The work presented in the thesis was carried with the aim to understand how Widerborst (Wdb) regulate planar cell polarity in Drosophila wing. In search of proteins interacting with Wdb I carried a Yeast Two Hybrid screen and identified a protein, bitesize, with tandem C2 domains in its C terminus interacting with Wdb. Wdb also interacts with btsz genetically and removal of one copy each of Wdb and btsz enhances the truncated hair phenotype observed in Wdb EMS mutants and btsz P element insertion mutants. There are at least three predicted isoforms of bitesize and loss of the btsz-II isoform is lethal. Clonal analysis of a btsz mutant, btszJ5-2, which removes the btsz II isoform resulted in truncated wing hair outgrowth. On the other hand over expression of a myc-btsz-II construct resulted in hair duplication phenotype. However, over expression of the GFP-CT is sufficient to give wing hair duplication phenotype and this hair duplication phenotype is stronger than that caused by myc-btsz-II over expression. The Myc tagged btsz-II protein shows apical localization. Though most of the protein is confined to cytoplasm, btsz-II also marks the plasma membrane. The GFP-CT construct marks the plasma membrane strongly and is enriched in the apical region. The over expression of CT domain is sufficient to give hair duplication phenotype and the strong difference observed in the localization pattern of full length btsz-II protein and GFP-CT together suggest that regulation of membrane localization of btsz through its CT region is important to regulate hair morphogenesis. As the loss of function (truncated wing hair) and gain of function (hair duplication) both affect the process of hair morphogenesis, it can be said that btsz is a positive regulator of hair morphogenesis. Since no defect in cortical polarization of Fmi was observed in cells lacking btsz-II, btsz is required for establishment of cortical domains. However with the present study it remains unknown how exactly the C2 domains might regulate hair morphogenesis and whether Wdb targets btsz for dephophorylation to PP2A catalytic subunit.

The silica-based cell walls of diatoms are outstanding examples of nature’s capability to synthesize complex porous structures with genetically controlled patterns from the nanometer scale to the range of hundreds of micrometers. Formation of the cell wall building blocks (valves and girdle bands) occurs in membrane-bound compartments, termed silica deposition vesicles (SDVs), which are unique organelles in silica-forming protists. Isolation of the SDVs has not yet been achieved, which has severely hampered the efforts to understand the mechanisms of biological silica morphogenesis. The present thesis aimed to address this shortcoming. The foundation was the development of an improved cell cycle synchronization and a fluorescence labeling method for the model diatom Thalassiosira pseudonana that enabled rapid identification of valve SDVs in a cell lysate. Correlative fluorescence and electron microscopy allowed visualizing the development of valve silica with unprecedented spatio-temporal resolution. Elemental analysis and demineralization of immature valves provided the first direct chemical evidence that silica morphogenesis is an interplay of inorganic and organic molecules inside the valve SDVs. Cryo TEM imaging of valve SDVs indicated the formation of organic patterns that precede silica depostion. From these observations, an organic biomolecule dependent, liquid-liquid phase separation based model for pore formation in the diatom T. pseudonana was proposed. The second part of this thesis was focused on the enrichment of valve SDVs from T. pseudonana and the subsequent proteomics based identification of more than 40 potential valve SDV proteins. Among these, three diatom-specific proteins contained conserved protein protein interaction domains (ankyrin-repeats) and were surprisingly predicted to be located in the cytoplasm. The fluorescent tagging of the three proteins (termed dANK1-3) confirmed their association with the valve SDVs. When the respective dank genes were knocked out by CRISPR/Cas9, the valves displayed permanent anomalies in the quantity and the pattern of ~22 nm sized pores. Double knockout mutants lacking both dank1 and dank3 were almost completely devoid of pores. The analysis of valve morphogenesis in the single and double knockout mutants revealed phenotypic changes that were consistent with the liquid-liquid phase separation based model for pore pattern formation in diatom biosilica. The work of this thesis has provided for the first time direct access to valve SDVs, which has opened entirely new possibilities for studying the composition, properties, and working mechanism of an organelle that forms a complex shaped mineral.

