TY - JOUR A1 - Franke, Werner W. A1 - Scheer, Ulrich T1 - Some structural differentiations in the HeLa cell: heavy bodies, annulate lamellae and cotte de maillet endoplasmic reticulum N2 - A small fraction of HeLa cells within an exponentially growing culture showed cisternal differentiations, such as cytoplasmic as well as intranuclear annulate lamellae and special smooth surfaced endoplasmic reticulum aggregates with a typical "Cotte de maillet" appearance. Additionally, clusters of dense granules were observed in the cytoplasm which were often associated with polysomes and strongly resembled the so-called "heavy bodies" known in particular in diverse oocytes. The functional meaning of these structures is discussed. Moreover, it is deduced from the ultrastructural identity of the pore complexes in the nuclear envelope and the cytoplasmic and intranuclear annulate lamellae that the pore complex material with its highly ordered arrangement is not a structure characteristic for nucleocytoplasmically migrating material, but rather is a general structural expression of a tight binding of ribonucleoprotein (RNP) to cisternal membranes. The pore complexes are thought of as representing sites of a RNP-storage. A similar functioning is hypothesized for the "heavy body"like aggregates. To the current hypotheses on the formation of annulate lamellae and the nuclear envelope, which are based on the concept of membrane continuities and constancies, the alternative view of a self assembly mechanism of membrane constituents on nucleoprotein structures is added. KW - Cytologie Y1 - 1971 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40614 ER - TY - JOUR A1 - Franke, Werner W. A1 - Zentgraf, Hanswalter A1 - Scheer, Ulrich T1 - Membrane linkages at the nuclear envelope N2 - Electron-opaque material is shown in the perinuclear cisternae of various cell types to connect the inner and outer nuclear membrane faces. Similar bridges were observed between the outer nuclear membrane and the outer mitochondrial membrane. The intracisternal bridges of the nuclear envelope appear to be important for the structural stability of the perinuclear cisterna. Stable structural linkage of mitochondria to the outer nuclear membrane might be relevant to the understanding of the characteristic juxtanuclear accumulation of mitochondria and also provide arguments for the discussions of certain biochemical activities found in nuclear and nuclear membrane fractions. KW - Cytologie Y1 - 1973 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40596 ER - TY - JOUR A1 - Spring, Herbert A1 - Trendelenbrug, Michael F. A1 - Scheer, Ulrich A1 - Franke, Werner W. A1 - Herth, Werner T1 - Structural and biochemical studies of the primary nucleus of two green algal species, Acetabularia mediterranea and Acetabularia major N2 - Primary (giant) nuclei of the green algae Acetabularia mediterranea and A. major were studied by light and electron microscopy using in situ fixed material as well as manually isolated nuclear components. In addition, cytochemical reactions of nuclear structures and biochemical determinations of nuclear and cytoplasmic RNA and of genome DNA content were performed. The data obtained and the structures observed are interpreted as demonstralions of transcriptional activities of different gene classes. The most prominent class is the nucleolar cistrons of precursors of ribosomal RNA which occur highly repeated in clusters in the form of regularly alternating intercepts on deoxyribonucleoprotein axes of transcribed rDNA, the fibril-covered matrix units, and the fibril-free "spacer" segments. A description and a classification of the various structural complexes which seem to represent transcriptional activities is given. Quantitative evaluations of these arrangements are presented. The morphology and the dimensions of such structures are compared with the RNA molecular weight determinations and with the corresponding data reported from various animal cell systems. It is suggested that the formation of the giant nucleus is correlated with, and probably due to, an enormous amplification of transcriptionally active rDNA and packing of the extrachromosomal copies into the large nucleolar aggregate bodies. KW - Cytologie