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Single-molecule and biochemical characterization of the human myosin isoforms IIb, VI and IXa
Get PDFMyosins are actin-dependent molecular motors which convert chemical energy from ATP hydrolysis into mechanical work. They form a large superfamily but all of them share a common domain structure consisting of three parts: a head, neck and tail region. The catalytic head region is evolutionary conserved and includes a nucleotide-binding site together with an actin-binding site. The head is followed by the neck domain, which binds additional accessory proteins (light chains, e.g. calmodulin) and acts as a lever arm. Finally, the tail region is the most variable domain and regulates cellular functions and dynamics of the motor.
The myosin superfamily interacts with actin filaments to generate a variety of cellular functions including muscle contraction, exo- and endocytosis, cytokinesis and even signal transduction. Twelve different myosin classes have evolved in humans to adapt to their specific cellular function. In this work I selected isoforms from three myosin classes to investigate their biochemical and single-molecule properties. The isoforms IIb, VI and IXa are structurally, biochemically and kinetically tailored to their specific cellular tasks and not much is known about their regulation (e.g. calcium, phosphorylation) to adapt to changing cellular environments or external stimuli.
The aim of the study is to elucidate the molecular roles of the myosin isoforms IIb, VI and IXa and how their structural and regulatory characteristic lead to their specific cellular functions. Besides diverse biochemical methods and functional in vitro assays an optical tweezers transducer was used to study the single-molecule behaviour of the different myosin isoforms. These optical tweezers experiments are able to resolve the nanometre displacements and piconewton forces generated by single motor molecules and allow insights into the basic molecular mechanism of the chemo-mechanical energy transduction cycle.
Myosin IXa is a intriguing molecular motor and is localized in acto-myosin networks at cell-cell adhesions. Recent studies identified myosin IXa as a key requirement for collective cell migration, which is an important mechanism during epithelial morphogenesis, tissue regeneration and cancer metastasis. On a structural level, myosin IXa contains several unique features: The tail domain links this motor to signalling functions as it contains a Rho-GTPase activating domain (RhoGAP) which can alter the dynamics of the actin cytoskeleton. Additionally, the head domain comprises a unique, ∼200 aa insertion in the loop 2 region involved in actin binding. This work clearly demonstrates that this insertion allows myosin IXa to bundle actin filaments into actomyosin networks. Quantitative TIRF microscopy revealed that myosin IXa assembles actin into bundles of up to seven or even more filaments. A collaboration project using transmission electron microscopy combined with single particle image processing showed that the bundles consisted of a highly ordered 2D actomyosin network, with three distinct conformations.
The novelty found in this work is that the bundles were also present at micromolar ATP concentrations and bundle dissociation occurred in a stepwise manner, suggesting a dynamic assembly and disassembly of the actomyosin networks in a cellular context. Furthermore, the presence of a calmodulin-binding site in the unique loop 2 insertion indicates that the motor and bundling activity of myosin IXa is regulated by calcium/calmodulin, which was also in the focus of this work. At the cellular level, the actin lattices might introduce specific tracks for other myosin motors and represent platforms, which allows specific RhoGAP activity from the tail of myosin IXa to be focused to distinct intracellular locations. In contrast to myosin IXa, the non-muscle myosin isoform IIb (NMIIb) is ubiquitously expressed and has key functions in fundamental cellular processes such as cytokinesis, cell migration and control of cell morphology. This double-headed myosin contains a coiledcoil motif in the tail domain, enabling it to form large filaments. In this work I compared the single-molecule properties of single and double-headed human NMIIb to gain insights into the cooperative nature of the myosin heads.
The results indicate that the single myosin heads act in an independent, non-cooperative manner. Furthermore, I show that single and double headed NMIIb produces two types of power strokes: A ∼5 nm working stroke known also from other myosin II isoforms, and unusual close-to-zero attachment events. The results suggest that the latter events might represent transient, weak binding states of myosin to actin. These weak binding interactions could have important cellular relevance to bind and tether the NMIIb myosin filament to actin without the consumption of further ATP, especially as this NMII isoform is known to be involved in tension maintenance and structural anchoring. Myosin VI is the only known member of the myosin superfamily moving towards the minus end of actin filaments. This molecular motor is involved in diverse cellular functions such as vesicle transport, endocytosis and intracellular membrane trafficking.