Knochenwachstumsfaktoren (Bone Morphogenetic Proteins, BMPs) sind ubiquitäre, sekretierte Proteine mit vielfältigen biologischen Funktionen. Die Vielfalt an zellulären Prozessen, die durch BMPs reguliert werden, von der Knochenentwicklung und Organhomöostase bis hin zur Neurogenese, erstaunt – und wirft angesichts von teils redundanten, teils spezifischen Funktionen der BMPs Fragen zu den Mechanismen ihrer Signalübermittlung auf. Die Signaltransduktion von BMPs erfolgt wie bei den strukturell verwandten TGF-βs und Activinen durch die ligandeninduzierte Oligomerisierung von transmembranen Serin/Threonin-Kinaserezeptoren, von denen zwei Typen – Typ I und Typ II – existieren. Einer Vielzahl von mehr als 18 BMP-Liganden stehen nach derzeitigem Erkenntnisstand nur vier Typ I und drei Typ II Rezeptorsubtypen für die Bildung von heteromeren Rezeptorkomplexen zur Verfügung. Ein BMP-Ligand kann hochspezifisch nur einen bestimmten Rezeptorsubtyp oder in einer promisken Art und Weise mehrere Rezeptorsubtypen binden. Trotz dieser Bindungspromiskuität üben BMPs ihre biologische Funktion überwiegend hochspezifisch aus, d.h. abhängig vom Liganden werden spezifische zelluläre Prozesse reguliert. Somit stellt sich die Frage, wie die Bildung von heteromeren Ligand-Rezeptor-Komplexen und die Aktivierung definierter intrazellulärer Signalkaskaden zusammenhängen und wie letztlich ein bestimmtes BMP-Signal durch einen „Flaschenhals“, repräsentiert durch die begrenzte Anzahl an Rezeptorsubtypen, in das Zellinnere übermittelt wird. Die Interaktionen zwischen BMP-2 / GDF-5 und den Typ I Rezeptoren BMPR-IA / BMPR-IB sind ein Paradebeispiel für Bindungspromiskuität und -spezifität. Während BMP-2 beide Rezeptoren BMPR-IA und BMPR-IB mit gleicher Bindungsaffinität bindet („promiske Interaktion“), zeigt GDF-5 eine 15-20fach höhere Bindungsaffinität zu BMPR-IB („spezifische“ Interaktion). Dieser Unterschied ist scheinbar gering, aber physiologisch überaus relevant. Um Einblick in die Mechanismen der molekularen Erkennung zwischen den Bindungspartnern zu gewinnen, wurden binäre und ternäre Komplexe aus den Liganden BMP-2 oder GDF-5, den extrazellulären Domänen der Typ I Rezeptoren BMPR-IA oder BMPR-IB sowie der extrazellulären Domäne des Typ II Rezeptors ActR-IIB untersucht. Die hier vorliegende Arbeit beschreibt die strukturelle und funktionelle Analyse dieser Ligand-Rezeptor-Komplexe. Um den Einfluss struktureller Flexibilität auf die BMP Typ I Rezeptor Erkennung näher zu analysieren, wurde zudem die Struktur von BMPRIA in freiem Zustand mittels NMR-Spektroskopie aufgeklärt. Aus Mutagenesedaten und der Kristallstruktur des GDF-5•BMPR-IB-Komplexes lassen sich im Vergleich zu bekannten Kristallstrukturen Merkmale ableiten, mit denen die Ligand-Rezeptor-Bindung und -Erkennung charakterisiert werden kann: (1) Die Hauptbindungsdeterminanten in Komplexen von BMPR-IA und BMPR-IB mit ihren Liganden sind unterschiedlich. Während in Komplexen mit BMPR-IB ein hydrophobes Motiv die Bindungsaffinität bestimmt, trägt in Komplexen mit BMPR-IA eine polare Interaktion signifikant zur Bindungsenergie bei. Ein Vergleich der Strukturen von freien und gebundenen Liganden und Typ I Rezeptoren zeigt, dass interessanterweise diese Hauptbindemotive erst bei der Ligand-Rezeptor-Interaktion entstehen, sodass ein „induced fit“ vorliegt und die Moleküle entsprechend „aufeinander falten“. (2) Die Bindungsspezifität wird durch periphere Schleifen in den Typ I Rezeptoren bestimmt. Wie Untersuchungen von Punktmutationen in BMPR-IA zeigen, die einer krebsartigen Darmerkrankung (Juvenile Polyposis) zugrunde liegen, führt erst die „richtige“ Kombination aus Flexibilität in den Schleifen und Rigidität des Rezeptorgrundgerüsts zu signalaktiven Typ I Rezeptoren mit einer potentiell den Liganden komplementären Oberfläche. Die mangelnde sterische Komplementarität von Ligand- und Rezeptoroberflächen führt zu der niedrigeren Bindungsaffinität von GDF-5 zu BMPR-IA im Vergleich zu BMPR-IB. Interessanterweise zeigen die hier vorgestellten, hochaufgelösten Strukturdaten, dass die Orientierungen/Positionen der Typ I Rezeptoren BMPR-IA und BMPR-IB in den Bindeepitopen der Liganden BMP-2 und GDF-5 variieren. Unter der Voraussetzung, dass die extrazelluläre Domäne, das Transmembransegment und die intrazelluläre Domäne der Typ I Rezeptoren ein starres Element bilden, sollte sich die unterschiedliche Orientierung der extrazellulären Domänen der Typ I Rezeptoren in der Anordnung der Kinasedomänen widerspiegeln und sich auf die Signaltransduktion auswirken. Möglicherweise ist eine bestimmte Anordnung der Kinasedomänen der Typ I und Typ II Rezeptoren für eine effiziente Phosphorylierung bzw. Signaltransduktion erforderlich. Der