Regelmechanismen und Determinanten der Transkription in Polytänchromosomen von Chironomus und Drosophila

Die kumulative Habil.‐Schrift gründet sich auf 6 Originalpublikationen, die beschreiben: [Sass, H. (1982), Cell 28: 269‐278]. RNA polymerase B in polytene chromosomes: Immunofluorescent and autoradiographic analysis during stimulated and repressed RNA synthesis. Elektronenmikroskopie charakterisierte das C. tentans Balbianiring BR2‐Gen von Speicheldrüsenchromosomen als hoch aktives 5‐6 μm langes single‐copy Gen, das 33/μm RNAPolymerasen B (Pol II) transkribieren (Diss., Sass, H., 1978, Univ. Tübingen). Diese Immunfluoreszenzstudie ortet Pol II in allen Interbanden von Region IV‐3B10‐3B5 des nichtinduzierten BR2. Prominente Fluoreszenz im BR2‐Genort 3B9/10 zeigt, das BR2‐Gen ist präaktiv, wie erwartet. 3H‐Autoradiogramme beweisen, in allen fluoreszierenden BR2, BR1, BR3, Puffs, aufgelockerten Banden, Interbanden und Loci ohne Puffing, synthetisiert Pol II RNA. Die genomweite ständige Pol II‐Präsenz zeigt, dass, wie beim nichtinduzierten BR2‐Gen, bereits schon gebundene Pol II wohl auch andere Gene präaktiviert. So erfolgt die Regulation der Transkription mehr über die transkriptionelle Elongation. Auch durch α‐Amanitin, oder Actinomycin D, oder Hitzeschock in vivo kollabierte BR2, BR1, BR3 besitzen Pol II. [Sass, H. (1984), Chromosoma 90: 20‐25]. Gene identification in polytene chromosomes: some Balbiani ring 2 gene sequences are located in an interband‐like region of Chironomus tentans. Immunfluoreszenz und 3H‐Autoradiographie zeigen, dass Injektionen von DRB in Larven die Balbianiringe (BR) sowie andere Puffs und deren Pol II‐Konzentration dramatisch reduzieren. Trotzdem zeigen 3H‐Uridin markierte Speicheldrüsenchromosomen, dass RNA‐Synthese doch in nichtinduzierten BR2, BR1, BR3 erfolgt, aber nur auf reduziertem Level. Das widerspricht der von Egyházi E. (1975, PNAS 73:947‐950) propagierten „Inhibition of Balbiani ring RNA synthesis at the initiation level“ durch DRB. Vielmehr sieht es so aus, DRB wirkt bei der transkriptionellen Elongation inhibierend. Durch in situ‐Hybridisierung von Sequenzen klonierter BR2‐DNA wurde in Speicheldrüsenchromosom IV das BR2‐Gen in Region 3B9/10 direkt identifiziert. [Sass, H. and Pederson, T. (1984), J. Mol. Biol. 180: 911‐926]. Transcription‐dependent localization of U1 and U2 small nuclear ribonucleoproteins at major sites of gene activity in polytene chromosomes. Immunolokalisation von Sm‐, U1‐ und U2snRNP‐spezifischen Antigenen in Speicheldrüsenchromosomen von C. tentans hat zur Entdeckung der beim Spleißen von prä‐mRNA beteiligten U1/U2snRNPs in Balbianiringen BR2, BR1, BR3 sowie anderen Puffs und aufgelockerten Banden geführt. Die überraschenden BR‐Daten zeigen erstmals: (i) Der Spleiß‐Apparat ist in Genloci mit intensiver RNA‐Synthese schon vorhanden. (ii) Immunfluoreszenz reflektiert den Exon‐Intron‐Bau dieser BR‐Gene. (iii) Transkription und spleißosomales Ausschneiden von Introns sind koordiniert. [Sass, H. (1989), Nucleic Acids Research 17: 10508]. Hsp82‐neo transposition vectors to study insertional mutagenesis in Drosophila melanogaster and tissue culture cells; [Sass, H. (1990), Gene 89: 179‐186]. P‐transposable vectors expressing a constitutive and thermoinducible hsp82‐neo fusion gene for Drosophila germline transformation and tissue‐culture transfection. Beschrieben sind Design, Konstruktion und Expression der Genfusion hsp82‐neo als ein in vivo selektierbares Reporter‐/Markergen, die Transposons P{hsp82‐neo/Adh} sowie P{hsp82‐neo} und Transformations‐Vektoren pHS22, pHS24, pHS85, pHS103 und pHS104. Sie stellen das von der Fliege gebildete Enzym bakteriellen Ursprungs, Neomycin‐Phosphotransferase II, für die G418‐Selektion bereit, um die Position, Struktur, Expression und Funktion von Genen mittels hsp82‐neo‐Mutagenese zu erforschen. [Sass, H. and Meselson, M. (1991), Proc. Natl. Acad. Sci. USA 88: 6795‐6799]. Dosage compensation of the Drosophila pseudoobscura Hsp82 gene and the D. melanogaster Adh gene at ectopic sites in D. melanogaster. Quantitative Unterschiede in der Dosiskompensation des X‐chromosomalen hsp82‐Gens von D. pseudoobscura und autosomalen Adh‐Gens von D. melanogaster wurden als Erhöhung der RNAMenge in D. melanogaster gemessen. Beide Transgene sind dosiskompensiert, sprang P{hsp82‐ neo/Adh} in euchromatische Regionen des D. melanogaster X‐Chromosoms. Beide Transgene sind nicht dosiskompensiert, insertierte P{hsp82‐neo/Adh} ins β‐Heterochromatin in Region 20 an der Basis des X. Keine der zehn autosomalen Insertionen ist dosiskompensiert. Die Ergebnisse lassen vermuten, dass X‐chromosomale regulatorische Sequenzen, die für die Verstärkung der Genaktivität um Faktor 2 in Männchen verantwortlich sind, gehäuft im X vorkommen, jedoch im β‐ Heterochromatin und den Autosomen fehlen. Das Kompensationsverhalten der transponierten Gene wird durch das neue chromosomale Milieu des Insertionsortes bestimmt.

