RESUMO
Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.
Assuntos
Microscopia Eletrônica de Transmissão/métodos , Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Substituição ao CongelamentoRESUMO
The pattern of plant organ initiation at the shoot apical meristem (SAM), termed phyllotaxis, displays regularities that have long intrigued botanists and mathematicians alike. In the SAM, the central zone (CZ) contains a population of stem cells that replenish the surrounding peripheral zone (PZ), where organs are generated in regular patterns. These patterns differ between species and may change in response to developmental or environmental cues [1]. Expression analysis of auxin efflux facilitators of the PIN-FORMED (PIN) family combined with modeling of auxin transport has indicated that organ initiation is associated with intracellular polarization of PIN proteins and auxin accumulation [2-10]. However, regulators that modulate PIN activity to determine phyllotactic patterns have hitherto been unknown. Here we reveal that three redundantly acting PLETHORA (PLT)-like AP2 domain transcription factors control shoot organ positioning in the model plant Arabidopsis thaliana. Loss of PLT3, PLT5, and PLT7 function leads to nonrandom, metastable changes in phyllotaxis. Phyllotactic changes in plt3plt5plt7 mutants are largely attributable to misregulation of PIN1 and can be recapitulated by reducing PIN1 dosage, revealing that PLT proteins are key regulators of PIN1 activity in control of phyllotaxis.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Fatores de Transcrição/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The epidermal growth factor receptor (EGFR) has been shown to be a valid cancer target for antibody-based therapy. At present, several anti-EGFR monoclonal antibodies have been successfully used, such as cetuximab and matuzumab. X-ray crystallography data show that these antibodies bind to different epitopes on the ecto-domain of EGFR, providing a rationale for the combined use of these two antibody specificities. We have previously reported on the successful isolation of antagonistic anti-EGFR nanobodies. In our study, we aimed to improve the efficacy of these molecules by combining nanobodies with specificities similar to both cetuximab and matuzumab into a single biparatopic molecule. Carefully designed phage nanobody selections resulted in two sets of nanobodies that specifically blocked the binding of either matuzumab or cetuximab to EGFR and that did not compete for each others' binding. A combination of nanobodies from both epitope groups into the biparatopic nanobody CONAN-1 was shown to block EGFR activation more efficiently than monovalent or bivalent (monospecific) nanobodies. In addition, this biparatopic nanobody potently inhibited EGF-dependent cell proliferation. Importantly, in an in vivo model of athymic mice bearing A431 xenografts, CONAN-1 inhibited tumour outgrowth with an almost similar potency as the whole mAb cetuximab, despite the fact that CONAN-1 is devoid of an Fc portion that could mediate immune effector functions. Compared to therapy using bivalent, monospecific nanobodies, CONAN-1 was clearly more potent in tumour growth inhibition. These results show that the rational design of biparatopic nanobody-based anticancer therapeutics may yield potent lead molecules for further development.
Assuntos
Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Carcinoma de Células Escamosas/terapia , Epitopos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/imunologia , Anticorpos de Cadeia Única/uso terapêutico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Afinidade de Anticorpos , Linhagem Celular Tumoral , Cetuximab , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno-TEM experiments on THP-1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin-Darby canine kidney (MDCK-II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno-TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy.
Assuntos
Microscopia Imunoeletrônica/métodos , Fixação de Tecidos/métodos , Animais , Linhagem Celular , Cães , Humanos , Imuno-Histoquímica , Lipídeos/química , Fatores de TempoRESUMO
The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, â¼40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.
Assuntos
Receptores ErbB/química , Animais , Anisotropia , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/química , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Cinética , Ligantes , Camundongos , Células NIH 3T3 , Ligação Proteica , Proteínas Tirosina Quinases/química , Transdução de Sinais , Espectrometria de Fluorescência/métodosRESUMO
There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.