Vergleich mehrerer Ligand-Typ I Rezeptor-Komplexe zeigt, dass die unterschiedliche Orientierung dieser Rezeptoren möglicherweise vom Liganden abhängt. Angesichts der Bindungspromiskuität unter BMP-Liganden und -Rezeptoren könnten so spezifische Signale übermittelt und spezifische biologische Funktionen reguliert werden. Die in dieser Arbeit vorgestellten Erkenntnisse tragen wesentlich zur strukturellen Charakterisierung der Ligand-Rezeptor-Erkennung in der BMP-Familie bei. Die Frage, warum trotz strukturell hoch homologer Liganden und Rezeptoren und weitgehend konservierten Bindeepitopen eine teils promiske und teils spezifische Interaktion möglich ist, kann nun für die Liganden BMP-2 und GDF-5 sowie den beiden Typ I Rezeptoren BMPR-IA und BMPR-IB beantwortet werden. / Bone morphogenetic proteins (BMPs) are ubiquitous, secreted cytokines involved in a manifold of biological functions. The diversity of cellular processes regulated by BMPs, from bone development to tissue homeostasis and neuronal processes, is amazing – and raises questions about the mechanisms of signal transduction in the light of redundant functions on the one hand, and specific functions on the other hand. Similar to structurally related activins and TGF-βs, the signal transduction of BMPs is accomplished by ligand-induced oligomerization of transmembrane BMP type I and type II serine/threonine receptor kinases. According to current knowledge, only four type I and three type II receptor subtypes are available for BMP signal transduction, facing a multitude of more than 18 BMP ligands. Binding of BMP ligands to their receptors can be highly specific meaning that only one specific receptor of either subtype is used for signaling. In contrast, many BMP ligands can recruit more than one receptor subtype, which results in binding promiscuity. However, even though receptor subtypes are bound in a promiscuous manner, only certain biological functions are triggered. Dependent on the BMP ligand, specific cellular processes are activated and regulated. This discrepancy between unspecific binding and specific signaling events and the biological response raises the question how the formation of heteromeric ligand-receptor complexes is linked to the activation of defined intracellular signaling cascades, and finally, how a certain BMP signal is transduced into the interior of the cell through a „bottleneck“ represented by the limited number of receptor subtypes. The interaction between BMP-2 / GDF-5 and the BMP type I receptors BMPR-IA / BMPR-IB is a prime example for binding promiscuity and binding specificity. BMP-2 binds BMPR-IA and BMPRIB with almost equal binding affinity („promiscuous interaction“) while GDF-5 exhibits a 15-20fold higher binding affinity to BMPR-IB („specific interaction“). Although this difference is seemingly small, it is however of considerable relevance for the physiological role of these ligands. To gain insight into the mechanisms of molecular recognition between the binding partners, binary and ternary ligand-receptor complexes consisting of BMP-2 or GDF-5, the extracellular domains of the type I receptors BMPR-IA or BMPR-IB, and the extracellular domain of the type II receptor ActRIIB were investigated. The thesis presented here describes the structural and functional analysis of these ligand-receptor complexes. To analyse the effect of structural flexibility on BMP type I receptor recognition in more detail, the structure of free BMPR-IA was determined using NMR spectroscopy. Based on data from a limited mutagenesis and the crystal structure of the GDF-5•BMPR-IB complex several characteristics concerning ligand-receptor binding and recognition can be deduced: (1) The main binding determinants in complexes of BMPR-IA and BMPR-IB with their ligands BMP-2 and GDF-5 differ. A hydrophobic binding motif determines binding affinity in complexes of BMPR-IB, whereas a polar interaction significantly contributes to binding energy in complexes of BMPR-IA. These main binding motifs are only formed during complex formation as demonstrated by a comparison between structures of free and bound ligands as well as type I receptors. Both ligand and receptor fold „onto each other“ which suggests an induced fit mechanism. (2) Binding specificity is encoded on loops at the periphery of the binding epitope of the type I receptors. Only the „appropriate“ combination between structural flexibility in the receptor loops and structural rigidity of the receptor backbone results in signal active type I receptors, as shown by analysis of single polymorphisms in BMPR-IA causing juvenile polyposis syndrome, a cancerous disease of the intestine. A lack of steric surface complementarity between GDF-5 