In this work I investigate the role of myosin head phosphorylation on the mechanical functions of myosin VI. In vitro motility assays from a collaboration partner at the University of Cambridge (UK) have shown that phosphorylation of serine at position 267 had a huge impact on the actin gliding velocity. Using an optical tweezers transducer, I show that the differences observed in the ensemble assay can be explained by tuning the ADP release and ATP binding rate in the actomyosin cycle without a change in the power stroke size. This dramatic effects on the in vitro properties of myosin VI might have pronounced cellular consequences on myosin VI activity in membrane dynamics and cytoskeletal organisation
Auswirkungen einer erneuten Schwangerschaft auf den Blutzuckerverlauf bei Frauen nach Gestationsdiabetes
Get PDFHidden neozoans in macrozoobenthos
Get PDFCraspedacusta sowerbii is a freshwater jellyfish species that has been invading freshwater almost all over the world. In marine environments, which are usually associated with jellyfish, increasing jellyfish observations are related to eutrophication, temperature increases and habitat degradation. Reports of jellyfish observations in freshwater have also been increasing over recent decades. The question arises as to whether population dynamics of freshwater jellyfish are affected by similar factors than observed in marine systems. Difficulties in studying freshwater jellyfish are related to the fact that most scientific interest is focused on the easily visible medusa stage. However, in the complex life cycle of C. sowerbii medusae play only a minor role. The inconspicuous polyp stage is much more important, as it is present throughout the year and is almost exclusively responsible for reproduction. The polyp stage is therefore crucial for a successful invasion of new habitats, and for current and future distribution patterns of this species. In my thesis I investigated the following aspects: (1.) Distribution patterns of medusae and polyps of C. sowerbii, (2.) Factors affecting the growth of polyps of C. sowerbii (chemical environment, such as nitrate and biocides) (3.) Technical aspects of polyp handling during monitoring and experi-mental analyses.
With a “Citizen Science” project and a literature research, recent distribution patterns of C. sowerbii medusae in Germany were revealed and evaluated. Analyses of the distribution patterns show that rivers probably act as important pathways for distribution. To determine how well the observation of easily detectable medusae reflect the “real” distribution of the species, including the polyp stage, the distributions of both life stages were analysed in lakes in Upper Bavaria. The analysis revealed that the polyp stage is approximately twice as abundant as the medusa stage; many more lakes than previously thought are therefore inhabited by this species. Additional comparison of lakes inhabited by only the polyp stage with lakes inhabited by both polyps and medusae show a clear difference in mean alti-tude—lakes inhabited by polyps and medusae are located at considerably lower altitudes. This could reflect a predicted influence of temperature on the development of medusae.
The distribution of polyps of C. sowerbii is affected by environmental parameters. Recently, the im-portance of parameters related to the chemical constitution of freshwater environments has gained increased attention. Nitrate is seen as an abundant and common threat to freshwater organisms; la-boratory experiments on the effect of nitrate on the growth of the polyp population of C. sowerbii were therefore performed. The results of these experiments show that nitrate decreases the growth of polyp populations. This decrease was observable under both acute and chronic exposure to nitrate. A comparison with field data supports the potential importance of nitrate as shown in the laboratory. In two rivers in Upper Bavaria with differing nitrate concentrations, polyp distributions were quantified. In the river with higher nitrate concentrations the number of polyps was much lower than in the river with lower nitrate concentrations.
Additionally, experiments with certain agricultural pesticides known to enter freshwater systems by run-off were performed. Results show that the polyp stage of C. sowerbii is sensitive to at least some pesticides, also potentially affecting species distribution.
