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...

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.

The microtubular cytoskeleton is a ubiquitous and highly diverse biopolymer network present in all eukaryotic cells. Microtubules stochastically alternate between phases of growth and shrinkage. Cells take advantage of this dynamicity to generate forces for essential processes, such as cell division, motility or morphogenesis. Regulating the microtubule dynamics enables cells to adaptively respond to a wide range of tasks and conditions. Molecular mechanisms underpinning the regulation are not fully understood. Using a bottom-up approach and the combination of single molecule total internal reflection fluorescence (TIRF) microscopy and interference reflection microscopy (IRM), we here reconstituted and explored two dynamic cytoskeletal systems. (i) Microtubule doublets, comprising incomplete B-microtubule on the surface of a complete A- microtubule, provide an essential structural scaffold for flagella. Despite the fundamental role of microtubule doublets, the molecular mechanism governing their formation is unknown. We here demonstrate an inhibitory role of tubulin C-terminus in microtubule doublet assembly. By partial enzymatic digestion of polymerized microtubules followed by the addition of free tubulin in the presence of a stabilizing agent, we assembled microtubule doublets and revealed the B-...

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.

Eukaryontische Zellen besitzen ein Zytoskelett, ein zelluläres Netzwerk aus Biopolymeren. Unter diesen Biopolymeren sind die Mikrotubuli weitgehend konserviert. Diese aus Tubulin aufgebauten Filamente sind dynamisch und wechseln zwischen Phasen des Wachstums und der Schrumpfung. Die genauen Mechanismen, die die dynamische Instabilität der Mikrotubuli bestimmen, werden noch erforscht. Die Allgegenwart von Mikrotubuli wirft die Frage auf, wie sie in verschiedenen thermischen Umgebungen konservierte Funktionen ausführen können. Um dieser Fragestellung nachzugehen, habe ich verwandte Froscharten mit unterschiedlich temperierten Lebensräumen untersucht: Xenopus laevis (16-22 °C), Xenopus borealis (19-23 °C) und Xenopus tropicalis (22-30 °C). Um zu untersuchen, ob sich die biochemischen Eigenschaften von Tubulin und die Dynamik der Mikrotubuli bei den drei Arten an die Temperatur angepasst hat, habe ich die Methoden der Tubulin-Affinitätsreinigung und die temperaturgesteuerte TIRF-Mikroskopie zur Rekonstitution der Mikrotubuli-Dynamik kombiniert. Dabei habe ich festgestellt, dass bei einer Temperatur von 25°C die Wachstumsgeschwindigkeit der Mikrotubuli im Bezug zur thermischen Nische der einzelnen Arten negativ korreliert. Die Verwendung der Arrhenius-Gleichung zum Vergleich der Aktivierungsenergie der Mikrotubuli-Polymerisation für jede Spezies ergab, dass die freie Energie des Tubulins umso höher ist, je kälter die thermische Nische der Spezies ist. Die Mikrotubuli von X. laevis und X. borealis zeigten eine längere Lebensdauer und wurden häufiger zerstört als die von X. tropicalis. Die Tubuline von X. laevis und X. borealis sind phosphoryliert, im Gegensatz zu X. tropicalis. Die Ergebnisse zeigen, dass sich Xenopus Tubulin und die Dynamik der Mikrotubuli an die Temperatur angepasst haben. Kalt lebende Arten kommen mit der niedrigeren Energie des Milieus zurecht, durch verbessertes Wachstum und Stabilität. / Eukaryotic cells hold a cytoskeleton, a cellular network of biopolymers. Among the filaments of the cytoskeleton, microtubules are widely conserved. Built from tubulin, those filaments are dynamic, alternating between phases of growth and shrinkage. The biochemical properties of tubulin shape the dynamic behavior of microtubules, which is crucial for many cellular processes. The precise mechanisms determining microtubule dynamic instability are still under investigation. The ubiquity of microtubules raises the question of how they can perform conserved functions within various thermal environments. To address this, I turned to closely related frog species living at different temperatures, Xenopus laevis (niche: 16-22°C), Xenopus borealis (19-23°C) and Xenopus tropicalis (22-30°C). To probe whether the biochemical properties of tubulin and microtubule dynamics adapted to temperature across those three species, I combined tubulin affinity purification and temperature-controlled TIRF microscopy of in vitro reconstitution of microtubule dynamics. I found that at 25°C, the microtubule growth velocity inversely correlates with the thermal niche of each species. Adjusting temperature to each species’ endogenous condition modulates the growth rate differences across species. Using the Arrhenius equation to compare the activation energy of microtubule polymerization for each species suggested that the colder the thermal niche of the species, the higher the free energy of its tubulin. Microtubules from the cold-adapted species X. laevis and X. borealis have longer lifetimes and rescue more often than those of X. tropicalis, both at 25°C and at each species’ endogenous condition. X. laevis and X. borealis tubulins are phosphorylated, contrary to X. tropicalis. My results show that Xenopus tubulin and microtubule dynamics have adapted to temperature. Cold-living species cope with the lower energy of the milieu by facilitating growth and stability.

