RESUMEN
The retina comprises numerous cells forming diverse neuronal circuits, which constitute the first stage of the visual pathway. Each circuit is characterized by unique features and distinct neurotransmitters, determining its role and functional significance. Given the intricate cell types within its structure, the complexity of neuronal circuits in the retina poses challenges for exploration. To better investigate retinal circuits and cross-talk, such as the link between cone and rod pathways, and precise molecular localization (neurotransmitters or neuropeptides), such as the presence of substance P-like immunoreactivity in the mouse retina, we employed a pre-embedding immunoelectron microscopy (immuno-EM) method to explore synaptic connections and organization. This approach enables us to pinpoint specific intercellular synaptic connections and precise molecular localization and could play a guiding role in exploring its function. This article describes the protocol, reagents used, and detailed steps, including (1) retina fixation preparation, (2) pre-embedding immunostaining, and (3) post-fixation and embedding.
Asunto(s)
Microscopía Inmunoelectrónica , Retina , Animales , Retina/metabolismo , Retina/química , Ratones , Microscopía Inmunoelectrónica/métodos , Adhesión del Tejido/métodos , Sinapsis/metabolismo , Sinapsis/ultraestructura , Sinapsis/químicaRESUMEN
Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold-electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.
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Anticuerpos Monoclonales , Sinapsis , Animales , Ratones , Sinapsis/ultraestructura , Sinapsis/metabolismo , Anticuerpos Monoclonales/inmunología , Oxidorreductasas de Alcohol/metabolismo , Oxidorreductasas de Alcohol/química , Proteínas Co-Represoras/metabolismo , Inmunohistoquímica/métodos , Dominios Proteicos , Microscopía Inmunoelectrónica/métodos , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/inmunologíaRESUMEN
Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.
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Microscopía Electrónica de Transmisión , Humanos , Microscopía Electrónica de Transmisión/métodos , Animales , Microscopía por Crioelectrón/métodos , Microscopía Electrónica/métodos , Microscopía Inmunoelectrónica/métodos , Microscopía Fluorescente/métodos , Substitución por Congelación/métodosRESUMEN
Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at -25 °C for several years (4-6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.
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Criopreservación/métodos , Sistema Hipotálamo-Hipofisario/citología , Neuronas/ultraestructura , Fijación del Tejido/métodos , Animales , Criopreservación/normas , Femenino , Fijadores , Formaldehído , Macaca fuscata , Masculino , Microscopía Inmunoelectrónica/métodos , Microscopía Inmunoelectrónica/normas , Neuronas/metabolismo , Fijación del Tejido/normas , Vasopresinas/metabolismo , Proteínas de Transporte Vesicular de Glutamato/metabolismoRESUMEN
α-Synuclein (α-syn) is a major component of abnormal protein deposits observed in the brains of patients with synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy (MSA). The synaptic protein α-syn is water-soluble under normal physiological conditions, but in these patients' brains, we see accumulation of insoluble amyloid-like α-syn fibrils with prion-like properties. Intracerebral accumulation of these fibrils is correlated with disease onset and progression. Recombinant α-syn protein also forms amyloid-like fibrils that are structurally akin to those extracted from patients' brains. Recent cryo-electron microscopic studies have identified the core structures of synthetic α-syn fibrils and α-syn fibrils extracted from the brains of patients with MSA at the atomic level. In this chapter, we describe negative staining and immunoelectron microscopy protocols for ultrastructural characterization of synthetic α-syn fibrils and pathological α-syn fibrils.
Asunto(s)
Amiloide/metabolismo , Microscopía Electrónica/métodos , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Progresión de la Enfermedad , Humanos , Microscopía Inmunoelectrónica/métodos , Atrofia de Múltiples Sistemas/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.
Asunto(s)
Amiloide/química , Microscopía por Crioelectrón/métodos , Espectroscopía de Resonancia Magnética/métodos , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Química Encefálica , Humanos , Microscopía Inmunoelectrónica/métodos , Conformación Proteica , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Identifying endogenous tissue stem cells remains a key challenge in developmental and regenerative biology. To distinguish and molecularly characterise stem cell populations in large heterogeneous tissues, the combination of cytochemical cell markers with ultrastructural morphology is highly beneficial. Here, we realise this through workflows of multi-resolution immuno-correlative light and electron microscopy (iCLEM) methodologies. Taking advantage of the antigenicity preservation of the Tokuyasu technique, we have established robust protocols and workflows and provide a side-by-side comparison of iCLEM used in combination with scanning EM (SEM), scanning TEM (STEM), or transmission EM (TEM). Evaluation of the applications and advantages of each method highlights their practicality for the identification, quantification, and characterization of heterogeneous cell populations in small organisms, organs, or tissues in healthy and diseased states. The iCLEM techniques are broadly applicable and can use either genetically encoded or cytochemical markers on plant, animal and human tissues. We demonstrate how these protocols are particularly suited for investigating neural stem and progenitor cell populations of the vertebrate nervous system.
