Les multicouches de polyelectrolytes, systèmes auto-assemblés par adsorption successive de polycations et de polyanions, constituent un matériau d’intérêt pour la fonctionnalisation de surface. Ce type de revêtement possède toutefois quelques limitations majeures : leur construction est lente et fastidieuse ; leur tenue mécanique et chimique est faible. Récemment, une méthode basée sur l’auto-construction de films par l’utilisation d’un morphogène, i.e. un gradient de catalyseur généré depuis une surface, a été développé permettant ainsi une rapide construction de revêtements robustes. Cette technique reste toutefois limitée à des systèmes particuliers basés sur la chimie click ou sur des interactions hôtes-invités. Nos travaux ont permis de diversifier cette approche de construction tout-en-un par l’utilisation de morphogènes. Dans un premier temps, des brosses de polymères ont été construites en une étape depuis une surface par la réaction de polymérisation ATRP. Cette réaction a été catalysée par la formation d’ions Cu(I) (le morphogène) par électrochimie depuis la surface de travail. L’approche morphogénique a également été utilisée afin de construire des films de polyélectrolytes et de polyampholytes en une étape par la génération électrochimique d’un gradient de protons (le morphogène) depuis la surface de travail. Ces films ont été fonctionnalisés pour présenter une activité enzymatique. / Polyelectrolyte multilayers, i.e. self-assembled systems based on successive polycation and polyanion adsorptions, constitute interesting materials for surface functionalization. These coatings possess several limitations: they are weak towards chemical and mechanical constraint and their buildup is long and tedious. Recently, a new method based on the self-construction of films by the means of a morphogen (a catalyst gradient generated from a surface) has attracted attention since it allows the quick self-assembly of robust films. Nevertheless, this technique was quite limited to peculiar systems based on click chemistry or on host-guest interactions.This present work generalize the one-pot morphogenic approach to other systems. In the first place, polymer brushes were built up from a surface by ATRP polymerization. The Cu(I) catalyst (the morphogen) was electrochemically generated at the interface.The morphogenic approach was later used to buildup polyelectrolytes and polyampholyte films in a one-pot manner by electrochemically generating protons (the morphogens) at the interface. These films exhibited an enzymatic activity.

