RESUMEN
Aggregation of the Tau protein into fibrils defines progression of neurodegenerative diseases, including Alzheimer's Disease. The molecular basis for potentially toxic reactions of Tau aggregates is poorly understood. Here we show that π-stacking by Arginine side-chains drives protein binding to Tau fibrils. We mapped an aggregation-dependent interaction pattern of Tau. Fibrils recruit specifically aberrant interactors characterised by intrinsically disordered regions of atypical sequence features. Arginine residues are key to initiate these aberrant interactions. Crucial for scavenging is the guanidinium group of its side chain, not its charge, indicating a key role of π-stacking chemistry for driving aberrant fibril interactions. Remarkably, despite the non-hydrophobic interaction mode, the molecular chaperone Hsp90 can modulate aberrant fibril binding. Together, our data present a molecular mode of action for derailment of protein-protein interaction by neurotoxic fibrils.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Arginina/metabolismo , Unión Proteica , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Animales , Arginina/química , Progresión de la Enfermedad , Guanidina/metabolismo , Proteínas HSP90 de Choque Térmico , Humanos , Espectrometría de Masas , Chaperonas Moleculares , Agregado de Proteínas , Dominios Proteicos , Pliegue de Proteína , Proteoma , Ratas , Análisis de Secuencia de Proteína , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
Selective cargo transport into axons and dendrites over the microtubule network is essential for neuron polarization. The axon initial segment (AIS) separates the axon from the somatodendritic compartment and controls the microtubule-dependent transport into the axon. Interestingly, the AIS has a characteristic microtubule organization; it contains bundles of closely spaced microtubules with electron dense cross-bridges, referred to as microtubule fascicles. The microtubule binding protein TRIM46 localizes to the AIS and when overexpressed in non-neuronal cells forms microtubule arrays that closely resemble AIS fascicles in neurons. However, the precise role of TRIM46 in microtubule fasciculation in neurons has not been studied. Here we developed a novel correlative light and electron microscopy approach to study AIS microtubule organization. We show that in cultured rat hippocampal neurons of both sexes, TRIM46 levels steadily increase at the AIS during early neuronal differentiation and at the same time closely spaced microtubules form, whereas the fasciculated microtubules appear at later developmental stages. Moreover, we localized TRIM46 to the electron dense cross-bridges and show that depletion of TRIM46 causes loss of cross-bridges and increased microtubule spacing. These data indicate that TRIM46 has an essential role in organizing microtubule fascicles in the AIS.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is a specialized region at the proximal axon where the action potential is initiated. In addition the AIS separates the axon from the somatodendritic compartment, where it controls protein transport to establish and maintain neuron polarity. Cargo vesicles destined for the axon recognize specialized microtubule tracks that enter the AIS. Interestingly the microtubules entering the AIS form crosslinked bundles, called microtubule fascicules. Recently we found that the microtubule-binding protein TRIM46 localizes to the AIS, where it may organize the AIS microtubules. In the present study we developed a novel correlative light and electron microscopy approach to study the AIS microtubules during neuron development and identified an essential role for TRIM46 in microtubule fasciculation.