and BMPR-IA, that cannot be overcome by structural flexibility, leads to the lower binding affinity in comparison to BMPR-IB. Interestingly, the high resolution structure of the GDF-5•BMPR-IB complex shows that the orientations/positions of BMPR-IA in the binding epitope of BMP-2 and of BMPR-IB in the binding epitope of GDF-5 vary. Assuming that the extracellular domain, the transmembrane segment, and the intracellular domain of the type I receptors form a rigid element, the different orientations of the extracellular domains should also reflect the assembly of the kinase domains and therefore, affect signal transduction. One can assume that a defined arrangement of the kinase domains of type I and type II receptors is required to allow for efficient phosphorylation and signal transduction, respectively. The comparison of several BMP ligand-type I receptor complexes suggests that the different orientations of these receptors are likely dependent on the ligand. Considering the binding promiscuity among BMP ligands and receptors such a mechanism would represent a possible way for the transmission of specific signals and regulation of specific biological functions. The insights into molecular structure and function of BMP ligands and receptors presented in this thesis contribute significantly to a more detailed understanding of their binding properties. The question why the interaction of BMP ligands and receptors is promiscuous on the one hand and specific on the other hand in spite of structurally highly homologous molecules can now be answered for BMP-2 and GDF-5 as well as BMPR-IA and BMPR-IB.

Die Detektion von Umweltsignalen und die gezielte zelluläre Reaktion ist eine zentrale und für das Überleben aller Lebewesen essentielle Fähigkeit. Candida albicans, als dominierender humanpathogener Pilz, ist hochgradig verschiedenen biochemischen und physikalischen Umweltbedingungen ausgesetzt, welche sowohl die Zellmorphologie als auch die Virulenz dieses Erregers beeinflussen. In der vorliegenden Arbeit wurde der Einfluss von Kohlendioxid, als ubiquitär vorkommendes Gasmolekül, auf die Zellmorphologie und Virulenz untersucht. Erhöhte Konzentrationen von Kohlendioxid stellen ein äußerst robustes Umweltsignal dar, welches die morphologische Transition vom Hefewachstum zum hyphalen Wachstum, einem Hauptvirulenzfaktor, in Candida albicans stimuliert. In diesem Zusammenhang wurde die Rolle der putativen Carboanhydrase Nce103 durch die Generation von knock – out Mutanten untersucht. Die Disruption von NCE103 in C. albicans führt zu einem Kohlendioxid – abhängigen Phänotyp, welcher Wachstum unter aeroben Bedingungen (ca. 0,033% CO2) nicht zulässt, jedoch unter Bedingungen mit einem erhöhten CO2 Gehalt von ca. 5% ermöglicht. NCE103 ist also für das Wachstum von C. albicans in Wirtsnischen mit aeroben Bedingungen essentiell. Durch Untersuchungen zur Enzymkinetik mittels Stopped – flow wurde in dieser Arbeit gezeigt, dass Nce103 die Funktion einer Carboanhydrase erfüllt. Die biochemische Funktion dieser Carboanhydrase besteht in der Fixation von CO2 bzw. HCO3ˉ in der Zelle zur Unterhaltung der wesentlichen metabolischen Reaktionen. Weiterhin konnte gezeigt werden, dass die Induktion hyphalen Wachstums durch CO2 in C. albicans nicht durch den Transport von CO2 mittels des Aquaporins Aqy1 beeinflusst wird. CO2 bzw. HCO3ˉ aktiviert in der Zelle direkt eine Adenylylcyclase (Cdc35), welche sich grundlegend von den bisher gut charakterisierten G-Protein gekoppelten Adenylylcylasen unterscheidet. Die Generation von cAMP beeinflusst in der Folge direkt die Transkription hyphenspezifischer Gene und nachfolgend die morphologische Transition vom Hefewachstum zum elongierten, hyphalen Wachstum. Dieser Mechanismus konnte sowohl in Candida albicans als auch in Cryptococcus neoformans nachgewiesen werden, was auf einen panfungal konservierten Signaltransduktionsmechanismus schliessen lässt. Die Inhibition dieser spezifischen Kaskade eröffnet neue Ansätze zur Entwicklung spezifischer antimykotischer Wirkstoffe. / Detection of environmental signals and subsequently directed reaction is essential for the survival of all living organisms. Candida albicans, as the predominant human fungal pathogen is exposed to severely different physical and chemical conditions, which influence cell morphology as well as virulence in human. In the present work, the influence of carbon dioxide as ubiquitous gaseous molecule on virulence and cell morphology was analysed. Elevated concentrations of carbon dioxide are a robust signal to induce the morphological transition from yeast growth to an elongated hyphal growth form, which is believed to be one of the main virulence factors in Candida albicans. The