Uptake mechanism, intracellular trafficking and endo-lysosomal pH monitoring of polystyrene nanoparticles

What is the intracellular fate of nanoparticles (NPs) taken up by the cells? This question has been investigated for polystyrene NPs of different sizes with a set of molecular biological and biophysical techniques.rnTwo sets of fluorescent NPs, cationic and non-ionic, were synthesized with three different polymerization techniques. Non-ionic particles (132 – 846 nm) were synthesized with dispersion polymerization in an ethanol/water solution. Cationic NPs with 120 nm were synthesized by miniemulsion polymerization Particles with 208, 267 and 603 nm were produced by seeding the 120 nm particle obtained by miniemulsion polymerization with drop-wise added monomer and polymerization of such. The colloidal characterization of all particles showed a comparable amount of the surface groups. In addition, particles were characterized with regard to their size, morphology, solid content, amount of incorporated fluorescent dye and zeta potential. The fluorescent intensities of all particles were measured by fluorescence spectroscopy for calibration in further cellular experiments. rnThe uptake of the NPs to HeLa cells after 1 – 24 h revealed a much higher uptake of cationic NPs in comparison to non-ionic NPs. If the same amount of NPs with different sizes is introduced to the cell, a different amount of particles is present in the cell medium, which complicates a comparison of the uptake. The same conclusion is valid for the particles’ overall surface area. Therefore, HeLa cells were incubated with the same concentration, amount and surface area of NPs. It was found that with the same concentration always the same polymer amount is taking up by cells. However, the amount of particles taken up decreases for the biggest. A correlation to the surface area could not be found. We conclude that particles are endocytosed by an excavator-shovel like mechanism, which does not distinguish between different sizes, but is only dependent on the volume that is taken up. For the decreased amount of large particles, an overload of this mechanism was assumed, which leads to a decrease in the uptake. rnThe participation of specific endocytotic processes has been determined by the use of pharmacological inhibitors, immunocytological staining and immunofluorescence. The uptake of NPs into the endo-lysosomal machinery is dominated by a caveolin-mediated endocytosis. Other pathways, which include macropinocytosis and a dynamin-dependent mechanism but exclude clathrin mediated endocytosis, also occur as competing processes. All particles can be found to some extent in early endosomes, but only bigger particles were proven to localize in late endosomes. No particles were found in lysosomes; at least not in lysosomes that are labeled with Lamp1 and cathepsin D. However, based on the character of the performed experiment, a localization of particles in lysosomes cannot be excluded.rnDuring their ripening process, vesicles undergo a gradual acidification from early over late endosomes to lysosomes. It is hypothesized that NPs in endo-lysosomal compartments experience the same change in pH value. To probe the environmental pH of NPs after endocytosis, the pH-sensitive dye SNARF-4F was grafted onto amino functionalized polystyrene NPs. The pH value is a ratio function of the two emission wavelengths of the protonated and deprotonated form of the dye and is hence independent of concentration changes. The particles were synthesized by the aforementioned miniemulsion polymerization with the addition of the amino functionalized copolymer AEMH. The immobilization of SNARF-4F was performed by an EDC-coupling reaction. The amount of physically adsorbed dye in comparison to covalently bonded dye was 15% as determined by precipitation of the NPs in methanol, which is a very good solvent for SNARF-4F. To determine influences of cellular proteins on the fluorescence properties, a intracellular calibration fit was established with platereader measurements and cLSM imaging by the cell-penetrable SNARF-4F AM ester. Ionophores equilibrated the extracellular and intracellular pH.rnSNARF-4F NPs were taken up well by HeLa cells and showed no toxic effects. The pH environment of SNARF-4F NPs has been qualitatively imaged as a movie over a time period up to 1 h in pseudo-colors by a self-written automated batch program. Quantification revealed an acidification process until pH value of 4.5 over 24 h, which is much slower than the transport of nutrients to lysosomes. NPs are present in early endosomes after min. 1 h, in late endosomes at approx. 8 h and end up in vesicles with a pH value typical for lysosomes after > 24 h. We therefore assume that NPs bear a unique endocytotic mechanism, at least with regards to the kinetic involvedrn