Asunto(s)
Fasciculación Axonal/fisiología , Segmento Inicial del Axón/metabolismo , Microtúbulos/metabolismo , Neuronas/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Animales , Polaridad Celular/fisiología , Células Cultivadas , Citoesqueleto/metabolismo , Femenino , Hipocampo/citología , Hipocampo/metabolismo , Masculino , Neuronas/citología , Ratas , Proteínas de Motivos Tripartitos/genéticaRESUMEN
Hypoxia is a characteristic feature of solid tumors and an important cause of resistance to radiotherapy. Hypoxic cell radiosensitizers have been shown to increase radiotherapy efficacy, but dose-limiting side effects prevent their widespread use in the clinic. We propose the encapsulation of hypoxic cell radiosensitizers in temperature-sensitive liposomes (TSL) to target the radiosensitizers specifically to tumors and to avoid unwanted accumulation in healthy tissues. The main objective of the present study is to develop and characterize TSL loaded with the radiosensitizer pimonidazole (PMZ) and to evaluate the in vitro efficacy of free PMZ and PMZ encapsulated in TSL in combination with hyperthermia and radiotherapy. PMZ was actively loaded into TSL at different drug/lipid ratios, and the physicochemical characteristics and the stability of the resulting TSL-PMZ were evaluated. PMZ release was determined at 37 °C and 42 °C in HEPES buffer saline and fetal bovine serum. The concentration-dependent radiosensitizing effect of PMZ was investigated by exposing FaDu cells to different PMZ concentrations under hypoxic conditions followed by exposure to ionizing irradiation. The efficacy of TSL-PMZ in combination with hyperthermia and radiotherapy was determined in vitro, assessing cell survival and DNA damage by means of the clonogenic assay and histone H2AX phosphorylation, respectively. All TSL-PMZ formulations showed high encapsulation efficiencies and were stable for 30 d upon storage at 4 °C and 20 °C. Fast PMZ release was observed at 42 °C, regardless of the drug/lipid ratio. Increasing the PMZ concentration significantly enhanced the effect of ionizing irradiation. Pre-heated TSL-PMZ in combination with radiotherapy caused a 14.3-fold increase in cell death as compared to radiotherapy treatment alone. In conclusion, our results indicate that TSL-PMZ in combination with hyperthermia can assist in improving the efficacy of radiotherapy under hypoxic conditions.
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Quimioradioterapia/métodos , Hipertermia Inducida/métodos , Neoplasias Hipofaríngeas/metabolismo , Nitroimidazoles/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Composición de Medicamentos , Estabilidad de Medicamentos , Humanos , Neoplasias Hipofaríngeas/terapia , Liposomas/química , TemperaturaRESUMEN
Blood platelets play a central role in the arrest of bleeding and the development of thrombosis. Unraveling the complex processes of platelet biogenesis from megakaryocytes, platelet adhesion, aggregation, and secretory responses are important topics in the field of hemostasis and thrombosis. Analysis of the ultrastructural changes that occur during these processes is essential for understanding the rapid membrane dynamics and has contributed substantially to our present knowledge of platelet formation and functioning. Recent developments in real-time imaging, correlative light and electron microscopy imaging (CLEM), and 3D (cryo) electron microscopy and tomography offer exciting opportunities to improve studies of the platelet adhesive responses and secretion at the ultrastructural level in a close to native environment. In this chapter we discuss and illustrate cryo preparation techniques (high-pressure freezing, vitrification), correlative LM and EM workflows, and 3D cryo-electron tomography that we apply in our current research projects.
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Plaquetas/citología , Tomografía con Microscopio Electrónico/métodos , Animales , Microscopía por Crioelectrón , Humanos , Programas InformáticosRESUMEN
The epidermal growth factor receptor (EGFR) represents one of the most common target proteins in anti-cancer therapy. To directly examine the structural and dynamical properties of EGFR activation by the epidermal growth factor (EGF) in native membranes, we have developed a solid-state nuclear magnetic resonance (ssNMR)-based approach supported by dynamic nuclear polarization (DNP). In contrast to previous crystallographic results, our experiments show that the ligand-free state of the extracellular domain (ECD) is highly dynamic, while the intracellular kinase domain (KD) is rigid. Ligand binding restricts the overall and local motion of EGFR domains, including the ECD and the C-terminal region. We propose that the reduction in conformational entropy of the ECD by ligand binding favors the cooperative binding required for receptor dimerization, causing allosteric activation of the intracellular tyrosine kinase.
