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1.
Wien Med Wochenschr ; 168(11-12): 322-329, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30084092

RESUMO

Electron tomography (ET) was developed to overcome some of the problems associated reconstructing three-dimensional (3D) images from 2D election microscopy data from ultrathin slices. Virtual sections of semithin sample are obtained by incremental rotation of the target and this information is used to assemble a 3D image. Herein, we provide an instruction to ET including the physical principle, possibilities, and limitations. We review the development of innovative methods and highlight important investigations performed in our department and with our collaborators. ET has opened up the third dimension at the ultrastructural level and represents a milestone in structural molecular biology.


Assuntos
Tomografia com Microscopia Eletrônica , Imageamento Tridimensional , Biologia Celular , Técnicas Citológicas , Humanos
2.
PeerJ ; 5: e3923, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29093995

RESUMO

BACKGROUND: Biomineralization, e.g., in sea urchins or mollusks, includes the assembly of mesoscopic superstructures from inorganic crystalline components and biopolymers. The resulting mesocrystals inspire biophysicists and material scientists alike, because of their extraordinary physical properties. Current efforts to replicate mesocrystal synthesis in vitro require understanding the principles of their self-assembly in vivo. One question, not addressed so far, is whether intracellular crystals of proteins can assemble with biopolymers into functional mesocrystal-like structures. During our electron microscopy studies into Artemia franciscana (Crustacea: Branchiopoda), we found initial evidence of such proteinaceous mesostructures. RESULTS: EM preparations with high-pressure freezing and accelerated freeze substitution revealed an extraordinary intracellular source of mesostructured inclusions in both the cyto-and nucleoplasm of the epidermal lining of ovisacs of A. franciscana. Confocal reflection microscopy not only confirmed our finding; it also revealed reflective, light dispersing activity of these flake-like structures, their positioning and orientation with respect to the ovisac inside. Both the striation of alternating electron dense and electron-lucent components and the sharp edges of the flakes indicate self-assembly of material of yet unknown origin under supposed participation of crystallization. However, selected area electron diffraction could not verify the status of crystallization. Energy dispersive X-ray analysis measured a marked increase in nitrogen within the flake-like inclusion, and the almost complete absence of elements that are typically involved in inorganic crystallization. This rise in nitrogen could possibility be related to higher package density of proteins, achieved by mesostructure assembly. CONCLUSIONS: The ovisac lining of A. franciscana is endowed with numerous mesostructured inclusions that have not been previously reported. We hypothesize that their self-assembly was from proteinaceous polycrystalline units and carbohydrates. These mesostructured flakes displayed active optical properties, as an umbrella-like, reflective cover of the ovisac, which suggests a functional role in the reproduction of A. franciscana. In turn, studies into ovisac mesostructured inclusions could help to optimizing rearing Artemia as feed for fish farming. We propose Artemia ovisacs as an in vivo model system for studying mesostructure formation.

3.
PLoS One ; 12(9): e0185557, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28957389

RESUMO

Principal epididymal cells have one of the largest and more developed Golgi complex of mammalian cells. In the present study, we have used this cell as model for the study of the three-dimensional architecture of the Golgi complex of highly secretory and endocytic cells. Electron tomography demonstrated the presence in this cell type of some unknown or very unusual Golgi structures such as branched cisternae, pocket-like cisternal invaginations or tubular connections. In addition, we have used this methodology and immunoelectron microscopy to analyze the close relationship between this organelle and both the endoplasmic reticulum and microtubules, and to describe in detail how these elements interact with compact and non-compact regions of the ribbon.


Assuntos
Epididimo/metabolismo , Complexo de Golgi/metabolismo , Animais , Epididimo/citologia , Complexo de Golgi/ultraestrutura , Masculino , Microscopia Eletrônica , Ratos Sprague-Dawley , Tomografia/métodos
4.
Histochem Cell Biol ; 147(4): 415-438, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27975144