role of the putative carbonic anhydrase Nce103p in carbon dioxide signalling is reviewed by generating knockout mutant strains, which exhibited a carbon dioxide dependent phenotype. Growth under aerobic conditions (0,033 % carbon dioxide) is inhibited but feasible in 5% carbon dioxide. Therefore, Nce103p is essential for growth in host niches with aerobic conditions. Analysis of the biochemical properties of Nce103p by stopped – flow kinetics revealed carbonic anhydrase activity. It is hypothesised, that Nce103p is essential for fixation of carbon dioxide and bicarbonate within the cell in order to sustain basic metabolic reactions. Furthermore, the induction of hyphal growth was independent of aquaporine-mediated transport of carbon dioxide. Bicarbonate rather carbon dioxide activates directly the adenylyl cyclase Cdc35p generating cyclic AMP as second messenger and influencing the transcription of hyphal specific genes in Candida albicans thus promoting the morphological transition from yeast growth to elongated hyphal growth. This signal transduction cascade is present in Candida albicans as well as Cryptococcus neoformans and it is believed to be a pan fungal signal transduction cascade. The specific inhibition of carbon dioxide mediated polymorphism may serve as a new target for antifungal therapeutic agents.

Die Familie der ErbB-Rezeptor-Tyrosinkinasen besteht aus vier Mitgliedern, dem EGF-Rezeptor (ErbB-1), ErbB-2, ErbB-3 und ErbB-4. ErbB-Rezeptoren spielen eine wichtige Rolle bei der Entwicklung des Nervensystems, des Herzens und der Brustdrüsen. Ein Teil dieser Differenzierungsvorgänge läßt sich in vitro nachvollziehen: so ist zum Beispiel die Aktivierung des ErbB-2 Rezeptors ausreichend für alveoläre Morphogenese der Brustdrüsenepithelzellinie EpH4. Intrazelluläre Moleküle, die dieses ErbB2-Signal übertragen, sind allerdings noch unbekannt. Mit Hilfe eines neuen, modifizierten Hefe-2-Hybrid-Systems wurde in der vorliegenden Arbeit Vav2 als neuer Interaktionspartner von ErbB-2 identifiziert. Vav2 assoziiert mit aktiviertem ErbB-2 über eine SH2-Domäne. Die Interaktion ist direkt und ist von zwei Phosphotyrosinen in ErbB-2 abhängig. Vav2 kann den GDP/GTP-Austausch bei GTPasen der Rho-Familie vermitteln. Dadurch kann der Umbau des Zytoskeletts und Veränderungen der Transkription sowie Zelltransformation induziert werden. In einem dreidimensionalen Zellkultursystem kann aktiviertes Vav2 in EpH4 Zellen die Bildung von alveolären Zellaggregaten induzieren. In diesen Alveolen umgibt eine Schicht polarisierter milchproduzierender Zellen ein zentrales Lumen. Diese Vav2-vermittelte Morphogenese ist abhängig von der katalytischen GDP/GTP-Austausch Aktivität von Vav2. Katalytisch-inaktives Vav2 kann die morphogenetische Aktivität von ErbB-2 in EpH4-Zellen verhindern, ohne die mitogene Aktivität von ErbB-2 zu beeinflussen. Vav2 ist mit ErbB-2 coexprimiert und interagiert mit dem Rezeptor in Brustdrüsenzellen schwangerer Mäuse. Diese Untersuchungen deuten darauf hin, dass Vav2 eine wichtige Funktion bei der durch ErbB-2 induzierten alveolären Morphogenese der Brustdrüse spielt. / The ErbB receptor tyrosine kinases constitute a subfamily of four structurally related members, the EGF receptor (ErbB-1), ErbB-2, ErbB-3 and ErbB-4. ErbB receptor tyrosine kinases are critical for embryonic development of central and peripheral neural structures and heart. In addition, ErbB receptors play an important role in the postnatal development of the mammary gland. Previous studies showed that activated ErbB-2 receptor induces alveolar morphogenesis of EpH4 mammary epithelial cells that are cultured on a three-dimensional matrix (termed Matrigel). However, the downstream signaling proteins that mediate this biological activity of ErbB-2 were unknown. In this work, Vav2 was identified as a direct interaction partner of tyrosine-phosphorylated ErbB receptors using the yeast two-hybrid system. Vav2 is a member of a family of guanine nucleotide exchange factors that induce cytoskeletal rearrangements, transcriptional alterations, and have oncogenic potential when activated. To test the ability of Vav2 to mediate morphogenic signals of ErbB-2, EpH4 cells overexpressing Vav2 protein were cultured on Matrigel. Indeed, Vav2 induces alveolar morphogenesis of EpH4 cells when activated either by oncogenic mutation or tyrosine phosphorylation by ErbB-2. The morphogenic activity of Vav2 requires the Dbl homology domain, which mediates GDP/GTP exchange. Dominant-negative Vav2 specifically blocks the morphogenic signals of ErbB-2 in EpH4 cells without interfering with ErbB2-induced mitogenesis. Importantly, Vav2 is co-expressed and interacts with ErbB-2 in the mammary glands of pregnant mice. Taken together, these results point to Vav2 as a candidate to mediate ErbB-2 signals for alveolar morphogenesis in vivo, which is a relevant step in the development of the mammary gland during pregnancy.