Asunto(s)
Receptores ErbB/química , Receptores ErbB/metabolismo , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/aislamiento & purificación , Humanos , Membranas Intracelulares/química , Resonancia Magnética Nuclear Biomolecular , Multimerización de Proteína , Termodinámica , Vesículas Transportadoras/químicaRESUMEN
Mast cells (MC) are well known for their effector role in allergic disorders; moreover, they are associated with diverse modulatory effects in innate and adaptive immunity. It is largely unclear how MC exert these modulating functions. In this article, we show that IgE-mediated MC degranulation leads to a rapid release of high quantities of extracellular vesicles (EV), comparable to the release of preformed mediators. EV are submicron structures composed of lipid bilayers, proteins, and nucleic acids that are released by cells in a regulated fashion and are involved in intercellular communication. Primary murine mucosal-type MC and connective tissue-type MC released phenotypically different EV populations depending on the stimulus they received. Although unstimulated MC constitutively released CD9+ EV, degranulation was accompanied by the release of CD63+ EV, which correlated with release of the soluble mediator ß-hexosaminidase. This CD63+ EV subset was smaller and exhibited a higher buoyant density and distinct phospholipid composition compared with CD9+ EV. Marked differences were observed for phosphatidylinositol, phosphatidic acid, and bis(monoacylglycero)phosphate species. Strikingly, proteomic analysis of CD63+ EV from connective tissue-type MC unveiled an abundance of MC-specific proteases. With regard to carboxypeptidase A3, it was confirmed that the enzyme was EV associated and biologically active. Our data demonstrate that, depending on their activation status, MC release distinct EV subsets that differ in composition and protease activity and are indicative of differential immunological functions. Concerning the strategic tissue distribution of MC and the presence of degranulated MC in various (allergic) disorders, MC-derived EV should be considered potentially important immune regulators.
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Degranulación de la Célula , Vesículas Extracelulares/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Péptido Hidrolasas/metabolismo , Animales , Degranulación de la Célula/inmunología , Células Cultivadas , Vesículas Extracelulares/inmunología , Ratones , Ratones Endogámicos C57BL , Péptido Hidrolasas/inmunologíaRESUMEN
Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) - a prominent extracellular matrix component - it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20-200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.
RESUMEN
Encapsulation of anticancer drugs in triggerable nanocarriers can beneficially modify pharmacokinetics and biodistribution of chemotherapeutic drugs, and consequently increase tumor drug concentration and efficacy, while reducing side effects. Thermosensitive liposomes release their contents triggered by hyperthermia, which can be, for example, precisely delivered using an MR Imaging-guided focused ultrasound procedure. In such a scenario, it is attractive to demonstrate the accumulation of liposomes before applying hyperthermia, as well as to document the release of liposome content using MRI. To address this need, thermosensitive liposomes were developed and characterized, which were doubly loaded by iron oxide nanoparticles and Gd-chelate, as opposed to loading with a single contrast agent. When intact, the transverse relaxivity of the liposomes is high allowing detection of carriers in tissue. After heating the longitudinal relaxivity steeply increases indicating release of the small molecular contents. By choosing the appropriate MR sequences, availability and release can be evaluated without interference of one contrast agent with the other. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Portadores de Fármacos/análisis , Liberación de Fármacos/efectos de la radiación , Liposomas/química , Imagen por Resonancia Magnética/métodos , Medios de Contraste/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Gadolinio , Calor , Nanopartículas de Magnetita , Ondas UltrasónicasRESUMEN
Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50-60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant.
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Membranas Intracelulares/química , Fusión de Membrana , Modelos Biológicos , Animales , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Transporte de Proteínas , Ratas , Ratas WistarRESUMEN
Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three-dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno-electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy-electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.
