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Zellen sind darauf angewiesen, ihre Lebensbedingungen wahrzunehmen und darauf zu reagieren. Der TOR („Target of Rapamycin“)-Signalweg spielt dabei eine wichtige Rolle, indem er das Wachstum in Abhängigkeit von Nährstoffen und Hormonen reguliert. In dieser Arbeit wurde die Spalthefe Schizosaccharomyces pombe (S. pombe) als Modellorganismus für die Untersuchungen des TOR-Signalweges verwendet. Dabei zeigte sich, dass in S. pombe, wie in Säugern und der Bäckerhefe, zwei TOR-Komplexe existieren. Ko-Immunpräzipitationsexperimente zeigten, dass sich der TOR-Komplex 1 aus SpTor2, SpMip1 und SpWat1 zusammensetzt. Bei SpTor1, SpSin1, SpSte20 und SpWat1 handelt es sich um Mitglieder des TOR-Komplex 2. Phänotypische Analysen von Mutanten in Genen, die für TOR-Komplex 2-Komponenten kodieren, unterstreichen, dass diese Proteine in der Zelle ähnliche Funktionen bei der Antwort auf verschiedene Stresssituationen ausüben. Die heterologe Expression von wat1+ in einer S. cerevisiae delta lst8-Mutante komplementiert deren Wachstumsdefekt, was untermauert, dass diese beiden Proteine tatsächlich gleiche Funktionen in der Zelle ausüben. Eine Membranassoziation der TOR-Komplexe, wie sie in Säugern und S. cerevisiae bereits beschrieben wurde, konnte in dieser Arbeit für die TOR-Komplex 2-Komponente SpSte20 nachgewiesen werden. Möglicherweise spielt dabei die Interaktion zwischen der leichten Kette des Clathrins mit SpSte20 eine Rolle. Auch für die homologen Proteine aus S. cerevisiae, ScCLC1 und ScAVO3, konnte mittels des "Zwei-Hybrid"-Systems eine Bindung nachgewiesen werden. Dieses deutet eine Konservierung dieser Proteininteraktion innerhalb von Eukaryonten an. Obwohl das vegetative Wachstum von S. pombe durch Rapamycin nicht gehemmt wird, zeigen die hier aufgeführten Daten eine in vivo-Bindung von SpFkh1 an sowohl SpTor1 als auch SpTor2 in Anwesenheit von Rapamycin. Das Phosphorylierungslevel von SpGad8, dem bisher einzigen postulierten TOR-Komplex 2-Zielprotein, wird durch die Bindung des SpFkh1-Rapamycin-Komplexes an SpTor1 jedoch nicht beeinflusst. Dies und die Tatsache, dass delta gad8-Mutanten ein Rapamycin-sensitives Wachstum zeigen, lassen vermuten, dass noch weitere bisher unbekannte SpTOR-Komplex-Zielproteine durch Rapamycin beeinflusst werden. Zusammengenommen unterstreichen die Daten dieser Arbeit die Konservierung der Komplexe und des von ihnen vermittelten Signaltransduktionsweges. Sie zeigen aber auch, dass die Wirkung von Rapamycin nicht einfach durch eine generelle Hemmung der Aktivität der Komplexe beschrieben werden kann, was insbesondere für die klinische Anwendung von Rapamycin von Bedeutung ist.

