Spir proteins are the founding members of the novel class of WH2-actin nucleators. A C-terminal modified FYVE zinc finger motif is necessary to target Spir proteins towards intracellular membranes. The function and regulation of the Spir actin organizers at vesicular membranes is almost unknown. Live cell imaging analyses performed in this study show that Spir-2 is localized at tubular vesicles. Cytoplasmic Spir-2-associated vesicles branch and form protrusions, which can make contacts to the microtubule network, where the Spir-2 vesicles stretch and slide along the microtubule filaments. The analysis of living HeLa cells expressing eGFP-tagged Spir-2, Spir-2-ΔKIND and Spir-2-ΔKW (lacking the 4 WH2 domains and the KIND domain) showed Spir-2-associated tubular structures which differ in their length and motility. Throughout the course of that study it could be shown that the tail domain of the actin motor protein myosin Vb, as a force-generating molecule, is colocalizing and co-immunoprecipitating with Spir-2-ΔKW. By using the tail domain of myosin Vb as a dominant negative mutant for myosin Vb-dependent vesicle transport processes it could be shown that Spir-2-ΔKW/MyoVb-cc-tail- associated vesicles exhibit an increased elongation. Moreover, using the microtubule depolymerizing drug nocodazole it could be shown that the elongation and the motility of Spir-2-ΔKW-associated vesicles depends on an intact microtubule cytoskeleton. Motility and morphological dynamics of Spir-2-associated vesicles is therefore dependent on actin, actin motorproteins and microtubule filaments. These results propose a model in which myosin/F-actin forces mediate vesicle branching, allowing the vesicles to move to and in between the microtubule filaments and thereby providing a new degree of freedom in vesicular motility. To determine the exact subcellular localization of Spir-2, colocalization studies were performed. It could be shown that Spir-2 shows a partial colocalization to Rab11a-positive compartments. Furthermore, Spir-2 exhibits an almost identical localization to Arf1 and the Arf1 small G protein but not Rab11a could be immunoprecipitated with Spir-2-ΔKW. This suggests, that Arf1 recruits Spir-2 to Arf1/Rab11a-positive membranes. Another important function of the Spir-2 C-terminus is the membrane targeting by the FYVE domain. By performing a protein-lipid overlay assay, it has been shown that purified GST- and 6xHis-tagged Spir-2-ΔKW bind phosphatidic acid suggesting a mechanism in which Spir-2 is recruited to phosphatidic acid-enriched membranes. To further elucidate the mechanism in which Spir-2 membrane-targeting could be regulated, interaction studies of C-terminal parts of Spir-2 revealed that the Spir-2 proteins interact directly. / Spir Proteine sind die ersten beschriebenen Mitglieder der neuen Klasse der WH2-Aktin Nukleatoren. Ein C-terminaler modifizierter FYVE Zinkfinger ist notwendig um Spir Proteine an intrazelluläre Membranen zu bringen. Die Funktion und die Regulation dieser Aktin Nukleatoren an vesikulären Membranen ist bis jetzt noch nahezu unbekannt. In dieser Studie durchgeführte “Live-cell-Imaging” Experimente zeigten, dass Spir-2 an tubulären Vesikeln lokalisiert ist. Zytoplasmatische Spir-2-assoziierte Vesikel formen Ausläufer, die Kontakte zum Mikrotubuli Netzwerk bilden. Spir-2 Vesikel haben die Fähigkeit sich entlang des Mikrotubuli Zytoskeletts auszudehnen und daran entlang zu gleiten. Die Analyse von lebenden HeLa Zellen, welche eGFP-Spir-2, eGFP-Spir-2-ΔKIND und eGFP-Spir-2-ΔKW (Deletion der 4 WH2 Domänen sowie der KIND Domäne) Fusionsproteine exprimieren, zeigen Spir-2-assoziierte tubuläre Vesikel, die sich in Länge und Beweglichkeit unterscheiden. Während dieser Studie konnte außerdem gezeigt werden, dass die “tail” Domäne des Aktinmotors myosin Vb mit Spir-2-ΔKW kolokalisiert und koimmunopräzipitiert. Die Verwendung der “tail” Domäne als dominant negative Mutante für myosin Vb-abhängigen Vesikeltransport zeigte, dass Spir-2-ΔKW/MyoVb-cc-tail-assoziierte Vesikel eine stark erhöhte Elongation aufweisen. Desweiteren konnte duch die Verwendung von Nocodazol, welches spezifisch Mikrotubulifilamente depolymerisiert, gezeigt werden, dass die Elongation und die Motilität der Spir-2-ΔKW-assoziierten Vesikel von einem intakten Mikrotubuli Zytoskelett abhängig ist. Motilität und morphologische Dynamik der