Asunto(s)
Tomografía con Microscopio Electrónico/métodos , Imagenología Tridimensional/métodos , Saccharomyces cerevisiae/metabolismo , Inmunohistoquímica/métodos , Microscopía Fluorescente/métodos , Transporte de ProteínasRESUMEN
"Candidatus Methylomirabilis oxyfera" is a newly discovered denitrifying methanotroph that is unrelated to previously known methanotrophs. This bacterium is a member of the NC10 phylum and couples methane oxidation to denitrification through a newly discovered intra-aerobic pathway. In the present study, we report the first ultrastructural study of "Ca. Methylomirabilis oxyfera" using scanning electron microscopy, transmission electron microscopy, and electron tomography in combination with different sample preparation methods. We observed that "Ca. Methylomirabilis oxyfera" cells possess an atypical polygonal shape that is distinct from other bacterial shapes described so far. Also, an additional layer was observed as the outermost sheath, which might represent a (glyco)protein surface layer. Further, intracytoplasmic membranes, which are a common feature among proteobacterial methanotrophs, were never observed under the current growth conditions. Our results indicate that "Ca. Methylomirabilis oxyfera" is ultrastructurally distinct from other bacteria by its atypical cell shape and from the classical proteobacterial methanotrophs by its apparent lack of intracytoplasmic membranes.
Asunto(s)
Proteobacteria/ultraestructura , Membrana Celular , Forma de la Célula , Criopreservación , Tomografía con Microscopio Electrónico , Resinas Epoxi , Grabado por Congelación , Regulación Bacteriana de la Expresión Génica/fisiología , Genoma Bacteriano , Imagenología Tridimensional , Microscopía Electrónica de Rastreo , Microtomía , Adhesión en Plástico , Proteobacteria/clasificación , Proteobacteria/metabolismo , TranscriptomaRESUMEN
Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium. In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear. Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body. We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.
Asunto(s)
Cilios/fisiología , Cilios/ultraestructura , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Morfogénesis , Venas Umbilicales/ultraestructura , Técnicas de Cultivo de Célula , Células Cultivadas , Tomografía con Microscopio Electrónico , Humanos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Modelos BiológicosRESUMEN
Specialized cell types exploit endosomal trafficking to deliver protein cargoes to cell type-specific lysosome-related organelles (LROs), but how endosomes are specified for this function is not known. In this study, we show that the clathrin adaptor AP-1 and the kinesin motor KIF13A together create peripheral recycling endosomal subdomains in melanocytes required for cargo delivery to maturing melanosomes. In cells depleted of AP-1 or KIF13A, a subpopulation of recycling endosomes redistributes to pericentriolar clusters, resulting in sequestration of melanosomal enzymes like Tyrp1 in vacuolar endosomes and consequent inhibition of melanin synthesis and melanosome maturation. Immunocytochemistry, live cell imaging, and electron tomography reveal AP-1- and KIF13A-dependent dynamic close appositions and continuities between peripheral endosomal tubules and melanosomes. Our results reveal that LRO protein sorting is coupled to cell type-specific positioning of endosomes that facilitate endosome-LRO contacts and are required for organelle maturation.
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Endosomas/metabolismo , Cinesinas/metabolismo , Melanosomas/metabolismo , Factor de Transcripción AP-1/metabolismo , Línea Celular , Endosomas/ultraestructura , Humanos , Cinesinas/genética , Melanosomas/ultraestructura , Microscopía Electrónica , ARN Interferente Pequeño/genética , Factor de Transcripción AP-1/genéticaRESUMEN
Anammox bacteria are members of the phylum Planctomycetes that oxidize ammonium anaerobically and produce a significant part of the atmosphere's dinitrogen gas. They contain a unique bacterial organelle, the anammoxosome, which is the locus of anammox catabolism. While studying anammox cell and anammoxosome division with transmission electron microscopy including electron tomography, we observed a cell division ring in the outermost compartment of dividing anammox cells. In most Bacteria, GTP hydrolysis drives the tubulin-analogue FtsZ to assemble into a ring-like structure at the cell division site where it functions as a scaffold for the molecular machinery that performs cell division. However, the genome of the anammox bacterium 'Candidatus Kuenenia stuttgartiensis' does not encode ftsZ. Genomic analysis of open reading frames with potential GTPase activity indicated a possible novel cell division ring gene: kustd1438, which was unrelated to ftsZ. Immunogold localization specifically localized kustd1438 to the cell division ring. Genomic analyses of other members of the phyla Planctomycetes and Chlamydiae revealed no putative functional homologues of kustd1438, suggesting that it is specific to anammox bacteria. Electron tomography also revealed that the bacterial organelle was elongated along with the rest of the cell and divided equally among daughter cells during the cell division process.