RESUMO

We studied Golgi apparatus disorganizations and reorganizations in human HepG2 hepatoblastoma cells by using the nonmetabolizable glucose analogue 2-deoxy-D-glucose (2DG) and analyzing the changes in Golgi stack architectures by 3D-electron tomography. Golgi stacks remodel in response to 2DG-treatment and are replaced by tubulo-glomerular Golgi bodies, from which mini-Golgi stacks emerge again after removal of 2DG. The Golgi stack changes correlate with the measured ATP-values. Our findings indicate that the classic Golgi stack architecture is impeded, while cells are under the influence of 2DG at constantly low ATP-levels, but the Golgi apparatus is maintained in forms of the Golgi bodies and Golgi stacks can be rebuilt as soon as 2DG is removed. The 3D-electron microscopic results highlight connecting regions that interlink membrane compartments in all phases of Golgi stack reorganizations and show that the compact Golgi bodies mainly consist of continuous intertwined tubules. Connections and continuities point to possible new transport pathways that could substitute for other modes of traffic. The changing architectures visualized in this work reflect Golgi stack dynamics that may be essential for basic cell physiologic and pathologic processes and help to learn, how cells respond to conditions of stress.


Assuntos
Carcinoma Hepatocelular/ultraestrutura , Desoxiglucose/metabolismo , Tomografia com Microscopia Eletrônica , Complexo de Golgi/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Células Hep G2 , Humanos , Células Tumorais Cultivadas
5.
Histochem Cell Biol ; 143(4): 369-80, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25422148

RESUMO

The classic Golgi apparatus organization, an arrangement of highly ordered cisternal stacks with tubular-vesicular membrane specializations on both sides, is the functional image of a continuous flow of contents and membranes with input, metabolization, and output in a dynamic steady state. In response to treatment with 2-deoxy-D-glucose (2-DG), which lowers the cellular ATP level by about 70% within minutes, this organization is rapidly replaced by tubular-glomerular membrane convolutes described as Golgi networks and bodies. 2-DG is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis, which has become attractive in the context of therapeutic approaches for several kinds of tumors specifically targeting glycolysis in cancer. With the question of whether the functions of the Golgi apparatus in lipid synthesis would be influenced by the 2-DG-induced Golgi apparatus reorganization, we focused on lipid metabolism within the Golgi bodies. For this, we applied a fluorophore-labeled short-chain ceramide (BODIPY-Cer) in various combinations with 2-DG treatment to HepG2 cell cultures and followed uptake, enrichment and metabolization to higher ordered lipids. The cellular ATP status in each experiment was controlled with a bioluminescence assay, and the response of the Golgi apparatus was tracked by immunostaining of the trans-Golgi network protein TGN46. For electron microscopy, the fluorescent BODIPY-Cer signals were converted into electron-dense precipitates by photooxidation of diaminobenzidine (DAB); DAB precipitates labeled trans-Golgi areas in control cultures but also compartments at the periphery of the Golgi bodies formed in response to 2-DG treatment, thus indicating that concentration of ceramide takes place in spite of the Golgi apparatus reorganization. Lipid analyses by thin-layer chromatography (TLC) performed in parallel showed that BODIPY-Cer is not only concentrated in compartments of the 2-DG-induced Golgi bodies but is partly metabolized to BODIPY-sphingomyelin. Both, uptake and condensation of BODIPY-Cer and its conversion to complex lipids indicate that functions of the Golgi apparatus in the cellular lipid metabolism persist although the classic Golgi apparatus organization is abolished.


Assuntos
Desoxiglucose/farmacologia , Complexo de Golgi/efeitos dos fármacos , Lipogênese/efeitos dos fármacos , Trifosfato de Adenosina/deficiência , Cromatografia em Camada Fina , Metabolismo Energético/efeitos dos fármacos , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células Hep G2 , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Fatores de Tempo , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismo , Rede trans-Golgi/ultraestrutura
6.
Contrast Media Mol Imaging ; 10(1): 18-27, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-24753451

