Die in der vorliegenden Habilitationsschrift zusammengefaßten Publikationen stellen Untersuchungen zu zwei Themenschwerpunkten dar: 1. Verankerungsmechanismen von Membranproteinen der basolateralen und der apikalen Plasmamembrandomäne der Parietalzellen mit dem Membranzytoskelett und 2. die regulierte Fusion von zytoplasmatischen Vesikeln mit der apikalen Plasmamembran dieser Zellen. Die strukturell und molekular sehr unterschiedlich gestaltete apikale und basolaterale Membrandomäne der Parietalzellen sollte funktionell charakterisiert und die Mechanismen der Membranumbauvorgänge aufgeklärt werden, die nach Aktivierung der Zellen im apikalen Membrankompartiment ablaufen. Für die strukturelle Stabilität der basolateralen Domäne spielt wahrscheinlich die Verankerung von AE2 über das Verknüpfungsprotein Ankyrin mit dem Membranzytoskelett eine wichtige Rolle. Die apikale Membrandomäne der Parietalzellen kann in drei Kompartimente unterteilt werden. Die freie apikale Membran, die canalikuläre Membran und die Membranen der tubulären Vesikel. Entlang der freien apikalen und der canaliculären Plasmamembran kommen wie auf der basolateralen Seite die Zytoskelett-Proteine Actin und Spectrin vor. Nach unseren Untersuchungen könnte es während der Sekretionsphase zu einer temporären Verbindung von H+,K+-ATPase Molekülen mit dem Membranzytoskelett kommen. Diese Verbindung wird wahrscheinlich durch das Verknüpfungsprotein Ezrin vermittelt. Der Mechanismus des Fusionsvorgangs der tubulären Vesikel mit der canaliculären Membran war bisher nicht bekannt. In Parietalzellen konnten die neuronalen SNARE-Proteine Synaptobrevin 2, Syntaxin 1 und SNAP25 sowie das zur Familie der kleinen G-Proteine gehörende Protein Rab3A und die Regulatorproteine NSF und alpha/beta SNAP nachgewiesen werden. Das in Parietalzellen gefundene Verteilungsmuster der SNARE-Proteine entspricht nicht der klassischen Vorstellung einer heterotypischen Membranfusion, vielmehr entspricht diese Verteilung einer homotypischen Fusion, wie sie für Vakuolen in Hefezellen beschrieben wurde. Die Bedeutung der SNARE-Proteine für die Fusion der tubulären Vesikel mit der canaliculären Membran und damit für die Steigerung der HCl-Sekretion konnte durch Inkubation der Zellen mit Tetanus Neurotoxin (TeNt) gezeigt werden. Die Behandlung der Parietalzellen mit TeNt führte zum vollständigen Ausbleiben der, nach Stimulation mit cAMP bei Kontrollzellen beobachteten Erhöhung, der Säuresekretion / The publications summarized here cover two topics: 1. the anchorage mechanism of membrane proteins of the basolateral and the apical plasma membrane with the membrane cytoskeleton of parietal cells and 2. the regulated fusion of cytoplasmic vesicles with the apical plasma membrane of these cells. It was the aim of these studies to characterize the structural and molecular differences between the apical and basolateral membrane domains in parietal cells. Moreover the mechanisms involved in membrane traffic within the apical membrane compartment following stimulation were investigated. We found that anchorage of AE2 with the membrane cytoskeleton through the linkage protein ankyrin seems to be important for the stability of the basolateral membrane. The apical membrane domain of parietal cells can be subdivided into three compartments. The free apical membrane, the canalicular membrane and the tubulovesicular membrane. The cytoskeletal proteins spectrin and actin can be found at the basolateral, the free apical and the canalicular membrane. We have shown that the H+K+-ATPase molecules appear to be temporary linked to the membrane cytoskeleton during acid-secretion. This contact is most likely mediated by the linker-protein ezrin. Until now the mechanism of fusion of the tubulovesicles with the canalicular membrane was unknown. In parietal cells the neuronal SNARE-proteins synaptobrevin 2, Syntaxin 1, SNAP25, the small G-protein rab3A, and the regulatory proteins NSF and alpha/beta-SNAP were detected. The subcellular distribution of these proteins does not support the notion of a neuron-like heterotypic fusion. Instead it shows similarity with the homotypic fusion process of vacuoles in yeast. The importance of SNARE-proteins for the fusion of tubulovesicles with the canalicular membrane and, by consequence also for the increase of acid-secretion was shown by incubation of the cells with tetanus neurotoxin (TeNt). The measurable increase of acid secretion by parietal cells after stimulation with c-AMP was inhibited completely through an incubation with TeNt.

