Hybride Mikroschwimmer, die einen biologischen Antrieb und eine künstlich hergestellte Mikrostruktur enthalten sind ein attraktiver Ansatz um kontrollierte Bewegung auf kleinstem Maßstab zu erreichen. In dieser Dissertation wird ein neuer hybrider Mikroschwimmer vorgestellt, der aus ferromagnetischen Nanomembranen besteht, die sich zu Mikroröhrchen aufrollen und in der Lage sind, einzelne Spermien einzufangen. Dieser Mikrobioroboter nutzt die starke Antriebskraft der Spermazelle um das magnetische Mikroröhrchen fortzubewegen. Die vorliegende Arbeit beschreibt, wie dieser Mikroschwimmer seine Bewegung vollzieht und wie verschiedene Faktoren wie Temperatur, Radius der Mikroröhrchen, Eindringtiefe der Spermien in das Röhrchen und Länge der Röhrchen einen Einfluss auf sein Verhalten haben. Richtungskontrolle wird durch externe magnetische Felder realisiert und es wird dargestellt, wie dies zur Trennung der Mikrobioroboter aus einer Mischung von Spermien und Mikroröhrchen genutzt werden kann. Weiterhin werden zwei Oberflächenmodifizierungsmethoden angewandt um die Kupplungseffizienz zwischen Mikroröhrchen und Spermien zu erhöhen. In diesen Methoden wird das extrazelluläre Protein Fibronektin auf die innere Röhrchenoberfläche aufgebracht und dient als Bindungsstoff für Spermien. Schließlich wird durch den Einbau temperatursensitiver Material in die Mikroröhrchen ein ferngesteuerter Freisetzungsmechanismus für die Spermazelle vorgestellt. Dabei falten sich die Mikroröhrchen bei kleinen Temperaturerhöhungen auf und setzen die Zelle frei. Diese Arbeit diskutiert letztendlich das Potential solch eines hybriden Mikroschwimmers für die Anwendung in assistierter Reproduktion. / The search for autonomously moving, highly functional and controllable microdevices is a purpose of current micro/nanobiotechnology research, especially in the area of biomedical applications, for which reason, biocompatible solutions are in demand. In this thesis, a novel type of hybrid microswimmer is fabricated by the combination of rolled up thin nanomembranes with bovine spermatozoa. The microbiorobot presented here uses the powerful motion of the sperm flagella as a propulsion source for the magnetic microtube. This work demonstrates how the microswimmer performs its motion and how several factors such as temperature, radius of the microtube, penetration of the cell inside the microtube and length of the tube have influence on its performance. Directional control mechanisms are offered by external magnetic fields and are presented to be useful for the on-chip separation of the microbiorobots from a mixture of cells and microtubes. Two surface modification methods are presented as means to improve the coupling efficiency between the microtubes and the sperm cells. By these surface functionalizations, the extracellular matrix protein fibronectin is attached on the inner microtube walls and serves as binding agent for the spermatozoa. Finally, a remote release mechanism for the sperm cells is demonstrated by the incorporation of thermoresponsive material into the microtubes, which makes them fold and unfold upon small temperature changes. This work discusses the potential of such microswimmers for the application in assisted reproduction techniques and gives an outlook on future perspectives.

