Eukaryotic cells depend on cargo uptake into the endocytic membrane system, which comprises a functionally interconnected network of endosomal compartments. The establishment and maintenance of such diverse compartments in face of the high rates of exchange between them, poses a major challenge for obtaining a molecular understanding of the endocytic system. Rab-GTPases have emerged as architectural key element thereof: Individual family members localize selectively to endosomal compartments, where they recruit a multitude of cytoplasmic effector proteins and coordinate them into membrane sub-domains. Such "Rab-domains" constitute modules of molecular membrane identity, which pattern the endocytic membrane system into a mosaic of Rab-domains. The main objective of this thesis research was to link such "static" mosaic-view with the highly dynamic nature of the endosomal system. The following questions were addressed: How are neighbouring Rab-domains coordinated? Are Rab-domains stable or can they undergo assembly and disassembly? Are the dynamics of Rab-domains utilized in cargo transport? The first part of this thesis research focused on the organization of Rab-domains in the recycling pathway. Utilizing Total Internal Reflection (TIRF) microscopy, Rab11-, but neither Rab4- nor Rab5-positive vesicles were observed to fuse with the plasma membrane. Rab4-positive membranes, however, could be induced to fuse in presence of Brefeldin A. Thus, these experiments complete the view of the recycling pathway by the following steps: a) Rab11-carriers likely mediate the return of recycling cargo to the surface; b) such carriers are presumably generated in an Arf-dependent fission reaction from Rab4-positive compartments. Rab11-chromatography was subsequently carried out in the hope of identifying Rab11-effectors functioning at the Rab4-Rab11 domain interface. An as yet uncharacterized ubiquitin ligase was identified, which selectively interacts with both Rab4 and Rab11. Contrary to expectations, however, the protein (termed RUL for *R*ab interacting *U*biquitin *L*igase) does not function in recycling,but appears to mediate trafficking between Golgi/TGN and endosomes instead.In order to address the dynamics of Rab-domains, fluorescently tagged Rab-GTPases were imaged during cargo transport reactions in living cells. Herefore high-speed/long-term imaging procedures and novel computational image analysis tools were developed. The application of such methodology to the analysis of Rab5-positive early endosomes showed that a) The amount of Rab5 associated with individual endosomes fluctuates strongly over time; b) such fluctuations can lead to the "catastrophic" loss of the Rab5-machinery from membranes; c) Rab5 catastrophe is part of a functional cycle of early endosomes, involving net centripetal motility, continuous growth and increase in Rab5 density. Next, the relevance of Rab5 catastrophe with respect to cargo transfer into either the recycling- or degradative pathway was examined. Recycling cargo (transferrin) could be observed to exit Rab5-positive early endosomes via the frequent budding of tubular exit carriers. Exit of degradative cargo (LDL) from Rab5-positive endosomes did not involve budding, but the rapid loss of Rab5 from the limiting membrane.Rab5-loss was further coordinated with the concomitant acquisition of Rab7, suggesting "Rab conversion" as mechanism of transport between early- and late endosomes.Altogether, this thesis research has shown that first, Rab-machineries can be acquired and lost from membranes. Second, such dynamics provide a molecular mechanism for cargo exchange between endosomal compartments. Jointly, these findings lead to the concept of Rab-domain dynamics modulation in /trans/ between neighbouring domains as mechanistic principle behind the dynamic organization of membrane trafficking pathways.

Endocytosis, a process of plasma membrane invaginations, is a fundamental cellular mechanism, ensuring uptake of nutrients, enhanced communication between cells, protective functions against invasive pathogens and remodelling of the plasma membrane composition. In turn, endocytic mechanisms are exploited by pathogens to enter their host cells. Endocytosis comprises multiple forms of which our molecular understanding has mostly advanced with respect to clathrin-mediated endocytosis and phagocytosis. Studies on the small GTPase Rab5 have provided important insights into the molecular mechanism of endocytosis and transport in the early stages of the endocytic pathways. Rab5 is a key regulator of clathrin-mediated endocytosis, but in addition, localises to several distinct endocytic carriers including phagosomes and pinocytic vesicles. On early endosomes, Rab5 coordinates within a spatially restricted domain enriched in phosphatidylinositol-3 phosphate PI(3)P a complex network of effectors, including PI3-Kinase (PI3-K), the FYVE-finger proteins EEA1 and Rabenosyn-5 that functionally cooperate in membrane transport. Moreover, Rab5 regulates endocytosis from the apical and basolateral plasma membrane in polarised epithelial cells. During my PhD thesis, I investigated the molecular mechanisms of endocytosis both in polarised and non-polarised cells. I obtained new insights into the molecular mechanisms of endocytosis and their coordination through the functional characterization of a novel Rab5 effector, termed Rabankyrin-5. I could demonstrated that Rabankyrin-5 is a novel PI(3)P-binding Rab5 effector that localises to early endosomes and stimulates their fusion activity in vitro. The latter activity depends on the oligomerisation of Rabankyrin-5 on the endosomal membrane via the N-terminal BTB/POZ domain. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid phase uptake whereas its downregulation through RNA interference inhibits these processes. In polarised epithelial cells, the function of Rabankyrin-5 is primarily restricted to the apical membrane. It localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells and its overexpression in polarised MDCK cells specifically stimulates apical but not basolateral, non-clathrin mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)-pinocytosis, my studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, the active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells. The results obtained in this thesis are central not only for our understanding of the basic principles underlying the regulation of multiple endocytic mechanisms. They are also relevant for the biomedical field, since actin-dependent (macro)-pinocytosis is an important mechanism for the physiology of cells and organisms and is upregulated under certain pathological conditions (e.g. cancer).