Gewebezellen sammeln ständig Informationen über die mechanischen Eigenschaften ihrer Umgebung, indem sie aktiv an dieser ziehen. Diese Kräfte werden an Zell-Matrix-Kontakten übertragen, die als Mechanosensoren fungieren. Jüngste Experimente mit Zellen auf elastischen Substraten zeigen, dass Zellen sehr empfindlich auf Veränderungen der effektiven Steifigkeit ihrer Umgebung reagieren, die zu einer Reorganisation des Zytoskeletts führen können. In dieser Arbeit wird ein theoretisches Model entwickelt, um die Selbstorganisation von Zellen in weichen Materialien vorherzusagen. Obwohl das Zellverhalten durch komplexe regulatorische Vorgänge in der Zelle gesteuert wird, scheint die typische Antwort von Zellen auf mechanische Reize eine einfache Präferenz für große effektive Steifigkeit der Umgebung zu sein, möglicherweise weil in einer steiferen Umgebung Kräfte an den Kontakten effektiver aufgebaut werden können. Der Begriff Steifigkeit umfasst dabei sowohl Effekte, die durch größere Härte als auch durch elastische Verzerrungsfelder in der Umgebung verursacht werden. Diese Beobachtung kann man als ein Extremalprinzip in der Elastizitätstheorie formulieren. Indem man das zelluläre Kraftmuster spezifiziert, mit dem Zellen mit ihrer Umgebung wechselwirken, und die Umgebung selbst als linear elastisches Material modelliert, kann damit die optimale Orientierung und Position von Zellen vorhergesagt werden. Es werden mehrere praktisch relevante Beispiele für Zellorganisation theoretisch betrachtet: Zellen in externen Spannungsfeldern und Zellen in der Nähe von Grenzflächen für verschiedene Geometrien und Randbedingungen des elastischen Mediums. Dafür werden die entsprechenden elastischen Randwertprobleme in Vollraum, Halbraum und Kugel exakt gelöst. Die Vorhersagen des Models stimmen hervorragend mit experimentellen Befunden für Fibroblastzellen überein, sowohl auf elastischen Substraten als auch in physiologischen Hydrogelen. Mechanisch aktive Zellen wie Fibroblasten können auch elastisch miteinander wechselwirken. Es werden daher optimale Strukturen als Funktion von Materialeigenschaften und Zelldichte bzw. der Geometrie der Zellpositionen berechnet. Schließlich wird mit Hilfe von Monte Carlo Simulationen der Einfluss stochastischer Störungen auf die Strukturbildung untersucht. Das vorliegende Model trägt nicht nur zu einem besseren Verständnis von vielen physiologischen Situationen bei, sondern könnte in Zukunft auch für biomedizinische Anwendungen benutzt werden, um zum Beispiel Protokolle für künstliche Gewebe im Bezug auf Substratgeometrie, Randbedingungen, Materialeigenschaften oder Zelldichte zu optimieren. / Adherent cells constantly collect information about the mechanical properties of their extracellular environment by actively pulling on it through cell-matrix contacts, which act as mechanosensors. In recent years, the sophisticated use of elastic substrates has shown that cells respond very sensitively to changes in effective stiffness in their environment, which results in a reorganization of the cytoskeleton in response to mechanical input. We develop a theoretical model to predict cellular self-organization in soft materials on a coarse grained level. Although cell organization in principle results from complex regulatory events inside the cell, the typical response to mechanical input seems to be a simple preference for large effective stiffness, possibly because force is more efficiently generated in a stiffer environment. The term effective stiffness comprises effects of both rigidity and prestrain in the environment. This observation can be turned into an optimization principle in elasticity theory. By specifying the cellular probing force pattern and by modeling the environment as a linear elastic medium, one can predict preferred cell orientation and position. Various examples for cell organization, which are of large practical interest, are considered theoretically: cells in external strain fields and cells close to boundaries or interfaces for different sample geometries and boundary conditions. For this purpose the elastic equations are solved exactly for an infinite space, an elastic half space and the elastic sphere. The predictions of the model are in excellent agreement with experiments for fibroblast cells, both on elastic substrates and in hydrogels. Mechanically active cells like fibroblasts could also interact elastically with each other. We calculate the optimal structures on elastic substrates as a function of material properties, cell density and the geometry of cell positioning, respectively, that allows each cell to maximize the effective stiffness in its environment due to the traction of all the other cells. Finally, we apply Monte Carlo simulations to study the effect of noise on cellular structure formation. The model not only contributes to a better understanding of many physiological situations. In the future it could also be used for biomedical applications to optimize protocols for artificial tissues with respect to sample geometry, boundary condition, material properties or cell density.