As mentioned above, the polyp stage of C. sowerbii is studied less frequently than its medusa stage. Detection in the field of this highly inconspicuous stage is extremely difficult, and needs some experi-ence. Experiments on staining the polyp stage show that neutral red is able to permeate and stain the species, showing a clear visible red colour in the polyp stage. This staining does not seriously harm the polyps and no higher mortality of polyps with staining was observed. After collecting polyps in the field, it is possible to successfully maintain and grow them in the laboratory and use individuals for further experiments. The necessary medium changes during cultivation have no influence on the reproduction of polyps, allowing experiments that need controlled environmental parameters that can only be achieved by regular growth medium changes. During such experiments polyps have to be transferred between culture and experimental environments and injuries of the fragile polyps can occur. However, my results show that almost 100% of the polyps used in experiments regenerated within 24 to 96 hours, even after heavy injuries. This demonstrates the high regeneration capabilities many cnidarian species are known for. My results provide important knowledge for planning and conducting further experi-ments investigating the ecology of the polyp stage of C. sowerbii.
In summary, my results help to understand recent and future distribution patterns of this highly mobile, invasive species. It became clear that the inconspicuous and under-investigated polyp stage of the jelly-fish is key to understanding the dynamics of C. sowerbii. My findings show that C. sowerbii is much more broadly distributed than originally thought from medusa observations. Additionally, temperature plays an important role for medusae production, hence increased abundances, and increasing food web effects, of C. sowerbii medusa with ongoing Global Change can be predicted
Power law signatures of cosmic rays, star formation, and interstellar turbulence
Get PDFPower laws are typical signatures of complex systems and examples in astrophysics are manifold. Understanding their origin is challenging and can be approached in very different ways. In this thesis, I first provide a mathematical treatment of power laws which starts from the defining property of scale-invariance. For physical systems this absence of scales holds only over a finite range. Power laws can be found as solutions to differential equations whereas their validity is usually constrained by initial and boundary conditions which I demonstrate with several examples. An almost physics-free interpretation for the origin of observed mass functions, with ~2, is given by a simple hierarchical fragmentation model which I discuss next to a more physical model of star formation.
Cosmic ray energy spectra are power laws over many orders of magnitude in contrast to exponentially decaying energy distribution functions which are typical for equilibrium statistical mechanichs. Superstatistics is a particular generalization of statistical mechanics for nonequilibrium systems. It generates power law distribution functions from a superposition of equilibrium distributions with variable temperatures. I carefully assess the physical motivation for this model in order to apply it to the observed energy spectra of cosmic rays. This requires a relation between the superstatistical distribution function and the observed differential intensity which has been treated inaccurately by previous studies of superstatistics. Hence, the provided derivation clarifies and improves the theoretical basis of superstatistical models applied to particle physics. I apply this model to recent AMS data for primary (He, C, O) and secondary (Li, Be, B) cosmic rays in order to determine the best fit parameters. Smolla et al. (2020) interpret the two observed universality classes of cosmic ray spectra as resulting from the characteristic energy scale ~ 200 MeV in QCD scattering processes and two distinct types of superpositions of temperature fluctuations. In addition to presenting these results, I also provide a critical discussion for this novel interpretation and the superstatistical model in general.
Interstellar turbulence, stellar initial mass function, star formation law, and far-infrared--radio correlation are examples for nearly universal power laws, in the sense that they are remarkably insensitive to variations in parameters of the respective physical system. For each case I review the available observational data and discuss models which account for their origin. Star formation turns out being an essential driver behind all these phenomena. I present a new schematic version of a galaxy model where thermal gas, turbulent gas, magnetic fields and cosmic rays all have comparable energy densities. The observed power laws characterize the interconnections between these four components and star formation. Due to the complexity and multi-scale nature of the galaxy, this model deliberately ignores many details. Its aim is to identify the self-regulating mechanisms which give rise to the observed power laws and the equipartition of energy.Potenzgesetze sind typische Signaturen von komplexen Systemen und in der Astrophysik gibt es zahlreiche Beispiele dafür. Die Ursachen dafür zu ergründen ist eine herausfordernde Aufgabe, für die es unterschiedliche Herangehensweisen gibt. In dieser Dissertation beginne ich mit einer mathematischen Betrachtung von Potenzgesetzen und der definierenden Eigenschaft der Skalen-Invarianz. Für physikalische Systeme ist diese Abwesenheit von charakteristischen Skalen lediglich in einem begrenzten Bereich erfüllt. Durch mehrere Beispiele zeige ich, dass Potenzgesetze als Lösungen von Differenzialgleichungen auftreten können, wobei ihre Gültigkeit üblicherweise durch die Anfangs- und Randbedingungen bestimmt wird. Für die beobachteten Massefunktionen, mit ~ 2, diskutiere ich ein sehr allgemeingültiges Modell hierarchischer Fragmentation gegenüber einem mehr physikbasierten Modell der Sternentstehung.