Spir-2-ΔKW-assoziierten Vesikel ist daher abhängig von Aktinfilamenten, Aktin Motorproteinen und Mikrotubulifilamenten. Anhand dieser Ergebnisse lässt sich ein Modell erstellen, in welchem eine Myosin/F-actin induzierte Bewegung eine Verzweigung der Vesikel bewirkt. Dadurch ist eine Bewegung der Vesikel zu Mikrotubulifilamenten aber auch zwischen verschiedenen Mikrotubulifilamenten möglich, welches einen ganz neuen Freiheitsgrad in der vesikulären Bewegung eröffnet. Um die genaue zelluläre Lokalisation von Spir-2 zu analysieren wurden Kolokalisationsstudien durchgeführt. Hierbei konnte gezeigt werden, dass Spir-2 eine partielle Kolokalisation mit Rab11a-positiven Kompartimenten zeigt. Außerdem weist Spir-2 eine nahezu identische Lokalisation zu Arf1 auf. Arf1, aber nicht Rab11a, konnte mit Spir-2-ΔKW koimmunpräzipitiert werden. Arf1 könnte daher für die Rekrutierung von Spir-2 an Arf1/Rab11a-positive Membranen ausschlaggebend sein. Eine weitere wichtige Funktion des Spir-2 C-Terminus ist die Membranlokalisation, welche durch die FYVE Domäne vermittelt wird. Mittels Protein-Lipid Bindungsstudien konnte gezeigt werden, dass aufgereinigte GST- bzw. 6xHis-Spir-2-ΔKW-Fusionsproteine an Phosphatidylsäure binden. Dies deutet darauf hin, dass Spir-2 spezifisch zu Phosphatidylsäure-positiven Membranen rekrutiert wird. Um die weitere Regulation der Spir-2 Membranlokalisation aufzuklären, wurden Protein-Protein-Interaktionsstudien durchgeführt, welche eine direkte Interaktion von Spir-2 Proteinen anhand ihrer C-Termini ergaben.

Hook-Proteine spielen eine wichtige Rolle im intrazellulären Transport. Sie binden n-terminal Mikrotubuli, haben eine coiled-coil-Domäne und binden c-terminal Organellen. Da aus humanen Hirnschnitten eine Kolokalisation mit den bei Alzheimerscher Erkrankung auftretenden neurofibrillären Tangles bekannt war, erscheint eine weitere Untersuchung der Expression und Lokalisation von Hook-Proteinen im zentralen Nervensystem interessant. Hierzu wurde die Expression der humanen Hook-Proteine mittels real-time RT-PCR quantifiziert und die Lokalisation der Hook-Proteine in verschiedenen transgenen Mausmodellen der Tauopathie mittels Immunhistochemie dargestellt. Außerdem wurde die cDNA der Hook-Proteine mittels PCR auf alternatives Spleißen untersucht.

Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.

In biological cells, the long-range intracellular traffic is powered by molecular motors which transport various cargos along microtubule filaments. The microtubules possess an intrinsic direction, having a 'plus' and a 'minus' end. Some molecular motors such as cytoplasmic dynein walk to the minus end, while others such as conventional kinesin walk to the plus end. Cells typically have an isopolar microtubule network. This is most pronounced in neuronal axons or fungal hyphae. In these long and thin tubular protrusions, the microtubules are arranged parallel to the tube axis with the minus ends pointing to the cell body and the plus ends pointing to the tip. In such a tubular compartment, transport by only one motor type leads to 'motor traffic jams'. Kinesin-driven cargos accumulate at the tip, while dynein-driven cargos accumulate near the cell body. We identify the relevant length scales and characterize the jamming behaviour in these tube geometries by using both Monte Carlo simulations and analytical calculations. A possible solution to this jamming problem is to transport cargos with a team of plus and a team of minus motors simultaneously, so that they can travel bidirectionally, as observed in cells. The presumably simplest mechanism for such bidirectional transport is provided by a 'tug-of-war' between the two motor teams which is governed by mechanical motor interactions only. We develop a stochastic tug-of-war model and study it with numerical and analytical calculations. We find a surprisingly complex cooperative motility behaviour. We compare our results to the available experimental data, which we reproduce qualitatively and quantitatively. / In biologischen Zellen transportieren molekulare Motoren verschiedenste Frachtteilchen entlang von Mikrotubuli-Filamenten. Die Mikrotubuli-Filamente besitzen eine intrinsische Richtung: sie haben ein "Plus-" und ein "Minus-"Ende. Einige molekulare Motoren wie Dynein laufen zum Minus-Ende, während andere wie Kinesin zum Plus-Ende laufen. Zellen haben typischerweise ein