München, Techn. Univ., Diss., 2010.

Universiẗat, Diss., 2001--Bonn.

Extracellular vesicle (EV)-mediated intercellular communication through exosomes, microvesicles (MVs) and apoptotic bodies has been shown to be implicated in various physiological as well as pathological processes such as the development and progression of atherosclerosis. While the cellular machinery controlling EV formation and composition has been studied extensively, little is known about the underlying morphological processes. This study focuses on a detailed ultrastructural analysis of the different steps of EV formation and release in Myocardial Endothelial (MyEnd) and Aortic Endothelial (AoEnd) cells cultured under serum starvation and inflammatory stimulation with TNF-α. Detailed morphological analyses were conducted applying and comparing different high- resolution light and electron microscopic methods. In this study, we could depict all steps of MV biogenesis named in literature. However, during the study of exosome biogenesis, we discovered a yet undescribed process: Instead of a direct fusion with the plasma membrane, multivesicular bodies were incorporated into a new distinct cellular compartment bound by fenestrated endothelium first. This may present a novel step in exosome biogenesis and warrants further study. Regarding the conditions of cell cultivation, we observed that the commonly used serum starvation causes MyEnd cells, but not AoEnd cells, to enter apoptosis after 48 hours. When preparing functional EV studies, we therefore recommend assessing the morphological condition of the serum-starved cells at different cultivation points first. When evaluating MV production, a statistical analysis showed that the more time AoEnd cells spent in cultivation under serum starvation, the higher the percentage of MV producing cells. However, additional TNF-α stimulation induced a significantly higher MV production than serum starvation alone. Lastly, our results show that TNF-α stimulation of AoEnd cells in vitro leads to the upregulation of CD44, an adhesion molecule critical in the early stages of atherosclerosis. CD44 was then depicted on the surface of generated MVs and exosomes. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells. This could be a novel mechanism by which MVs contribute to the development and progression of atherosclerotic disease and should be clarified by further studies. / Extrazelluläre Vesikel (EV), darunter Exosomen, Mikrovesikel (MV) und apoptotische Körperchen, werden von fast allen Zellen des Körpers freigesetzt, transportieren zellspezifische Informationen und sind von großer Bedeutung in der Zell-Zell-Kommunikation. Sie spielen eine zentrale Rolle in verschiedensten physiologischen sowie pathologischen Vorgängen, wie etwa der Atherosklerose. Während die zellulären Mechanismen hinter der Entstehung und Komposition der EV bereits intensiv erforscht wurden, ist noch wenig über die zugrundeliegenden morphologischen Prozesse bekannt. Diese Arbeit präsentiert eine detaillierte ultrastrukturelle Analyse der Bildung und Freisetzung von EV in myokardialen (MyEnd) und aortalen Endothelzellen (AoEnd), die unter Serumentzug sowie inflammatorischer Stimulation mit TNF-α kultiviert wurden. Dazu wendeten wir verschiedene hochauflösende licht- und elektronenmikroskopische Techniken an. Wir konnten alle in der Literatur beschriebenen Schritte der MV-Biogenese darstellen. Bei der Untersuchung der exosomalen Biogenese entdeckten wir jedoch einen bisher unbekannten Prozess: Anstelle einer direkten Fusion der multivesikulären Körperchen mit der Plasmamembran, wurden diese zunächst in ein neues, von fenestriertem Endothel begrenztes, zelluläres Kompartiment integriert. Ferner stellten wir fest, dass der häufig durchgeführte Serumentzug während der Kultivierung bei MyEnd- – allerdings nicht AoEnd- – Zellen nach 48 Stunden zur Apoptose führte. Daher empfehlen wir, bei funktionellen Studien von EV zunächst eine morphologische Untersuchung der unter Serumentzug kultivierten Zellen zu verschiedenen Zeitpunkten durchzuführen. Eine statistische Analyse der MV-Produktion zeigte, dass die Zellen umso mehr MV produzierten, je länger sie sich unter Serumentzug befanden. Jedoch induzierte eine zusätzliche Stimulation mit TNF-α eine signifikant höhere MV-Produktion als der alleinige Serumentzug. Wir konnten zeigen, dass eine TNF-α Stimulation von AoEnd Zellen in vitro zu einer vermehrten Expression von CD44 führte – einem vor allem in der Frühphase der Atherosklerose bedeutendem Adhäsionsmolekül. CD44 konnte ebenso auf der Oberfläche von produzierten MV und Exosomen nachgewiesen werden. Wir schließen daraus, dass MV unter inflammatorischen Bedingungen den Transfer von CD44 von Endothelzellen zu Zielzellen vermitteln und so zur Entstehung und Progression von Atherosklerose beitragen können.