KW - Nucleolus KW - electron microscopy KW - Acetabularia KW - transcription KW - gene activity KW - ribosomes Y1 - 1974 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40600 ER - TY - JOUR A1 - Franke, Werner W. A1 - Scheer, Ulrich A1 - Trendelenburg, Michael F. A1 - Spring, Herbert A1 - Zentgraf, Hanswalter T1 - Absence of nucleosomes in transcriptionally active chromatin N2 - The ultrastructure of twO kinds of transcription ally active chromatin, the lampbrush chromosome loops and the nucleoli from amphibian oocytes and primary nuclei of the green alga Acetabularia, has been examined after manual isolation and dispersion in low salt media of slightly alkaline pH using various electron microscopic staining techniques (positive staining, metal shadowing, negative staining, preparation on positively charged films, etc.) and compared with the appearance of chromatin from various somatic cells (hen erythrocytes, rat hepatocytes, ClIltured murine sarcoma cells) prepared in parallel. While typical nucleosomes were revealed with all the techniques for chromatin from the latter three cell system, no nucleosomes were identified in either the lampbrush chromosome structures or the nucleolar chromatin. Nucleosomal arrays were absent not only in maximally fibril-covered matrix units but also in fibril-free regions between transcriptional complexes, including the apparent spacer intercepts between different transcriptional units. Moreover, comparisons of the length of the repeating units of rDNA in the transcribed state with those determined in the isolated rDNA and with the lengths of the first stable product of rDNA transcription, the pre-rRNA, demonstrated that the transcribed rDNA was not significantly shortened and/or condensed but rather extended in the transcriptional units. Distinct granules of about nucleosomal size which were sometimes found in apparent spacer regions as well as within matrix units of reduced fibril density were shown not to represent nucleosomes since their number per spacer unit was not inversely correlated with the length of the specific unit and also on the basis of their resistance to treatment with the detergent Sarkosyl NL-30. It is possible to structurally distinguish between transcriptionally active chromatin in which the DNA is extended in a non-nucleosomal form of chromatin and condensed, inactive chromatin within the typical nucleosomal package. The characteristic extended structure of transcriptionally active chromatin is found not only in the transcribed genes but also in non-transcribed regions within or between ("spacer") transcriptional units as well as in transcriptional units that are untranscribed amidst transcribed ones and/or have been inactivated for relatively short time. It is hypothesized that activation of transcription involves a transition from a nucleosomal to an extended chromatin organisation and that this structural transition is not specific for single "activated" genes but may involve larger chromatin regions, including adjacent untranscribed intercepts. KW - Cytologie KW - Chromatin structure KW - nucleosomes KW - transcription KW - electron microscopy Y1 - 1976 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40646 ER - TY - JOUR A1 - Weber, Klaus A1 - Osborn, Mary A1 - Franke, Werner W. A1 - Seib, Erinita A1 - Scheer, Ulrich A1 - Herth, Werner T1 - Identification of microtubular structures in diverse plant and animal cells by immunological cross-reaction revealed in immunofluorescence microscopy using antibodies against tubulin from porcine brain N2 - Antibody against tubulin from porcine brain was used to evaluate the immunological cross reactivity of tubulin from a variety of animal and plant cells. Indirect immunofluorescence microscopy revealed microtubule-containing structures including cytoplasmic microtubules, spindle microtubules, cilia and fIagella. Thus tubulin from diverse species of both mammals and plants show immunological cross-reactivity with tubulin from porcine brain. Results obtained by immunofluorescence microscopy are whenever possible compared with previously known ultrastructural results obtained by electron microscopy. KW - Cytologie KW - Microtubules KW - immunofluorescence KW - evolution KW - antibody KW - sperm Y1 - 1977 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-41383 ER - TY - JOUR A1 - Scheer, Ulrich T1 - Structural organization of spacer chromatin between transcribed ribosomal RNA genes in amphibian oocytes N2 - Transcribed nucleolar chomatin, including the spacer regions interspersed between the rRNA genes, is different from the bulk of nontranscribed chromatin in that the DNA of these regions appears to be in an extended (B) conformation when examined by electron microscopy. The possibility that this may reflect artificial unfolding of nucleosomes during incubation in very low salt buffers as routinely used in such spread preparations has been examined by studying the influence of various ion concentrations on nucleolar chromatin structure. Amplified nucleolar chromatin of amphibian oocytes (Xenopus laevis, Pleurodeles waltlii, Triturus cristatus) was spread in various concentrations of NaCl (range 0 to 20 mM). Below 1 mM salt spacer chromatin frequently revealed a variable number of irregularly shaped beads, whereas above this concentration the chromatin axis appeared uniformly smooth. At all salt concentrations studied, however, the length distribution of spacer and gene regions was identical. Preparations fixed with glutaraldehyde instead of formaldehyde, or unftxed preparations, were indistinguishable in this respect. The observations indicate that (i) rDNA spacer regions are not compacted into nucleosomal particles and into supranucleosomal structures when visualized at chromatin stabilizing salt concentrations (e.g., 20 mM NaCl), and (ii) spacer DNA is covered by a uniform layer of proteins of unknown nature which, at very low salt concentrations (below 1 mM NaCl), can artificially give rise to the appearance of small granular particles of approximately nucleosome-like sizes. These particles, however, are different from nucleosomes in that they do not foreshorten the associated spacer DNA. The data support the concept of an altered nucleohistone conformation not only in transcribed chromatin but also in the vicinity of transcriptional events. KW - Cytologie KW - Chromatin structure KW - rDNA KW - amphibian oocytes Y1 - 1980 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-41057 ER - TY - JOUR A1 - Hügle, Barbara A1 - Hazan, Rachel A1 - Scheer, Ulrich A1 - Franke, Werner W. T1 - Localization of ribosomal protein S1 in the granular component of the interphase nucleolus and its distribution during mitosis N2 - Using antibodies to various nucleolar and ribosomal proteins, we define, by immunolocalization in situ, the distribution of nucleolar proteins in the different morphological nucleolar subcompartments. In the present study we describe the nucleolar localization of a specific ribosomal protein (51) by immunofluorescence and immunoelectron microscopy using a monoclonal antibody (R5 1-105). In immunoblotting experiments, this antibody reacts specifically with the largest and most acidic protein of the small ribosomal subunit (51) and shows wide interspecies cross-reactivity from amphibia to man. Beside its localization in cytoplasmic ribosomes, this protein is found to be specifically localized in the granular component of the nucleolus and in distinct granular aggregates scattered over the nucleoplasm. This indicates that ribosomal protein 51, in contrast to reports on other ribosomal proteins, is not bound to nascent pre-rRNA transcripts but attaches to preribosomes at later stages of rRNA processing and maturation. This protein is not detected in the residual nucleolar structures of cells inactive in rRNA synthesis such as amphibian and avian erythrocytes. During mitosis, the nucleolar material containing ribosomal protein 51 undergoes a remarkable transition and shows a distribution distinct from that of several other nucleolar proteins. In prophase, the nucleolus disintegrates and protein 51 appears in numerous small granules scattered throughout the prophase nucleus. During metaphase and anaphase, a considerable amount of this protein is found in association with the surfaces of all chromosomes and finely dispersed in the cell plasm. In telophase, protein 51-containing material reaccumulates in granular particles in the nucleoplasm of the