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Microscopía Electrónica/métodos , Microscopía Inmunoelectrónica/métodos , Células Madre/clasificación , Animales , Membrana Celular , Humanos , Microscopía Electrónica de Transmisión/métodos , Microscopía Fluorescente/métodos , Cintigrafía , Células Madre/inmunología , Células Madre/metabolismo , Flujo de TrabajoRESUMEN
OBJECTIVE: Non-alcoholic steatohepatitis (NASH) can progress to fibrosis, cirrhosis and end-stage liver disease. Glucagon-like peptide 1 receptor (GLP-1R) mediates ß cell function. Its receptor agonists, currently used to treat type 2 diabetes mellitus, might be effective against NASH. GLP-1R, a G protein-coupled receptor family member, preferentially localises to caveolae. Therefore, we ascertained the cellular localisation of GLP-1R and caveolin (CAV)-1 in NASH liver. METHODS: Liver biopsies were obtained from three patients with NASH and were compared with those of four normal patients. Immunohistochemistry (IHC) and immunoelectron microscopy (IEM) were used to compare GLP-1R and CAV-1 expression in the livers of patients with metastatic liver cancer and normal patients. RESULTS: IHC showed that GLP-1R localised to basolateral membranes of hepatocytes with macrovesicular steatosis and was expressed in monocytes infiltrating hepatic sinusoids. CAV-1 was minimally associated with low-electron density lipid droplets (LDs) in hepatocytes. IEM showed small clusters of GLP-1R molecules on the peripheral rims of LDs and on cytoplasmic leaflets of endoplasmic reticulum membranes and vesicles, whereas CAV-1 molecules were found in LD caveolae. CONCLUSIONS: GLP-1R is present in the lipid microdomains of hepatocytes with macrovesicular steatosis. These results may help inform future studies about the liver-specific mechanisms of GLP-1 modulation in NASH therapy.
Asunto(s)
Caveolina 1/metabolismo , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Hígado/patología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad Hepática en Estado Terminal/metabolismo , Receptor del Péptido 1 Similar al Glucagón/agonistas , Receptor del Péptido 1 Similar al Glucagón/uso terapéutico , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Inmunohistoquímica/métodos , Hígado/metabolismo , Hígado/ultraestructura , Cirrosis Hepática/complicaciones , Cirrosis Hepática/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Microscopía Inmunoelectrónica/métodos , Monocitos/metabolismo , Monocitos/patología , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.
Asunto(s)
Compuestos Epoxi/química , Técnicas Histológicas , Inmunohistoquímica/métodos , Microscopía Inmunoelectrónica/métodos , Tetróxido de Osmio/química , Anticuerpos/química , Desulfurococcaceae/ultraestructura , Microalgas/ultraestructura , Microtomía/métodosRESUMEN
Plasmodium parasites cause malaria in mammalian hosts and are transmitted by Anopheles mosquitoes. Gametocytes, which differentiate from asexual-stage parasites, are activated by environmental changes when ingested into the mosquito midgut, and are rapidly released from erythrocytes prior to fertilization. Secretory proteins localized to osmiophilic bodies (OBs), organelles unique to gametocytes, have been reported to be involved in female gametocyte egress. In this study, we investigate the dynamics of OBs in activated gametocytes of Plasmodium falciparum and Plasmodium yoelii using the female OB-specific marker protein, G377. After activation, female gametocyte OBs migrate to the parasite surface and fuse to form large vesicles beneath the parasite plasma membrane. At the marginal region of female gametocytes, fused vesicles secrete contents by exocytosis into the parasitophorous vacuole space, prior to parasite egress via the break-down of the erythrocyte membrane. This is the first detailed description of how proteins are transported through osmiophilic bodies.