In Saccharomyces cerevisiae, experiments with temperature-sensitive mutants of the PI4-kinase Pik1p revealed that the PI4P pool generated by this enzyme is essential for Golgi morphology and normal secretory function and that the PI4P pool at the Golgi represents a regulatory signal on its own. In order to function as a spatial and temporal regulator of membrane traffic, PI4P synthesis and turnover must be tightly regulated. It remains elusive which factors are involved in the targeting and regulation of Pik1p. Little is also known about PI4P binding proteins mediating the effects of this phosphoinositide on Golgi function. Since it has been shown that multiple pathways leave the Golgi towards the plasma membrane one can ask the question whether Pik1p and its product PI4P specifically control one pathway? Here we demonstrate an interaction of Pik1p with the 14-3-3 proteins Bmh1p and Bmh2p. Interestingly, overexpression of Bmh1p and Bmh2p results in multiple genetic interactions with genes involved in late steps of exocytosis and it affects the forward transport of the general amino acid permease Gap1p. The detected interaction depends on the phosphorylation state of Pik1p and Pik1p phosphorylation accompanies its shuttling out of the nucleus into the cytoplasm where presumably the binding to Bmh1/2p occurs. Therefore, we reason that these interactions might serve the sequestration of Pik1p away from the Golgi. This study reveals that Pik1p shows a strong effect on the delivery of Gap1p to the surface whereas the transport of exocytosis markers implicated in the direct Golgi-to-plasma membrane pathway are not significantly disturbed. Cells carrying a deletion of gga2 also show a strong defect in delivery of Gap1p to the surface. In addition, pik1-101 gga2[delta]double mutants display synthetic genetic and membrane transport phenotypes and recruitment of Gga2 to the TGN partially depends on functional Pik1p. Therefore, our results suggest a role of Pik1p in the TGN to endosome pathway.

Utilizing various systems such as cell-based assays but also multicellular organisms such as Drosophila melanogaster and C.elegans, for example, the endocytic system has been shown to consist of a network of biochemically and morphologically distinct organelles that carry out specialized tasks in the uptake, recycling and catabolism of growth factors and nutrients, serving a plethora of key biological functions (Mellman, 1996). Different classes of endosomes were found to exhibit a characteristic intracellular steady state distribution. This distribution pattern observed at steady state results from a dynamic interaction of endosomes with the actin and the microtubule cytoskeleton. It remains unclear, however, which microtubule-based motors besides Dynein control the intracellular distribution and motility of early endosomes and how their function is integrated with the sorting and transport of cargo. The first part of this thesis research outlines the search for such motor. I describe the identification of KIF16B which functions as a novel endocytic motor protein. This molecular motor, a kinesin-3, transports early endosomes to the plus end of microtubules, in a process regulated by the small GTPase Rab5 and its effector, the phosphatidylinositol-3-OH kinase hVPS34. In vivo, KIF16B overexpression relocated early endosomes to the cell periphery and inhibited transport to the degradative pathway. Conversely, expression of dominant-negative mutants or ablation of KIF16B by RNAi caused the clustering of early endosomes to the peri-nuclear region, delayed receptor recycling to the plasma membrane and accelerated degradation. These results suggest that KIF16B, by regulating the plus end motility of early endosomes, modulates the intracellular localization of early endosomes and the balance between receptor recycling and degradation. In displaying Rab5 and PI(3)P-containing cargo selectivity, a remarkable property of KIF16B is that it is subjected to the same regulatory principles governing the membrane tethering and fusion machinery (Zerial and McBride, 2001). Since KIF16B can modulate growth factor degradation, we propose that this motor could have also important implications for signaling. Importantly, KIF16B has provided novel insight into how intracellular localization of endosomes governs the transport activity of these organelles. The second part of this thesis describes the proof-of-principle of a genome-wide screening strategy aimed at gaining insights into the next level of understanding: How the spatial distribution of organelles is linked to their function in an experimental system which features cellular polarity, for example, a tissue or organ. The suitability of C. elegans as a model organism to identify genes functioning in endocytosis has been demonstrated by previous genetic screens (Grant and Hirsh 1999; Fares and Greenwald, 2001). Offering excellent morphological resolution and polarization, the nematode intestine represents a good system to study the apical sorting of a transmembrane marker. The steady state localization of such a marker is likely the result of a dynamic process that depends on biosynthetic trafficking to the apical surface, apical endocytosis and recycling occurring through apical recycling endosomes. Therefore, mis-sorting of this marker upon RNA-mediated interference will be indicative of a failure in one of the aforementioned processes. Furthermore, since it is still largely unclear why apical endosomes maintain their polarized localization, this screen will also monitor the morphology of this endocytic compartment using a second marker. Following image acquisition based on an automated confocal microscope, data can be analyzed using custom-built software allowing objective phenotypic analysis. The successful establishment of the proof-of-principle marks the current state-of-the-art of this large-scale screening project.