Morphogens are secreted signalling molecules that are expressed in restricted groups of cells within the developing tissue. From there, they are secreted and travel throughout the target field and form concentration gradients. These concentration profiles endow receiving cells with positional information. A number of experiments in Drosophila demonstrated that the morphogen Decapentaplegic (Dpp) forms activity gradients by inducing the expression of several target genes above distinct concentration thresholds at different distances from the source. This way, Dpp contributes to developmental fates in the target field such as the Drosophila wing disc. Although the tissue distribution as well as the actual shape and size of the Dpp morphogen concentration gradient has been visualized, the cell biological mechanisms through which the morphogen forms and maintains a gradient are still a subject of debate. Two hypotheses as to the dominant mechanism of movement have been proposed that can account for Dpp spreading throughout the Drosophila wing imaginal target tissue: extracellular diffusion and planar transcytosis, i. e. endocytosis and resecretion of the ligand that is thereby transported through the cells. Here, I present data indicating that implications of a theoreticalanalysis of Dpp spreading, where Dpp transport through the target tissue is solely based on extracellular diffusion taking into account receptor binding and subsequent internalization, are inconsistent with experimental results. By performing Fluorescence Recovery After Photobleaching (FRAP) experiments, I demonstrate a key role of Dynamin-mediated endocytosis for Dpp gradient formation. In addition, I show that most of GFP-Dpp traffics through endocytic compartments at the receiving epithelial cells, probably recycled through apical recycling endosomes (ARE). Finally, a Dpp recycling assay based on subcellular photouncage of ligand is presented to address specifically the Dpp recycling event at the receiving cells.

The work presented in the thesis was carried with the aim to understand how Widerborst (Wdb) regulate planar cell polarity in Drosophila wing. In search of proteins interacting with Wdb I carried a Yeast Two Hybrid screen and identified a protein, bitesize, with tandem C2 domains in its C terminus interacting with Wdb. Wdb also interacts with btsz genetically and removal of one copy each of Wdb and btsz enhances the truncated hair phenotype observed in Wdb EMS mutants and btsz P element insertion mutants. There are at least three predicted isoforms of bitesize and loss of the btsz-II isoform is lethal. Clonal analysis of a btsz mutant, btszJ5-2, which removes the btsz II isoform resulted in truncated wing hair outgrowth. On the other hand over expression of a myc-btsz-II construct resulted in hair duplication phenotype. However, over expression of the GFP-CT is sufficient to give wing hair duplication phenotype and this hair duplication phenotype is stronger than that caused by myc-btsz-II over expression. The Myc tagged btsz-II protein shows apical localization. Though most of the protein is confined to cytoplasm, btsz-II also marks the plasma membrane. The GFP-CT construct marks the plasma membrane strongly and is enriched in the apical region. The over expression of CT domain is sufficient to give hair duplication phenotype and the strong difference observed in the localization pattern of full length btsz-II protein and GFP-CT together suggest that regulation of membrane localization of btsz through its CT region is important to regulate hair morphogenesis. As the loss of function (truncated wing hair) and gain of function (hair duplication) both affect the process of hair morphogenesis, it can be said that btsz is a positive regulator of hair morphogenesis. Since no defect in cortical polarization of Fmi was observed in cells lacking btsz-II, btsz is required for establishment of cortical domains. However with the present study it remains unknown how exactly the C2 domains might regulate hair morphogenesis and whether Wdb targets btsz for dephophorylation to PP2A catalytic subunit.

