Biological information processing in living systems like cells, tissues and organs critically depends on the physical interactions of molecular signaling components in time and space. How endocytic transport of signaling molecules contributes to the regulation of developmental signaling in the complex in vivo environment of a developing organism is not well understood. In a previously performed genome-wide screen on endocytosis, several genes have been identified, that selectively regulate transport of signaling molecules to different types of endosomes, without disrupting endocytosis. My PhD thesis work provides the first functional in vivo characterization of one of these candidate genes, the novel, highly conserved Rab5 effector protein P95 (PPP1R21). Cell culture studies suggest that P95 is a novel endocytic protein important to maintain the balance of distinct endosomal sub-populations and potentially regulates the sorting of signaling molecules between them (unpublished work, Zerial lab). The scientific evidence presented in this study demonstrates that zebrafish P95 is essential for early zebrafish embryogenesis. Both, knockdown and overexpression of zebrafish P95 compromise accurate morphogenetic movements and patterning of the zebrafish gastrula, showing that P95 functions during zebrafish gastrulation. P95 is functionally required to maintain signaling activity of signaling pathways that control embryonic patterning, in particular for WNT/β-catenin signaling activity. Knockdown of zebrafish P95 amplifies the recruitment of β-catenin to early endosomes, which correlates with the limitation of β-catenin to translocate to the nucleus and function as transcriptional activator. The obtained results suggest that zebrafish P95 modulates the cytoplasmic pools of β-catenin in vivo, via endosomal transport of β-catenin. In conclusion, the data presented in this thesis work provides evidence that the cytoplasm-to-nucleus shuttling of β-catenin is modulated by endocytic trafficking of β-catenin in vivo. We propose the endocytic modulation of β-catenin cytoplasm-to-nucleus trafficking as potential new mechanism to fine-tune the functional output of WNT/β-catenin signaling during vertebrate gastrulation.

Die im Rahmen dieser Arbeit durchgeführten Untersuchungen zum Einfluss der Wachstumsfaktoren IGF-I, EGF und dem Zytokin IL-6 auf den Androgenrezeptor-Signalweg zeigten in verschiedenen Prostatakarzinom-zelllinien schon nach zwei Stunden eine deutliche Degradation des Androgenrezeptor-Proteins. Die ausschließlich auf Protein-Ebene stattfindende, Wachstumsfaktor-induzierte negative Regulation des Androgenrezeptors konnte durch einen schnellen Androgeneffekt wieder aufgehoben werden. Mittels Luziferase-Reportergen-Assays wurde kein Einfluss der Wachstums-faktorwirkung auf die transkriptionelle Aktivität des Androgenrezeptors nachgewiesen. Darüber hinaus konnte eine signifikant reprimierende Wirkung durch IGF-I und EGF in Kombination mit geringen Mengen DHT beobachtet werden. Weitere Resultate dieser Arbeit deuten auf einen, durch den PI3-Kinase-Signalweg vermittelten, proteasomalen Abbauprozess des Rezeptors hin. Da die Suppression der downstream gelegenen Proteinkinase Akt keine Veränderung hinsichtlich der Degradation aufwies, konzentrierte sich die weiterführende Arbeit auf eine mögliche direkte Regulation des Androgen-rezeptors durch die PI3-Kinase. Unter Verwendung von rekombinanten GST-Fusionsproteinen konnte in Interaktionsstudien unter in vitro Bedingungen eine Phosphotyrosin-unabhängige Bindung zwischen der C-SH2-Domäne der p85-Untereinheit der PI3-Kinase und dem N- und C-Terminus des Androgenrezeptors nachgewiesen werden. Durch die nähere Charakterisierung dieser Bindungsbereiche mit Hilfe von Peptidarrays und anschließenden Alanin-Substitutionen war es möglich, für den N-Terminus 18, für den C-Terminus des Androgenrezeptors 6 und für die p85-C-SH2-Domäne der PI3-Kinase 11 Aminosäuren zu identifizieren. Die durch gezielte Punktmutagenese an diesen Aminosäurepositionen hergestellten Androgenrezeptor-Einzel- und -Mehrfachmutanten wiesen in Bindungsstudien dennoch Interaktion zur PI3-Kinase auf. Eine von Anderson und Kollegen postulierte Phosphotyrosin-unabhängige Bindung der SH2-Domänen der p85-Untereinheit der PI3-Kinase durch sogenannte „basic-X-basic“-Motive wurde ebenfalls in Interaktionstests zwischen der PI3-Kinase und dem Androgenrezeptor überprüft. Aufgrund der Tatsache, dass einige der identifizierten Aminosäuren auf dem Androgenrezeptor Teil eines „basic-X-basic“-Bindungsmotives sind, wurden Kombinationsmutanten generiert, die sowohl im N-Terminus als auch im C﷓Terminus des Androgenrezeptors ein bzw. zwei zerstörte „basic-X-basic“-Motive enthielten. Untersuchungen zum Bindungsverhalten dieser Mutanten zeigten zwar weiterhin Interaktion zur p85-C-SH2-Domäne der PI3-Kinase, jedoch der durch Western-blot-Analyse überprüfte IGF-I-induzierte Degradationseffekt des Androgenrezeptor-Proteins konnte mit zwei der verwendeten Androgenrezeptor-Kombinationsmutanten nicht mehr beobachtet werden.