<p>RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. </p><p>We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The <i>Caenorhabditis elegans</i> homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the <i>par</i> (for partitioning defective) gene family, first identified in <i>C.elegans</i>. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex.</p><p>Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. </p><p>To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. </p><p>In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.</p>

RhoGTPases are proteins working as molecular switches as they bind and hydrolyze GTP. They are in their active conformation when GTP is bound and are then able to interact with their effector proteins, which relay the downstream signaling. When the GTP is hydrolyzed to GDP, the RhoGTPase is inactivated. RhoGTPases have been shown to be activated by a variety of stimuli and they are implicated in regulation of diverse cellular processes, including cell migration, cell cycle progression, establishment of cell polarity and transformation. We identified mammalian Par6 as a novel effector protein for the RhoGTPases Cdc42 and Rac1. The Caenorhabditis elegans homologue of Par6 had previously been shown to be essential for cell polarity development in the worm embryo. We found that endogenous Par6 colocalized with the tight junction protein ZO-1 in MDCKII epithelial cells. Par6 also interacted with mammalian Par3, another member of the par (for partitioning defective) gene family, first identified in C.elegans. Endogenous Par3 also localized to tight junctions in epithelial cells. This suggested that Par6 and Par3 are part of a complex regulating cell polarity also in mammalian cells. The interaction between Par6 and activated Cdc42 and Rac1 suggested a role for these RhoGTPases in the regulation of this complex. Co-expression of Par6 together with PKCζ, induced a dramatic change in cell morphology. The cells rounded up and long cellular extensions, resembling neurites, were formed. The ability to induce these changes in cell morphology was found to be dependent on the direct interaction between Par6 and PKCζ, as well as on the kinase activity of PKCζ. We observed that cells co-expressing mPar6C and PKCζ contained bundled microtubules and microtubules that hade been acetylated, indicating that the microtubules were stabilized. To investigate the roles of RhoGTPases in PDGF-induced gene expression we performed cDNA microarray analyses on AG01518 human foreskin fibroblasts in which we over-expressed the dominant negative forms of Cdc42, Rac1 and RhoA. We found that the expression of 16 genes, out of the 45 up-regulated by PDGF-BB, were inhibited ≥50% in the presence of dominant negative Cdc42, Rac1 or RhoA. 19 other genes were down-regulated by one or two of the dominant RhoGTPases. Our data implied that the expression of many PDGF-BB induced genes can be affected by RhoGTPase signaling. In conclusion, the work presented here has increased the knowledge of the involvement of RhoGTPase signaling in establishment of cell polarity and gene regulation.

The compositional technique of cross partitioning is one of Luigi Dallapiccola's most used twelve-tone devices. Through a detailed analysis of three contrapuntal canonic movements from Dallapiccola's Quaderno Musicale di Annalibera, I examine his use of cross partitioning as a motivic tool and as a referential collection. The development of the BACH motive and the derivation of tone-row statements reflects on Dallapiccola's extensive use of cross partitioning and his compositional principles used to achieve a sense of polarity. Upon a preliminary analysis based on set-theory analysis set out by Joseph Straus I draw an interpretive analysis through Alegant's cross partitioning model as well as develop my own set of parameters for interpretation in regards to polarity which is based on intervallic stability.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

Crumbs is a transmembrane protein and apical determinant in Drosophila epithelial cells. Its cytoplasmic tail contains a PDZ and a FERM domain-binding site through which Crumbs interacts with the FERM proteins Yurt, Moesin and Expanded. Recent evidence suggests that Crumbs can also interact with the uncharacterised FERM proteins CG34347 and Cdep. The main objective of my thesis was to generate mutations in CG34347 and Cdep to facilitate the functional analysis of these genes. I generated a mutation for Cdep that remains to be characterised and two mutant lines for CG34347; one lacking the first exon and one lacking the entire gene, using a FRT-based recombination strategy. Both CG34347 mutants cause severe ovarian defects. The most consistent defect is a multilayering of the interfollicular stalk. These defects are also observed when Notch, Hippo, Wingless and Hedgehog signalling pathways are overactive in ovaries suggesting that CG34347 participates in one of those pathways.