Arthropods typically show two types of segmentation: the embryonic parasegments and the adult segments that lie out of register with each other. Such a dual nature of body segmentation has not been described from Onychophora, one of the closest arthropod relatives. Hence, it is unclear whether onychophorans have segments, parasegments, or both, and which of these features was present in the last common ancestor of Onychophora and Arthropoda. To address this issue, we analysed the expression patterns of the "segment polarity genes" engrailed, cubitus interruptus, wingless and hedgehog in embryos of the onychophoran Euperipatoides rowelli. Our data revealed that these genes are expressed in repeated sets with a specific anterior-to-posterior order along the body in embryos of E. rowelli. In contrast to arthropods, the expression occurs after the segmental boundaries have formed. Moreover, the initial segmental furrow retains its position within the engrailed domain throughout development, whereas no new furrow is formed posterior to this domain. This suggests that no re-segmentation of the embryo occurs in E. rowelli. Irrespective of whether or not there is a morphological or genetic manifestation of parasegments in Onychophora, our data clearly show that parasegments, even if present, cannot be regarded as the initial metameric units of the onychophoran embryo, because the expression of key genes that define the parasegmental boundaries in arthropods occurs after the segmental boundaries have formed. This is in contrast to arthropods, in which parasegments rather than segments are the initial metameric units of the embryo. Our data further revealed that the expression patterns of "segment polarity genes" correspond to organogenesis rather than segment formation. This is in line with the concept of segmentation as a result of concerted evolution of individual periodic structures rather than with the interpretation of \"segments\" as holistic units.

Kieselalgen (Diatomee) sind eukaryotische einzellige Algen die hochspezifische Proteine (sogenannte Silaffine) erzeugen, um ‘nanopatterned’ Silica-Zellwände herzustellen. Diese Proteine zeigen geringe oder gar keine Homologie innerhalb der Diatomeen Gattung und sind ausgiebig (extensiv) posttranslatorisch modifiziert. Zum Unterschied zu konventioneller Modifikation (z.B. Phosphorylierung und Glykosylierung) weisen Lysinreste von Silaffinen einige Polyaminketten mit sehr heterogenen molekularen Strukturen auf. Diese Modifikationen sind spezifisch für Kieselalgen und spielen somit hypothetisch eine Rolle in der Biosilica-Synthese. Allerdings sind Lysin Polyamin Modifikationen, modifizierte Proteine und modifizierte Stellen kaum charakterisiert. Um diese Frage zu beantworten entwickelten wir eine Methode Polyamine zu quantifizieren und die Position von Polyamin-Modifikationen in engverwandte Proteine zu identifizieren (in morphologisch unterschiedliche Diatomeen Thalassiosira pseudonana, T. oceanica und Cyclotella cryptica). Wir zeigten, dass das Gesamtmuster von Polyaminender phylogenetischen Nähe dieser Kieselalgenarten folgt und dass diese Polyaminmodifikationen an Konsensusstellen sogar in Proteinen auftraten, die keine Sequenzähnlichkeit zeigten.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung / Diatoms are eukaryotic unicellular algae that employ highly specialized proteins called silaffins for making nanopatterned silica-based cell walls. These proteins share little or no homology across diatom species and are extensively post-translationally modified. Apart from conventional modifications (e. g., phosphorylation and glycosylation) lysine residues of silaffins bear polyamine chains with highly heterogeneous molecular structure. The latter appear to be specific for silicifying organisms and therefore hypothesized to play a key role in biosilica synthesis. However, polyamine modifications of lysines, modified proteins, and modification sites remain poorly characterized. To address these questions, we developed a method to quantify polyamines and identify sites of polyamine modifications in proteins from phylogenetically closely related, yet morphologically distinct diatoms Thalassiosira pseudonana, T. oceanica, and Cyclotella cryptica. We demonstrated that the overall pattern of polyamines followed the phylogenetic proximity across these diatom species and showed that polyamine modifications occurred at consensus sites even in proteins showing no sequence similarity.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung