Univ., Diss., 2006--Frankfurt (Main) / Zsfassung in engl. und dt. Sprache

Scaffolding protein Disheveled (Dvl) is a key component of Wnt signaling cascades. Dvl participates in a number of biological processes, such as cell proliferation, differentiation and migration, determination of cell polarity, and also stem cell self-renewal. It is therefore indispensable for the correct embryo development and tissue homeostasis in adulthood. The protein regulator of cytokinesis (PRC1) is a microtubule-associated protein. PRC1 is involved in spindle midzone formation during cell division. Spindle midzone precedes the contractile ring assembly and is essential for normal cell cleavage. In our laboratory, PRC1 was identified as a putative interaction partner of DVL3. This master thesis is focused on delineation of the interaction interface between DVL3 and PRC1 using TIRF microscopy (Total Internal Reflection Fluorescence microscopy). To this end, full-length DVL and PRC1 proteins together with their truncated variants were designed, expressed and purified. It was discovered that PRC1 interacts with all three DVL isoforms and the N-terminal part of PRC1 is required for the interaction between PRC1 and DVL3. Furthermore, the DEP domain of DVL3 is likely involved in PRC1interactions. Key words: Dishevelled 3, DVL3, Protein regulator of cytokinesis 1, PRC1, interaction interface, TIRF...

No description available.

Fluorescence Correlation Spectroscopy (FCS) is a powerful tool to measure important physical quantities such as concentrations, diffusion coefficients, diffusion modes or binding parameters, both in solution and in membranes. However, it can suffer from severe artifacts, especially in non-ideal systems. Here we develop several novel implementations of FCS which overcome these limitations and facilitate accurate and quantitative determination of dynamic parameters in membranes. Two-focus FCS with camera-detection allows for accurate and calibration-free determination of diffusion coefficients. Confocal FCS using a laser scanning microscope provides an unprecedented positioning accuracy which enabled us to study, for the first time with FCS, dynamics in bacterial membranes. Scanning FCS with a scan path perpendicular to the membrane plane allows to correct for instabilities permitting long measurement times necessary to study slow diffusion. It can easily be extended to measure calibration-free diffusion coefficients with two-focus scanning FCS and to quantify binding with dual color scanning FCS. Spectral crosstalk can be avoided effectively by using alternating excitation. Using this method we were able to perform measurements in systems previously not accessible with FCS, such as yeast cell membranes or membranes of living zebrafish embryos. Line-scan FCS with a scan path in the membrane plane uses the parallel acquisition along the line to increase the statistical accuracy and decrease the measurement times. Knowledge of the scan speed serves as an internal calibration, enabling accurate diffusion and concentration measurements within seconds, hardly affected by photobleaching. Both realizations of scanning FCS can be easily implemented with commercial laser scanning microscopes. Often, a fluorescence background around the membrane cannot be avoided. The high surface selectivity needed in this case can be achieved efficiently by using a novel objective for FCS, the supercritical angle objective, which produces a very flat and laterally confined detection volume. Another technique with similar surface selectivity is FCS with total internal reflection excitation (TIRFCS). Due to the lack of a correct model, the accurate analysis of TIR-FCS data was previously not possible. In this work we develop such a model, enabling quantitative measurements of membrane dynamics with TIR-FCS. The novel FCS techniques developed here will have a high impact on the use of FCS to address key questions in biological systems, previously inaccessible by other methods. / Fluoreszenz-Korrelations-Spektroskopie (FCS) ist eine mächtige Methode, um wichtige physikalische Parameter wie Konzentrationen, Diffusionskoeffizienten, Diffusionsarten oder Bindungsparameter in Lösung und in Modell- oder Zellmembranen zu bestimmen. In nichtidealen Systemen ist FCS fehleranfällig. In dieser Arbeit entwickeln wir mehrere neuartige Realisierungen von FCS, welche diese Fehlerquellen umgehen und die genaue und quantitative Messung dynamischer Parameter in Membranen ermöglichen. Zwei-Fokus FCS mit Kamera-Detektion erlaubt eine genaue und kalibrationsfreie Messung von Diffusionskoeffizienten. Konfokale FCS mit einem Laserscanningmikroskop besitzt eine bislang unerreichte Positionsgenauigkeit, welche uns erstmals dynamische Messungen in Bakterienmembranen mit FCS ermöglichte. Scanning FCS mit einem Scanweg senkrecht zur Membran ermöglicht eine Korrektur von Instabilitäten und damit lange Messzeiten, die zur Bestimmung langsamer Diffusionskoeffizienten notwendig sind. Eine Erweiterung zur kalibrationsfreien Messung von Diffusionskoeffizienten mit Zwei-Fokus Scanning FCS und von Bindungsparametern mit Zwei-Farben Scanning FCS ist einfach. Mit diesen Methoden konnten wir in Systemen messen, die bislang FCS nicht zugänglich waren, so in Hefezellmembranen oder in Membranen lebender Zebrafischembryonen. Line-scan FCS besitzt einen Scanweg parallel zur Membran. Die parallele Messung entlang der ganzen Linie führt zu einer deutlichen Verbesserung der Statistik und damit zu kurzen Messzeiten. Die Kenntnis der Scangeschwindigkeit dient einer internen Kalibration und erlaubt eine akkurate Bestimmung von Diffusionskoeffizienten und Konzentrationen innerhalb weniger Sekunden, kaum beeinflusst vom Bleichen von Fluorophoren. Beide Arten von Scanning FCS können mit einem kommerziellen Laserscanningmikroskop realisiert werden. Häufig kann bei FCS Messungen ein fluoreszierender Hintergrund nicht vermieden werden. Hier ist eine hohe Oberflächenselektivitiät nötig, welche effizient mit einem neuartigen Objektiv erreicht werden kann. Dieses Supercritical Angle-Objektiv erzeugt ein sehr flaches und lateral begrenztes Detektionsvolumen. Eine weitere Methode mit einer ähnlich guten Oberflächenselektivität ist FCS mit Anregung über totale interne Reflektion (TIR-FCS). Bislang war eine quantitative Analyse der TIR-FCS Daten kaum möglich, da keine ausreichend genaue theoretische Beschreibung existierte. In dieser Arbeit entwickeln wir ein akkurates Modell, welches quantitative Messungen mit TIR-FCS erlaubt. Die hier entwickelten neuartgien FCS-Techniken ermöglichen die Untersuchung biologischer Fragestellungen, welche bislang keiner anderen Methode zugänglich sind.