Cellular localization and mechanism of amyloid precursor protein (APP) homodimer formation in an oxidizing environment

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein, which resembles a cell surface receptor, comprising a large ectodomain, a single spanning transmembrane part and a short C-terminal, cytoplasmic domain. It belongs to a conserved gene family, with over 17 members, including also the two mammalian APP homologues proteins APLP1 and APLP2 („amyloid precursor like proteins“). APP is encoded by 19 exons, of which exons 7, 8, and 15 can be alternatively spliced to produce three major protein isoforms APP770, APP751 and APP695, reflecting the number of amino acids. The neuronal APP695 is the only isoform that lacks a Kunitz Protease Inhibitor (KPI) domain in its extracellular portion whereas the two larger, peripheral APP isoforms, contain the 57-amino-acid KPI insert. rnRecently, research effort has suggested that APP metabolism and function is thought to be influenced by homodimerization and that the oligomerization state of APP could also play a role in the pathology of Alzheimer's disease (AD), by regulating its processing and amyloid beta production. Several independent studies have shown that APP can form homodimers within the cell, driven by motifs present in the extracellular domain, as well as in the juxtamembrane (JM) and transmembrane (TM) regions of the molecule, whereby the exact molecular mechanism and the origin of dimer formation remains elusive. Therefore, we focused in our study on the actual subcellular origin of APP homodimerization within the cell, an underlying mechanism, and a possible impact on dimerization properties of its homologue APLP1. Furthermore, we analyzed homodimerization of various APP isoforms, in particular APP695, APP751 and APP770, which differ in the presence of a Kunitz-type protease inhibitor domain (KPI) in the extracellular region. In order to assess the cellular origin of dimerization under different cellular conditions, we established a mammalian cell culture model-system in CHO-K1 (chinese hamster ovary) cells, stably overexpressing human APP, harboring dilysine based organelle sorting motifs at the very C-terminus [KKAA-Endoplasmic Reticulum (ER); KKFF-Golgi]. In this study we show that APP exists as disulfide-bound, SDS-stable dimers, when it was retained in the ER, unlike when it progressed further to the cis-Golgi, due to the KKFF ER exit determinant. These stable APP complexes were isolated from cells, and analyzed by SDS–polyacrylamide gel electrophoresis under non-reducing conditions, whereas strong denaturing and reducing conditions completely converted those dimers to monomers. Our findings suggested that APP homodimer formation starts early in the secretory pathway and that the unique oxidizing environment of the ER likely promotes intermolecular disulfide bond formation between APP molecules. We particularly visualized APP dimerization employing a variety of biochemical experiments and investigated the origin of its generation by using a Bimolecular Fluorescence Complementation (BiFC) approach with split GFP-APP chimeras. Moreover, using N-terminal deletion constructs, we demonstrate that intermolecular disulfide linkage between cysteine residues, exclusively located in the extracellular E1 domain, represents another mechanism of how an APP sub-fraction can dimerize within the cell. Additionally, mutational studies revealed that cysteines at positions 98 and 105, embedded in the conserved loop region within the E1 domain, are critical for interchain disulfide bond formation. Using a pharmacological treatment approach, we show that once generated in the oxidative environment of the ER, APP dimers remain stably associated during transport, reaching the plasma membrane. In addition, we demonstrate that APP isoforms, encompassing the KPI domain, exhibit a strongly reduced ability to form cis-directed dimers in the ER, whereas trans-directed cell aggregation of Drosophila Schneider (S2)-cells was isoform independent, mediating cell-cell contacts. Thus, suggesting that steric properties of KPI-APP might be the cause for weaker cis-interaction in the ER, compared to APP695. Finally, we provide evidence that APP/APLP1 heterointeractions are likewise initiated in the ER, suggesting a similar mechanism for heterodimerization. Therefore, dynamic alterations of APP between monomeric, homodimeric, and possibly heterodimeric status could at least partially explain some of the variety in the physiological functions of APP.rn