Asunto(s)
Plasmodium falciparum , Plasmodium yoelii , Proteínas Protozoarias/ultraestructura , Eritrocitos/parasitología , Malaria/parasitología , Microscopía Inmunoelectrónica/métodos , Orgánulos/metabolismo , Orgánulos/ultraestructura , Plasmodium falciparum/metabolismo , Plasmodium falciparum/ultraestructura , Plasmodium yoelii/metabolismo , Plasmodium yoelii/ultraestructura , Proteínas Protozoarias/metabolismoRESUMEN
The CRISPR-Cas9 system is a powerful and yet precise DNA-editing tool in rapid development. By combining immunogold labeling and electron microscopy with the novel CRISPR-Cas9 system, we propose a new method to gain insight into the biology of this tool. In this study, we analyzed different Cas9-induced systems such as HEK293T cell line, murine oligodendrocyte progenitor cells, brain and liver to detect Cas9 expression by immunoelectron microscopy. Our results show that while Cas9 expression could be found in the nuclei and nucleopores of transfected HEK293T cells, in transfected oligodendrocyte precursor cells, Cas9 was found in cytoplasmic vesicles. In Cas9 constitutively expressing oligodendrocyte precursors, the enzyme was located in the cytoplasm of nondividing cells. Finally, while in the liver Cas9 was detected in different cell types, in the brain we found no specifically labeled cells. In conclusion, immunoelectron microscopy opens a new spectrum of opportunities to study the CRISPR-Cas9 system in a more precise manner.
Asunto(s)
Proteína 9 Asociada a CRISPR/genética , Edición Génica/métodos , Streptococcus pyogenes/genética , Animales , Encéfalo , Sistemas CRISPR-Cas/inmunología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/inmunología , ADN , Vectores Genéticos , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica/métodos , Microscopía Inmunoelectrónica/métodos , ARN Guía de KinetoplastidaRESUMEN
Vesicle-associated membrane protein-associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and nonspecific hits. Furthermore, we identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.
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ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , Endonucleasas/metabolismo , Enzimas Multifuncionales/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al ADN/metabolismo , Retículo Endoplásmico/metabolismo , Células HeLa , Humanos , Marcaje Isotópico , Proteínas de la Membrana/metabolismo , Microscopía Inmunoelectrónica/métodos , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Mapeo de Interacción de Proteínas/métodos , Mapas de Interacción de Proteínas , Transporte de Proteínas , Proteómica , Sirolimus/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The chromosome scaffold is considered to be a key structure of the mitotic chromosome. It plays a vital role in chromosome condensation, shaping the X-shaped structure of the mitotic chromosome, and also provides flexibility for chromosome movement during cell division. However, it remains to be elucidated how the chromosome scaffold organizes the mitotic chromosome and how it supports shaping the structure of the chromosome during metaphase. Here we present a new technique that enables the observation of the chromosome scaffold structure in metaphase chromosomes from any direction, by transferring an isolated chromosome to a 360° rotational holder for electron tomography (ET). The chromosome was stained with immunogold-labeled condensin complex, one of the major chromosome scaffold proteins and then observed in three dimensions using ET. Using the locations of gold nanoparticles to visualize the underlying structure, the tomograms we obtained reveal the patterns of chromosome scaffold organization, which appears to consist of a helical structure that serves to organize chromatin loops into the metaphase chromosome.
Asunto(s)
Cromosomas/ultraestructura , Tomografía con Microscopio Electrónico/métodos , Proteínas Cromosómicas no Histona/química , Oro , Células HeLa , Humanos , Nanopartículas del Metal , Metafase , Microscopía Inmunoelectrónica/métodos , Manejo de EspecímenesRESUMEN
Immunoelectron microscopy is a powerful technique for identifying viral antigens and determining their structural localization and organization within vaccines and viruses. While traditional negative staining transmission electron microscopy provides structural information, identity of components within a sample may be confounding. Immunoelectron microscopy allows for identification and visualization of antigens and their relative positions within a particulate sample. This allows for simple qualitative analysis of samples including whole virus, viral components, and viral-like particles. This article describes methods for immunogold labeling of viral antigens in a liquid suspension, with examples of immunogold-labeled influenza virus glycoproteins, and also discusses the important considerations for sample preparation and determination of morphologies. Together, these methods allow for understanding the antigenic makeup of viral particulate samples, which have important implications for molecular virology and vaccine development. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Asunto(s)
Antígenos Virales/ultraestructura , Microscopía Inmunoelectrónica/métodos , Coloración y Etiquetado/métodos , Cultivo de Virus/métodos , Virus/ultraestructura , Animales , Antígenos Virales/química , Antígenos Virales/inmunología , Línea Celular , Virus/química , Virus/crecimiento & desarrollo , Virus/inmunologíaRESUMEN
Many enveloped viruses utilize the cellular ESCRT pathway for budding, even flaviviruses, which form viral particles inside replication organelles derived from the endoplasmic reticulum (ER). In this section, we introduce methods for detecting several ESCRT subunit proteins in virus-infected cells by immunofluorescence microscopy and immunoelectron microscopy (immuno-EM). We also introduce a new method; correlative light microscopy and electron microscopy (CLEM), which allows the observation of target structures with both high-resolution EM and fluorescence labeling.