isopolares Mikrotubuli-Netzwerk. Dies ist besonders ausgeprägt in neuronalen Axonen oder Pilz-Hyphen. In diesen langen röhrenförmigen Ausstülpungen liegen die Mikrotubuli parallel zur Achse mit dem Minus-Ende zum Zellkörper und dem Plus-Ende zur Zellspitze gerichtet. In einer solchen Röhre führt Transport durch nur einen Motor-Typ zu "Motor-Staus". Kinesin-getriebene Frachten akkumulieren an der Spitze, während Dynein-getriebene Frachten am Zellkörper akkumulieren. Wir identifizieren die relevanten Längenskalen und charakterisieren das Stauverhalten in diesen Röhrengeometrien mit Hilfe von Monte-Carlo-Simulationen und analytischen Rechnungen. Eine mögliche Lösung für das Stauproblem ist der Transport mit einem Team von Plus- und einem Team von Minus-Motoren gleichzeitig, so dass die Fracht sich in beide Richtungen bewegen kann. Dies wird in Zellen tatsächlich beobachtet. Der einfachste Mechanismus für solchen bidirektionalen Transport ist ein "Tauziehen" zwischen den beiden Motor-Teams, das nur mit mechanischer Interaktion funktioniert. Wir entwickeln ein stochastisches Tauzieh-Modell, das wir mit numerischen und analytischen Rechnungen untersuchen. Es ergibt sich ein erstaunlich komplexes Motilitätsverhalten. Wir vergleichen unsere Resultate mit den vorhandenen experimentellen Daten, die wir qualitativ und quantitativ reproduzieren.

Cargo transport by molecular motors is ubiquitous in all eukaryotic cells and is typically driven cooperatively by several molecular motors, which may belong to one or several motor species like kinesin, dynein or myosin. These motor proteins transport cargos such as RNAs, protein complexes or organelles along filaments, from which they unbind after a finite run length. Understanding how these motors interact and how their movements are coordinated and regulated is a central and challenging problem in studies of intracellular transport. In this thesis, we describe a general theoretical framework for the analysis of such transport processes, which enables us to explain the behavior of intracellular cargos based on the transport properties of individual motors and their interactions. Motivated by recent in vitro experiments, we address two different modes of transport: unidirectional transport by two identical motors and cooperative transport by actively walking and passively diffusing motors. The case of cargo transport by two identical motors involves an elastic coupling between the motors that can reduce the motors’ velocity and/or the binding time to the filament. We show that this elastic coupling leads, in general, to four distinct transport regimes. In addition to a weak coupling regime, kinesin and dynein motors are found to exhibit a strong coupling and an enhanced unbinding regime, whereas myosin motors are predicted to attain a reduced velocity regime. All of these regimes, which we derive both by analytical calculations and by general time scale arguments, can be explored experimentally by varying the elastic coupling strength. In addition, using the time scale arguments, we explain why previous studies came to different conclusions about the effect and relevance of motor-motor interference. In this way, our theory provides a general and unifying framework for understanding the dynamical behavior of two elastically coupled molecular motors. The second mode of transport studied in this thesis is cargo transport by actively pulling and passively diffusing motors. Although these passive motors do not participate in active transport, they strongly enhance the overall cargo run length. When an active motor unbinds, the cargo is still tethered to the filament by the passive motors, giving the unbound motor the chance to rebind and continue its active walk. We develop a stochastic description for such cooperative behavior and explicitly derive the enhanced run length for a cargo transported by one actively pulling and one passively diffusing motor. We generalize our description to the case of several pulling and diffusing motors and find an exponential increase of the run length with the number of involved motors. / Lastentransport mittels Motorproteinen ist ein grundlegender Mechanismus aller eukaryotischen Zellen und wird üblicherweise von mehreren Motoren kooperativ durchgeführt, die zu einer oder zu verschiedenen Motorarten wie Kinesin, Dynein oder Myosin gehören. Diese Motoren befördern