newly formed nuclei and, finally, in the re-forming nucleoli. These observations indicate that the nucleolus-derived particles containing ribosomal protein 51 are different from cytoplasmic ribosomes and, in the living cell, are selectively recollected after mitosis into the newly formed nuclei and translocated into a specific nucleolar subcompartment, i.e ., the granular component. The nucleolar location of ribosomal protein 51 and its rearrangement du'ring mitosis is discussed in relation to the distribution of other nucleolar proteins. KW - Cytologie Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-39695 ER - TY - JOUR A1 - Scheer, Ulrich A1 - Hansmann, Paul A1 - Falk, Heinz A1 - Sitte, Peter T1 - Ultrastructural localization of DNA in two Cryptomonas species by use of a monoclonal DNA-antibody N2 - Immunogold cytochemistry - DNA localization - Cryptomonas nucleomorph The distribution and subcellular localization of DNA in the unicellular alga Cryptomonas has been investigated electron-microscopically by indirect immunocytochemistry, using a monoclonal DNA antibody and a gold-Iabeled secondary antibody. This technique proved to be very sensitive and entirely specific. DNA could be demonstrated in four different compartments (nucleus, nucleomorph, plastid, and mitochondrion). Within the plastid, DNA is concentrated in stroma regions that are localized preferentially around the center of the organelle. The mitochondrion contains several isolated DNA-containing regions (nucleoids). Within the nucleus, most of the DNA is localized in the 'condensed' chromatin. DNA was also detectable in small areas of the nucleolus, whereas the interchromatin space of the nucleus appeared almost devoid of DNA. Within the nucleomorph, DNA is distributed inhomogeneously in the matrix. DNA could furthermore be detected in restricted areas of the 'fibrillogranular body' of the nucleomorph, resembling the situation encountered in the nucleol us. The presence of DNA and its characteristic distribution in the nucleomorph provide additional, strong evidence in favour of the interpretation of that organelle as the residual nucleus of a eukaryotic endosymbiont in Cryptomonas. KW - Cytologie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-39746 ER - TY - JOUR A1 - Scheer, Ulrich A1 - Messner, Karin A1 - Hazan, Rachel A1 - Raska, Ivan A1 - Hansmann, Paul A1 - Falk, Heinz A1 - Spiess, Eberhard A1 - Franke, Werner W. T1 - High sensitivity immunolocalization of double and single-stranded DNA by a monoclonal antibody N2 - A monoclonal antibody (AK 30-10) is described which specifically reacts with DNA both in double and single-stranded forms but not with other molecules and structures, including deoxyribonucleotides and RNAs. When used in immunocytochemical experiments on tissue sections and permeabilized cultured cells, this antibody detects DNA-containing structures, even when the DNA is present in very small amounts. Examples of high resolution detection include the DNA present in amplified extrachromosomal nucleoli, chromomeres of lampbrush chromosomes, mitochondria, chloroplasts and mycoplasmal particles. In immunoelectron microscopy using the immunogold technique, the DNA was localized in distinct substructures such as the "fibrillar centers" of nucleoli and certain stromal centers in chloroplasts. The antibody also reacts with DNA of chromatin of living cells, as shown by microinjection into cultured mitotic cells and into nuclei of amphibian oocytes. The potential value and the limitations of immunocytochemical DNA detection are discussed. KW - Cytologie KW - DNA antibodies KW - monoclonal antibodies KW - DNA immunolocalization KW - chromatin KW - mycoplasma tests Y1 - 1987 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-41063 ER - TY - JOUR A1 - Thiry, Marc A1 - Scheer, Ulrich A1 - Goessens, Guy T1 - Immunoelectron microscopic study of nucleolar DNA during mitosis in Ehrlich tumour cells N2 - In order to investigate the DNA localization within Ehrlich tumor cell nucleoli during mitosis, two recent immunocytochemical methods using either an anti-DNA or an anti-bromodeoxyuridine (BrdU) monoclonal antibody have been applied. In both cases, the immunogold labeling has been performed on ultrathin