Movement of motor proteins along cytoskeletal filaments is fundamental for various cellular processes ranging from muscle contraction over cell division and flagellar movement to intracellular transport. Not surprisingly, the impairment of motility was shown to cause severe diseases. For example, a link between impaired intracellular transport and neurodegenerative diseases, such as Alzheimer’s, has been established. There, the movement of kinesin-1, a neuronal motor protein transporting vesicles along microtubules toward the axonal terminal, is thought to be strongly affected by roadblocks leading to malfunction and death of the nerve cell. Detailed information on how the motility of kinesin-1 deteriorates in the presence of roadblocks and whether the motor has a mechanism to circumvent such obstructions is scarce. In this thesis, kinesin-1 motility was studied in vitro in the presence of rigor kinesin-1 mutants, which served as permanent roadblocks, under controlled single-molecule conditions. The 25 nm wide microtubule track, consisting of 13 individual protofilaments, resembles a multi-lane environment for transport by processive kinesin-1 motors. The existence of multiple traffic-lanes, allows kinesin-1 to utilize different paths for cargo transport and potentially also for the circumvention of roadblocks. However, direct observation of motor encounters with roadblocks has been intricate in the past, mainly due to limitations in both, spatial and temporal resolution. These limitations, intrinsic to fluorescent probes commonly utilized to report on the motor positions, originate from a low rate of photon generation (low brightness) and a limited photostability (short observation time). Thus, studying kinesin-1 encounters with microtubule-associated roadblocks requires alternative labels, which explicitly avoid the shortcomings of fluorescence and consequently allow for a higher localization precision. Promising candidates for replacing fluorescent dyes are gold nanoparticles (AuNPs), which offer an enormous scattering cross-section due to plasmon resonance in the visible part of the optical spectrum. Problematic, however, is their incorporation into conventionally used (fluorescence) microscopes, because illumination and scattered light have the same wavelength and cannot be separated spectrally. Therefore, an approach based on total internal reflection (TIR) utilizing a novel parabolically shaped quartz prism for illumination was developed within this thesis. This approach provided homogenous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, single-molecule fluorescence as well as single-particle scattering were detectable with high signal-to-noise ratios. Importantly, AuNPs with a diameter of 40 nm provided sub-nanometer localization accuracies within millisecond integration times and reliably reported on the characteristic 8 nm stepping of individual kinesin-1 motors moving along microtubules. These results highlight the potential of AuNPs to replace fluorescent probes in future single-molecule experiments. The newly developed parabolic prism-type TIR microscope is expected to strongly facilitate such approaches in the future. To study how the motility of kinesin-1 is affected by permanent roadblocks on the microtubule lattice, first, conventional objective-type TIRF microscopy was applied to GFP-labeled motors. An increasing density of roadblocks caused the mean velocity, run length, and dwell time to decrease exponentially. This is explained by (i) the kinesin-1 motors showing extended pausing phases when confronted with a roadblock and (ii) the roadblocks causing a reduction in the free path of the motors. Furthermore, kinesin-1 was found to be highly sensitive to the crowdedness of microtubules as a roadblock decoration as low as 1 % sufficed to significantly reduce the landing rate. To study events, where kinesin-1 molecules continued their runs after having paused in front of a roadblock, AuNPs were loaded onto the tails of the motors. When observing the kinesin-1 motors with nanometer-precision, it was interestingly found that about 60 % of the runs continued by movements to the side, with the left and right direction being equally likely. This finding suggests that kinesin-1 is able to reach to a neighboring protofilament in order to ensure ongoing transportation. In the absence of roadblocks, individual kinesin-1 motors stepped sideward with a much lower, but non-vanishing probability (0.2 % per step). These findings suggest that processive motor proteins may possess an intrinsic side stepping mechanism, potentially optimized by evolution for their specific intracellular tasks.