Eukaryotic cells are populated by membrane-enclosed organelles possessing discrete molecular and biochemical properties. Communication between organelles is established by shuttling vesicles that transport proteins and other molecules. Vesicles bud from a donor organelle, travel in the cytosol, and are delivered to a target organelle. All these steps are regulated to ensure that cargoes are transported in a specific and directed manner. The focus of this thesis is on the last part of the journey of a vesicle: the process of vesicle targeting. Two phases can be distinguished in this process: vesicle tethering, defined as the first interaction between the shuttling vesicle and the target membrane, and membrane fusion, which is the mixing of the lipid bilayers and of lumen content. Both phases are mediated by a minimal set of molecular components that include one member of the family of Rab GTPases, a vesicle tethering factor, a phosphoinositide lipid, and four SNAREs together with their regulatory proteins. While many studies have investigated the molecular details of how SNAREs mediate membrane fusion, the process of vesicle tethering is less well understood. The overall scope of my study is to describe the molecular details of vesicle tethering and how they can contribute to the general process of vesicle targeting. To address this question I developed an in vitro assay where I reconstitute in vitro the process of vesicle tethering. This bottom-up approach allows the molecular dissection of cellular processes outside of the complex context of the cell. With this assay I have characterized the vesicle tethering abilities of individual proteins involved in vesicle tethering on early endosomes. I show that a minimal vesicle tethering machinery can be formed by the concomitant interaction between one vesicle tethering factor and a phosphoinositide on the membrane of one vesicle, and by a vesicle tethering factor and a Rab GTPase on the membrane of another vesicle. These results provide an explanation for how vesicle tethering contributes to the specificity of vesicle targeting and to the directionality of cargo transport. In particular, specificity of vesicle targeting can arise from the specific interaction between a Rab and a vesicle tethering factor that is an effector of the Rab. I show that the asymmetric distribution of binding sites in the structure of a vesicle tethering factor can generate a heterotypic vesicle tethering reaction that can account for the directionality of cargo transport. The outcome of this thesis emphasizes the role that vesicle tethering factors have in the self-organized system of vesicle trafficking of eukaryotic cells. To identify novel Rab5 effectors implicated in vesicle tethering, I carried out a Rab5-chromatography on mouse liver. Amongst other novel Rab5 effectors, I identify a multi-subunit vesicle tethering complex that was not previously characterized in mammalian cells. The complex, named CORVET, is conserved from yeast to humans and plays a major role in cell physiology since its removal causes embryonic death in mice. I define its subunits composition, determine its subcellular localization, and elucidate its role in cargo transport. This finding reconciles a disharmony between findings in mammals and yeast regarding the molecular machinery responsible for the conversion from early to late endosomes. I also show that the newly identified subunit of the mammalian CORVET complex is the only Rab5 effector to localize to autophagosomes. I hypothesise that it is through the CORVET complex that Rab5 is involved in the formation and maturation of autophagosomes.

The Rab family of small GTPases orchestrates intracellular endomembrane transport through the recruitment of diverse effector proteins. Since its first discovery in 1987, almost 70 Rab proteins have been identified in humans to date and their perturbed function is implicated in several hereditary and acquired diseases. In this Ph.D. thesis, I systematically characterize cell type expression and subcellular localization of all Rab proteins present in Drosophila melanogaster utilizing a genetic resource that represents a major advance for studying membrane trafficking in vivo: the ’Drosophila YRab library’. This collection comprises 27 different D. melanogaster knock-in lines that harbor YFPMyc fusions to each Rab protein, referred to as YRab. For each YRab, I present a comprehensive data set of quantitative and qualitative expression profiles across six larval and adult tissues that include 23 annotated cell types. The whole image data set, along with its annotations, is publicly accessible through the FLYtRAB database that links to CATMAID for online browsing of tissues. I exploit this data set to address basic cell biological questions. i) How do differentiating cells reorganize their transport machinery to perform cell type-specific functions? My data indicates that qualitative and quantitative changes in YRab protein expression facilitate the functional specialization of differentiated cells. I show that about half of the YRab complement is ubiquitously expressed across D. melanogaster tissues, while others are missing from some cell types or reflect strongly restricted cell type expression, e.g. in the nervous system. I also depict that relative YRab expression levels change as cells differentiate. ii) Are specific Rab proteins dedicated to apical or basolateral protein transport in all epithelia? My data suggests that the endomembrane architecture reflects specific tasks performed by particular epithelial tissues, rather than a generalized apicobasal organization. I demonstrate that there is no single YRab that is similarly polarized in all epithelia. Rather, different epithelial tissues dynamically polarize the subcellular localization of many YRab compartments, producing membrane trafficking architectures that are tissue- and stage-specific. I further discuss YRab cell type expression and subcellular localization in the context of Rab family evolution. I report that the conservation of YRab protein expression across D. melanogaster cell types reflects their evolutionary conservation in eukaryotes. In addition, my data supports the assumption that the flexible deployment of an expanded Rab family triggered cell differentiation in metazoans. The FLYtRAB database and the ’Drosophila Rab Library’ are complementary resources that facilitate functional predictions based on YRab cell type expression and subcellular localization, and to subsequently test them by genetic loss-of-function experiments. I demonstrate the power of this approach by revealing new and redundant functions for Rab23 and Rab35 in wing vein patterning. My data collectively highlight that in vivo studies of endomembrane transport pathways in different D. melanogaster cell types is a valuable approach to elucidate functions of Rab family proteins and their potential implications for human disease.