Cosmic Rays haben Energieverteilungen welche über viele Größenordnungen durch Potenzgesetze gekennzeichnet sind, im Gegensatz zu den exponentiell abfallenden Verteilungsfunktionen, die in der statistischen Mechanik für Systeme im Gleichgewicht charakteristisch sind. Superstatistics ist eine mögliche Verallgemeinerung der statistischen Mechanik für Nichtgleichgewichts-Systeme. Hier sind die auftretenden Potenzgesetze in den Verteilungsfunktionen das Ergebnis einer Überlagerung von Gleichgewichtsverteilungen mit unterschiedlichen Temperaturen. Ich untersuche die physikalischen Grundlagen dieses Modells, um es auf die beobachteten Energieverteilungen der Cosmic Rays anzuwenden. Dafür ist ein mathematischer Zusammenhang zwischen den beobachteten differentiellen Intensitäten und der aus Superstatistics hergeleiteten Verteilungsfunktion notwendig, was in vorausgehenden Studien fehlerhaft behandelt wurde. Insofern bietet diese Abhandlung eine Verbesserung der theoretischen Grundlagen des Superstatistics-Modells mit Anwendung in der Teilchenphysik. Dieses Modell nutze ich, um damit aus den Beobachtungsdaten von AMS für primäre (He, C, O) und sekundäre (Li, Be, B) Cosmic Rays die besten Fit-Parameter zu bestimmten. Smolla et al. (2020) führen die zwei beobachten Universalitätsklassen an Cosmic Ray Spektren auf die für QCD-Streuprozessen charakteristische Energieskala ~ 200 MeV und zwei unterschiedlichen Arten der Überlagerung von Temperaturfluktuationen zurück. Zu den Ergebnissen dieser Arbeit ergänze ich eine kritische Diskussion dieser neuartigen Interpretation und dem Superstatistics-Modell im Allgemeinen.
Interstellare Turbulenz, die initiale Massenverteilung der Sterne, das Sternentstehungsgesetz, und die Ferninfrarot-Radio Korrelation sind Beispiele für nahezu universelle Potenzgesetze, in dem Sinn, dass sie überraschend schwach auf die Variation der detaillierten Parameter des jeweiligen physikalischen Systems reagieren. Ich fasse jeweils die Beobachtungsdaten zusammen und diskutiere physikalische Modelle, um das Auftreten der Potenzgesetze zu erklären. Die Sternentstehung zeigt sich als ein zentraler Treiber hinter all diesen Phänomenen. Ich präsentiere ein neues schematisches Galaxien-Modell, bei dem thermisches Gas, turbulentes Gas, Magnetfelder und Cosmic Rays vergleichbare Energiedichten haben. Die beobachteten Potenzgesetze kennzeichnen die Kopplungen zwischen diesen vier Hauptkomponenten und der Sternentstehung. Aufgrund der Komplexität und der Bandbreite an physikalischen Skalen in unserer Galaxie, ignoriert dieses Modell viele Details absichtlich. Das Ziel ist auf diese Weise die selbstregulierenden Mechanismen aufzudecken, welche für die beobachteten Potenzgesetze und die Gleichverteilung der Energie verantwortlich sind
