The majority of bacteria are organized in surface-associated communities, the so called biofilms. Crucial processes that drive the formation of such biofilms are the motility of bacteria on a substrate, enabling cells to reach each others vicinity, and attractive cell-cell-interactions, driving the formation of microcolonies. These colonies, aggregates consisting of thousands of cells, are the precursors of biofilms. In this thesis we investigate the role of cell appendages, called type IV pili, in the substrate motion of bacteria and the formation of bacterial microcolonies. Therefore, we study the bacterial dynamics with the help of experiments and theoretical models. We introduce a novel simulation tool in the tradition of Brownian dynamics simulations. In this computational model, that was developed alongside experimental observations, we study how explicit pili dynamics, pili-substrate and pili–pili interactions drive the cell dynamics. First, we apply our model to investigate how individual cells move on a substrate due to cycles of protrusion and retraction of type IV pili. We show that the characteristic features, in particular persistent motion, can solely originate from collective interactions of pili. Next, we perform experiments to study the coalescence of bacterial microcolonies. With the help of experiments and our computational model, we identify a spatially-dependent gradient of motility of cells within the colony as the origin of a separation of time scale, a feature which is in disagreement with the coalescence dynamics of fluid droplets. Additionally, we show that altering the force generation of pili can cause demixing of cells within bacterial aggregates. Finally, we combine our knowledge of the substrate motion of cells and of the pili-mediated interactions of colonies to identify the main processes (aggregation, fragmentation and cell divisions) that drive assembly of colonies. Starting from experiments, we develop a mathematical model and observe excellent qualitative and quantitative agreement to experimental data of the density of colonies of different sizes. In summary, hand in hand with experiments, we develop theoretical frameworks to unravel the role of type IV pili in bacterial surface motility, microcolony dynamics and colony formation.

The majority of bacteria are organized in surface-associated communities, the so called biofilms. Crucial processes that drive the formation of such biofilms are the motility of bacteria on a substrate, enabling cells to reach each others vicinity, and attractive cell-cell-interactions, driving the formation of microcolonies. These colonies, aggregates consisting of thousands of cells, are the precursors of biofilms. In this thesis we investigate the role of cell appendages, called type IV pili, in the substrate motion of bacteria and the formation of bacterial microcolonies. Therefore, we study the bacterial dynamics with the help of experiments and theoretical models. We introduce a novel simulation tool in the tradition of Brownian dynamics simulations. In this computational model, that was developed alongside experimental observations, we study how explicit pili dynamics, pili-substrate and pili–pili interactions drive the cell dynamics. First, we apply our model to investigate how individual cells move on a substrate due to cycles of protrusion and retraction of type IV pili. We show that the characteristic features, in particular persistent motion, can solely originate from collective interactions of pili. Next, we perform experiments to study the coalescence of bacterial microcolonies. With the help of experiments and our computational model, we identify a spatially-dependent gradient of motility of cells within the colony as the origin of a separation of time scale, a feature which is in disagreement with the coalescence dynamics of fluid droplets. Additionally, we show that altering the force generation of pili can cause demixing of cells within bacterial aggregates. Finally, we combine our knowledge of the substrate motion of cells and of the pili-mediated interactions of colonies to identify the main processes (aggregation, fragmentation and cell divisions) that drive assembly of colonies. Starting from experiments, we develop a mathematical model and observe excellent qualitative and quantitative agreement to experimental data of the density of colonies of different sizes. In summary, hand in hand with experiments, we develop theoretical frameworks to unravel the role of type IV pili in bacterial surface motility, microcolony dynamics and colony formation.:1. Introduction 2. Computational model of bacterial motility and mechanics 3. Motility of single bacteria on a substrate 4. Coalescence and internal dynamics of bacterial microcolonies 5. Demixing of bacterial microcolonies 6. Self-assembly of microcolonies 7. Summary and Outlook A. Details of the Simulation model B. Experimental protocols C. Geometric estimation of the parameters of the stochastic model D. Solutions for simplified models of pili-mediated cell motion E. Image analysis of experimental data F. Simulations and data analysis G. The mean squared relative distance (MSRD)

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.

Motile cilia are hair-like cell appendages that actively bend themselves, thus driving the surrounding fluid in motion. For many microorganisms, such as unicellular Paramecium, cilia are essential for their motility. Higher animals, including mammals, utilize cilia for transporting fluids. For example, in humans, large collections of cilia, called cilia carpets, remove mucus and pathogens from the airways. Cilia constitute an example of biological oscillators that can spontaneously synchronize their beat in the form of metachronal waves, i.e., traveling waves of cilia phase. These waves may arise purely by hydrodynamic interactions between the cilia and supposedly enhance fluid transport. Our goal is to theoretically understand how the properties of individual cilia, e.g., cilia beat pattern, determine the emergent behavior, e.g., the direction of the metachronal wave. Additionally, we address the robustness of hydrodynamically-induced synchronization with respect to intrinsic active fluctuations of the cilia beat and disorder of intrinsic cilia frequencies. Both of these effects are not yet well understood. In this thesis, we studied metachronal synchronization in cilia carpets using a theoretical physicist’s toolbox. First, we proposed a novel multi-scale modeling framework Lagrangian Mechanics of Active Systems (LAMAS) to describe fluid-structure interactions for active elastic structures, such as cilia. We quantified hydrodynamic interactions between cilia using detailed hydrodynamic simulations with a realistic cilia beat pattern. In the dynamical simulations for N = 2 cilia, we found that cilia would synchronize either in-phase or anti-phase, depending on their relative positions. For a lattice of N ≫ 1 cilia, we found the emergence of metachronal waves, many of which are locally stable. Nevertheless, just a single wave has a predominantly large basin of attraction, i.e., it is likely to be selected from a random initial condition. In the presence of noise, synchronization abruptly breaks up beyond a characteristic noise strength. Likewise, for cilia with non-identical intrinsic frequencies, synchronization is lost beyond a characteristic level of frequency disorder. In large cilia carpets, noise excites long-wavelength perturbations, whose relaxation times are proportional to the square of the system length. Thus, in large systems, we predict locally synchronized domains, instead of the global synchronization.