sections of cells embedded either in Lowicryl K4M or in Epon, respectively. Identical results are observed with both immunocytochemical approaches. In the interphase nucleolus, besides the labeling of the perinucleolar chromatin shell and of its intranucleolar invaginations which penetrate into the nucleolar body and often terminate at the fibrillar centers, a few gold particles are also preferentially found towards the peripheral region of the fibrillar centers. In contrast, the dense fibrillar component and the granular component are never labeled. During mitosis, the fibrillar centers persist at the chromosomal nucleolus organizing regions (NOR's) and can be selectively stained by the silver method. However, these metaphase fibrillar centers are no longer decorated by the DNA- or BrdU antibodies. These results indicate that until the end of prophase, rRNA genes are present inside the fibrillar center material, disappear during metaphase and reappear in reconstituting nucleoli during telophase. Thus, fibrillar centers appear to represent structures sui generis, which are populated by rRNA genes only when the nucleolus is functionally active. In segregated nucleoli after actinomycin D treatment, the DNA labeling is exclusively restricted to the perinucleolar chromatin blocks. These findings also suggest that the DNA content of the fibrillar center material varies according to the rRNA transcription level of the cells. The results are discussed in the light of the present knowledge of the functional organization of the nucleolus. KW - Cytologie KW - Nucleolus KW - DNA KW - mitosis Y1 - 1988 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40745 ER - TY - JOUR A1 - Weber, Thomas A1 - Schmidt, Erwin A1 - Scheer, Ulrich T1 - Mapping of transcription units on Xenopus laevis lampbrush chromosomes by in situ hybridization with biotin-labeled cDNA probes N2 - A non-radioactive in situ hybridization method is described for the localization of transcription units of defined genes to lateral loops of Xenopus laevis lampbrush chromosomes. Two Xenopus cONA probes were used encoding the nucleolar protein N038/ B23 and cytokeratin 1(8). Both proteins are known to be synthesized in Xenopus oocytes, and Northern blot analysis revealed the presence of the corresponding mRNAs in different oogenic stages. The probes were enzymatically labeled with biotin-dCTP and hybridized to lampbrush chromosomes. The sites of hybridization were detected either by indirect immunofluorescence microscopy using rabbit antibodies against biotin and fluorescein-conjugated antirabbit IgG or enzymatically using peroxidase-conjugated streptavi din. The probe encoding the nucleolar protein hybridized to two sets of lateral loops on different bivalents, the cytokeratin probe to at least four. Our finding that each probe hybridized to more than one chromosomal locus may reflect the tetraploid nature of the Xenopus laevis genome or results from cross-hybridization to other transcriptionally active members of the N038/ B23-nucleoplasmin or the cytokeratin-Iamin gene families. The method described should facilitate further in situ hybridization studies with appropriate genomic clones in order to map specific DNA sequences to defined loop regions and to come to a better understanding of the relationship between loop organization and gene transcription unit. KW - Cytologie KW - Lampbrush chromosomes KW - in situ hybridization KW - transcription units KW - Xenopus oocytes Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40763 ER - TY - JOUR A1 - Benavente, Ricardo A1 - Scheer, Ulrich A1 - Chaly, Nathalie T1 - Nucleocytoplasmic sorting of macromolecules following mitosis: fate of nuclear constituents after inhibition of pore complex function N2 - PtK2 cells in which pore complex-mediated transport is blocked by microinjection early in mitosis of a monoclonal antibody (specific for an Mr 68000 pore complex glycoprotein) or of wheat germ agglutinin (WGA) complete cytokinesis. However, their nuclei remain stably arrested in a telophase-like organization characterized by highly condensed chromatin and the absence of nucleoli, indicating a requirement for pore-mediated transport for the reassembly of interphase nuclei. We have now examined