Software tools and efficient algorithms for the feature detection, feature tracking, event localization, and visualization of large sets of atmospheric data

Data sets describing the state of the earth's atmosphere are of great importance in the atmospheric sciences. Over the last decades, the quality and sheer amount of the available data increased significantly, resulting in a rising demand for new tools capable of handling and analysing these large, multidimensional sets of atmospheric data. The interdisciplinary work presented in this thesis covers the development and the application of practical software tools and efficient algorithms from the field of computer science, aiming at the goal of enabling atmospheric scientists to analyse and to gain new insights from these large data sets. For this purpose, our tools combine novel techniques with well-established methods from different areas such as scientific visualization and data segmentation. In this thesis, three practical tools are presented. Two of these tools are software systems (Insight and IWAL) for different types of processing and interactive visualization of data, the third tool is an efficient algorithm for data segmentation implemented as part of Insight.Insight is a toolkit for the interactive, three-dimensional visualization and processing of large sets of atmospheric data, originally developed as a testing environment for the novel segmentation algorithm. It provides a dynamic system for combining at runtime data from different sources, a variety of different data processing algorithms, and several visualization techniques. Its modular architecture and flexible scripting support led to additional applications of the software, from which two examples are presented: the usage of Insight as a WMS (web map service) server, and the automatic production of a sequence of images for the visualization of cyclone simulations. The core application of Insight is the provision of the novel segmentation algorithm for the efficient detection and tracking of 3D features in large sets of atmospheric data, as well as for the precise localization of the occurring genesis, lysis, merging and splitting events. Data segmentation usually leads to a significant reduction of the size of the considered data. This enables a practical visualization of the data, statistical analyses of the features and their events, and the manual or automatic detection of interesting situations for subsequent detailed investigation. The concepts of the novel algorithm, its technical realization, and several extensions for avoiding under- and over-segmentation are discussed. As example applications, this thesis covers the setup and the results of the segmentation of upper-tropospheric jet streams and cyclones as full 3D objects. Finally, IWAL is presented, which is a web application for providing an easy interactive access to meteorological data visualizations, primarily aimed at students. As a web application, the needs to retrieve all input data sets and to install and handle complex visualization tools on a local machine are avoided. The main challenge in the provision of customizable visualizations to large numbers of simultaneous users was to find an acceptable trade-off between the available visualization options and the performance of the application. Besides the implementational details, benchmarks and the results of a user survey are presented.

Intraflagellar transport in the developing and mature vertebrate retina

Intraflagellar transport (IFT) is required for the assembly and maintenance of cilia. In this study we analyzed the subcellular localization of IFT proteins in retinal cells by correlative high-resolution immunofluorescence and immunoelectron microscopy. The rod photoreceptor cell was used as a model system to analyze protein distribution in cilia. To date the expression of IFT proteins has been described in the ciliary region without deciphering the precise spatial and temporal subcellular localization of IFT proteins, which was the focus of my work. rnThe establishment of the pre-embedding immunoelectron method was an important first step for the present doctoral thesis. Results of this work reveal the differential localization of IFT20, IFT52, IFT57, IFT88, IFT140 in sub-ciliary compartments and also their presence in non-ciliary compartments of retinal photoreceptor cells. Furthermore, the localization of IFT20, IFT52 and IFT57 in dendritic processes of non-ciliated neurons indicates that IFT protein complexes also operate in non-ciliated cells and may participate in intracellular vesicle trafficking in eukaryotic cells in general.rnIn addition, we have investigated the involvement of IFT proteins in the ciliogenesis of vertebrate photoreceptor cilia. Electron microscopy analyses revealed six morphologically distinct stages. The first stages are characterized by electron dense centriolar satellites and a ciliary vesicle, while the formation of a ciliary shaft and of the light sensitive outer segment disks are features of the later stages. IFT proteins were expressed during all stages of photoreceptor cell development and found to be associated with the ciliary apparatus. In addition to the centriole and basal body IFT proteins are present in the photoreceptor cytoplasm, associated with centriolar satellites, post-Golgi vesicles and with the ciliary vesicle. Therewith the data provide an evidence for the involvement of IFT proteins during ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium of photoreceptor cells. Moreover, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis indicates roles of IFT proteins beyond their well-established function for IFT in mature cilia and flagella. rn