Das stäbchenförmige Bakterium Escherichia coli teilt sich in zwei gleich große Tochterzellen. Dies ist nur möglich, wenn sich die Zelle in der Mitte teilt. Bei E. coli wird die Zellteilung durch den Zusammenschluss der FtsZ-Proteine an der Membran zum Z-Ring eingeleitet. Topologische Regulierung des Z-Ringes erfolgt durch räumlich-zeitliche Oszillationen von Min-Proteinen zwischen den beiden Zellpolen. MinC, MinD und MinE binden an und lösen sich von der Membran unter Hydrolyse von ATP und in antagonistischer Art und Weise, was zu einer alternierenden Ansammlung von MinC und MinD an den Zellpolen führt. Gemittelt über die Zeit ergibt sich somit ein MinD-Verteilungsprofil, das maximale Konzentration an den Zellpolen und ein Minimum in der Zellmitte aufweist. MinC bindet an MinD und folgt somit seiner Verteilung. Der Zusammenschluss von FtsZ-Proteinen wird durch MinC unterbunden, und somit kann sich der Z-ring nur an einer Position herausbilden, die ein Minimum an MinC aufweist - der Zellmitte. Das Min-system wurde in der Vergangenheit auch mit einem in-vitro-Ansatz untersucht, indem Min-Proteine in künstliche, aufliegende Lipiddoppelschichten (supported lipid bilayers, SLB) rekonstitutiert wurden. Dabei bildeten die Min-Proteine kein oszillierendes Muster aus, sondern organisierten sich vielmehr in parallelen und propagierenden Wellen (Loose, 2008, Science, 320). In diesen in-vitro-Experimenten war das Membransubstrat wesentlich größer als die Wellenlänge der Min-Proteinwellen. In vivo hingegen ist die Länge der Zelle in der gleichen Größenordnung wie die charakteristische Länge des Oszillationsmusters der Min-Proteine. Daher war es das Ziel dieser Arbeit, den Einfluß einer beschränkten Fläche und geometrischer Formgebung der künstlichen Lipiddoppelschichten auf die Wellenpropagation der Min-Protein zu untersuchen. Flächige Beschränkung künstlicher Membranen erfolgte durch Mikrostrukturtechnologie. Deckglässchen wurden mit einer Goldschicht und mikroskopischen Aussparungen unterschiedlicher geometrischer Formen strukturiert. Funktionale SLBs bildeten sich nur auf Glasflächen ohne Goldbeschichtung aus. Nach der Rekonstitution der Min-Proteine, organisierten sich diese auf den Membranstücken in parallele Wellen. Dabei bestimmte die flächige Beschränkung der künstlichen Membranen die Ausbreitungsrichtung der Min-Proteinwellen. Min-Proteinwellen konnten entlang gekrümmter Membranstreifen, in Ring- und sogar in Slalomstrukturen geleitet werden. In geraden, länglichen Strukturen richteten sich die Wellen entlang der längsten Achse aus. Kopplung von Proteinwellen auf räumlich getrennten Membranstücken in Abhängigkeit des Abstandes und des sogenannten Molecular Crowdings in der wässrigen Lösung konnte ebenfalls beobachtet werden. Diese Kopplung ist ein Indiz für inhomogene Proteinverteilungen in der Lösung oberhalb der Membran. Desweiteren konnten Min-Proteinwellen auch in diversen dreidimensionalen künstlichen Membranen rekonstitituiert werden. Im Wildtyp von E. coli ähneln die Min-Proteindynamiken der einer Oszillation mit einer charakteristischen Länge von 5 µm. Auf SLBs, bilden Min-Proteine Wellen mit einer Wellenlänge aus, die ca. zehnmal größer ist als in vivo. Dieser Unterschied zwischen der in-vivo- und der in-vitro-Welt wurde untersucht und diskutiert. In vitro konnte die Wellenlänge um 50 % durch Erhöhung des Molecular Crowding in der Lösung sowie um 33 % durch Temperaturerhöhung verkleinert werden. Das oszillierende