Dans les cellules il existe deux grandes sources d’énergie : l’ATP et la force proton-motrice, produites au niveau du cytoplasme et de la membrane interne respectivement. La mise en place de processus actifs dans la membrane externe ou à la surface des bactéries à Gram négatif requière la présence de machineries protéiques transmettant les forces de leur lieu de production à leur lieu d’utilisation. Durant ma thèse j’ai étudié une de ces machines : la machinerie de motilité (Agl-Glt) de Myxococcus xanthus. Plus précisément, j’ai cherché à comprendre comment les composants de cette machine s’organisent pour permettre le déplacement d’une bactérie. J’ai montré que l’assemblage de la machinerie de motilité au pôle avant des cellules nécessite la formation d’une plateforme cytosolique sur laquelle vient se fixer la machine Agl-Glt. Sous l’action du moteur, le complexe interne de la machine se déplace en direction du pôle arrière en suivant une trajectoire hélicoïdale de main droite. Au niveau de la surface les protéines de membrane externe sont recrutées au niveau d’adhésions focales et permettent l’ancrage de la machinerie au substrat. Enfin, la transmission des forces de la membrane interne à la surface par la machinerie de motilité génère le déplacement des cellules selon une trajectoire hélicoïdale de main gauche. Finalement, cette étude a révélé l’existence d’une machine protéique de l’enveloppe dont l’activité repose sur l’association d’un moteur linéaire et du cytosquelette bactérien. De par l’homologie qu’il existe entre les systèmes il est possible de proposer que ce type de machines peut-être retrouvé associées à d’autres fonctions que la motilité cellulaire. / Two energy sources are present in cells: the ATP and the Proton Motive Force, produced in the cytoplasm and inner membrane respectively. Active processes in the outer membrane or on the surface of Gram negative bacteria require the presence of a proteic machinery to transduce the forces from their production site, in the cytoplasm or inner membrane, to their usage site. During my thesis I have studied one of these machineries: the motility machinery (Agl-Glt) of Myxococcus xanthus. More precisely, I try to understand how the components of this transmembrane machinery interact with each other to promote cell motility. I have shown that the assembly of the motility machinery at the leading pole requires the formation of a cytoplasmic platform onto which the Agl-Glt machinery is going to nucleate. The inner-membrane motor complex moves intracellularly along a right-handed path in the cell and becomes stationary at focal adhesion sites on the surface through the connection of the motor to the outer membrane proteins of the complex. This powers the left-handed helical motion of the bacteria. Finally, this study reveals the existence of a dynamic transmembrane machinery which associates the bacterial cytoskeleton to a linear motor to promote cell movement. The homology between the systems tells us that this type of motor is likely to be found associate with other function than cell motility.

