centrosome cycles are uncoupled, revealed that the effects of RhoD and its mutants on centrosome duplication and cell cycle are independent. Enhancement of G1/S-phase progression was mediated via Diaph1, a novel effector of RhoD, which we have identified using a two-hybrid screen. These results indicate that RhoD participates in the regulation of cell-cycle progression and centrosome duplication.
Asunto(s)
Centrosoma/fisiología , Fase G1/fisiología , Mutación/genética , Fase S/fisiología , Piel/patología , Proteínas de Unión al GTP rho/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Apoptosis , Western Blotting , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Proliferación Celular , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Forminas , Humanos , Hiperplasia/metabolismo , Hiperplasia/patología , Ratones , Ratones Transgénicos , Osteosarcoma/genética , Osteosarcoma/metabolismo , Osteosarcoma/patología , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Técnicas del Sistema de Dos Híbridos , Proteínas de Unión al GTP rho/metabolismoRESUMEN
The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubule-kinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.
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Proteínas de Ciclo Celular/fisiología , Huso Acromático/fisiología , Antígenos Nucleares/análisis , Proteínas de Unión al Calcio/fisiología , Células HeLa , Humanos , Proteínas Mad2 , Quinasas Relacionadas con NIMA , Proteínas Asociadas a Matriz Nuclear/análisis , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Proteínas Represoras/fisiología , Tubulina (Proteína)/análisis , Quinasa Tipo Polo 1Asunto(s)
Aorta/fisiología , Caveolas/fisiología , Caveolinas/fisiología , Pulmón/citología , Transducción de Señal , Animales , Astenia/etiología , Transporte Biológico , Señalización del Calcio , Caveolas/ultraestructura , Caveolina 1 , Caveolinas/deficiencia , Caveolinas/genética , División Celular , Colesterol/metabolismo , Endotelio Vascular/fisiología , Pulmón/anomalías , Pulmón/patología , Ratones , Ratones Noqueados , Músculo Liso Vascular/fisiología , Óxido Nítrico/metabolismo , FenotipoRESUMEN
Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.
Asunto(s)
Caveolas/fisiología , Glicosilfosfatidilinositoles/fisiología , Animales , Células CHO , Caveolina 1 , Caveolinas/biosíntesis , Línea Celular , Colesterol/análisis , CricetinaeRESUMEN
The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta-dystroglycan, dystrophin, and caveolin-3. These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta-dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta-dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expresseddomain-specific markers. We found that each marker protein was targeted to distinct microdomains. The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division.
Asunto(s)
Membrana Celular/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Animales , Caveolina 3 , Caveolinas , Membrana Celular/ultraestructura , Proteínas del Citoesqueleto , Distroglicanos , Distrofina , Femenino , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Glicoproteínas de Membrana , Microdominios de Membrana , Ratones , Fibras Musculares Esqueléticas/ultraestructura , Proteínas Musculares/aislamiento & purificación , Músculo Esquelético/ultraestructura , Unión Neuromuscular , Señales de Clasificación de Proteína , Transporte de Proteínas , Ratas , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Experimental protocols that allow confident assignment of signaling proteins to specific subdomains of the plasma membrane are essential for a full understanding of the complexities of signal transduction. This is especially relevant for Ras proteins, where the different membrane anchors of the Ras isoforms target them to functionally distinct microdomains that in turn allow quantitatively different signal outputs from otherwise highly homologous proteins. The methods outlined in this chapter, in addition to being invaluable in addressing Ras function, should also have wide utility in the study of many mammalian signal transduction pathways.
Asunto(s)
Caveolinas/metabolismo , Proteínas ras/fisiología , Animales , Caveolina 1 , Caveolinas/química , Caveolinas/genética , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Células Cultivadas , Cricetinae , Inmunohistoquímica , Microscopía Electrónica , Mutación , Estructura Terciaria de Proteína , Transfección , Proteínas ras/química , Proteínas ras/metabolismoRESUMEN
Different sites of plasma membrane attachment may underlie functional differences between isoforms of Ras. Here we show that palmitoylation and farnesylation targets H-ras to lipid rafts and caveolae, but that the interaction of H-ras with these membrane subdomains is dynamic. GTP-loading redistributes H-ras from rafts into bulk plasma membrane by a mechanism that requires the adjacent hypervariable region of H-ras. Release of H-ras-GTP from rafts is necessary for efficient activation of Raf. By contrast, K-ras is located outside rafts irrespective of bound nucleotide. Our studies identify a novel protein determinant that is required for H-ras function, and show that the GTP/GDP state of H-ras determines its lateral segregation on the plasma membrane.