Muster könnte dahingegen eine Folge der Kompartimentierung sein. Erste Versuche, das Min-System in geschlossene Membrankompartimente zu rekonstitutieren, wurden getestet. / Escherichia coli, a rod-like bacterium, divides by binary fission. Cell division into two daughter cells of equal size requires that fission takes place at a midcell position. In E. coli, cell division is initiated by assembly of the FtsZ-proteins at the inner membrane to the Z-ring. Topological regulation of the Z-ring is achieved by spatiotemporal pole-to-pole oscillations of Min-proteins. MinC, MinD and MinE bind to and detach from - under hydrolysis of ATP - the membrane in an antagonistic manner leading to an alternating accumulation of MinC and MinD at the cell poles. Averaged over time, the distribution profile of MinD exhibits maximal concentration at the cell poles and a minimum at the cell center. MinC binds to MinD and thus follows its distribution. FtsZ assembly is inhibited by MinC and therefore the Z-ring can only form at a cell position low in MinC - at the cell center. In the past, the Min-system was also investigated in an in vitro approach by reconstitution of Min-proteins into a supported lipid bilayer (SLB). Here, Min-proteins did not self-organize into an oscillatory pattern but into parallel and propagating waves (Loose, 2008, Science, 320). In this in vitro assay, the membrane substrate was infinitely large compared to the wavelength. However, in vivo, the cell length is on the same order of magnitude as the respective length scale of the oscillatory pattern of Min-proteins. Therefore, we wished to investigate the effect of lateral confinement and geometric structuring of artificial lipid bilayers on the Min-protein wave propagation. Lateral confinement of artificial membranes was achieved by microfabrication technology. Glass slides were patterned by a gold coating with microscopic windows of different geometries, and functional SLBs were only formed on uncoated areas. Upon reconstitution, Min-proteins organized into parallel waves on the geometric membrane patches. Confinement of the artificial membranes determined the direction of propagation of Min-protein waves. Min-protein waves could be guided along curved membrane stripes, in rings and even along slalom-geometries. In elongated membrane structures, the protein waves always propagate along the longest axis. Coupling of protein waves across spatially separated membrane patches was observed, dependent on gap size and level of molecular crowding of the aqueous media above the bilayer. This indicates the existence of an inhomogeneous and dynamic protein gradient in the solution above the membrane. Furthermore, reconstitution of Min-protein waves in various three-dimensional artificial membranes was achieved. In wild-type E. coli, Min-protein dynamics resemble that of an oscillation with a characteristic length scale of 5 µm. On supported lipid bilayers, Min-proteins self-organize into waves with a wavelength approximately 10-fold larger than in vivo. These discrepancies between the in vivo and in vitro world were investigated and discussed. In vitro, the wavelength could be decreased by a factor of 50 % by increase of the molecular crowding in solution and by 33 % through temperature increase. The oscillatory pattern is thought to be a consequence of compartmentalization and first attempts to encapsulate the Min-system in closed bilayer compartments are presented.