The trafficking of insulin secretory granules(SGs) of pancreatic b-cells is a tightly controlled complex network. Increasing evidence indicates that the cortical actin cytoskeleton modulates the mobility and exocytosis of SGs,yet the mechanisms anchoring SGs to the cytoskeleton is not completely understood.It has been shown by Ort et al.(2000,2001) that the cytoplasmic tail of an intrinsic membrane protein of the SGs named ICA512/IA-2 binds the PDZ domain of b2-syntrophin,which in turn binds to the F-actin-binding protein utrophin. These data also indicate that stimulation of SG exocytosis affects the phosphorylation of b2-syntrophin,hence altering its binding to ICA512.Therefore a model was proposed whereby SGs are anchored to the actin cytoskeleton through the ICA512/b2-syntrophin complex, whose dynamics are regulated by phosphorylation.To test this model GFP-b2-syntrophin stable INS-1 cell clones were generated.GFP-b2-syntrophin expression and localization pattern were similar to those of the endogenous protein. Electron microscopy showed that in GFP-b2-syntrophin INS-1 cells the number of SGs with a pear-like shape was increased relative to control cells. Insulin content and stimulated secretion were increased in three GFP-â2-syntrophin INS-1 cell clones,compared to non-transfected INS-1 cells and INS-1 cells expressing GFP. These increments correlated with the different expression levels of GFP-b2-syntrophin in the three GFP-b2-syntrophin INS-1 cell clones. These findings support the hypothesis that b2-syntrophin regulates the trafficking and exocytosis of SGs by modulating their tethering to the actin cytoskeleton.In order to confirm the proposed model, the phosphorylation of b2-syntrophin was investigated in more detail. Similar to endogenous b2-syntrophin,GFP-b2-syntrophin underwent Ca2+-dependent and okadaic acid-sensitive dephosphorylation upon stimulation of insulin secretion. Stimulation-dependent dephosphorylation was confirmed by immunoprecipitation of 32P-labeled GFP-b2-syntrophin.Mass spectrometry of immunoprecipitated GFP-b2-syntrophin allowed the identification of four serine-phosphorylation sites (S75,S90,S213,S373) that could affect the binding to ICA512.Mutants,in which all four phosphoserines, were replaced by either asp or ala to mimic(S/D) or prevent(S/A) phosphorylation were expressed in INS-1 cells. All S/D mutants retained a cortical localization,but by immunoblotting the pattern of the S75D allele differed from wild type and all other S/D alleles.Conversely, all S/A alleles were diffused cytosolically, except S213A,which was still restricted to the cortex. Finally, pull down assays showed increased binding of ICA512 to the S75A and S90D alleles compared to wild type b2-syntrophin,while the opposite was observed with the S75D and S90A mutants.Additionally,both the S75 and the S213 allele conform a consensus for phosphorylation by Cdk5,which is known to modulate insulin secretion. The phosphorylation of GFP-b2-syntrophin and particularly the S75 allele by Cdk5 was exhibited with pharmacological inhibitors,by in vitro phosphorylation and by RNAi. Taken together, these findings are consistent with the model by which phosphorylation of b2-syntrophin modulates the tethering of SGs to the cytoskeleton, and thereby their mobility and exocytosis. Specifically, the data of this thesis suggest that Cdk5-dependent phosphorylation of the S75 site of GFP-b2-syntrophin facilitates insulin secretion by reducing the interaction of b2-syntrophin with ICA512,thereby decreasing the actin cytoskeleton constrain on SG mobility. This process could occur in combination with the phosphatase-dependent dephosphorylation of b2-syntrophin at phosphosites other than S75. / Der Transport Insulin-gefüllter sekretorische Granula(SG) ist ein streng kontrollierter komplexer Prozess.Es gibt vermehrt Beweise,dass das kortikale Actinzytoskelett die Ausschüttung der SGs beeinflusst.Bisher ist der Mechanismus der Verankerung von SGs am Zytoskelett noch nicht vollständig aufgeklärt.Ort et al.