Movement of motor proteins along cytoskeletal filaments is fundamental for various cellular processes ranging from muscle contraction over cell division and flagellar movement to intracellular transport. Not surprisingly, the impairment of motility was shown to cause severe diseases. For example, a link between impaired intracellular transport and neurodegenerative diseases, such as Alzheimer’s, has been established. There, the movement of kinesin-1, a neuronal motor protein transporting vesicles along microtubules toward the axonal terminal, is thought to be strongly affected by roadblocks leading to malfunction and death of the nerve cell. Detailed information on how the motility of kinesin-1 deteriorates in the presence of roadblocks and whether the motor has a mechanism to circumvent such obstructions is scarce. In this thesis, kinesin-1 motility was studied in vitro in the presence of rigor kinesin-1 mutants, which served as permanent roadblocks, under controlled single-molecule conditions. The 25 nm wide microtubule track, consisting of 13 individual protofilaments, resembles a multi-lane environment for transport by processive kinesin-1 motors. The existence of multiple traffic-lanes, allows kinesin-1 to utilize different paths for cargo transport and potentially also for the circumvention of roadblocks. However, direct observation of motor encounters with roadblocks has been intricate in the past, mainly due to limitations in both, spatial and temporal resolution. These limitations, intrinsic to fluorescent probes commonly utilized to report on the motor positions, originate from a low rate of photon generation (low brightness) and a limited photostability (short observation time). Thus, studying kinesin-1 encounters with microtubule-associated roadblocks requires alternative labels, which explicitly avoid the shortcomings of fluorescence and consequently allow for a higher localization precision. Promising candidates for replacing fluorescent dyes are gold nanoparticles (AuNPs), which offer an enormous scattering cross-section due to plasmon resonance in the visible part of the optical spectrum. Problematic, however, is their incorporation into conventionally used (fluorescence) microscopes, because illumination and scattered light have the same wavelength and cannot be separated spectrally. Therefore, an approach based on total internal reflection (TIR) utilizing a novel parabolically shaped quartz prism for illumination was developed within this thesis. This approach provided homogenous and spatially invariant illumination profiles in combination with a convenient control over a wide range of illumination angles. Moreover, single-molecule fluorescence as well as single-particle scattering were detectable with high signal-to-noise ratios. Importantly, AuNPs with a diameter of 40 nm provided sub-nanometer localization accuracies within millisecond integration times and reliably reported on the characteristic 8 nm stepping of individual kinesin-1 motors moving along microtubules. These results highlight the potential of AuNPs to replace fluorescent probes in future single-molecule experiments. The newly developed parabolic prism-type TIR microscope is expected to strongly facilitate such approaches in the future. To study how the motility of kinesin-1 is affected by permanent roadblocks on the microtubule lattice, first, conventional objective-type TIRF microscopy was applied to GFP-labeled motors. An increasing density of roadblocks caused the mean velocity, run length, and dwell time to decrease exponentially. This is explained by (i) the kinesin-1 motors showing extended pausing phases when confronted with a roadblock and (ii) the roadblocks causing a reduction in the free path of the motors. Furthermore, kinesin-1 was found to be highly sensitive to the crowdedness of microtubules as a roadblock decoration as low as 1 % sufficed to significantly reduce the landing rate. To study events, where kinesin-1 molecules continued their runs after having paused in front of a roadblock, AuNPs were loaded onto the tails of the motors. When observing the kinesin-1 motors with nanometer-precision, it was interestingly found that about 60 % of the runs continued by movements to the side, with the left and right direction being equally likely. This finding suggests that kinesin-1 is able to reach to a neighboring protofilament in order to ensure ongoing transportation. In the absence of roadblocks, individual kinesin-1 motors stepped sideward with a much lower, but non-vanishing probability (0.2 % per step). These findings suggest that processive motor proteins may possess an intrinsic side stepping mechanism, potentially optimized by evolution for their specific intracellular tasks.