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Bioensayo/métodos , Retículo Endoplásmico/ultraestructura , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Microscopía Electrónica de Transmisión/métodos , Imagen Molecular/métodos , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular , Virus del Dengue/inmunología , Virus de la Encefalitis Japonesa (Especie)/inmunología , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/virología , Complejos de Clasificación Endosomal Requeridos para el Transporte/química , Complejos de Clasificación Endosomal Requeridos para el Transporte/inmunología , Oro/química , Humanos , Mesocricetus , Nanopartículas del Metal/química , Microscopía Fluorescente/métodos , Microscopía Inmunoelectrónica/métodos , Coloración y Etiquetado/métodos , Virión/químicaRESUMEN
The aerobic mitochondrion had undergone evolutionary diversification, most notable among lineages of anaerobic protists. Entamoeba is one of the genera of parasitic protozoans that lack canonical mitochondria, and instead possess mitochondrion-related organelles (MROs), specifically mitosomes. Entamoeba mitosomes exhibit functional reduction and divergence, most exemplified by the organelle's inability to produce ATP and synthesize iron-sulfur cluster. Instead, this organelle is capable of sulfate activation, which has been linked to amoebic stage conversion. In order to understand other unique features and components of this MRO, we utilized an in silico prediction tool to screen transmembrane domain containing proteins in the mitosome proteome. Here, we characterize a novel lineage-specific mitosomal membrane protein, named Entamoeba transmembrane mitosomal protein of 30 kDa (ETMP30; EHI_172170), predicted to contain five transmembrane domains. Immunofluorescence analysis demonstrated colocalization of hemagglutinin (HA)-tagged ETMP30 with the mitosomal marker, adenosine-5'-phosphosulfate kinase. Mitosomal membrane localization was indicated by immunoelectron microscopy analysis, which was supported by carbonate fractionation assay. Transcriptional gene silencing successfully repressed RNA expression by 60%, and led to a defect in growth and partial elongation of mitosomes. Immunoprecipitation of ETMP30 from ETMP30-HA-expressing transformant using anti-HA antibody pulled down one interacting protein of 126 kDa. Protein sequencing by mass spectrometry revealed this protein as a cation-transporting P-type ATPase, previously reported to localize to vacuolar compartments/Golgi-like structures, hinting at a possible mitosome-vacuole/Golgi contact site.
Asunto(s)
Entamoeba/metabolismo , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Secuencia de Aminoácidos , Evolución Biológica , ATPasas Transportadoras de Calcio/metabolismo , Simulación por Computador , Entamoeba histolytica/genética , Entamoeba histolytica/metabolismo , Aparato de Golgi/metabolismo , Microscopía Inmunoelectrónica/métodos , Mitocondrias/metabolismo , Orgánulos/metabolismo , Transporte de Proteínas , Proteínas ProtozoariasRESUMEN
Helicobacter pylori possesses intrabacterial nanotransportation system (ibNoTS) for transporting CagA, VacA, and urease within the bacterial cytoplasm, which is controlled by the extrabacterial environment. The route of ibNoTS for CagA is reported to be associated with the MreB filament, whereas the route of ibNoTS for urease is not yet known. In this study, we demonstrated by immunoelectron microscopy that urease along the route of ibNoTS localizes closely with the FtsZ filament in the bacterium. Supporting this, we found by enzyme immunoassay and co-immunoprecipitation analysis that urease interacted with FtsZ. These findings indicate that urease along the route of ibNoTS is closely associated with the FtsZ filament. Since these phenomena were not observed in ibNoTS for CagA, the route of ibNoTS for CagA is different from that of ibNoTS for urease. We propose that the route of ibNoTS for urease is associated with the FtsZ filament in H. pylori.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Transporte Biológico/fisiología , Helicobacter pylori/metabolismo , Humanos , Microscopía Inmunoelectrónica/métodos , Ureasa/metabolismoRESUMEN