Die räumliche und zeitliche Organisation von Zellen während Embryonalentwicklung, Regeneration oder Erneuerung von Geweben ist eine faszinierende Fähigkeit lebender Organismen. Dazu benötigen die Zellen Informationen über ihre Position im Organismus. Diese Informationen werden oft in Form von Signal- oder Morphogengradienten bereitgestellt, also von Signalmolekülen, die Konzentrationsprofile im Raum bilden. Plattwürmer (Planarien) sind ein sehr geeigneter Modellorganismus, um solche Gewebeorganisationsprozesse zu erforschen, weil sie kontinuierlich alle Zellen ihres Körpers erneuern und aus kleinsten Gewebestücken regenerieren können. Bei einer Körperlänge von bis zu 2 cm muss Gewebe auf größeren Längenskalen organisiert werden, als es für die Embryonalentwicklung in anderen Spezies nötig ist. Trotzdem treten auch in Planarien Signalgradienten auf. Ihre Hauptkörperachse wird, wie bei anderen Tieren auch, von einem Wnt-Signalgradienten organisiert. Experimentelle Beobachtungen legen nahe, dass ein positiver Feedbackmechanismus, in dem ein Wnt-Signal zur Erzeugung von mehr Wnt-Molekülen führt, wesentlich zur Bildung dieses Gradienten beiträgt. Inspiriert durch diese Beobachtungen stellen wir in dieser Arbeit einen Mechanismus zur Ausbildung von Signalgradienten vor, der auf positivem Feedback basiert. Um die besondere Bedeutung der Zellen für dieses Feedback berücksichtigen zu können, ist das hier präsentierte Modell diskret und besteht aus Zellen und Extrazellularräumen. Das positive Feedback sorgt für eine Signalübertragung von Zelle zu Zelle, wobei die Konzentration der extrazellulären Signalmoleküle die Konzentration des intrazellulären Effektors positiv reguliert, was wiederum zur Bildung von mehr Signalmolekülen führt. Wir zeigen, dass dieser Signalübertragungsmechanismus langreichweitige Signalgradienten mit einer Längenskala von mehreren hundert Zellen, also in der Größenordnung von Millimetern, ausbildet. Die Längenskala wird durch die Stärke des positiven Feedbacks reguliert. Eine entsprechende Regulation der Feedbackstärke ermöglicht es, die Längenskala des Signalgradienten an die Größe des Systems anzupassen. Erfolgt die Sekretion der Signalmoleküle, die die Zellen als Antwort auf das Feedback produzieren, gerichtet, führt das zu einer gerichteten Ausbreitung der Signalmolekülkonzentration im System, also zu Drift. Auf diese Weise können bei biologisch relevanten Werten des Diffusionskoeffizienten und der Degradationsrate der Signalmoleküle Signalgradienten mit einer Längenskala von mehreren zehn bis hundert Zellen in Stunden bis Tagen gebildet werden. Im Unterschied zum Diffusions/Degradations-Mechanismus, der häufig zur Erklärung von Gradientenbildung im Kontext von Embryonalentwicklung herangezogen wird, benötigt der in dieser Arbeit präsentierte Signalübertragungsmechanismus also weder sehr schnell diffundierende noch sehr langlebige Moleküle, um die Bildung von langreichweitigen Signalgradienten auf biologisch relevanten Zeitskalen zu erklären. Da viele Morphogene langsam diffundieren, macht das den Zell-zu-Zell-Signalübertragungsmechanismus zu einem attraktiven Konzept, um die Bildung von langreichweitigen Morphogengradienten zu erklären. / Embryonic development, regeneration, and tissue renewal are spectacular tissue-patterning events. Tissue patterning requires information. This information is often provided by signalling molecules that form graded concentration profiles in space, referred to as signalling gradients or morphogen gradients. Planarian flatworms are an ideal model organism to study tissue patterning as they constantly turn over all of their tissues and are able to regenerate from arbitrary amputation fragments. At a body length of up to 2 cm, planarians are orders of magnitudes larger than tissues organised during embryonic development in other species. Yet, flatworms employ signalling gradients for tissue patterning. Like in other organisms throughout the animal kingdom, their main body axis is patterned by a Wnt signalling gradient. Experiments have suggested a positive feedback mechanism of Wnt-mediated Wnt expression to be implicated in the formation of this Wnt signalling gradient in planarians. Inspired by these observations, in this thesis we present a cell-to-cell relay mechanism based on positive feedback to explain long-ranged signalling gradient formation. To account for the cellular nature of the relay, we built a discrete model, that considers individual cells and extracellular spaces. The relay is generated by a positive feedback loop in which extracellular signalling levels positively regulate intracellular effector concentrations which in turn leads to production of more extracellular signalling molecules. We show that a cell-to-cell relay gives rise to steady-state gradients reaching length scales of the order of hundreds of cells, corresponding to millimetres. The length scale is regulated by the strength of the feedback, which allows scaling the steady-state gradient to tissue size by adapting the feedback strength. Polarised secretion of signalling molecules in response to the positive feedback leads to an effective drift of signalling molecule concentration through the system. This allows the formation of signalling gradients with a length scale of tens to hundreds of cells (millimetres) within hours to days for a physiologically relevant diffusion coefficient and degradation rate of the signalling molecules. Thus, in contrast to a diffusion/degradation-based mechanism that is widely used to explain signalling gradient formation during embryonic development, the relay mechanism requires neither extraordinarily quickly-diffusing nor very long-lived signalling molecules to explain the formation of long-ranged signalling gradients on biologically relevant time scales. The cell-to-cell relay mechanism is therefore an attractive concept to explain the long-ranged patterning effects of poorly diffusive morphogens.

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Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.

Morphogens of the Wnt and Hedgehog families are secreted signaling molecules that coordinate growth and patterning of many different tissues. Both, Wingless and Hedgehog spread across long distances in developing wing of Drosophila melanogaster. However, both proteins are covalently modified with lipid moieties. The mechanisms that allow long-range movement of such hydrophobic molecules are unclear. Like Wingles and Hedgehog, glycosylphosphatidylinositol (gpi)-linked proteins also transfer between cells with their lipid anchor intact. It has been speculated that gpi-linked proteins and lipid-linked morphogens travel together on a membranous particle, which was termed an argosome. As yet however, no functional link between argosome production and dispersal of lipid-linked proteins has been established. The topic of this thesis is to understand the cell biological nature of the argosome and thus contribute to understanding of morphogen gradient formation. To address the question of argosome biosynthesis, at least two models have been proposed. One possibility is that argosomes are membranous exovesicles with a complete membrane bilayer. Alternatively, argosomes might resemble lipoprotein particles that comprise on of a family of apolipoproteins scaffolded around a phospholipid monolayer that surrounds a core of esterified cholesterol and triglyceride. Lipid-modified proteins of the exoplasmic face of the membrane (like GFPgpi, Wingless or Hedgehog) might fit well into the outer phospholipid monolayer of such a particle. Here, I utilize biochemical fractionation to determine the sort of particle that lipid-linked proteins associate with. I show that Wingless, Hedgehog and gpi-linked proteins bind Drosophila lipoprotein particles in vitro, and colocalize with them in wing imaginal discs. Next, I use genetic means to address the functional importance of this association. I demonstrate that reducing Lipophorin levels in Drosophila larvae perturbs long-range but not shor-range Wingless and Hedgehog signaling, and increases the sequestration of Hedgehog by Patched. I propose that Lipophorin particles are vehicles for the long-range movement of lipid-linked morphogens and gpi-linked proteins.