Lasten wie zum Beispiel RNAs, Proteinkomplexe oder Organellen entlang Filamenten, von denen sie nach einer endlichen zurückgelegten Strecke abbinden. Es ist ein zentrales und herausforderndes Problem zu verstehen, wie diese Motoren wechselwirken und wie ihre Bewegungen koordiniert und reguliert werden. In der vorliegenden Arbeit wird eine allgemeine theoretische Herangehensweise zur Untersuchung solcher Transportprozesse beschrieben, die es uns ermöglicht, das Verhalten von intrazellularem Transport, ausgehend von den Transporteigenschaften einzelner Motoren und ihren Wechselwirkungen, zu verstehen. Wir befassen uns mit zwei Arten kooperativen Transports, die auch kürzlich in verschiedenen in vitro-Experimenten untersucht wurden: (i) gleichgerichteter Transport mit zwei identischen Motorproteinen und (ii) kooperativer Transport mit aktiv schreitenden und passiv diffundierenden Motoren. Beim Lastentransport mit zwei identischen Motoren sind die Motoren elastisch gekoppelt, was eine Verminderung ihrer Geschwindigkeit und/oder ihrer Bindezeit am Filament hervorrufen kann. Wir zeigen, dass solch eine elastische Kopplung im Allgemeinen zu vier verschiedenen Transportcharakteristiken führt. Zusätzlich zu einer schwachen Kopplung, können bei Kinesinen und Dyneinen eine starke Kopplung und ein verstärktes Abbinden auftreten, wohingegen bei Myosin Motoren eine verminderte Geschwindigkeit vorhergesagt wird. All diese Transportcharakteristiken, die wir mit Hilfe analytischer Rechnungen und Zeitskalenargumenten herleiten, können durch Änderung der elastischen Kopplung experimentell untersucht werden. Zusätzlich erklären wir anhand der Zeitskalenargumente, warum frühere Untersuchungen zu unterschiedlichen Erkenntnissen über die Auswirkung und die Wichtigkeit der gegenseitigen Beeinflussung der Motoren gelangt sind. Auf diese Art und Weise liefert unsere Theorie eine allgemeine und vereinheitlichende Beschreibung des dynamischen Verhaltens von zwei elastisch gekoppelten Motorproteinen. Die zweite Art von Transport, die in dieser Arbeit untersucht wird ist der Lastentransport durch aktiv ziehende und passiv diffundierende Motoren. Obwohl die passiven Motoren nicht zum aktiven Transport beitragen, verlängern sie stark die zurückgelegte Strecke auf dem Filament. Denn wenn ein aktiver Motor abbindet, wird das Lastteilchen immer noch am Filament durch den passiven Motor festgehalten, was dem abgebundenen Motor die Möglichkeit gibt, wieder an das Filament anzubinden und den aktiven Transport fortzusetzen. Für dieses kooperative Verhalten entwickeln wir eine stochastische Beschreibung und leiten explizit die verlängerte Transportstrecke für einen aktiv ziehenden und einen passiv diffundierenden Motor her. Wir verallgemeinern unsere Beschreibung für den Fall von mehreren ziehenden und diffundierenden Motoren und finden ein exponentielles Anwachsen der zurückgelegten Strecke in Abhängigkeit von der Anzahl der beteiligten Motoren.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

No description available.

Here we review viral and cellular requirements for entry and intracellular trafficking of foamy viruses (FVs) resulting in integration of viral sequences into the host cell genome. The virus encoded glycoprotein harbors all essential viral determinants, which are involved in absorption to the host membrane and triggering the uptake of virus particles. However, only recently light was shed on some details of FV’s interaction with its host cell receptor(s). Latest studies indicate glycosaminoglycans of cellular proteoglycans, particularly heparan sulfate, to be of utmost importance. In a species-specific manner FVs encounter endogenous machineries of the target cell, which are in some cases exploited for fusion and further egress into the cytosol. Mostly triggered by pH-dependent endocytosis, viral and cellular membranes fuse and release naked FV capsids into the cytoplasm. Intact FV capsids are then shuttled along microtubules and are found to accumulate nearby the centrosome where they can remain in a latent state for extended time periods. Depending on the host cell cycle status, FV capsids finally disassemble and, by still poorly characterized mechanisms, the preintegration complex gets access to the host cell chromatin. Host cell mitosis finally allows for viral genome integration, ultimately starting a new round of viral replication.