Asunto(s)
Guanosina Trifosfato/metabolismo , Microdominios de Membrana/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Cricetinae , Activación Enzimática , Metabolismo de los Lípidos , Microscopía Inmunoelectrónica , Proteínas Proto-Oncogénicas c-raf/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
We have identified a novel, highly conserved protein of 14 kD copurifying with late endosomes/lysosomes on density gradients. The protein, now termed p14, is peripherally associated with the cytoplasmic face of late endosomes/lysosomes in a variety of different cell types. In a two-hybrid screen with p14 as a bait, we identified the mitogen-activated protein kinase (MAPK) scaffolding protein MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) partner 1 (MP1) as an interacting protein. We confirmed the specificity of this interaction in vitro by glutathione S-transferase pull-down assays and by coimmunoprecipitation, cosedimentation on glycerol gradients, and colocalization. Moreover, expression of a plasma membrane-targeted p14 causes mislocalization of coexpressed MP1. In addition, we could reconstitute protein complexes containing the p14-MP1 complex associated with ERK and MEK in vitro.The interaction between p14 and MP1 suggests a MAPK scaffolding activity localized to the cytoplasmic surface of late endosomes/lysosomes, thereby combining catalytic scaffolding and subcellular compartmentalization as means to modulate MAPK signaling within a cell.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Compartimento Celular , Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/aislamiento & purificación , Secuencia Conservada , Endosomas/ultraestructura , Membranas Intracelulares/ultraestructura , Lisosomas/ultraestructura , Sistema de Señalización de MAP Quinasas , Proteínas de la Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Unión Proteica , Análisis de Secuencia de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV), specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.
Asunto(s)
Caveolinas/metabolismo , Colesterol/metabolismo , Vesículas Citoplasmáticas/metabolismo , Metabolismo de los Lípidos , Mutación/genética , Secuencia de Aminoácidos , Androstenos/farmacología , Animales , Anticuerpos , Transporte Biológico/efectos de los fármacos , Biomarcadores/análisis , Brefeldino A/farmacología , Caveolina 1 , Caveolinas/química , Caveolinas/genética , Línea Celular , Membrana Celular/química , Membrana Celular/metabolismo , Cricetinae , Vesículas Citoplasmáticas/química , Vesículas Citoplasmáticas/efectos de los fármacos , Vesículas Citoplasmáticas/ultraestructura , Retículo Endoplásmico/química , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Endosomas/química , Endosomas/metabolismo , Fibroblastos , Técnica del Anticuerpo Fluorescente , Genes Dominantes/genética , Homeostasis , Microscopía Electrónica , Microscopía por Video , Ácido Oléico/farmacología , Transducción de SeñalRESUMEN
Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes. These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1). The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes. Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta-subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.
Asunto(s)
Macrófagos/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Macrófagos/ultraestructura , Ratones , Fagosomas , Transducción de SeñalRESUMEN
Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (WND:) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The tx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell. Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multi-vesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of tx mice.
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Adenosina Trifosfatasas/fisiología , Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión , Cobre/metabolismo , Líquido Intracelular/metabolismo , Leche , Mutación , Proteínas Recombinantes de Fusión , Homología de Secuencia de Aminoácido , Adenosina Trifosfatasas/biosíntesis , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/ultraestructura , Animales , Células CHO , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/ultraestructura , ATPasas Transportadoras de Cobre , Cricetinae , Femenino , Degeneración Hepatolenticular/genética , Degeneración Hepatolenticular/metabolismo , Humanos , Síndrome del Pelo Ensortijado/genética , Ratones , Ratones Endogámicos BALB C , Leche/toxicidad , TransfecciónRESUMEN
Phosphatidylinositol 3-kinase (PI3K) regulates several vital cellular processes, including signal transduction and membrane trafficking. In order to study the intracellular localization of the PI3K product, phosphatidylinositol 3-phosphate [PI(3)P], we constructed a probe consisting of two PI(3)P-binding FYVE domains. The probe was found to bind specifically, and with high affinity, to PI(3)P both in vitro and in vivo. When expressed in fibroblasts, a tagged probe localized to endosomes, as detected by fluorescence microscopy. Electron microscopy of untransfected fibroblasts showed that PI(3)P is highly enriched on early endosomes and in the internal vesicles of multivesicular endosomes. While yeast cells deficient in PI3K activity (vps15 and vps34 mutants) were not labelled, PI(3)P was found on intralumenal vesicles of endosomes and vacuoles of wild-type yeast. vps27Delta yeast cells, which have impaired endosome to vacuole trafficking, showed a decreased vacuolar labelling and increased endosome labelling. Thus PI(3)P follows a conserved intralumenal degradation pathway, and its generation, accessibility and turnover are likely to play a crucial role in defining the early endosome and the subsequent steps leading to multivesicular endosome formation.
Asunto(s)
Fosfatos de Fosfatidilinositol/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Línea Celular , Cricetinae , Humanos , Microscopía Electrónica , Sondas Moleculares , Mutación , Unión Proteica , Saccharomyces cerevisiae/genética , TransfecciónRESUMEN
Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.