Le suivi des interactions entre protéines, localisées à la membrane plasmique ou à l’intérieur de cellules, a été réalisé au cours de cette thèse par imagerie de fluorescence et par l’analyse de processus dits de FRET (Forster Resonance Energy Transfer). Pour quantifier le FRET entre nos protéines d’intérêt, nous avons choisi le contraste de durée de vie de fluorescence car cette méthode est indépendante de la concentration et de l’intensité de fluorescence. Afin d’obtenir une résolution suffisante pour des problématiques neurobiologiques, un microscope TIRFLIM (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) avait préalablement été développé. Celui-ci nous permet de faire de l’imagerie en plein champ avec une résolution axiale sub-longueur d’onde. Ce dispositif a été calibré et optimisé au cours de cette thèse pour répondre au mieux à des problématiques biologiques. Différentes approches ont ainsi été testées dans le but de calibrer la profondeur de pénétration de l’onde évanescente. Des surfaces plasmoniques ont entre autres été utilisées pour augmenter la sélectivité axiale du montage. Notre microscope a été dédié à l’étude de l’effet du cholestérol sur l’interaction entre la protéine précurseur de l’amyloïde APP, protéine transmembranaire impliquée dans la maladie d’Alzheimer et une de ses enzymes de clivage BACE1. Nous avons ainsi effectué un suivi dynamique de l’effet du cholestérol sur l’interaction entre APP et BACE1 dans des cellules HEK-293 et dans des cultures primaires de neurones d’hippocampe d’embryons de rat, de la membrane plasmique à l’intérieur des cellules grâce à notre dispositif TIRFLIM. La mesure d’anisotropie de fluorescence résolue en temps a également été implémentée sur notre montage. Ces mesures résolues en temps et en polarisation ont permis de mesurer le temps de corrélation rotationnelle de fluorophores et de mettre en évidence de manière qualitative différents niveaux d’homodimérisation de protéines impliquées dans la maladie d’Alzheimer. / In the framework of this thesis, we have used FRET (Forster Resonance Energy Transfer) as a mechanism to follow the interaction of proteins from the plasma membrane to the cytoplasm of cells. To quantify FRET, we have chosen Fluorescence Lifetime Imaging Microscopy (FLIM) since this method is independent of the concentration and intensity of the fluorophores. To have a good axial resolution, a TIRFLIM set-up (Total Internal Reflection Fluorescence Lifetime Imaging Microscopy) was developed and this allowed us to perform wide-field imaging with sub-wavelength axial resolution. This set-up was calibrated and optimized in order to answer biological questions. Different approaches were tested in order to measure the penetration depth of the evanescent field and especially plasmonic surfaces were used to further enhance the axial resolution. Our set-up was dedicated to the study of the effect of cholesterol on the interaction between the Amyloid Precursor Protein (APP), a transmembrane protein involved in Alzheimer Disease, and one of its cleaving enzyme (BACE1). We performed a dynamic tracking of APP and BACE1 proximity under the effect of cholesterol, in HEK-293 cells and primary cultures of embryonic rat hippocampal neurons, thanks to our TIRFLIM set-up.Time-resolved fluorescence anisotropy has been implemented on our set-up. This has enabled us to measure the rotational correlation time of fluorophores and to investigate quantitatively different states of homodimerization of proteins involved in Alzheimer’s disease.