Analysen zur molekularen Charakterisierung von Proteinen des humanen Usher-Syndroms und Evaluation genbasierter Therapiestrategien

Analysen zur molekularen Charakterisierung von Proteinen des humanen Usher-Syndroms und Evaluation genbasierter Therapiestrategien rnDas humane Usher Syndrom (USH) ist die häufigste Form vererbter Taub-Blindheit. In der vorliegenden Dissertation wurde diese komplexe Erkrankung auf verschiedenen Ebenen analysiert: in Arbeiten zur Expression und Lokalisation von USH-Proteinen, der Analyse der USH-Proteinnetzwerke und deren Funktionen sowie darauf aufbauend die Entwicklung von Therapiestrategien für USH.rnIm Rahmen der Arbeit wurde die Expression und (sub)-zelluläre Lokalisation des USH1D-Genproduktes CDH23 in der Retina und Cochlea analysiert. CDH23-Isoformen werden in der Maus zeitlich und räumlich differentiell exprimiert. In den Retinae von Mäusen, nicht humanen Primaten und Menschen zeigten Analysen eine unterschiedliche Expression und Lokalisation des Zell-Zelladhäsionsmoleküls CDH23, was auf Funktions-unterschiede der einzelnen Isoformen in den analysierten Spezies hindeutet.rnAnalysen zur Aufklärung der USH-Proteinnetzwerke ergaben eine potentielle Interaktion des USH1G-Gerüstproteins SANS mit dem Golgi- und Centrosom-assoziierten Protein Myomegalin. Die direkte Interaktion der Proteine konnte durch unabhängige Experimente verifiziert werden. Beide Interaktionspartner sind in den Retinae verschiedener Spezies partiell ko-lokalisiert und partizipieren im periciliären USH-Proteinnetzwerk. Die Assoziation von SANS und Myomegalin mit dem Mikrotubuli-Cytoskelett weist auf eine Funktion des Proteinkomplexes in gerichteten Transportprozessen innerhalb der Photorezeptoren hin und bekräftigt die Hypothese einer Rolle von SANS und assoziierten Netzwerken mit Transportprozessen.rnDas hier gewonnene erweiterte Verständnis der molekularen Grundlagen sowie die Aufklärung der zellulären Funktion der Proteinnetzwerke ermöglichen die Entwicklung therapeutischer Strategien für USH. Ein Fokus der vorliegenden Arbeit lag auf der Entwicklung genbasierter Therapiestrategien und deren Evaluation, wobei der Schwerpunkt auf der Therapiestrategie der Genreparatur lag. Die mit Hilfe von Zinkfinger-Nukleasen (ZFN) induzierte Homologe Rekombination für die Genkorrektur wurde exemplarisch an der 91C>T/p.R31X-Mutation im USH1C-Gen gezeigt. Effiziente ZFN wurden identifiziert, generiert und erfolgreich im Zellkulturmodellsystem eingesetzt. Die Analysen demonstrierten eine Reparatur der Mutation durch Homologe Rekombination auf genomischer Ebene und die Expression des wiederhergestellten Proteins. Durch die Genkorrektur im endogenen Lokus sind Größe des Gens, Isoformen oder die Art der Mutation keine limitierenden Faktoren für die Therapie. Die in der vorliegenden Arbeit durchgeführten Experimente unterstreichen das enorme Potential ZFN-basierter Therapiestrategien hin zu personalisierten Therapieformen nicht nur für USH sondern auch für andere erbliche Erkrankungen, deren genetische Grundlagen bekannt sind.rn