Since the pioneering work of Kiyoteru Tokuyasu in the 70ths the use of thawed cryosections prepared according to the "Tokuyasu-method" for immunoelectron microscopy did not lose popularity. We owe this method a whole subcellular world described by discrete gold particles pointing at cargo, receptors and organelle markers on delicate images of the inner life of a cell. Here we explain the procedure of sample preparation, sectioning and immunolabeling in view of recent developments and the reasoning behind protocols including some historical perspective. Cryosections are prepared from chemically fixed and sucrose infiltrated samples and labeled with affinity probes and electron dense markers. These sections are ideal substrates for immunolabeling, since antigens are not exposed to organic solvent dehydration or masked by resin. Instead, the structures remain fully hydrated throughout the labeling procedure. Furthermore, target molecules inside dense intercellular structural elements, cells and organelles are accessible to antibodies from the section surface. For the validation of antibody specificity several approaches are recommended including knock-out tissue and reagent controls. Correlative light and electron microscopy strategies involving correlative probes are possible as well as correlation of live imaging with the underlying ultrastructure. By applying stereology, gold labeling can be quantified and evaluated for specificity.
Asunto(s)
Criopreservación/métodos , Crioultramicrotomía/métodos , Microscopía Inmunoelectrónica/métodos , Fijación del Tejido/métodos , Animales , Humanos , Hielo , SacarosaRESUMEN
Caveolins (CAVs) regulate intracellular cholesterol transport by a complex process involving caveolae, endoplasmic reticulum (ER), and the Golgi network. Hepatic stellate cells (HSCs) are the central site for retinoid storage in the liver and indeed the entire body. Herein, we attempted to elucidate the ultrastructural localization and expression of caveolin-1 (CAV-1) in human HSCs during the progression of liver cirrhosis (LC). Normal and hepatitis C-related cirrhotic liver samples were prepared using a modified perfusion-fixation method to fix organelle structures and molecules in their in vivo positions, and examined using immunoelectron microscopy. In control liver specimens, CAV-1 was minimally associated with low electron density lipid droplets (LDs) segregated around zones 1-2, and specifically associated with membranes surrounding LDs. CAV-1 was segregated in high-density LDs, consistent with the formation of membrane-enclosed lipid-rich vesicular structures, as well as caveolae on plasma membranes around zones 2-3. In cirrhotic liver specimens, CAV-1 molecules were inserted into the cytoplasmic leaflets of ER membranes for transportation to LDs. Thus, CAV-1 transport to LDs might represent an intracellular pathway from the ER in cirrhotic liver tissue.
Asunto(s)
Caveolina 1/metabolismo , Retículo Endoplásmico/ultraestructura , Células Estrelladas Hepáticas/ultraestructura , Gotas Lipídicas/ultraestructura , Humanos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/patología , Microscopía Inmunoelectrónica/métodosRESUMEN
The locus coeruleus (LC)-norepinephrine (NE) system is a key nucleus in which endogenous opioid and stress systems intersect to regulate the stress response. LC neurons of male rats become sensitized to stress following chronic morphine administration. Whether sex dictates this pattern of opioid-induced plasticity has not been demonstrated. Delineating the neurobiological adaptations produced by chronic opioids will enhance our understanding of stress vulnerability in opioid-dependent individuals, and may reveal how stress negatively impacts addiction recovery. In the present study, the effect of chronic morphine on the subcellular distribution of mu-opioid (MOR) and CRF receptors (CRFR) was investigated in the LC of male and female rats using immunoelectron microscopy. Results showed that placebo-treated females exhibited higher MOR and CRFR cytoplasmic distribution ratio when compared to placebo-treated males. Chronic morphine exposure induced a shift in the distribution of MOR immunogold-silver particles from the plasma membrane to the cytoplasm selectively in male LC neurons. Interestingly, chronic morphine exposure induced CRFR recruitment to the plasma membrane of both male and female LC neurons. These findings provide a potential mechanism by which chronic opioid administration increases stress vulnerability in males and females via an increase in surface availability of CRFR in LC neurons. However, our results also support the notion that cellular adaptations to chronic opioids differ across the sexes as redistribution of MOR following morphine exposure was only observed in male LC neurons.