Asunto(s)
Bacterias/ultraestructura , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/metabolismo , División Celular , Sustancias Macromoleculares , Orgánulos/ultraestructura , Compuestos de Amonio Cuaternario/metabolismo , Anaerobiosis , Bacterias/metabolismo , Tomografía con Microscopio Electrónico , Orden Génico , Genes Bacterianos , Microscopía Electrónica de Transmisión , Microscopía Inmunoelectrónica , Orgánulos/metabolismo , Oxidación-ReducciónRESUMEN
Toxoplasma gondii is an obligate intracellular parasite from the phylum Apicomplexa. A hallmark of these protozoans is the presence of a unique apical complex of organelles that includes the apicoplast, a plastid acquired by secondary endosymbiosis. The apicoplast is indispensible for parasite viability. It harbours a fatty acid biosynthesis type II (FAS II) pathway and plays a key role in the parasite lipid metabolism. Possibly, the apicoplast provides components for the establishment and the maturation of the parasitophorous vacuole, ensuring the successful infection of the host cell. This implies the presence of a transport mechanism for fast and accurate allocation of lipids between the apicoplast and other membrane-bound compartments in the parasite cell. Using a combination of high-pressure freezing, freeze-substitution and electron tomography, we analysed the ultrastructural organization of the apicoplast of T. gondii in relation with the endoplasmic reticulum (ER). This allowed us to clearly show the presence of four continuous membranes surrounding the apicoplast. We present, for the first time, the existence of membrane contact sites between the apicoplast outermost membrane and the ER. We describe the morphological characteristics of these structures and discuss their potential significance for the subcellular distribution of lipids in the parasite.
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Retículo Endoplásmico/ultraestructura , Acido Graso Sintasa Tipo II/biosíntesis , Interacciones Huésped-Parásitos , Membranas Intracelulares/ultraestructura , Orgánulos/ultraestructura , Toxoplasma/ultraestructura , Animales , Transporte Biológico , Chlorocebus aethiops , Crioultramicrotomía , Tomografía con Microscopio Electrónico , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/parasitología , Ácidos Grasos/biosíntesis , Membranas Intracelulares/metabolismo , Metabolismo de los Lípidos , Modelos Biológicos , Orgánulos/metabolismo , Simbiosis/fisiología , Toxoplasma/enzimología , Toxoplasma/metabolismo , Vacuolas/metabolismo , Vacuolas/parasitología , Vacuolas/ultraestructura , Células VeroRESUMEN
Melanosomes are lysosome-related organelles (LROs) in which melanins are synthesized and stored. Early stage melanosomes are characterized morphologically by intralumenal fibrils upon which melanins are deposited in later stages. The integral membrane protein Pmel17 is a component of the fibrils, can nucleate fibril formation in the absence of other pigment cell-specific proteins, and forms amyloid-like fibrils in vitro. Before fibril formation Pmel17 traffics through multivesicular endosomal compartments, but how these compartments participate in downstream events leading to fibril formation is not fully known. By using high-pressure freezing of MNT-1 melanoma cells and freeze substitution to optimize ultrastructural preservation followed by double tilt 3D electron tomography, we show that the amyloid-like fibrils begin to form in multivesicular compartments, where they radiate from the luminal side of intralumenal membrane vesicles. The fibrils in fully formed stage II premelanosomes organize into sheet-like arrays and exclude the remaining intralumenal vesicles, which are smaller and often in continuity with the limiting membrane. These observations indicate that premelanosome fibrils form in association with intralumenal endosomal membranes. We suggest that similar processes regulate amyloid formation in pathological models.