In mammalian cells, homeostasis and fate during development relies on the proper transport of membrane-bound cargoes to their designated cellular locations. The hetero-tetrameric adaptor protein complexes (APs) are required for sorting and concentration of cargo at donor membranes, a crucial step during targeted transport. AP2, which functions at the plasma membrane during clathrin-mediated endocytosis, is well characterized. In contrast, AP1 a clathrin adaptor mediating the delivery of lysosomal hydrolases via mannose 6-phosphate receptors (MPRs) and AP3 an adaptor ensuring the proper targeting of lysosomal membrane protein are difficult to study by classic cell biology tools. To gain new insights on these APs, our lab has previously designed an in vitro system. Reconstituted liposomes were modified with small peptides mimicking the cytosolic domains of bona fide cargoes for AP1 and AP3 respectively and thereby enabling the selective recruitment of these APs and the identification of the interacting protein network. In the study at hand we utilize above-described liposomes to generate supported lipid bilayers and Giant Unilamellar Vesicles (GUVs), large-scale membrane systems suited for analysis by fluorescence microscopy. By using cytosol containing fluorescently-tagged subunits, we visualized clathrin coats on artificial membranes under near physiological conditions for the first time. Moreover, we demonstrated clathrin-independent recruitment of AP3 coats on respective GUVs. Presence of active ARF1 was sufficient for the selective assembly of AP1-dependent clathrin coats and AP3 coats on GUVs. By using dye-conjugated ARF1, we show that ARF1 colocalized with AP3 coats on GUVs and that increased association of ARF1 with GUVs coincided with AP1-dependent clathrin coats. Our previous study identified members of the septin family together with AP3 coats on liposomes. Here we show on GUVs, that active ARF1 stimulated the assembly of septin7 filaments, which may constrain the size and mobility of AP3 coats on the surface. Subsequent cell biology studies in HeLa cells linked septins to actin fibers on which they may control mobility of AP3-coated endosomes and thus their maturation. An actin nucleation complex, based on CYFIP1 was identified together with AP1 on liposomes before. Here we show on GUVs, that CYFIP1 is recruited on the surface surrounding clathrin coats. Upon supply of ATP, sustained actin polymerization generated a thick shell of actin on the GUV surface. The force generated by actin assembly lead to formation of long tubular protrusions, which projected from the GUV surface and were decorated with clathrin coats. Thereby the GUV model illustrated a possible mechanism for tubular carriers formation. The importance of CYFIP1-reliant actin polymerization for the generation of MPR-positive tubules at the trans-Golgi network (TGN) of HeLa cells was subsequently demonstrated in our lab. The notion that tubulation of artificial membranes could be triggered by actin polymerization allowed us to perform a comparative mass spectrometry screen. By comparing the abundance of proteins on liposomes under conditions promoting or inhibiting actin polymerization, candidates possibly involved in stabilization, elongation or fission of membrane tubules could be identified. Among the proteins enriched under conditions promoting tubulation, we identified type I phosphatidylinositol-4-phosphate 5-kinases. Their presence suggested an involvement of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in tubule formation. By cell biology studies in HeLa we show, that down regulation of these enzymes altered the dynamics of fluorescently-tagged MPRs, illustrating the importance of locally confined PI(4,5)P2 synthesis during formation of coated carriers at the TGN. Bin–Amphiphysin–Rvs (BAR) domains are known to sense membrane curvature and induce membrane tubulation. Among various BAR domain proteins, Arfaptin2 was enriched under conditions allowing tubulation of liposomes. By microscopy studies on HeLa cells we show, that Arfaptin2 as well as its close paralog Arfaptin1 were present on AP1-coated MPR tubules emerging from the TGN. We further show, that tubule fission occurred at regions were Arfaptin1 is concentrated and that simultaneous down regulation of both Arfaptins lead to increased number and length of MPR tubules. Since fission of coated transport intermediates at the TGN is poorly understood, our findings contribute a valuable component towards a model describing the entire biogenesis of coated post-Golgi carriers. In conclusion, combining artificial membrane systems and cell biology studies allowed us to propose new models for formation as wall as for fission of AP1-coated transport intermediates at the TGN. Further we gained new insights on AP3 coats and the possible involvement of septin filaments in AP3-dependent endosomal maturation.

Hook-Proteine spielen eine wichtige Rolle im intrazellulären Transport. Sie binden n-terminal Mikrotubuli, haben eine coiled-coil-Domäne und binden c-terminal Organellen. Da aus humanen Hirnschnitten eine Kolokalisation mit den bei Alzheimerscher Erkrankung auftretenden neurofibrillären Tangles bekannt war, erscheint eine weitere Untersuchung der Expression und Lokalisation von Hook-Proteinen im zentralen Nervensystem interessant. Hierzu wurde die Expression der humanen Hook-Proteine mittels real-time RT-PCR quantifiziert und die Lokalisation der Hook-Proteine in verschiedenen transgenen Mausmodellen der Tauopathie mittels Immunhistochemie dargestellt. Außerdem wurde die cDNA der Hook-Proteine mittels PCR auf alternatives Spleißen untersucht.