(2000,2001) haben gezeigt,daß der zytosoplasmatische Teil des trans-membranen SG-Proteins ICA512 mit der PDZ-Domäne von b2-Syntrophin interagiert.Dieses Protein bindet das F-Actin-Bindeprotein Utrophin.Die Ergebnisse zeigen außerdem,daß durch Stimulation der SG-Exozytose der Phosphorilierungsstatus von b2-Syntrophin beeinflusst wird,woraus ein verändertes Bindungsvermögen zu ICA512 resultiert.Es wurde ein Funktionsmodel vorgestellt,in dem sich SGs durch die Interaktion des ICA512/b2-Syntrophin Komplexes an das Actinzytoskelett binden.Dabei wird die Bindedynamik durch Phosphorilierung reguliert.Um dieses Model zu etablieren,wurden stabile GFP-b2-Syntrophin produzierende INS-1-Zellklone erzeugt.Die zelluläre Lokalisation und das Expressionsmuster von GFP-b2-Syntrophin stimmen mit dem des endogenen Proteins überein.Elektronenmikroskopie zeigte eine größe Anzahl oval-verformter SGs in GFP-b2-Syntrophin INS-1-Zellen im Vergleich zu Kontrollzellen.Verglichen mit nicht-transfizierten INS-1 Zellen waren in drei GFP-b2-Syntrophin INS-1-Zellklonen der Insulingehalt der Zellen und die stimulierte Insulinsekretion erhöht.Die Werte korrelierten mit den unterschiedlichen GFP-b2-Syntrophin Expressionsmengen der Klone.Diese Ergebnisse untermauern die Hypothese,daß b2-Syntrophin den Transport und die Sekretion der SGs durch Modulation ihres Bindevermögens an Actin reguliert.Um das postulierte Model genauer zu prüfen,wurde die Phosphorilierung von b2-Syntrophin detaillierter untersucht.Das GFP-Protein wurde,ähnlich dem endogenen b2-Syntrophin,durch Stimulation der Insulinausschüttung dephosphoriliert.Diese Dephosphorilierung ist Ca2+-abhängig und Okadeinsäuresensitiv.Die stimulationsabhängige Dephosphorilierung wurde durch Immunoprezipitation von 32P-markiertem GFP-b2-Syntrophin bestätigt.Massenspektrometrie des präzipitierten Proteins ermöglichte die Identifikation von vier Serin-Phosphorilierungsstellen(S75,S90,S213,S373),welche die Bindung zu ICA512 beeinflussen könnten.Mutanten,in denen die vier Phosphoserine durch Asp beziehungsweise Ala ersetzt wurden,um entweder eine Phosphorilierung(S/D) oder Dephosphorilierung(S/A) nachzuahmen,wurden in INS-1-Zellen exprimiert.Alle S/D Mutanten blieben kortikal lokalisiert.Das Expressionsmuster des S75D Allels unterschied sich jedoch von denen des Wild-Typs(wt).Im Gegensatz dazu waren alle S/A Allele zytosolisch verteilt.Eine Ausnahme bildete S213A,das an der Zellkortex lokalisiert blieb.Im Vergleich zu wt b2-Syntrophin zeigten PullDown-Assays eine erhöhte Bindung von ICA512 zu den S75A und S90D Allelen.Das Gegenteil konnte für die S75D und S90A Mutanten nachgewiesen werden.S75,S90 und S213 sind in einer Konsensussequenz für Cdk5-Phosphorilierung enthalten.Diese Kinase kann die Insulinsekretion regulieren.Die Phosphorilierung von b2-Syntrophin,insbesondere des S75 Allels durch Cdk5 wurde durch pharmakologische Inhibitoren,in vitro-Phosphorilierung und RNAi demonstriert.Zusammenfassend stimmen diese Erkenntnisse mit dem Model überein,daß die Phosphorilierung von b2-Syntrophin die Vernetzung von SGs mit Actin und dadurch deren Mobilität und Exozytose moduliert.Im Speziellen postulieren die Ergebnisse dieser Arbeit eine Cdk5-abhängige Phosphorilierung der S75 Stelle des b2-Syntrophins.Durch eine verminderte Interaktion von b2-Syntrophin und ICA512 erleichtert diese Mutante vermutlich die Insulinsekretion,da der Einfluss des Actinzytoskeletts auf die Granulamobilität vermindert ist.Dieser Prozess ereignet sich möglicherweise in Kombination mit einer Dephosphorilierung des b2-Syntrophins.in Kombination mit einer Dephosphorilierung des b2-Syntrophins.