Neste trabalho foi sintetizado o corante (E)-2-[3-[4-(difenilamina)-fenil]-1-(p-tolil)- alilideno]-malononitrila (DFTAM), a partir da reação de condensação entre 4- (difenilamino)-benzaldeído e 2- [1- (4- metilfenil)-etilideno]-malononitrila, com catálise básica de piperidina. O produto obtido foi purificado por cromatografia líquida de alta eficiência (HPLC) e caracterizado pelas técnicas de espectrometria de massas, ressonância magnética nuclear de 13C e 1H e espectroscopia no infravermelho com transformada de Fourier. Para estudar suas propriedades fotofísicas, espectros de absorção e emissão de fluorescência, decaimento de fluorescência e espectro de absorção de transientes foram feitos em diferentes solventes, variando-se a polaridade e viscosidade do meio. Duas bandas de absorção foram observadas, uma em 303 nm e outra em cerca de 490 nm, a qual apresentou deslocamento batocrômico com o aumento da polaridade do solvente. Para essa região de excitação a banda de emissão variou entre 517 e 630 nm, com o aumento da polaridade do meio. Os decaimentos de fluorescência mostraram duas componentes, uma na ordem de picossegundos e a outra de nanossegundos. Os experimentos de absorção de transientes apresentaram três espécies, uma mais longa (maior que 10 ms) e duas outras de cerca 2 e 22 μs. Surfactantes catiônicos, não iônico, e aniônico também foram usados para produzir micelas e fazer os experimentos já citados. Pôde-se observar que o corante interagiu com as micelas, melhorando sua fluorescência e aumentando o tempo de vida do estado singleto. Por fim, acompanhou-se in situ, através da técnica de microscopia TIRF, a reação de formação de DFTAM a nível single molecule com catalise básica de nanopartículas de MgO e lamínulas de vidro funcionalizadas com piperazina. Através da intermitência de fluorescência dos filmes feitos de ambas as amostras, observou-se a formação de moléculas do corante através de ciclos de catálise da piperazina. / In this project the synthesis of (E) -2- [3- [4- (diphenylamine) phenyl] -1- (p-tolyl) - allylidene] -malononitrile (DFTAM) dye, from the condensation reaction between 4- (diphenylamino) benzaldehyde and 2- [1- (4-methylphenyl) ethylidene]-malononitrile using piperidine basic catalysis has been achieved. The dye was purified by high-performance liquid chromatography (HPLC) and characterized by mass spectrometry, nuclear magnetic resonance 13C and 1H and Fourier Transform infrared spectroscopy techniques. To study DFTAM photophysical properties, absorption and fluorescence emission spectra, fluorescence decay and transient absorption spectrum were recorded in solvents with different polarity and viscosity. Two absorption bands of DFTAM were observed, the first one at 303 nm was solvent independent while the second one at about 490 nm, had bathochromic shift with increasing polarity of the medium. In the visible region of excitation the maximum of the dye emission band observed varied between 517 and 630 nm, upon increasing solvent polarity. Fluorescence decays showed two distinct components, a fast one in picosecond time scale and a slow one in nanoseconds. Transient absorption experiments indicated the presence of three species with different lifetimes, one longer than 10 ms and the other two with lifetimes about 2 and 22 μs. Cationic, nonionic, anionic surfactants were also used to produce micelles for easy solubilization of DFTAM. It was observed that the dye interacted with the micelles, improving its fluorescence yield and lifetime. Finally, the DFTAM formation reaction was monitored in situby TIRF wide field microscopy technique at single molecule level. The basic catalysis was tested for MgO nanoparticles and glass surface functionalized with bound piperazine. Through the fluorescence intermittency time trace obtained from TIRF movies, the discrete formation of dye molecules was only observed in the case of piperazine catalytic cycles.