RESUMO

We present a new synthesis protocol for a multivalent, multimodality, nucleophilic nanoparticle ideal for in vivo imaging. Stability requirements necessitated covalent cross-linking of the carbohydrate cage, easy functionalization the introduction of sterically accessible amine groups. The new protocol aimed at more uniform particle size, less clustering and superior magnetic properties compared with commercial nanoparticles. Particles were precipitated from Fe(2+) and Fe(3+) in the presence of 10 kDa dextran monodispersed from the aerosol phase. Cross-linking was achieved with epichlorhydrin, nuclophilication with NH3, purification with ultrafiltration and dialysis. Particles and a commercial product (Rienso®, Takeda Pharma) underwent physicochemical characterizations. Biocompatibility was assessed by Resazurin on LLC-PK1 cells; the internalization rate was measured for three cell lines (HAEC, HASMC, HT29). Core size was 5.61 ± 1.25 nm; hydrodynamic size was 49.56 ± 11.73 nm. The number of sterically accessible amine groups averaged 9.9. The cores showed cubic magnetite structure. Values of r1 and r2 were 10.9 and 148.17 mM(-1) s(-1). Cellular viability was unchanged after incubation. Introduction of aerosol phase dextran resulted in a reduction of the overall hydrodynamic diameter and a narrower size distribution of the synthesized particles. Electron tomography visualized for the first time the postulated 'hairy layer' of the dextran coating and enabled the measurement of the overall diameter of 100.2 ± 7.92 nm. The resulting nanoparticle is biocompatible, functionalizable and detectable at nanomolar concentrations with MRI and optical imaging. It can potentially serve as a platform for multimodal molecular imaging and targeted therapy approaches.


Assuntos
Meios de Contraste , Compostos Férricos , Nanopartículas de Magnetita , Imagem Molecular , Meios de Contraste/química , Dextranos/química , Compostos Férricos/química , Humanos , Imageamento por Ressonância Magnética , Nanopartículas de Magnetita/química , Tamanho da Partícula
7.
In Vitro Cell Dev Biol Anim ; 50(1): 56-65, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23982913

RESUMO

The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines were examined for their growth and morphological characteristics, in vitro and in vivo oncogenic potential, chromosome numbers, and expression of melanocytic antigens and tumor suppressors. Both cell lines exhibited malignant characteristics; however, the metastatic HoMel-A1 showed a more aggressive phenotype characterized by higher proliferation rates, invasiveness, and a stronger tumorigenic potential both in vitro and in vivo. HoMel-A1 displayed a near-haploid karyotype, whereas HoMel-L1 was near-diploid. The cell lines expressed melanocytic lineage markers such as TYR, TRP1, MITF, PMEL, ASIP, MC1R, POMC, and KIT. The tumor suppressor p53 was strongly expressed in both cell lines, while the tumor suppressors p16 and PTEN were absent in HoMel-A1, potentially implicating significance of these pathways in the melanoma progression. This in vitro model system will not only aid in understanding of the Grey horse melanoma pathogenesis, but also in unraveling the steps during melanoma progression in general as well as being an invaluable tool for development of new therapeutic strategies.


Assuntos
Linhagem Celular Tumoral , Cavalos , Melanoma/veterinária , Animais , Proliferação de Células , Cromossomos de Mamíferos , Cariótipo , Melanoma/genética , Melanoma/patologia , Metástase Neoplásica/genética , Metástase Neoplásica/patologia
8.
Transfus Med Hemother ; 40(2): 101-7, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23652838

RESUMO

OBJECTIVE: The quality of platelet concentrates (PC) is important for the in vivo recovery of thrombostasis in patients suffering from bleeding disorders and in tumor patients after chemotherapy. In this respect, activated platelets (PLT) cannot display their full functionality in the recipient and even can cause adverse effects. Therefore, we developed a transmission electron microscopy (TEM) method for quality assessment of PC. METHODS: Score values taken from panorama TEM images describe the progress of PLT activation. To exemplify this method, i) 19 apheresis PC isolated with the Baxter Amicus system (BA) were compared with 14 PC obtained from pooled buffy coats (BC). ii) The score values of 33 PC derived from BA as well from BC were compared with flow-cytometric CD62P determinations by cross correlation. iii) Changes in the score value profiles during storage of a single pathogen-reduced BA PC were monitored over a period of 7 days. RESULTS: The TEM evaluation described allows for demonstrating particular PLT activation stages. i) Significant differences between the percentages of the score values 0, 1 and 2 could be demonstrated in both processing groups. No significant differences were found comparing these two groups. ii) A weak correlation could be shown when comparing the percentages of score values 2 plus 3 with the percentage of CD62P-positive PLT. iii) The pathogen reduction affected slightly the score profiles during storage due to an increase of dead PLT. CONCLUSION: Our investigations demonstrate the unique detailed quality information of PC obtained by the TEM method. This method can be performed in every routine electron microscopy laboratory.