Filtration of grape juice was investigated. Effects of process parameters of filtration were examined. The usage of precoating material and filter aid were found to be necessary to prolong the filter life. Filter cake was found to be incompressible with the effect of increasing pressure. Increase in temperature caused increase in flow rate due to the reduction in viscosity. The influences of depectinization and clarification on filtration process were also examined. Depectinization and clarification decreased the resistance and increased the flow rate. Improvement in the quality of the grape juice was observed when these pretreatments were employed. The effects of temperature and soluble solid concentration on physical properties of clarified fruit juices were investigated. High temperature and soluble solid content dependencies of density, viscosity and heat capacity were detected. Experimental data were fitted as a function of temperature and soluble solid content. Models being valid for studied clarified fruit juices were achieved for density and viscosity with the regression coefficients (R2) higher than 0.90. Optimization of the solid-liquid extraction conditions for trans-resveratrol, trans-&amp / #949 / -viniferin, ferulic acid, and total phenolics from milled grape canes has been investigated. Temperature and ethanol concentration were found to be major process variables for all responses. Maximum yields of trans-resveratrol, trans-&amp / #949 / -viniferin, ferulic acid, and total phenolics were predicted as 4.25 mg/g dw, 2.03 mg/g dw, 1.05 mg/g dw, and 9.28 mg/g dw, respectively. Optimization of extraction conditions for antioxidant activity of grape cane extracts measured by the Trolox equivalent antioxidant capacity (TEAC) and the oxygen radical absorbance capacity (ORACFL) assays was carried out using solid-liquid extraction and response surface methodology. Ethanol concentration and temperature employed for the extraction of antioxidant agents from grape cane samples were found to be statistically significant process variables affecting antioxidant activity measured by the TEAC and ORAC methods. trans-Resveratrol and trans-&amp / #949 / -viniferin were extracted from milled grape canes using pressurized low polarity water (PLPW). The extraction temperature was significant for both compounds: extraction at 160oC resulted in a 40% loss of trans-resveratrol compared to 95oC while reduction of trans-&amp / #949 / -viniferin at both temperatures remained at 30%. Increasing ethanol concentration from 0 to 25% increased the extraction of total phenolics and trans-&amp / #949 / -viniferin by 44% and 489%, respectively. Solvent flow rate also influenced trans-&amp / #949 / -viniferin extraction. Effective diffusivities of trans-resveratrol increased by three times with increasing temperature. The modified Gompertz equation satisfactorily explained the extraction of the stilbenes investigated.

Compartmentalization of cell membranes, in particular of the apical membrane of columnar epithelia, is the topic of this thesis. The first part characterizes the apical membrane and its specialized organization and morphology, whereas the second part focuses on the formation of this unique plasma membrane domain during epithelial polarization. The apical membrane of columnar epithelia is enriched in glycosphingolipids, a class of lipids that are known to interact with cholesterol to form liquid ordered domains, also termed &amp;quot;rafts&amp;quot;, in cell membranes. Imaging the apical surface of untreated and raft lipid depleted MDCK cells with atomic force microscopy revealed that raft lipids are involved in the formation and/or maintenance of microvilli, actin based protrusions of the apical plasma membrane, indicating a regulatory link between membrane domains and the cytoskeleton. Furthermore, antibody patching and photobleaching experiments performed during the work of this thesis suggest that the organization into raft and non-raft domains is very different in the apical membrane of MDCK cells compared to the plasma membrane of a fibroblast. In fact, the data support the hypothesis that the apical membrane could be a percolating raft membrane in which rafts constitute the major phase and non-raft domains exist as isolated entities. The second part of this thesis analyses the segregation of apical and basolateral membrane domains during epithelial polarization. This segregation can either be achieved by generating scaffolded domains prior to junction formation or by polarized secretory and endocytic membrane traffic after the establishment of cell junctions. While most apical and basolateral marker proteins in MDCK cells follow the latter mechanism, this thesis reports that the apical marker gp135 is confined to the dorsal face already in single attached cells. The unknown antigen was purified and identified as podocalyxin. Analysis of a series of domain mutants revealed that the C-terminal PDZ-binding motif of podocalyxin is mainly responsible for its special localization, which it shares with the PDZ protein NHERF-2. Knocking down podocalyxin by RNA interference resulted in retardation of cell growth and epithelial polarization. Taken together, the data suggest that podocalyxin and NHERF-2 could be part of an early apical polarity scaffolding system based on PDZ-binding and PDZ-containing proteins.

The studies presented here were focused on the understanding of the principles for protein sorting from the Golgi to the cell surface. As a marker protein we used Fus1p, a type I plasma membrane protein that is O-glycosylated on the extracellular domain and plays a role in cell fusion during yeast mating. Additionally, we analyzed mechanisms responsible for asymmetric distribution of Fus1p in mating cells. We demonstrated that the glycans attached to the protein act as a sorting determinant for protein transport to the cell surface. In cells lacking PMT4, encoding a mannosyltransferase involved in the initial step of O-glycosylation, Fus1p was not glycosylated and accumulated in late Golgi structures. A similar defect in exocytosis was observed when a Fus1p mutant lacking the O-glycosylated domain was expressed in wild-type cells, however, the cell surface delivery could be rescued if the 33 amino acid portion of the Fus1p ectodomain, containing 15 potentially glycosylated sites was added to the protein. It was previously well documented in epithelial cells that different types of protein glycosylation and association with lipid rafts play a role of determinants for protein delivery to the apical plasma membrane. However, otherwise the machinery responsible for cargo sorting to the apical membrane is poorly understood. Our finding that also in yeast, protein glycosylation can function as a sorting determinant provides a new possibility to investigate underlying mechanisms...

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