Decapentaplegic (Dpp), a member of the TGF-[Beta] superfamily, acts as a morphogen to direct cell differentiation, determine cell fate and promote cell survival and proliferation in Drosophila wing development. To investigate the role of Dpp in Drosophila wing development, three aspects of the patterning role of Dpp have been analyzed. First, I investigated the cellular responses to Dpp signaling by a loss of function strategy. The consequences of lacking Dpp signal transduction on cell morphology and tissue integrity were analyzed. Second, I investigated whether Dpp signaling is down-stream of Hh signaling to maintain the normal cell segregation at the A/P boundary by clonal analysis. Third, I investigated whether cross talk among the Hh, Dpp and Wg signaling pathways exists and what its relevance for wing patterning is. To investigate the role of Dpp in Drosophila wing development, the general strategies are to look at the phenotypes of loss-of-function and gain-of-function. Mutant clones lacking Dpp signal transduction by knock down Dpp receptor Thick veins (Tkv) do not survive in wing blade due to JNK dependent apoptosis. To get larger mutant clones for analysis, JNK pathway was inhibited by knock down bsk (encodes JNK) in mutant clones lacking Dpp signaling using FLP-FRT system. Clones double mutant for tkv and bsk did not undergo apoptosis, but recovered at very low frequencies compared to sibling clones. Here, I showed that the low recovery of tkv bsk double mutant clones are due to the extrusion of mutant cells. The extrusion of tkv bsk double mutant cells correlated with changes in the actin cytoskeleton and a dramatic loss of the apical microtubule web normally present in these cells. These results suggest that Dpp signaling is required for cell morphogenesis in Drosophila wing development. We propose that Dpp acts as a survival factor in the wing disc epithelium by orchestrating proper cytoskeletal organization and maintaining normal cell-cell contact. Drosophila wing is subdivided into anterior (A) and posterior (P) compartments. This developing into adjacent compartments is crucial for the patterning of Drosophila wing. Previous study has shown that Hedgehog (Hh) signaling is required in A cells to maintain the A/P boundary and is sufficient to specify A type cell sorting. A previous study has in addition implicated the signaling molecule Decapentaplegic (Dpp) in maintaining the A/P boundary. However, this study did not address whether and in which cells, A and/or P, Dpp signal transduction was required to maintain this boundary. Here, I have analyzed the role of components of the Dpp signal transduction pathway and the relation of Dpp and Hh signaling in maintaining the A/P boundary by clonal analysis. I showed that Dpp signaling mediated by the Dpp target gene, T-box protein Optomotor-blind (Omb), is required in A cells, but not in P cells, to maintain the normal position of the A/P boundary. During patterning formation, it is essential for cells to receive precise positional information to pattern the tissue. It has been proposed for a long time that different signaling pathways such as Hedgehog (Hh), Dpp and Wingless (Wg) signaling pathways provide positional information for tissue patterning in an integrated manner. Recently, evidence of interactions between Hh and Dpp as well as Wg and Hh signaling pathways has been reported in Drosophila wing. Here, I have identified additional interactions among Hh, Dpp and Notch/Wg signaling. We propose that the selector gene engrailed, Hh and Dpp signaling interact with each other to regulate target genes expression and thus to pattern the wing along the A/P axis. Further more, I showed that Dpp signaling is also participating in the patterning along the D/V axis by interaction with the selector gene apterous and Notch/Wg signaling.

Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.