Assuntos
Microscopia Crioeletrônica/métodos , Secções Congeladas/instrumentação , Secções Congeladas/métodos , Microscopia Crioeletrônica/instrumentação , Tomografia com Microscopia Eletrônica/métodos , Microscopia Eletrônica de Transmissão/métodos , Saccharomyces cerevisiae/ultraestruturaRESUMO
Caveolae are invaginations of the plasma membrane involved in multiple cellular processes, including transcytosis. In this paper we present an extensive 3-D electron tomographic study of the endothelial caveolar system in situ. Analysis of large cellular volumes of (high-pressure frozen, freeze-substituted and epon-embedded) human umbilical vein endothelial cells (HUVECs) provided a notable view on the architecture of the caveolar system that comprises--as confirmed by 3-D immunolabeling for caveolin of 'intact' cells--bona fide caveolae, free plasmalemmal vesicles, racemose invaginations and free multi-caveolar bodies. Application of template matching to tomograms allowed the 3-D localization of caveolar membrane coatings in a robust manner. In this way we observed that bona fide endothelial caveolae, cryofixed and embedded in their cellular context, show a spiral organization of the coating as shown in the past for chemically fixed and freeze-etched caveolae from fibroblasts. Meticulous 3-D analysis further revealed that the coatings are distributed in triads of spirals over the caveolar bulb and neck. Remarkably, this coating distribution is consistently present over the membranes of the other members of the caveolar system in HUVECs. The novel observations that we present clarify the ultrastructural complexity of the 'intact' caveolar system, setting a detailed morphological basis for its functional diversity.
Assuntos
Cavéolas/ultraestrutura , Tomografia com Microscopia Eletrônica , Células Endoteliais/ultraestrutura , Cavéolas/metabolismo , Caveolinas/metabolismo , Linhagem Celular , Células Endoteliais/metabolismo , Humanos , Microscopia Eletrônica de Transmissão , Transporte ProteicoRESUMO
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.
Assuntos
Endossomos/metabolismo , Cinesinas/metabolismo , Melanossomas/metabolismo , Fator de Transcrição AP-1/metabolismo , Linhagem Celular , Endossomos/ultraestrutura , Humanos , Cinesinas/genética , Melanossomas/ultraestrutura , Microscopia Eletrônica , RNA Interferente Pequeno/genética , Fator de Transcrição AP-1/genéticaRESUMO
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.
Assuntos
Bactérias/ultraestrutura , Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Divisão Celular , Substâncias Macromoleculares , Organelas/ultraestrutura , Compostos de Amônio Quaternário/metabolismo , Anaerobiose , Bactérias/metabolismo , Tomografia com Microscopia Eletrônica , Ordem dos Genes , Genes Bacterianos , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Organelas/metabolismo , OxirreduçãoRESUMO
In endothelial cells specifically, cPLA2alpha translocates from the cytoplasm to the Golgi complex in response to cell confluence. Considering the link between confluence and cell-cell junction formation, and the emerging role of cPLA2alpha in intracellular trafficking, we tested whether Golgi-associated cPLA2alpha is involved in the trafficking of junction proteins. Here, we show that the redistribution of cPLA2alpha from the cytoplasm to the Golgi correlates with adherens junction maturation and occurs before tight junction formation. Disruption of adherens junctions using a blocking anti-VE-cadherin antibody reverses the association of cPLA2alpha with the Golgi. Silencing of cPLA2alpha and inhibition of cPLA2alpha enzymatic activity using various inhibitors result in the diminished presence of the transmembrane junction proteins VE-cadherin, occludin, and claudin-5 at cell-cell contacts, and in their accumulation at the Golgi. Altogether, our data support the idea that VE-cadherin triggers the relocation of cPLA2alpha to the Golgi and that in turn, Golgi-associated cPLA2alpha regulates the transport of transmembrane junction proteins through or from the Golgi, thereby controlling the integrity of endothelial cell-cell junctions.
Assuntos
Complexo de Golgi/enzimologia , Fosfolipases A2 do Grupo IV/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Junções Aderentes/metabolismo , Anticorpos/imunologia , Anticorpos/farmacologia , Antígenos CD/imunologia , Antígenos CD/metabolismo , Western Blotting , Caderinas/imunologia , Caderinas/metabolismo , Células Cultivadas , Claudina-5 , Citoplasma/enzimologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Complexo de Golgi/efeitos dos fármacos , Fosfolipases A2 do Grupo IV/genética , Humanos , Microscopia de Fluorescência , Ocludina , Transporte Proteico/efeitos dos fármacos , Interferência de RNARESUMO
In this paper, we present a comprehensive study of the sol-gel transitions and liquid crystal phase transitions in aqueous suspensions of positively charged colloidal gibbsite platelets at pH 4-5 over a wide range of particle concentrations (50-600 g/L) and salt concentrations (10(-4)-10(-1) M NaCl). A detailed sol-gel diagram was established by oscillatory rheological experiments. These demonstrate the presence of kinetically arrested states both at high and at low salt concentrations, enclosing a sol region. Birefringence and iridescence show that in the sol state nematic and hexagonal columnar liquid crystal phases are formed. The gel and liquid crystal structures are studied in further detail using small-angle X-ray scattering (SAXS) and cryo-focused ion beam/scanning electron microscopy (cryo-FIB-SEM). The gel formed at high salt concentration shows signatures of a sponge-like structure and does not display birefringence. In the sol region, by lowering the salt concentration and/or increasing the gibbsite concentration, the nematic phase gradually transforms from the discotic nematic (ND) into the columnar nematic (NC) with much stronger side-to-side interparticle correlations. Subsequently, this NC structure can be either transformed into the hexagonal columnar phase or arrested into a birefringent repulsive gel state with NC structure.
RESUMO
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.
Assuntos
Retículo Endoplasmático/ultraestrutura , Ácido Graxo Sintase Tipo II/biossíntese , Interações Hospedeiro-Parasita , Membranas Intracelulares/ultraestrutura , Organelas/ultraestrutura , Toxoplasma/ultraestrutura , Animais , Transporte Biológico , Chlorocebus aethiops , Crioultramicrotomia , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/parasitologia , Ácidos Graxos/biossíntese , Membranas Intracelulares/metabolismo , Metabolismo dos Lipídeos , Modelos Biológicos , Organelas/metabolismo , Simbiose/fisiologia , Toxoplasma/enzimologia , Toxoplasma/metabolismo , Vacúolos/metabolismo , Vacúolos/parasitologia , Vacúolos/ultraestrutura , Células VeroRESUMO
The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales. It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.
Assuntos
Basidiomycota/ultraestrutura , Carpóforos/ultraestrutura , Basidiomycota/classificação , Microscopia Crioeletrônica , Microscopia Eletrônica de TransmissãoRESUMO
Binary nanocrystal superlattices, that is, ordered structures of two sorts of nanocolloids, hold promise for a series of functional materials with novel collective properties. Here we show that based on electron tomography a comprehensive, quantitative, three-dimensional characterization of these systems down to the single nanocrystal level can be achieved, which is key in understanding the emerging materials properties. On four binary lattices composed of PbSe, CdSe, and Au nanocrystals, we illustrate that ambiguous interpretations based on two-dimensional transmission electron microscopy can be prevented, nanocrystal sizes and superlattice parameters accurately determined, individual crystallographic point and plane defects studied, and the order/disorder at the top and bottom surfaces imaged. Furthermore, our results suggest that superlattice nucleation and growth occurred at the suspension/air interface and that the unit cells of some lattices are anisotropically deformed upon drying.
Assuntos
Nanopartículas/química , Compostos de Cádmio/química , Cristalografia por Raios X , Tomografia com Microscopia Eletrônica , Ouro/química , Chumbo/química , Compostos de Selênio/químicaRESUMO
Membranes play a crucial role in many cellular processes, and it is therefore not surprising that many electron tomographic studies in life sciences concern membranous structures. While these tomographic studies provide many new insights into membrane connections and continuities in three dimensions, they are mostly limited to a macro-morphological level. In this paper, we demonstrate that by combining electron tomography and three-dimensional template matching we are able to investigate membrane morphology at a new level: membrane domains in three dimensions. To test this, temperature induced lipid phase separation in the biological model system of the Escherichia coli bacteria was used. We compared the inner (containing phospholipids) and outer (containing lipopolysaccharides) leaflet of the E. coli outer membrane at both 37 and -20 degrees C, and could visualize how these leaflets react differently to temperature shifts. These findings can be explained by the physico-chemical nature of the building blocks and are in line with earlier published data. This study shows that the combination of electron tomography and template matching is robust enough to visualize membrane domains that are beyond the perception of manual annotation.
Assuntos
Membrana Celular/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Escherichia coli/ultraestruturaAssuntos
Tomografia com Microscopia Eletrônica , Nanoestruturas/ultraestrutura , Carbono/química , Fibra de Carbono , Catálise , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão e Varredura , Nanoestruturas/química , Óxidos/química , Zeolitas/químicaRESUMO
In this technical note we report a tannic acid-mediated osmium impregnation method that, applied after freeze-substitution, increases membrane contrast in cells for transmission electron microscopy and tomography studies. The general staining that is achieved allows visualization of organelles, plasma membrane and associated specializations (e.g. caveolae) in non-post-stained plastic sections by conventional transmission electron microscopy. In combination with electron tomography it results in membranes with a proper contrast and equal staining pattern through the depth of the tomograms. The protocol that we contribute can serve as starting point for those willing to improve the membrane contrast of their specimens or to make 3D studies on the architecture of membranous compartments by electron tomography.
Assuntos
Membrana Celular/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Substituição ao Congelamento/métodos , Tetróxido de Ósmio/química , Coloração e Rotulagem/métodos , Taninos/química , Soluções Tampão , Linhagem Celular , Membrana Celular/química , Células Endoteliais/ultraestrutura , Células Eucarióticas/ultraestrutura , Fixadores/química , Humanos , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica de TransmissãoRESUMO
BACKGROUND INFORMATION: Treatment of cells with UVC radiation leads to the formation of DNA cross-links which, if not repaired, can lead to apoptosis. gamma-H2AX and cleaved caspase 3 are proteins formed during UVC-induced DNA damage and apoptosis respectively. The present study sets out to identify early morphological markers of apoptosis using a new method of correlative microscopy, ILEM (integrated laser electron microscopy). Cleaved caspase 3 and gamma-H2AX were immunofluorescently labelled to mark the cells of interest. These cells were subsequently searched in the fluorescence mode of the ILEM and further analysed at high resolution with TEM (transmission electron microscopy). RESULTS: Following the treatment of HUVECs (human umbilical vein endothelial cells) with UVC radiation, in the majority of the cells gamma-H2AX was formed, whereas only in a subset of cells caspase 3 was activated. In severely damaged cells with high levels of gamma-H2AX a round, electron-dense nuclear structure was found, which was hitherto not identified in UV-stressed cells. This structure exists only in nuclei of cells containing cleaved caspase 3 and is present during all stages of the apoptotic process. Energy-loss imaging showed that the nuclear structure accumulates phosphorus, indicating that it is rich in nucleic acids. Because the nuclear structure did not label for DNA and was not affected by regressive EDTA treatment, it is suggested that the UV-induced nuclear structure contains a high amount of RNA. CONCLUSIONS: Because the UV-induced nuclear structure was only found in cells labelled for cleaved caspase 3 it is proposed as an electron microscopic marker for all stages of apoptosis. Such a marker will especially facilitate the screening for early apoptotic cells, which lack the well-known hallmarks of apoptosis within a cell population. It also raises new questions on the mechanisms involved in the UV-induced apoptotic pathway.
Assuntos
Apoptose/efeitos da radiação , Caspase 3/análise , Núcleo Celular/ultraestrutura , Histonas/análise , Microscopia Confocal/métodos , Microscopia Eletrônica/métodos , Biomarcadores/análise , Caspase 3/efeitos da radiação , Núcleo Celular/química , Núcleo Celular/efeitos da radiação , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/efeitos da radiação , Células Endoteliais/ultraestrutura , Imunofluorescência , Histonas/efeitos da radiação , Humanos , Microscopia Confocal/instrumentação , Microscopia Eletrônica/instrumentação , RNA/análise , Raios UltravioletaRESUMO
The filamentous fungus Aspergillus niger is widely exploited for industrial production of enzymes and organic acids. An integrated genomics approach was developed to determine cellular responses of A. niger to protein production in well-controlled fermentations. Different protein extraction methods in combination with automated sample processing and protein identification allowed quantitative analysis of 898 proteins. Three different enzyme overproducing strains were compared to their isogenic fungal host strains. Clear differences in response to the amount and nature of the overproduced enzymes were observed. The corresponding genes of the differentially expressed proteins were studied using transcriptomics. Genes that were up-regulated both at the proteome and transcriptome level were selected as leads for generic strain improvement. Up-regulated proteins included proteins involved in carbon and nitrogen metabolism as well as (oxidative) stress response, and proteins involved in protein folding and endoplasmic reticulum-associated degradation (ERAD). Reduction of protein degradation through the removal of the ERAD factor doaA combined with overexpression of the oligosaccharyl transferase sttC in A. niger overproducing beta-glucuronidase (GUS) strains indeed resulted in a small increase in GUS expression.
Assuntos
Aspergillus niger/genética , Aspergillus niger/metabolismo , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Genômica , Microbiologia Industrial , Perfilação da Expressão Gênica , Glucuronidase/biossíntese , Glucuronidase/genética , Proteoma/análiseRESUMO
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.