9.
Methods Mol Biol ; 931: 437-47, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23027016

RESUMO

Detailed insight into the fine structure and 3D-architecture of the complex and dynamic compartments of the endocytic system is essential for a morpho-functional analysis of retrograde traffic from the cell surface to different intracellular destinations. Here, we describe a cytochemical approach for electron microscopic exploration of endocytic pathways with the use of wheat germ agglutinin (WGA) in combination with either conventional chemical fixation or ultrafast physical fixation of the cells by high pressure-freezing. Horseradish peroxidase-labeled WGA endocytozed by human hepatoma cells for various periods of time served as a marker. Its intracellular routes were visualized by means of diaminobenzidine oxidation either done conventionally after chemical fixation or in living cells prior to physical fixation. The latter protocol permits the combination of peroxidase-catalyzed cytochemistry with high pressure-freezing (HPF), which is state of the art for ultrastructural studies of complex and dynamic organelles at high spatial and temporal resolutions. The technique yields distinct cytochemical reactions and excellently preserved fine structures well qualified for detailed electron microscopic and 3D-studies of the complex endocytic architectures.


Assuntos
Endocitose , Endossomos/ultraestrutura , Rede trans-Golgi/ultraestrutura , 3,3'-Diaminobenzidina/química , Soluções Tampão , Técnicas de Cultura de Células , Precipitação Química , Vesículas Revestidas por Clatrina/ultraestrutura , Criopreservação , Fixadores/química , Glutaral/química , Células Hep G2 , Humanos , Indicadores e Reagentes/química , Microscopia Eletrônica de Transmissão , Oxirredução , Fixação de Tecidos , Aglutininas do Germe de Trigo/metabolismo
10.
Methods Mol Biol ; 931: 423-36, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23027015

RESUMO

Correlative microscopic approaches combine the advantages of both light and electron microscopy. Here we show a correlative approach that uses the photooxidation capacity of fluorescent dyes. Through illumination with high energetic light, the chromogen diaminobenzidine is oxidized and stable deposits are formed at the sites of the former fluorescent signals, which after osmification are then visible in the electron microscope. The potential of the method is illustrated by tracing the endocytic pathway of three different ligands: the lipid ceramide, high density lipoproteins, and the lectin wheat germ agglutinin. The ligands were labeled either with BODIPY or Alexa dyes. Following cell surface binding, uptake, and time-dependent intracellular progression, the route taken by these molecules together with the organelles that have been visited is characterized. Correlative microscopic data are recorded at various levels. First, by fluorescence and phase contrast illumination with the light microscope, followed by the analysis of semithin sections after photooxidation, and finally of thin sections at the ultrastructural level.


Assuntos
Microscopia Eletrônica de Transmissão/métodos , 3,3'-Diaminobenzidina/química , Compostos de Boro/química , Técnicas de Cultura de Células , Células Cultivadas , Ceramidas/química , Ceramidas/metabolismo , Precipitação Química/efeitos da radiação , Compostos Cromogênicos/química , Células Endoteliais/ultraestrutura , Corantes Fluorescentes/química , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Microscopia de Fluorescência , Microtomia , Oxirredução/efeitos da radiação , Processos Fotoquímicos
11.
PLoS One ; 8(12): e83189, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24386159

RESUMO

Endothelial progenitor cells (EPCs) originate either directly from hematopoietic stem cells or from a subpopulation of monocytes. Controversial views about intracellular lipid traffic prompted us to analyze the uptake of human high density lipoprotein (HDL), and HDL-cholesterol in human monocytic EPCs. Fluorescence and electron microscopy were used to investigate distribution and intracellular trafficking of HDL and its associated cholesterol using fluorescent surrogates (bodipy-cholesterol and bodipy-cholesteryl oleate), cytochemical labels and fluorochromes including horseradish peroxidase and Alexa Fluor® 568. Uptake and intracellular transport of HDL were demonstrated after internalization periods from 0.5 to 4 hours. In case of HDL-Alexa Fluor® 568, bodipy-cholesterol and bodipy-cholesteryl oleate, a photooxidation method was carried out. HDL-specific reaction products were present in invaginations of the plasma membrane at each time of treatment within endocytic vesicles, in multivesicular bodies and at longer periods of uptake, also in lysosomes. Some HDL-positive endosomes were arranged in form of "strings of pearl"- like structures. HDL-positive multivesicular bodies exhibited intensive staining of limiting and vesicular membranes. Multivesicular bodies of HDL-Alexa Fluor® 568-treated EPCs showed multilamellar intra-vacuolar membranes. At all periods of treatment, labeled endocytic vesicles and organelles were apparent close to the cell surface and in perinuclear areas around the Golgi apparatus. No HDL-related particles could be demonstrated close to its cisterns. Electron tomographic reconstructions showed an accumulation of HDL-containing endosomes close to the trans-Golgi-network. HDL-derived bodipy-cholesterol was localized in endosomal vesicles, multivesicular bodies, lysosomes and in many of the stacked Golgi cisternae and the trans-Golgi-network Internalized HDL-derived bodipy-cholesteryl oleate was channeled into the lysosomal intraellular pathway and accumulated prominently in all parts of the Golgi apparatus and in lipid droplets. Subsequently, also the RER and mitochondria were involved. These studies demonstrated the different intracellular pathway of HDL-derived bodipy-cholesterol and HDL-derived bodipy-cholesteryl oleate by EPCs, with concomitant.


