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Zugl.: München, Univ., Diss., 2002

Frankfurt (Main), Univ., Diss., 2008

Stuttgart, Univ., Diss., 2003.

Signal propagation across the membrane by cytokine receptor signalling involves a complex interplay of receptor-ligand interactions, allostery and conformational changes. Type I interferons (IFNs) exert their biological activities through binding to a shared receptor consisting of the type II cytokine receptor subunits IFNAR1 and IFNAR2. The aim of this thesis was to establish biochemical and biophysical approaches for exploring in vitro interactions and conformational changes accompanying the formation of type I IFN receptor complex. For these purposes, in this work a versatile combination of covalent vs. non-covalent reversible site-specific protein modification chemistries was exploited for their surface immobilization, incorporation of fluorescence probes or crosslinkers. The generic bioorthogonal strategy of PPTase-catalysed covalent modification of ybbR short peptide tag fused to a protein of interest enabled highly efficient site-directed fluorescence labelling of wild type IFNα2 and mutants, IFNAR1 and IFNAR2 receptors as well as their functional immobilization onto surfaces. These modified proteins were confirmed to be active by studying their interactions in ligand-receptor surface binding assays in real time by total internal reflection fluorescence spectroscopy and reflectance interference. A rapid quantitative surface assay for probing binding kinetics of proteins captured directly via His-tags from cell supernatants was established and employed for screening of IFNAR1 mutants in order to identify the residues responsible for differential recognition of various IFN subtypes. Thus the fine-tuned IFN binding chemistries through few ligand-specific interaction points as the basis for receptor plasticity was identified. Site-specific covalent immobilization allowed exploring cooperativity in ligand recognition by the receptor subunits. The observed small allosteric effect is apparently not related to the potential contact of membrane-proximal receptor domains but probably mediated through conformational cross-communication of binding sites on the ligand. Substantial conformational changes of IFNAR1 upon ligand binding were exploited as fluorescence readout to monitor the assembly of ternary complexes on artificial membranes. This enabled exploring the life times of ternary complexes with IFNα2 combined mutants targeting binding to IFNAR1 and IFNAR2 and corroborated to the suggestion that the differential physiological activity of IFN subtypes is related to the total ternary complex affinity and not to ligand affinity towards individual receptor subunits. Finally, in vitro stabilization of dual-colour labelled weakly interacting IFNα2/IFNAR1/IFNAR2 complex by means of an entropic clamp was implemented, enabling to analyze ternary complexes by fluorescence cross-correlation spectroscopy Förster resonance energy transfer on the single molecule level. These novel tools will prove valuable for unravelling the subtle interplay of interactions and conformational changes in cytokine receptor complexes.

The aim of my PhD research was to develop and establish techniques for surface functionalization of nanoparticles, which can be employed to study the dynamics, function and activity of recombinantly expressed as well as endogenous proteins inside living cells. A prerequisite to achieve this goal was the ability to bio-functionalize nanoparticles with proteins in the cytoplasm of living cells. The HaloTag technology was utilized for generic site-specific targeting of nanoparticles to proteins. Fast and efficient targeting of nanoparticles to proteins was then achieved by using an engineered clickHTL exhibiting fast reactivity towards the HaloTag-enzyme. Application of this approach to track individual proteins in the outer membrane of mitochondria revealed that the physicochemical properties of the nanoparticles biased the mobility of the targeted proteins. To circumvent this, a model nanoparticle was systematically engineered in order to identify physicochemical properties that are important for tracking intracellular membrane proteins without affecting their diffusion dynamics. Nanoparticles exhibiting stealth properties were finally obtained upon densely coating the nanoparticle surface with PEG2k. These particles were mono-functionalization with clickHTL, to ensure labeling in a 1:1 stoichiometry, and could be successfully used for unbiased tracking of individual membrane proteins. Beyond the observation of proteins, generic approaches that allow intracellular manipulation and probing of protein activities are desired. To this end, 500 nm superparamagnetic nanoparticles were used as mobile nanoscopic hotspots self-assebled into active signaling platforms. Inside living cells, precise and accurate manipulation of endogeneous Rac1 activity was possible at different subcellular locations and over extended time periods. These experiments demonstrated that Rac1 signaling is dependent on the subcellular-context by spatial isolation of distinct signaling pathways. Furthermore, these MNPs provided well defined platforms for selective spectroscopy in order to quantify bait-prey protein interactions in the cytoplasm as was demonstrated by the interaction of cdc42 and N-WASP.