Compartmentalization of cell membranes, in particular of the apical membrane of columnar epithelia, is the topic of this thesis. The first part characterizes the apical membrane and its specialized organization and morphology, whereas the second part focuses on the formation of this unique plasma membrane domain during epithelial polarization. The apical membrane of columnar epithelia is enriched in glycosphingolipids, a class of lipids that are known to interact with cholesterol to form liquid ordered domains, also termed "rafts", in cell membranes. Imaging the apical surface of untreated and raft lipid depleted MDCK cells with atomic force microscopy revealed that raft lipids are involved in the formation and/or maintenance of microvilli, actin based protrusions of the apical plasma membrane, indicating a regulatory link between membrane domains and the cytoskeleton. Furthermore, antibody patching and photobleaching experiments performed during the work of this thesis suggest that the organization into raft and non-raft domains is very different in the apical membrane of MDCK cells compared to the plasma membrane of a fibroblast. In fact, the data support the hypothesis that the apical membrane could be a percolating raft membrane in which rafts constitute the major phase and non-raft domains exist as isolated entities. The second part of this thesis analyses the segregation of apical and basolateral membrane domains during epithelial polarization. This segregation can either be achieved by generating scaffolded domains prior to junction formation or by polarized secretory and endocytic membrane traffic after the establishment of cell junctions. While most apical and basolateral marker proteins in MDCK cells follow the latter mechanism, this thesis reports that the apical marker gp135 is confined to the dorsal face already in single attached cells. The unknown antigen was purified and identified as podocalyxin. Analysis of a series of domain mutants revealed that the C-terminal PDZ-binding motif of podocalyxin is mainly responsible for its special localization, which it shares with the PDZ protein NHERF-2. Knocking down podocalyxin by RNA interference resulted in retardation of cell growth and epithelial polarization. Taken together, the data suggest that podocalyxin and NHERF-2 could be part of an early apical polarity scaffolding system based on PDZ-binding and PDZ-containing proteins.

This work addresses properties of two-component lipid membranes on the experimentally relevant spatial scales of order of a micrometer and time intervals of order of a second by means of lattice-based Monte Carlo (MC) simulations. To be able to do that with reasonable computational efforts the lipid membrane is modeled as a square lattice of lipid molecules with next-neighbor interaction. This allows for efficient computation and thus provides a large-scale simulation with which it was possible to obtain important results previously not reported in simulation studies of lipid membranes. After properly tuning the next-neighbor interaction energies the simulation reproduces the experimental phase diagram of the DMPC/DSPC lipid system which is used as a model system in this work. Beyond that, the MC simulation provides a more detailed description of the phase behavior of the lipid mixture than the experimental data. It is found that, within a certain range of lipid compositions, the phase transition from the fluid phase to the fluid–gel phase coexistence proceeds via near-critical fluctuations, while for other lipid compositions this phase transition has a quasi-abrupt character. The complete combined state and component phase diagram is constructed by structure function analysis which confirms the existence of a critical point in the system. The dynamics of membrane coarsening after an abrupt temperature quench to the fluid–gel coexistence region of the phase diagram are studied. In this context, it is found that lateral diffusion of lipids plays an important role in the fluid–gel phase separation process. Dynamic scaling is observed only if the ratio of gel and fluid phase in the membrane stays constant in time. The line tension characterizing lipid domains in the fluid–gel coexistence region is found to be in the pN range thus matching values both predicted theoretically and measured experimentally. When approaching the critical point, the line tension, the inverse correlation length of fluid–gel spatial fluctuations, and the corresponding inverse order parameter susceptibility of the membrane vanish in agreement with recent experimental findings for model lipid membranes. By simulating single particle tracking and fluorescence correlation spectroscopy experiments it is found that in the presence of near-critical fluctuations lipid molecules show transient subdiffusive behavior, which is a new result important for understanding the origins of subdiffusion in cell membranes which are believed to be close to a critical point. The membrane–cytoskeleton interaction strongly affects phase separation, enhances subdiffusion, and eventually leads to hop diffusion of lipids. Thus, a minimum realistic model for membrane rafts showing the features of both microscopic phase separation and subdiffusion is established.