Assuntos
Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Lipoproteínas HDL/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Tomografia com Microscopia Eletrônica , Células Endoteliais/ultraestrutura , Células-Tronco Hematopoéticas/ultraestrutura , Humanos , Lipoproteínas HDL/análise , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Receptores Depuradores Classe B/metabolismo
12.
PLoS One ; 7(3): e32935, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22427911

RESUMO

The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 µm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4-15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.


Assuntos
Vesículas Transportadoras/ultraestrutura , Bexiga Urinária/citologia , Urotélio/ultraestrutura , Animais , Tomografia com Microscopia Eletrônica , Técnica de Fratura por Congelamento , Imuno-Histoquímica , Camundongos , Urotélio/citologia
13.
J Comp Neurol ; 520(2): 384-400, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21826661

RESUMO

In a synaptic active zone, vesicles aggregate around a densely staining structure called the presynaptic density. We focus on its three-dimensional architecture and a major molecular component in the locust. We used electron tomography to study the presynaptic density in synapses made in the brain by identified second-order neuron of the ocelli. Here, vesicles close to the active zone are organized in two rows on either side of the presynaptic density, a level of organization not previously reported in insect central synapses. The row of vesicles that is closest to the density's base includes vesicles docked with the presynaptic membrane and thus presumably ready for release, whereas the outer row of vesicles does not include any that are docked. We show that a locust ortholog of the Drosophila protein Bruchpilot is localized to the presynaptic density, both in the ocellar pathway and compound eye visual neurons. An antibody recognizing the C-terminus of the Bruchpilot ortholog selectively labels filamentous extensions of the presynaptic density that reach out toward vesicles. Previous studies on Bruchpilot have focused on its role in neuromuscular junctions in Drosophila, and our study shows it is also a major functional component of presynaptic densities in the central nervous system of an evolutionarily distant insect. Our study thus reveals Bruchpilot executes similar functions in synapses that can sustain transmission of small graded potentials as well as those relaying large, spike-evoked signals.


Assuntos
Sistema Nervoso Central/anatomia & histologia , Gafanhotos/anatomia & histologia , Sinapses/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Tomografia com Microscopia Eletrônica/métodos , Imuno-Histoquímica , Microscopia Eletrônica/métodos , Junção Neuromuscular/metabolismo , Junção Neuromuscular/ultraestrutura
14.
Curr Pharm Biotechnol ; 13(2): 331-40, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21470121

RESUMO

Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.


Assuntos
3,3'-Diaminobenzidina/química , Metabolismo dos Lipídeos/fisiologia , Lipoproteínas HDL/metabolismo , Microscopia Eletrônica/métodos , Transporte Biológico/fisiologia , Compostos de Boro/química , Colesterol/metabolismo , Ésteres do Colesterol/metabolismo , Endossomos/metabolismo , Fluorescência , Células Hep G2 , Humanos , Luz , Lipoproteínas HDL/ultraestrutura , Lisossomos/metabolismo , Microscopia de Fluorescência/métodos , Corpos Multivesiculares/metabolismo , Oxirredução , Processos Fotoquímicos , Células Tumorais Cultivadas , Rede trans-Golgi/metabolismo
15.
PLoS One ; 6(8): e23636, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21887288