Cilien und eukariotische Flagellen sind lange, dünne Fortsaetze von Zellen. Sie enthalten eine Struktur namens Axonem. Die wesentlichen Komponenten des Axonems sind die Filamente und Motorproteine namens Mikrotubuli und Dynein. Die Motoren forcieren die Filamente, sich in oszillierender Weise gegeneinander zu verschieben, was zu einem Schlagmuster entlang des Axonemes fuehrt. Wie diese Motoren koordiniert werden und wie dieses Phaenomen quantitativ beschrieben werden kann, ist nicht verstanden. Wir studierten die Wellenformen an Spermienschwaenzen, welche ein solches Axonem enthalten, unter verschiedenen Bedingungen mit einer Hochgeschwindigkeitskamera. Wir entwickelten eine automatisierte Bildanalyse-Software, die es erlaubt, lange Zeitreihen solcher Wellenformen von Filmen zu extrahieren. In einer anschließenden Fourieranalyse erzielten wir eine gemittelte Wellenform mit erhoehter Präzision. Ein Vergleich von unseren Daten mit den Vorhersagen einer Theorie (Camalet, Julicher et al. 1999) führte zu einer Diskrepanz. Entsprechend schlugen wir eine Erweiterung der Theorie vor, indem wir annahmen, daß an der Basis des Axonems ein viskos-elastisches Element existiert. Dies führte zu einer zufrieden stellenden Übereinstimmung zwischen Theorie und Experiment. Abschließend diskutieren wir offene Fragen und zukünftige Experimente. Als ein Nebenprodukt entdeckten wir ein neues Phaenomen, bei welchem Spermien Anordnungen von dynamischen Strudeln (Vortices) bilden. Wir beschrieben dieses Phaenomen im Detail und führten einen neuen Ordnungsparameter ein, mit dem die Ordnung zwischen vielen Objekten quantifiziert werden kann. Mittels dieses Ordnungsparameters konnten wir zeigen, daß dieses Muster sich erst ab einer kritischen Dichte herausbildet. Wir schlugen ein Model vor, um den Ursprung des Musters zu erklären. Die Simulation des Models zeigte volle Uebereinstimmung mit den wesentlichen Eigenschaften dieses Musters. Weiterhin schaetzten wir die typische Wechselwirkungskraft zwischen aktiven Axonemen mit 0.1 pN ab. Abschließend ziehen wir Schlußfolgerungen über die kollektive Wirkung von Axonemen im Allgemeinen mit Hinblick auf Spermienkooperation und metachronale Cilienwellen. - Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: QuickTimeMovies (ca. 65 MB) Nutzung: PLAY32 - Übersicht über Inhalte siehe Dissertation S. 108 - 109 / Cilia and eukaryotic flagella are long, thin extensions of cells that contain a structure known as axoneme. The key components of the axoneme are microtubule filaments and the motor proteins dynein. These dynein motors force the microtubules to slide in an oscillatory fashion leading to a wave pattern along the axoneme. How these motors are coordinated and how this phenomenon can be described quantitatively is not understood. I therefore studied the waveforms of sperm tails that contain such an axoneme. I observed these waveforms under different conditions with a high-speed camera and developed an automated image analysis tool allowing the extraction of long time series of this waveform. In a subsequent Fourier analysis I increased the precision by obtaining an averaged waveform. I then compared the data to the predictions of a theoretical framework (Camalet, Julicher et al. 1999) and found that they do not agree. I suggested extending this theoretical framework by considering a visco-elastic element at the base of the axoneme, which leads to a satisfactory agreement. This project leaves open questions hence further work is discussed. As a side finding, I discovered a new phenomenon on how spermatozoa form dynamic vortex arrays. I described this pattern in detail and introduced a novel order parameter to quantify the order among many particles. I showed that the array only forms above a critical sperm density. I suggested a model to explain the origin of the pattern and showed by simulation that the model can account for the main features of the pattern. Finally I estimated the typical interaction force between beating axonemes to be 0.1 pN and drew conclusions about their collective action in general that might be relevant for sperm cooperation or metachronal waves of cilia.

The mechanical parameters of biological cells are relevant indicators of their function or of disease. For example, certain cancerous cells are more deformable than healthy cells. The challenge consists in developing methods that can measure these parameters while not affecting the cell. The Optical Stretcher is a microfluidic system that deforms single suspended cells without contact using lasers and determines the cells’ viscoelastic properties. The advantage compared to standard methods of molecular biology is that cells do not need to be treated with additional markers. Basic versions of the Optical Stretcher have existed for some years. These allow the measurement of homogeneous cell populations. Up until now, it was only possible to calculate average population values of compliance. To characterize inhomogeneous populations however, it is necessary to consider each single cell and measure additional mechanical or optical parameters such as the refractive index. This work highlights various extensions of the Optical Stretcher. A novel procedure, including an improved image processing algorithm, is presented to analyze mechanical data in real time. In combination with measurements of the optical refractive index, single cells can now be characterized in more detail. Moreover, it is now possible to extract interesting subpopulations that can be further examined with molecular biology techniques. Depending on the intended purpose, novel devices for cell measurements, based on microfluidic and optical considerations, are presented. The fundamental concept involves microstructured chips that can be integrated into a commercial microscope. These chips offer the possibility of separating measured cell populations according to their mechanical properties. This separation, including mathematical classification, is demonstrated. These methods are tested with cell types of differing mechanical properties to prove their applicability in practice. Single cells are sorted into their respective population of origin. These novel methods offer the possibility of a versatile device to be applied in biophysical research.

Stuttgart, Univ., Diss., 2008.

University, Diss., 2005--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.