Cytokines activate cell surface receptors to control and regulate immunity and hematopoiesis. Despite its enormous potential, pharmaceutical use of cytokines is in most cases hampered by their pleiotropic functionality, which renders cytokine-based therapies exceptionally difficult to control. Although there is growing evidence that the functional plasticity of cytokine receptors is largely encoded in the spatiotemporal dynamics of receptor complexes, no mechanistic correlation has hitherto been achieved. Two related aspects, the spatiotemporal organization and the activation mechanism of cytokine receptors in the plasma membrane, have further remained a topic of intensive and controversial debate. To shed to light into the mechanistic principles responsible for functional selectivity, this thesis aimed to quantitatively explore the molecular and cellular determinants governing cytokine receptor assembly and signaling using the type II interleukin-4 (IL-4) receptor as model system. To this end, by taking advantage of IL-4 and interleukin 13 (IL-13) agonists binding the receptor subunits IL-4Rα and IL-13Rα1 with different affinities and rate constants, an in vitro kinetic characterization of the receptor system was combined with live cell microscopy on the single molecule level and flow cytometry as well as in silico modeling approaches. The quantification of kinetics by a dedicated solid-phase detection method with the extracellular receptor domains tethered onto artificial membranes confirmed that the affinity and stability of the two-dimensional molecular interactions determine receptor dimerization levels and dynamics. Single molecule localization microscopy at physiological cell surface expression levels, however, revealed efficient ligand-induced receptor dimerization, largely independent of the two-dimensional receptor binding affinities, in line with similar STAT6 activation potencies observed for different IL-4 variants. Detailed spatiotemporal analyses and single molecule co-tracking of receptor subunits and ligands in conjunction with spatial-stochastic modeling identified confinement by actin-dependent membrane micro-compartments as an important cellular determinant for sustaining transient receptor dimers. By correlating downstream cellular responses with various three-dimensional binding affinities and kinetics of engineered IL-13 variants, distinct roles of ligand association and dissociation kinetics were uncovered. Whereas the extent of membrane-proximal effector activation is dependent on the association rate by controlling the number of formed receptor complexes in the plasma membrane, the lifetime of receptor complexes determines the potency of a ligand for inducing more distal responses and is, due to accumulation of signaling complexes in endosomes, directly connected to the kinetics of early signaling events.

Drosophila insulin like peptides (DILPs) and their human homolog insulin act as messengers to control many physiological processes in the body. Fields in which insulin signaling is crucial are e.g. growth, stress responses and aging. Consequently, many diseases are caused by disturbed insulin signaling, of which diabetes is the most prominent. During the last decades the functions of insulins and their signaling pathways have been studied in detail; what remains less well understood is how the production of insulin and insulin like peptides is regulated. The family of Neprilysins (Neps) belongs to the M13-zinc ion binding metallopeptidases. Neprilysins cleave peptides that regulate a wide range of cellular processes and are therefore linked to a variety of diseases like cancer, analgesia, hypertension or Alzheimer’s disease. In the fruit fly Drosophila melanogaster, five Neprilysins are expressed; but their in vivo substrates have not yet been identified. One of the Drosophila Neprilysins, Nep4, is expressed in the CNS, in muscle tissue, in cardiac tissue and in male reproductive organs. Nep4 is expressed in two isoforms, Nep4A and Nep4B. Isoform A is composed of a short intracellular domain, a transmembrane domain and a large extracellular domain containing the catalytically active center, whereas soluble Nep4B only consists of the extracellular domain. This thesis reveals that overexpression of catalytically active Nep4A in muscle tissue leads to animals with impaired insulin expression, decreased size and weight, affected feeding behavior and reduced locomotion speed. Further phenotypes are an impaired energy metabolism and larval lethality. Knockdown of the whole enzyme or knockout of its catalytic activity also interferes with feeding and locomotion speed and, in addition, causes pupal lethality. As an explanation for the phenotypes, Nep4 mediated hydrolysis of different short neuropeptide F (sNPF) species, which were identified as novel substrates of the peptidase, is proposed. sNPF is known to regulate insulin signaling and knockdown of sNPF phenocopies the Nep4 overexpression phenotypes, which suggests that Nep4 mediated hydrolysis of sNPF regulates insulin expression in the fly. Based on these results additional regulatory peptides were identified as novel Nep4 substrates. Among them are peptides that do not only regulate insulin signaling, but also feeding behavior (Hallier et al., 2016). These findings represent good evidence that muscle bound Nep4 is key to regulate homeostasis of distinct hemolymph circulating peptide hormones. Nep4 localizing to the surface of the central nervous system is likely necessary to ensure effective ligand clearance and thus proper regulation of corresponding peptide receptors.

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The fusion of yeast vacuoles requires a Rab-GTPase (Ypt7), a tethering complex termed HOPS (homotypic fusion and vacuole protein sorting) and SNAREs. The HOPS complex consists of six subunits and is involved in the initial contact between late endosome (multi vesicular body) and the vacuole. The homologous CORVET complex shares four subunits with the HOPS complex and is required at the endosome. Upon overexpression, I was able to isolate the entire HOPS and stable subcomplexes consisting of two to six subunits. These subcomplexes might represent the core for the assembly, or may be transition intermediates. They could arise when the CORVET complex at the endosome matures into the HOPS complex at the vacuole. Using a structure-function approach, I analysed the HOPS structure via electron microscopy and its function via vacuole fusion assay.