this requirement more closely by monitoring the behavior of individual nuclear macromolecules in microinjected cells using immunofluorescence microscopy and have investigated the effect of microinjecting the antibody or WGA on cellular ultrastructure. The absence of nuclear transport did not affect the sequestration into daughter nuclei of components such as DNA, DNA topoisomerase I and the nucleolar protein fibrillarin that are carried through mitosis on chromosomes. On the other hand, lamins, snRNAs and the p68 pore complex glycoprotein, all cytoplasmic during mitosis, remained largely cytoplasmic in the telophase-arrested cells. Electron microscopy showed the nuclei to be surrounded by a doublelayered membrane with some inserted pore complexes. In addition, however, a variety of membranous structures with associated pore complexes was regularly noted in the cytoplasm, suggesting that chromatin may not be essential for the postmitotic formation of pore complexes. We propose that cellular compartmentalization at telophase is a two-step process. First, a nuclear envelope tightly encloses the condensed chromosomes, excluding non-selectively all macromolecules not associated with the chromosomes. Interphase nuclear organization is then progressively restored by selective pore complex-mediated uptake of nuclear proteins from the cytoplasm. KW - Cytologie KW - Nucleocytoplasmic transport KW - nuclear organization KW - nuclear envelope KW - nucleologenesis KW - mitosis Y1 - 1989 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-40777 ER - TY - JOUR A1 - Weisenberger, Dieter A1 - Scheer, Ulrich A1 - Benavente, Ricardo T1 - The DNA topoisomerase I inhibitor camptothecin blocks postmitotic reformation of nucleoli in mammmalian cells N2 - No abstract available KW - Cytologie KW - Nucleolus-DNA KW - opoisomerase I KW - camptothecin KW - mitosis Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-41434 ER - TY - THES A1 - Kukat, Christian T1 - Fusion, Fission und Nucleoids in Megamitochondrien T1 - Fusion, fission and nucleoids in megamitochondria N2 - In rho0-Zellen, die über keine mitochondriale DNA (mtDNA) mehr verfügen, entstehen während der Kultivierung Megamitochondrien durch endogene Milchsäure-Azidifizierung des Kulturmediums. Diese Riesenorganellen bilden sich dabei durch mitochondriale Fusionsereignisse und/oder eine Hemmung der Fission. In Zellen mit mitochondrialem Genom ist es ebenso möglich Megamitochondrien durch artifizielles Ansäuern des Kulturmediums zu induzieren. Diese Erkenntnisse wurden im Rahmen dieser Arbeit als Werkzeug verwendet, um Einblicke in mitochondriale Fusions- und Fissionsereignisse zu erlangen. Zunächst wurde die Fusion mitochondrialer Matrixkompartimente mithilfe der photoaktivierbaren Variante des grünen fluoreszierenden Proteins (PA-GFP) untersucht. Hiermit konnte gezeigt werden, dass das Vermischen der Matrixkompartimente nach der Fusion ein sehr schneller Prozess ist. Die Analyse der Bildung und Rückbildung der Megamitochondrien erfolgte sowohl konfokal- als auch elektronenmikroskopisch, wobei sich zeigte, dass die Matrix der Riesenorganellen kaum mehr Cristae beinhaltet. Die Rückbildung der Megamitochondrien zum normalen Netzwerk ist ein sehr schneller Prozess, bei dem schon nach 15 min keine vergrößerten Organellen mehr sichtbar sind. Dies indiziert, dass der Rückbildungsprozess wahrscheinlich durch Veränderungen von verfügbaren Proteinen durchgeführt wird, ohne die Induzierung von Proteinneusynthese. Untersuchungen auf ultrastruktureller Ebene zeigten, dass es während der Rückbildung zur Formation von drei unterschiedlichen Mitochondrientypen kam, die sich in ihrer Morphologie stark unterschieden. Weiterhin wurden vergleichende Studien zur Bildung der Megamitochondrien durchgeführt, bei denen der Einfluss von Atmungsketten-Inhibitoren auf die Bildung von Milchsäure-induzierten Riesenorganellen untersucht wurde. Die Resultate deuten für die Megamitochondrieninduktion auf eine Abhängigkeit auf ein intaktes Membranpotential hin. Immunzytochemisch wurde die endogene Lokalisation der mitochondrialen Fusions- und Fissionsproteine Mitofusin 2, hFis1 und Drp1/DNM1L am Modellsystem der Megamitochondrieninduktion aufgeklärt. Es zeigte sich, dass diese Proteine punktförmig an der äußeren Membran der Riesenorganellen lokalisieren Um das Modellsystem an lebenden Zellen zu nutzen, wurden Vektoren konstruiert, die fluoreszenzmarkierte Proteine der mitochondrialen Fusions- und Fissionsmaschinerie exprimierten. Hiermit konnte einerseits die Lokalisation von Mitofusin 1, Mitofusin 2, hFis1 und Drp1/DNM1L in lebenden Zellen nach Induktion der Megamitochondrien analysiert werden und andererseits der Einfluss der Überexpression dieser Proteine auf die Bildung der Riesenorganellen dokumentiert werden. Die Ergebnisse machten deutlich, dass nur die Überepxression von hFis1 die Bildung der Megamitochondrien verhinderte. Ein weiterer Schwerpunkt der vorliegenden Arbeit lag in der Visualisierung und Dynamik mitochondrialer Nucleoids in lebenden Zellen. Nucleoids sind Protein-DNA-Komplexe, in denen mitochondriale Genome organisiert sind. Mit dem Farbstoff PicoGreen gelang es mtDNA in lebenden Zellen zu färben und Dynamikstudien der punktförmigen Strukturen mikroskopisch festzuhalten. Während sich mtDNA im mitochondrialen Netzwerk nur marginal aufgrund stattfindender Fusions- und Fissionsereignisse bewegte kam es in den Milchsäure-induzierten Megamitochondrien zu einer extensiven und extrem schnellen Bewegung von mitochondrialer DNA. In anschließenden Versuchen wurde der mitochondriale Transkriptions- und Verpackungsfaktor TFAM als fluoreszentes Fusionsprotein in Zellen transfiziert und Kolokalisationsstudien zeigten, dass das Fusionsprotein mit mtDNA kolokalisiert. In den Riesenorganellen präsentierten punktförmige TFAM-gefärbte Nucleoids ein sehr dynamisches Verhalten mit schneller Bewegung. In rho0-Zellen ohne mitochondriale DNA war die TFAM-Fluoreszenz hingegen gleichmäßig verteilt. Ein weiterer Nucleiodbestandteil ist das mitochondriale DNA-Einzelstrangbindeprotein SSBP1, welches in Megamitochondrien ebenso ein sehr dynamisches Verhalten aufwies. Eine mitochondrial-zielgesteuerte und EGFP-markierte Restriktionsendonuklease wies ebenfalls das typische, punktförmige Nucleoidmuster im mitochondrialen Netzwerk auf, was auf eine Interaktion mit der mtDNA schließen lässt. In rho0-Zellen ohne mtDNA kam es jedoch zur gleichmäßigen Verteilung des Konstruktes in den Mitochondrien. Zusammenfassend wurden in dieser Arbeit sowohl Einblicke in die Biologie der Megamitochondrien gewonnen, als auch Erkenntnisse über die Dynamik mitochondrialer Protein-DNA-Komplexe, wobei der Schwerpunkt hierbei auf einer Analyse mit Hilfe optischer Methoden lag. N2 - During the cultivation of rho0 cells, which are devoid of mitochondrial DNA (mtDNA), excessive endogenous lactic acid production during the fermentation process drives the development of megamitochondria. These giant organelles are formed by mitochondrial fusion events and/or the repression of fission. Megamitochondria formation is inducible in cells containing mtDNA by an artificial acidification of the culture medium by lactic acid. This work exploits these findings in order to investigate mitochondrial fusion and fission events. Fusion of mitochondrial matrix compartments was examined with the aid of the photoactivatable variant of the green fluorescent protein (PA-GFP), showing that the mixing of matrix components after fusion is an extremely fast process. Formation and reversion of megamitochondria was analyzed by confocal and electron microscopy, revealing that the matrix of giant organelles contains only rudiment structures of cristae organization. Reversion of the megamitochondria to a normal network displays fast kinetic characteristics. This indicates that the restoration process most likely is performed by alterations of novel protein expression. Additional assessment on an ultrastructural level displayed the occurrence of three different types of mitochondria during the reversion, which strongly differed in their morphology. To gain insights into the mechanism of megamitochondria formation and reversion comparative studies were performed with various inhibitors of the respiratory chain. The results indicated a dependence on an intact membrane potential for the induction of megamitochondria. Localization of the endogenous mitochondrial fusion and fission proteins mitofusin 1, hFis1 and Drp1/DNM1L were analyzed in megamitochondria by immunocytochemistry, demonstrating that these proteins localize foci-like to the outer membrane of the giant organelles. Fluorescently tagged proteins of the mitochondrial fusion and fission apparatus (mitofusin 1, mitofusin 2, hFis1 and Drp1/DNM1L) were used in localization and overexpression experiments in living cells by transient transfection. The results revealed that only the overexpression of hFis1 inhibited the formation of megamitochondria. Another main focus of this thesis was the visualization and dynamics of mitochondrial nucleoids in living cells. Nucleoids are protein-DNA complexes representing organizational units for the mitochondrial genomes The dye PicoGreen was used to stain mtDNA in living cells and the dynamics of nucleoid movement was analyzed by fluorescent and confocal microscopy. While mitochondrial DNA showed only marginal movements due to ongoing fusion and fission events in a mitochondrial network, an extensive and extremely fast movement in the lactic acid-induced megamitochondria could be observed. In subsequent experiments the mitochondrial transcription and packaging factor TFAM was fluorescently tagged and transfected into cells. Colocalization studies showed that this fusion protein colocalizes with mitochondrial DNA. In the giant organelles foci-like TFAM-stained nucleoids displayed a very dynamic performance with fast movement. However, in rho0 cells without mitochondrial DNA the TFAM-fluorescence was uniformly distributed. An additional nucleoid constituent and marker for mitochondrial DNA is the mitochondrial single-stranded DNA-binding protein SSBP1, and it could be shown that SSBP1 also exhibits very dynamic characteristics in megamitochondria. A mitochondrial-targeted and EGFP-tagged restriction endonuclease also exhibited the typical, punctual nucleoid pattern in the mitochondrial network, suggesting an mtDNA interaction. In rho0 cells, however, the construct was uniformly distributed in the mitochondrial matrix. In summary, in the present thesis optical and mechanistic insights into the biology of megamitochondria were obtained. This approach was further exploited to study the dynamics of mitochondrial protein-DNA complexes with optical methods. KW - Mitochondrium KW - Cytologie KW - Mitochondrien KW - mitochondria Y1 - 2008 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-30467 ER - TY - JOUR A1 - Schramm, Sabine A1 - Fraune, Johanna A1 - Naumann, Ronald A1 - Hernandez-Hernandez, Abrahan A1 - Höög, Christer A1 - Cooke, Howard J. A1 - Alsheimer, Manfred A1 - Benavente, Ricardo T1 - A Novel Mouse Synaptonemal Complex Protein Is Essential for Loading of Central Element Proteins, Recombination, and Fertility N2 - The synaptonemal complex (SC) is a proteinaceous, meiosis-specific structure that is highly conserved in evolution. During meiosis, the SC mediates synapsis of homologous chromosomes. It is essential for proper recombination and segregation of homologous chromosomes, and therefore for genome haploidization. Mutations in human SC genes can cause infertility. In order to gain a better understanding of the process of SC assembly in a model system that would be relevant for humans, we are investigating meiosis in mice. Here, we report on a newly identified component of the murine SC, which we named SYCE3. SYCE3 is strongly conserved among mammals and localizes to the central element (CE) of the SC. By generating a Syce3 knockout mouse, we found that SYCE3 is required for fertility in both sexes. Loss of SYCE3 blocks synapsis initiation and results in meiotic arrest. In the absence of SYCE3, initiation of meiotic recombination appears to be normal, but its progression is severely impaired resulting in complete absence of MLH1 foci, which are presumed markers of crossovers in wild-type meiocytes. In the process of SC assembly, SYCE3 is required downstream of transverse filament protein SYCP1, but upstream of the other previously described CE–specific proteins. We conclude that SYCE3 enables chromosome loading of the other CE–specific proteins, which in turn would promote synapsis between homologous chromosomes. KW - Maus KW - Genetik KW - Cytologie Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68895 ER -