Asunto(s)
Amiloide/biosíntesis , Melaninas/biosíntesis , Melanocitos/ultraestructura , Melanosomas/ultraestructura , Amiloide/química , Animales , Línea Celular Tumoral , Microscopía por Crioelectrón , Citocalasina D/química , Citocalasina D/metabolismo , Endosomas/química , Endosomas/metabolismo , Endosomas/ultraestructura , Congelación , Humanos , Melanocitos/metabolismo , Melanosomas/química , Melanosomas/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Ratones , Miosina Tipo I/química , Miosina Tipo I/metabolismo , Presión , Estructura Secundaria de Proteína , Antígeno gp100 del MelanomaRESUMEN
Dictyostelium HMX44A cells can withstand starvation under monolayer conditions for a few days without dying. They die only when the differentiation factor DIF-1 is exogenously added. Still, when HMX44A were subjected to starvation without addition of DIF-1 they showed, by electron microscopy and electron tomography, gross mitochondrial lesions including marked cristae alterations with frequent "holes" probably originating from dilated cristae. Since these cells did not die as shown for instance by FACS analysis, these results showed unexpected resilience of cells bearing markedly altered mitochondria, and thus showed that apparently destructive mitochondrial alterations may not lead to cell death. Also, these marked mitochondrial lesions could not be caused by caspases or bcl-2 family members, which these cells do not encode.
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Dictyostelium/citología , Mitocondrias/metabolismo , Mitocondrias/patología , Animales , Caspasas/metabolismo , Muerte Celular , Células Cultivadas , Medios de Cultivo , Dictyostelium/enzimología , Dictyostelium/metabolismo , Microscopía Electrónica , Mitocondrias/ultraestructura , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Anaerobic ammonium oxidation (anammox) is an ecologically and industrially important process and is performed by a clade of deeply branching Planctomycetes. Anammox bacteria possess an intracytoplasmic membrane-bounded organelle, the anammoxosome. In the present study, the ultrastructures of four different genera of anammox bacteria were compared with transmission electron microscopy and electron tomography. The four anammox genera shared a common cell plan and contained glycogen granules. Differences between the four genera included cell size (from 800 to 1,100 nm in diameter), presence or absence of cytoplasmic particles, and presence or absence of pilus-like appendages. Furthermore, cytochrome c proteins were detected exclusively inside the anammoxosome. This detection provides further support for the hypothesis that this organelle is the locus of anammox catabolism.
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Bacterias/ultraestructura , Citocromos c/análisis , Glucógeno/metabolismo , Bacterias/clasificación , Proteínas Bacterianas/análisis , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/ultraestructura , Fimbrias Bacterianas/ultraestructura , Glucógeno/análisis , Microscopía Electrónica de Transmisión , Complejos Multiproteicos/química , Tomografía Computarizada por Rayos XRESUMEN
Anammox bacteria have unique intracellular membranes that divide their cytoplasm into three separate compartments. The largest and innermost cytoplasmic compartment, the anammoxosome, is hypothesized to be the locus of all catabolic reactions in the anammox metabolism. Electron tomography showed that the anammoxosome and its membrane were highly folded. This finding was confirmed by a transmission electron microscopy study using different sample preparation methods. Further, in this study electron-dense particles were observed and electron tomography showed that they were confined to the anammoxosome compartment. Energy dispersive X-ray analysis revealed that these particles contained iron. The functional significance of a highly folded anammoxosome membrane and intracellular iron storage particles are discussed in relation to their possible function in energy generation.
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Bacterias/química , Compartimento Celular , Citoplasma/ultraestructura , Criopreservación , Crioultramicrotomía , Lípidos/análisis , Microscopía Electrónica de Transmisión , Especificidad de la Especie , TomografíaRESUMEN
Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno-electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three-dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.