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Endosomas/fisiología , Lisosomas/fisiología , Fusión de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Animales , Línea Celular , Perros , Endocitosis , Endosomas/ultraestructura , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Riñón/fisiología , Riñón/ultraestructura , Hígado/fisiología , Hígado/ultraestructura , Lisosomas/ultraestructura , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratas , Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/ultraestructura , Red trans-Golgi/fisiología , Red trans-Golgi/ultraestructuraRESUMEN
Growth arrest specific (gas) 1 gene product is expressed in non-transformed fibroblasts in response to stimuli driving cells into Go phase. Gas1 has been demonstrated to inhibit cell proliferation when over-expressed in proliferating fibroblasts. This activity depends on a function of the p53 protein independent of its transactivating ability. To better define the pathway leading from Gas1, which is located on the plasma membrane, to p53, we have undertaken a detailed characterization of its topology. We demonstrate that the protein undergoes cotranslational modifications in the endoplasmic reticulum, consisting of signal peptide cleavage, N-linked glycosylation and glycosyl-phosphatidylinositol anchor addition. Immunoelectron microscopy shows that, in its mature form, Gas1 is randomly distributed over the outer leaflet of the plasma membrane and that upon antibody-induced clustering it relocalizes to caveolae.
Asunto(s)
Membrana Celular/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Células 3T3 , Animales , Células COS , Proteínas de Ciclo Celular , División Celular , Secuencia de Consenso/fisiología , Retículo Endoplásmico/metabolismo , Proteínas Ligadas a GPI , Glutaral , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestructura , Proteínas de la Membrana , Ratones , Microscopía Inmunoelectrónica , Ácido Palmítico/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Pruebas de Precipitina , Unión Proteica , Señales de Clasificación de Proteína/fisiología , Fijación del Tejido , Transfección , Fosfolipasas de Tipo C/metabolismoRESUMEN
EEA1 is an early endosomal Rab5 effector protein that has been implicated in the docking of incoming endocytic vesicles before fusion with early endosomes. Because of the presence of complex endosomal pathways in polarized and nonpolarized cells, we have examined the distribution of EEA1 in diverse cell types. Ultrastructural analysis demonstrates that EEA1 is present on a subdomain of the early sorting endosome but not on clathrin-coated vesicles, consistent with a role in providing directionality to early endosomal fusion. Furthermore, EEA1 is associated with filamentous material that extends from the cytoplasmic surface of the endosomal domain, which is also consistent with a tethering/docking role for EEA1. In polarized cells (Madin-Darby canine kidney cells and hippocampal neurons), EEA1 is present on a subset of "basolateral-type" endosomal compartments, suggesting that EEA1 regulates specific endocytic pathways. In both epithelial cells and fibroblastic cells, EEA1 and a transfected apical endosomal marker, endotubin, label distinct endosomal populations. Hence, there are at least two distinct sets of early endosomes in polarized and nonpolarized mammalian cells. EEA1 could provide specificity and directionality to fusion events occurring in a subset of these endosomes in polarized and nonpolarized cells.
Asunto(s)
Endosomas/metabolismo , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/metabolismo , Animales , Línea Celular , Polaridad Celular , Células Cultivadas , Dendritas/metabolismo , Perros , Endosomas/ultraestructura , Células Epiteliales/citología , Fibroblastos/ultraestructura , Hipocampo/citología , Hipocampo/metabolismo , Proteínas de la Membrana/ultraestructura , Microscopía Fluorescente , Microscopía Inmunoelectrónica , Neuronas/citología , Ratas , Proteínas de Transporte VesicularRESUMEN
We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.
Asunto(s)
Endosomas/metabolismo , Endosomas/ultraestructura , Receptores de Transferrina/metabolismo , ATPasas de Translocación de Protón Vacuolares , Animales , Caveolina 1 , Caveolinas/metabolismo , Caveolinas/ultraestructura , Línea Celular , Colesterol/metabolismo , Perros , Endocitosis , Células Epiteliales/química , Células Epiteliales/citología , Células Epiteliales/fisiología , Concentración de Iones de Hidrógeno , Membranas Intracelulares/química , ATPasas de Translocación de Protón/metabolismo , Receptores de Transferrina/genética , Esfingomielinas/metabolismo , Fracciones Subcelulares/química , Transfección , Transferrina/metabolismoRESUMEN
In the present study, we show that in human endothelial cells the tetraspanin CD63/lamp3 distributes predominantly to the internal membranes of multivesicular-multilamellar late endosomes, which contain the unique lipid lysobisphosphatidic acid. Some CD63/lamp3 is also present in Weibel-Palade bodies, the characteristic secretory organelle of these cells. We find that CD63/lamp3 molecules can be transported from late endosomes to Weibel-Palade bodies and thus that CD63/lamp3 cycles between endocytic and biosynthetic compartments; however, movement of CD63/lamp3 is much slower than that of P-selectin, which is known to cycle between plasma membrane and Weibel-Palade bodies. When cells are treated with U18666A, a drug that mimics the Niemann-Pick type C syndrome, both proteins accumulate in late endosomes and fail to reach Weibel-Palade bodies efficiently, suggesting that P-selectin, like CD63/lamp3, cycles via late endosomes. Our data suggest that CD63/lamp3 partitions preferentially within late endosome internal membranes, thus causing its accumulation, and that this mechanism contributes to CD63/lamp3 retention in late endosomes; however, our data also indicate that the protein can eventually escape from these internal membranes and recycle toward Weibel-Palade bodies to be reused. Our observations thus uncover the existence of a selective trafficking route from late endosomes to Weibel-Palade bodies.