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.

During cell communication, cells produce secreted signals termed morphogens, which traffic through the tissue until they are received by target, responding cells. Using the fruit fly Drosophila melanogaster as a model organism, I have studied transforming growth factor-beta (TGF-beta) signal from the secreting to the receiving cells in the developing wing epithelial cells and at the neuromuscular junctions. Cell culture studies have suggested that cells modulate morphogenetic signaling by expressing the receptors and secreting the ligand in spatially defined areas of the cell. Indeed, I have found that TGF-beta ligands, receptors and R-Smads show a polarized distribution both in the epithelial cells and at the synapses. My results indicate that the cellular junctions define a signaling domain within the plasma membrane, to which TGF-beta signaling machinery is targeted. In the context of epithelial cells, the junctions play a role in TGF-beta signaling regulation through their component beta-cat. A complex forms between beta-cat and the R-Smad Mad, but the mechanism by which beta-cat modulates signaling is not yet understood. At the synapse, the sub-cellular localization of TGF-beta pathway components indicates the occurrence of an anterograde signal. Moreover, my results suggest a scenario in which TGF-beta signaling is coupled with synaptic activity: quanta of growth factor, released upon neurostimulation together with neurotransmitter quanta, could modulate therefore the development and the function of the synapse.

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.:Summary 1 List of publications 3 1 Introduction 9 Aims of the thesis 15 2 Generation of a local BMP signal in testis and wing disc 17 2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.1.1 Stem cells and stem cell niches . . . . . . . . . . . . . . 19 2.1.2 The Drosophila testis stem cell niche . . . . . . . . . . 20 2.1.3 BMP signalling in the fly . . . . . . . . . . . . . . . . . 23 2.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2.1 The BMP niche signal is transduced locally at adherens junctions 25 2.2.2 Generation of the local BMP niche signal . . . . . . . . 30 2.2.3 Exocyst involvement in long-range BMP signalling . . 34 2.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 3 Hedgehog pathway overactivation in the testicular niche 41 3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 3.1.1 The role of Hedgehog in the fly . . . . . . . . . . . . . 43 3.2 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 3.2.1 Overexpression of Hh increases the CySC number and expands their range 45 3.3 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 4 Visualization of Smo phosphorylation and biophysical detection of Smo clustering 49 4.1 Introduction (part I) . . . . . . . . . . . . . . . . . . . . . . . 51 4.1.1 Hedgehog signalling in the fly . . . . . . . . . . . . . . 51 4.1.2 Reception and transduction of the Hh signal by Ptc and Smo 54 4.2 Results (part I) . . . . . . . . . . . . . . . . . . . . . . . . . . 56 4.2.1 A fluorescent reporter for Drosophila Smo tail phosphorylation 56 4.2.2 Smo phosphorylation and localisation in the salivary gland 61 4.2.3 Smo localisation in cultured insect cells . . . . . . . . . 63 4.2.4 Smo membrane localisation and phosphorylation . . . . 65 4.3 Introduction (part II) . . . . . . . . . . . . . . . . . . . . . . . 67 4.3.1 Fluorescence correlation spectroscopy (FCS) . . . . . . 67 4.3.2 Dual-color fluorescence cross-correlation spectroscopy (FCCS) 72 4.3.3 Artefacts in FCS/FCCS . . . . . . . . . . . . . . . . . 73 4.4 Results (part II) . . . . . . . . . . . . . . . . . . . . . . . . . . 79 4.4.1 Smo clustering measured by FCCS . . . . . . . . . . . 79 4.5 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

During cell communication, cells produce secreted signals termed morphogens, which traffic through the tissue until they are received by target, responding cells. Using the fruit fly Drosophila melanogaster as a model organism, I have studied transforming growth factor-beta (TGF-beta) signal from the secreting to the receiving cells in the developing wing epithelial cells and at the neuromuscular junctions. Cell culture studies have suggested that cells modulate morphogenetic signaling by expressing the receptors and secreting the ligand in spatially defined areas of the cell. Indeed, I have found that TGF-beta ligands, receptors and R-Smads show a polarized distribution both in the epithelial cells and at the synapses. My results indicate that the cellular junctions define a signaling domain within the plasma membrane, to which TGF-beta signaling machinery is targeted. In the context of epithelial cells, the junctions play a role in TGF-beta signaling regulation through their component beta-cat. A complex forms between beta-cat and the R-Smad Mad, but the mechanism by which beta-cat modulates signaling is not yet understood. At the synapse, the sub-cellular localization of TGF-beta pathway components indicates the occurrence of an anterograde signal. Moreover, my results suggest a scenario in which TGF-beta signaling is coupled with synaptic activity: quanta of growth factor, released upon neurostimulation together with neurotransmitter quanta, could modulate therefore the development and the function of the synapse.