RESUMO

Urothelial plaques are specialized membrane domains in urothelial superficial (umbrella) cells, composed of highly ordered uroplakin particles. We investigated membrane compartments involved in the formation of urothelial plaques in mouse umbrella cells. The Golgi apparatus did not contain uroplakins organized into plaques. In the post-Golgi region, three distinct membrane compartments containing uroplakins were characterized: i) Small rounded vesicles, located close to the Golgi apparatus, were labelled weakly with anti-uroplakin antibodies and they possessed no plaques; we termed them "uroplakin-positive transporting vesicles" (UPTVs). ii) Spherical-to-flattened vesicles, termed "immature fusiform vesicles" (iFVs), were uroplakin-positive in their central regions and contained small urothelial plaques. iii) Flattened "mature fusiform vesicles" (mFVs) contained large plaques, which were densely labelled with anti-uroplakin antibodies. Endoytotic marker horseradish peroxidase was not found in these post-Golgi compartments. We propose a detailed model of de novo urothelial plaque formation in post-Golgi compartments: UPTVs carrying individual 16-nm particles detach from the Golgi apparatus and subsequently fuse into iFV. Concentration of 16-nm particles into plaques and removal of uroplakin-negative membranes takes place in iFVs. With additional fusions and buddings, iFVs mature into mFVs, each carrying two urothelial plaques toward the apical surface of the umbrella cell.


Assuntos
Compartimento Celular , Complexo de Golgi/metabolismo , Microdomínios da Membrana/metabolismo , Uroplaquinas/metabolismo , Urotélio/metabolismo , Animais , Complexo de Golgi/ultraestrutura , Microdomínios da Membrana/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestrutura , Uroplaquinas/ultraestrutura , Urotélio/ultraestrutura
16.
Histochem Cell Biol ; 135(2): 159-71, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21225431

RESUMO

In this study, the ceramide-enriched trans-Golgi compartments representing sites of synthesis of sphingomyelin and higher organized lipids were visualized in control and ATP-depleted hepatoma and endothelial cells using internalization of BODIPY-ceramide and the diaminobenzidine photooxidation method for combined light-electron microscopical exploration. Metabolic stress induced by lowering the cellular ATP-levels leads to reorganizations of the Golgi apparatus and the appearance of tubulo-glomerular bodies and networks. The results obtained with three different protocols, in which BODIPY-ceramide either was applied prior to, concomitantly with, or after ATP-depletion, revealed that the ceramide-enriched compartments reorganize together with other parts of the Golgi apparatus under these conditions. They were found closely associated with and integrated in the tubulo-glomerular bodies formed in response to ATP-depletion. This is in line with the changes of the staining patterns obtained with the Helix pomatia lectin and the GM130 and TGN46 immuno-reactions occurring in response to ATP-depletion and is confirmed by 3D electron tomography. The 3D reconstructions underlined the glomerular character of the reorganized Golgi apparatus and demonstrated continuities of ceramide positive and negative parts. Most interestingly, BODIPY-ceramide becomes concentrated in compartments of the tubulo-glomerular Golgi bodies, even though the reorganization took place before BODIPY-ceramide administration. This indicates maintained functionalities although the regular Golgi stack organization is abolished; the results provide novel insights into Golgi structure-function relationships, which might be relevant for cells affected by metabolic stress.


Assuntos
Trifosfato de Adenosina/metabolismo , Ceramidas/metabolismo , Complexo de Golgi/metabolismo , Tomografia com Microscopia Eletrônica , Células Endoteliais/metabolismo , Células Hep G2 , Humanos , Microscopia Eletrônica , Esfingosina/análogos & derivados
17.
J Struct Biol ; 169(3): 286-93, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19857575

RESUMO

Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.


Assuntos
Endocitose/fisiologia , Congelamento , Histocitoquímica/métodos , Pressão , Tomografia com Microscopia Eletrônica , Complexo de Golgi/metabolismo , Células Hep G2 , Peroxidase do Rábano Silvestre/metabolismo , Humanos
18.
Histochem Cell Biol ; 133(3): 261-72, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20039053