An essential prerequisite for the existence of multi-cellular life is the organization of cells into tissues. In this thesis, we theoretically study how large-scale tissue properties can emerge from the collective behavior of individual cells. To this end, we focus on the properties of epithelial tissue, which is one of the major tissue types in animals. We study how rheological properties of epithelia emerge from cellular processes, and we develop a physical description for the dynamics of an epithelial cell polarity. We apply our theoretical studies to observations in the developing wing of the fruit fly, Drosophila melanogaster. In order to study epithelial mechanics, we first develop a geometrical framework that rigorously describes the deformation of two-dimensional cellular networks. Our framework decomposes large-scale deformation into cellular contributions. For instance, we show how large-scale tissue shear decomposes into contributions by cell shape changes and into contributions by different kinds of topological transitions. We apply this framework in order to quantify the time-dependent deformation of the fruit fly wing, and to decompose it into cellular contributions. We also use this framework as a basis to study large-scale rheological properties of epithelia and their dependence on cellular fluctuations. To this end, we represent epithelial tissues by a vertex model, which describes cells as elastic polygons. We extend the vertex model by introducing fluctuations on the cellular scale, and we develop a method to perform perpetual simple shear simulations. Analyzing the steady state of such simple shear simulations, we find that the rheological behavior of vertex model tissue depends on the fluctuation amplitude. For small fluctuation amplitude, it behaves like a plastic material, and for high fluctuation amplitude, it behaves like a visco-elastic fluid. In addition to analyzing mechanical properties, we study the reorientation of an epithelial cell polarity. To this end, we develop a simple hydrodynamic description for polarity reorientation. In particular, we account for polarity reorientation by tissue shear, by another polarity field, and by local polarity alignment. Furthermore, we develop methods to quantify polarity patterns based on microscopical images of the fly wing. We find that our hydrodynamic description does not only account for polarity reorientation in wild type fly wings. Moreover, it is for the first time possible to also account for the observed polarity patterns in a number of genetically altered flies. / Eine wesentliche Voraussetzung für die Existenz mehrzelligen Lebens ist, dass sich einzelne Zellen sinnvoll zu Geweben ergänzen können. In dieser Dissertation untersuchen wir, wie großskalige Eigenschaften von Geweben aus dem kollektiven Verhalten einzelner Zellen hervorgehen. Dazu konzentrieren wir uns auf Epitheliengewebe, welches eine der Grundgewebearten in Tieren darstellt. Wir stellen theoretische Untersuchungen zu rheologischen Eigenschaften und zu zellulärer Polarität von Epithelien an. Diese theoretischen Untersuchungen vergleichen wir mit experimentellen Beobachtungen am sich entwickelnden Flügel der schwarzbäuchigen Taufliege (Drosophila melanogaster). Um die Mechanik von Epithelien zu untersuchen, entwickeln wir zunächst eine geometrische Beschreibung für die Verformung von zweidimensionalen zellulären Netzwerken. Unsere Beschreibung zerlegt die mittlere Verformung des gesamten Netzwerks in zelluläre Beitrage. Zum Beispiel wird eine Scherverformung des gesamten Netzwerks auf der zellulären Ebene exakt repräsentiert: einerseits durch die Verformung einzelner Zellen und andererseits durch topologische Veränderungen des zellulären Netzwerks. Mit Hilfe dieser Beschreibung quantifizieren wir die Verformung des Fliegenflügels während des Puppenstadiums. Des Weiteren führen wir die Verformung des Flügels auf ihre zellulären Beiträge zurück. Wir nutzen diese Beschreibung auch als Ausgangspunkt, um effektive rheologische Eigenschaften von Epithelien in Abhängigkeit von zellulären Fluktuationen zu untersuchen. Dazu simulieren wir Epithelgewebe mittels eines Vertex Modells, welches einzelne Zellen als elastische Polygone abstrahiert. Wir erweitern dieses Vertex Modell um zelluläre Fluktuationen und um die Möglichkeit, Schersimulationen beliebiger Dauer durchzuführen. Die Analyse des stationären Zustands dieser Simulationen ergibt plastisches Verhalten bei kleiner Fluktuationsamplitude und visko-elastisches Verhalten bei großer Fluktuationsamplitude. Neben mechanischen Eigenschaften untersuchen wir auch die Umorientierung einer Zellpolarität in Epithelien. Dazu entwickeln wir eine einfache hydrodynamische Beschreibung für die Umorientierung eines Polaritätsfeldes. Wir berücksichtigen dabei insbesondere Effekte durch Scherung, durch ein anderes Polaritätsfeld und durch einen lokalen Gleichrichtungseffekt. Um unsere theoretische Beschreibung mit experimentellen Daten zu vergleichen, entwickeln wir Methoden um Polaritätsmuster im Fliegenflügel zu quantifizieren. Schließlich stellen wir fest, dass unsere hydrodynamische Beschreibung in der Tat beobachtete Polaritätsmuster reproduziert. Das gilt nicht nur im Wildtypen, sondern auch in genetisch veränderten Tieren.