Der zerstörungsfreien Charakterisierung und Analyse von lebenden humanen Zellen über längere Zeiträume kommt zukünftig in Medizin und Biotechnologie eine zentrale Rolle zu. Für eine therapeutische Nutzung müssen die Zellen nach der Analyse unverändert und vital vorliegen. Ein Ansatz beruht auf der Analyse von nanoskopischen Zellrückständen, die von Zellen während der Migration hinterlassen werden, den Zellspuren. Diese spiegeln in repräsentativer Weise die Merkmale der Erzeugerzelle wider. Für die technische Nutzung muss der Entstehungsprozess reproduzierbar kontrolliert werden können. Im Zusammenhang wurde ein Versuchsaufbau zur Beobachtung der dynamischen Prozesse Adhäsion, Migration und substratnahe Organisation des Zytoskeletts von lebenden Zellen entwicklet, der hochauflösende Langzeitbeobachtungen mittels Totaler Interner Reflexions Fluoreszenz (TIRF-) Mikroskopie ermöglicht. Zur Auswertung wurde eine auf Falschfarben beruhende Darstellungsweise der dynamischen Prozesse entwickelt. Es konnte eine Korrelation der Eigenschaften der Zellspuren mit dem Adhäsions- und Migrationsverhalten, sowie dem Aufbau der substratnahen Bereiche des Zytoskeletts der Erzeugerzellen nachgewiesen werden. Ebenso wurde der Einfluss der oberflächenspezifischen Substrateigenschaften (Beschichtung oder topografische Strukturierung) auf die Zellspurablage gezeigt. / Nondestructive characterisation and analysis of human living cells over a long period will be an important issue for medicin and biotechnology in the near future. In order to use the cells after analysis for therapeutical applications, the cells have to be unmodified and still alive after the analytical procedure. The analysis of nanoscopic cell residues, called cell traces, which are left behind during cell migration represent an appropriate approach. Attributes of the donor cell are shown by the cell trace characteristically. In order to use cell traces for biotechnological applications the formation and deposition of cell traces has to be repeatable. Thus, an experimental set up using Total Internal Fluorescence Microscopy (TIRF) has been established to observe the dynamic processes of cell adhesion, cell migration and the organisation of the cytoskeleton used therein. Using miscolours a new embodiment has been developed to evaluate dynamic processes. Cell adhesion, cell migration and the organisation of the actin cytoskeleton of the donor cells have been found to influence the attributes of cell traces. Further specific modified surfaces have been used to influence the deposition of cell traces. Effects have been shown for coated surfaces or surfaces with a topographic structure.

Many genetic processes require enzymes or enzyme complexes that interact simultaneously with distant sites along the genome. Such long-range DNA-enzyme interactions are important for example in gene regulation, DNA replication, repair and recombination. In addition many restriction enzymes depend on interactions between two recognition sites and form therefore a model system for studying long-range communications on DNA. Topic of the present work are Type III restriction enzymes. For these enzymes the communication mechanism between their distant target sites has not been resolved and conflicting models including 3D diffusion, 1D translocation and 1D diffusion have been proposed. Also the role of ATP hydrolysis by their superfamily 2 helicase domains which catalyse functions of many enzyme systems is still poorly understood. To cleave DNA, Type III restriction enzymes sense the relative orientation of their distant target sites and cleave DNA only if at least two of them are situated in an inverted repeat. This process strictly depends on ATP hydrolysis. The aim of this PhD thesis was to elucidate this long-range communication. For this a new single molecule assay was developed using a setup combining magnetic tweezers and objective-type total internal reflection fluorescence microscopy. In addition of being able to mechanically manipulate individual DNA molecules, this assay allows to directly visualize the binding and movement of fluorescently labelled enzymes along DNA. Applying this assay to quantum dot labelled Type III restriction enzymes, a 1D diffusion of the enzymes after binding at their target sites could be demonstrated. Furthermore, it was found that the diffusion depends on the nucleotide that is bound to the ATPase domains of these enzymes. This suggested that ATP hydrolysis acts as a switch to license diffusion from the target site which leads to cleavage. In addition to the direct visualization of the enzyme-DNA interaction, the cleavage site selection, the DNA end influence (open or blocked) and the DNA binding kinetics were measured in bulk solution assays (not part of this thesis). The experimental results were compared to Monte Carlo simulations of a diffusion-collision-model which is proposed as long-range communication in this thesis.

No description available.