RESUMO

Holo-high density lipoprotein (HDL) particle uptake, besides selective lipid uptake, constitutes an alternative pathway to regulate cellular cholesterol homeostasis. In the current study, the cellular path of holo-HDL particles was investigated in human liver carcinoma cells (HepG2) using combined light and electron microscopical methods. The apolipoprotein moiety of HDL was visualized with different markers: horseradish peroxidase, colloidal gold and the fluorochrome Alexa(568), used in fluorescence microscopy and after photooxidation correlatively at the ultrastructural level. Time course experiments showed a rapid uptake of holo-HDL particles, an accumulation in endosomal compartments, with a plateau after 1-2 h of continuous uptake, and a clearance 1-2 h upon replacement by unlabeled HDL. Correlative microscopy, using HDL-Alexa(568)-driven diaminobenzidine (DAB) photooxidation, identified the fluorescent organelles as DAB-positive multivesicular bodies (MVBs) in the electron microscope; their luminal contents but not the internal vesicles were stained. Labeled MVBs increased in numbers and changed shapes along with the duration of uptake, from polymorphic organelles with multiple surface domains and differently shaped protrusions dominating at early times of uptake to compact bodies with mainly tubular appendices and densely packed vesicles after later times. Differently shaped and labeled surface domains and appendices, as revealed by three dimensional reconstructions, as well as images of homotypic fusions indicate the dynamics of the HDL-positive MVBs. Double staining visualized by confocal microscopy, along with the electron microscopic data, shows that holo-HDL particles after temporal storage in MVBs are only to a minor degree transported to lysosomes, which suggests that different mechanisms are involved in cellular HDL clearance, including resecretion.


Assuntos
Endocitose , Endossomos/metabolismo , Lipoproteínas HDL/metabolismo , Endossomos/química , Ouro/química , Ouro/metabolismo , Células Hep G2 , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Lipoproteínas HDL/química , Tamanho da Partícula
19.
Ann Biomed Eng ; 38(2): 308-18, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19937468

RESUMO

In the present study, descriptors from the theory of random heterogeneous media were used to characterize the morphology of the myocardial interstitial space in histological sections from hearts of healthy subjects and of patients with idiopathic dilated cardiomyopathy (DCM). Histological sections from resected DCM hearts (n = 9) were compared with donor hearts showing no signs of cardiac disease (n = 6). From control to DCM, the area fraction phi(1) of the interstitial space increased from 0.13 +/- 0.05 to 0.27 +/- 0.08, the chord-length z from 1.67 +/- 0.61 to 5.56 +/- 1.78 microm, the pore-size delta from 0.72 +/- 0.13 to 1.73 +/- 0.40 microm, the distance r (min) of the first local minimum in the two-point correlation function from 10.99 +/- 1.09 to 18.57 +/- 4.36 mum, whereas specific interface length s and decay-rate gamma of the lineal-path function decreased from 0.20 +/- 0.07 to 0.16 +/- 0.04 microm(-1) and from 0.39 +/- 0.09 to 0.16 +/- 0.05 microm(-1), respectively. All descriptors (except for s) were significantly different (p < 0.05) between control and DCM, reflecting an increasingly heterogeneous morphology in DCM hearts. Our results suggest that (1) descriptors originally developed to characterize the morphology of random heterogeneous media are well suited for histomorphometry of DCM, and (2) among the descriptors studied, either pore-size delta or chord-length z qualify best to discriminate between control and DCM hearts.


Assuntos
Cardiomiopatias/patologia , Modelos Anatômicos , Modelos Cardiovasculares , Miocárdio/patologia , Miócitos Cardíacos/patologia , Adulto , Simulação por Computador , Interpretação Estatística de Dados , Feminino , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos
20.
Cell Biol Int ; 33(2): 217-23, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19056504

RESUMO

A novel organelle-like membrane specialization has been found in an epithelial cell line. Characteristically, the helical membrane arrays (referred to hereafter as TUHMAs) are organized around tubular, proteinaceous electron-dense cores of 80 nm in diameter. Depending on the cell status, up to 8 of these cores provide the basis for an intermingled membrane scaffold of an overall length of 3-5 microm. TUHMAs exist as single organelles in transient association with the nucleus, the rough endoplasmic reticulum, patches of annulate lamellae, and the Golgi complex. While most of the constituents are still unknown, evidence for an involvement of nucleoporins in TUHMA organization is presented, as shown by fluorescence immunochemistry. This should make TUHMAs more easily accessible for future studies on their structure and function.


Assuntos
Membranas Intracelulares/ultraestrutura , Animais , Linhagem Celular , Núcleo Celular/ultraestrutura , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático Rugoso/ultraestrutura , Complexo de Golgi/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Potoroidae , Ratos
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