Calcium (Ca2+) ist ein in eukaryotischen Zellen allgegenwärtiger sekundärer Botenstoff. Durch Inositoltrisphosphat (IP3) ausgelöste Ca2+-Signale von IP3-Rezeptoren (IP3Rs) sind eines der universellsten Zell Signalübertragungssysteme. Ca2+ Signale sind fundamental stochastisch. Dennoch hat sich die Modellierung dieser Ca2+-Signale bisher stark auf deterministische Ansätze mit gewöhnlichen Differentialgleichungen gestützt. Diese wurden als Ratengleichungen etabliert und beruhen auf räumlich gemitteltem Ca2+ Werten. Diese Ansätze vernachlässigen Rauschen und Zufall. In dieser Dissertation präsentieren wir ein stochastisches Modell zur Erzeugung von Ca2+ Spikes in Form einer linearen Zustands-Kette. Die Anzahl offener Cluster ist die Zustandsvariable und Erholung von negativem Feedback wird berücksichtigt. Wir identifizieren einen Ca2+ Spike mit dem ersten Erreichen eines kritischen Zustands und sein Interspike Intervall mit der first-passage time (FPT) zu diesem Zustand. Dafür entwickeln wir einen allgemeinen mathematischen Rahmen zur analytischen Berechnung von FPTs auf solch einer Kette. Wir finden z.B. einen allgemein verringerten CV, der ein deutliches Minimum in Abhängigkeit der Zustandskettenlänge N aufweist. Dies nennen wir resonante Länge. Danach ergänzen wir positives Feedback und wenden das Modell auf verschiedene Zelltypen an. Es erfasst alle verfügbaren allgemeinen Beobachtungen zu Ca2+ Signalvorgängen. Es erlaubt uns Einblicke in den Zusammenhang von Agonistenstärke und Puffraten. Auch werden einzelne Ca2+ Spikes in Purkinje Neuronen, welche eine Rolle für Lernen und Erinnerung spielen, als stochastisches reaction-diffusion Model in einer 3D Dornenfortsatz Geometrie modelliert. Ataxia, eine Krankheit, die zum Verlust der Feinmotorik führt, wird auf defekte IP3R zurückgeführt, die abnormale Ca2+ Spikes erzeugen. Dieser Zustand wird ebenfalls untersucht und es wird ein Weg zur Wiederherstellung normaler Ca2+ Spikes vorgeschlagen. / Calcium (Ca2+) is a ubiquitous 2nd messenger molecule in all eukaryotic cells. Inositol trisphosphate (IP3)-induced Ca2+ signalling via IP3 receptors (IP3Rs) is one of the most universal signalling systems used by cells to transmit information. Ca2+ signalling is noisy and a fundamentally stochastic system. Yet, modelling of IP3-induced Ca2+ signalling has relied heavily on deterministic approaches with ordinary differential equations in the past, established as rate equations using spatially averaged Ca2+. These approaches neglect the defining features of Ca2+ signalling, noise and fluctuations. In this thesis, we propose a stochastic model of Ca2+ spike generation in terms of a linear state chain with the number of open clusters as its state variable, also including recovery from negative feedback. We identify a Ca2+ spike with reaching a critical state for the first time, and its interspike interval with the first passage time to that state. To this end, a general mathematical framework for analytically computing first-passage times of such a linear chain is developed first. A substantially reduced CV with a pronounced minimum, dependent on the chain length N, termed resonant length, are found. Positive feedback is then included into the model, and it is applied directly to various cell types. The model is fundamentally stochastic and successfully captures all available general observations on Ca2+ signalling. Also, we specifically study single Ca2+ spikes in spines of Purkinje neurons, assumed to be important for motor learning and memory, using MCell to simulate a reaction-diffusion system in a complex 3D Purkinje spine geometry. The model successfully reproduces experimentally findings on properties of Ca2+ spikes. Ataxia, a pathological condition resulting in, e.g., a loss of fine motor control, assumed to be caused by malfunctioning IP3Rs, is modelled and a possible way of recovery is suggested.