Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 05/06 Archiv

Bacteria necessitate multiple signal transduction systems to sense the ever-changing environments and mediate the cellular response accordingly. The major bacterial signal transduction systems are one-component system (1CS), two-component system (2CS) and extracytoplasmic function (ECF) σ factor. Compared to 1CSs and 2CSs, ECF σ factors have only been identified much later and therefore the knowledge about their molecular mechanisms and physiological roles is less profound. This thesis mainly focuses on the study of ECF σ factors from the bacterial phyla, Planctomycetes and Actinobacteria.

Polarity is a fundamental feature of almost all cells. It generally refers to the asymmetric organization of several cellular components. The plasma membrane, for example, exhibits both a transbilayer and a lateral asymmetry in most eukaryotic cells. Lipids are asymmetrically distributed between the cytoplasmic and the extracellular leaflet of the membrane and segregate laterally together with specific proteins to form dynamic nanoscale assemblies, known as rafts. Polarity can also specifically describe the asymmetric distribution of key molecules within a cell. These molecules, known as polarity determinants, can orient a multitude of specialized cellular functions, such as cell shape, cell division and fate determination. In the framework of this thesis, we aimed to reconstitute essential features of membrane unmixing and cell polarity with a "bottom-up" synthetic biology approach. We worked with both: pure lipid systems, whose unmixing is driven by the asymmetric distribution of lipids in the two leaflets, and a lipid-protein system, whose polarization is instead due to reaction-diffusion mechanisms. In both cases, we used Giant Unilamellar Vesicles (GUVs) and Sup- ported Lipid Bilayers (SLBs) to model biological membranes and employed modern biophys- ical techniques, such as fluorescence correlation spectroscopy, to quantitatively characterize lipid bilayers and protein-lipid interactions. In the pure lipid systems, we first reconstituted membrane transbilayer asymmetry, applying a cyclodextrin-mediated lipid exchange method, which enables us to enrich membranes with lipids of choice. The enrichment of the membrane with sphingomyelin and/or cholesterol triggers the segregation of lipids into two coexisting asymmetric phases both in SLBs and GUVs, whereas exchanging different amounts of phosphatidylglycerol with the outer leaflet of the GUV membranes controls vesicle shape. Tuning the lipid content of model membranes revealed that small changes in the composition of one leaflet affect the overall lipid miscibility of the bilayer and that membrane shape transformations are possible also in absence of a protein machinery and as a consequence of the lipid redistribution in the membrane. In the protein-lipid system, we aimed to reconstitute a minimal polarization system inspired by the C. elegans embryo at one-cell stage, which polarize along the anterior-posterior axis by sorting the PARtitioning defective (PAR) proteins into two distinct cortical domains. In this system polarity is maintained by the mutual inhibition between anterior (aPARs: PAR-3, PAR-6 and PKC-3) and posterior (pPARs: PAR-1, PAR-2 and LGL-1) PARs, which reciprocally antagonize their binding to the cortex, mutually excluding each other. We focused on LGL-1, which acts directly on PAR-6. Submitting LGL-1 to model membranes allowed us to identify a conserved region of the protein that binds negatively-charged membranes and to determine its lipid binding affinity and specificity. Selected LGL-1 mutants were then gen- erated to better understand the electrostatic mechanism involved in the membrane binding. LGL-1 was finally combined with PKC-3 to generate a functional membrane binding switch.

Adulte Weichgewebesarkome (engl. soft tissue sarcoma; STS) werden zu einer Gruppe seltener maligner und teilweise aggressiver Tumoren klassifiziert, die eine Tendenz zur Bildung von hämatogenen Fernmetastasen aufweisen. Die Kombination der Regionalen Hyperthermie mit einer Chemotherapie erwies sich in vorangegangenen Studien als eine vielversprechende Behandlungsoption beim lokalisierten Hochrisiko STS. Es wurde gezeigt, dass eine neoadjuvante Chemotherapie mit Regionaler Hyperthermie bei diesen Sarkomen das Tumoransprechen, das lokale progressionsfreie und das krankheitsfreie Überleben im Vergleich zu einer alleinigen Chemotherapie signifikant verbessert. Auf zellulärer Ebene induziert ein Hitzeschock (HS) bei klinisch relevanten Temperaturen (41,8°C/43°C) unter anderem eine temporäre Defizienz der Homologen Rekombinationsreparatur (HR), einem essentiellen Mechanismus für die fehlerfreie Reparatur von DNA-Doppelstrangbrüchen (DSB). Dies steht im Zusammenhang mit einer hitzeinduzierten proteosomalen Degradierung von BRCA2, einer unerlässlichen Komponente der HR. Trabectedin (Tr) ist eine antiproliferativ wirksame Substanz, die ursprünglich aus dem marinen Tunikat Ecteinascidia turbinata isoliert wurde. Die vielfältigen zytotoxischen Aktivitäten von Tr umfassen neben dem Interferieren mit der aktivierten Transkription und der Modulation der Tumor-Mikroumgebung hauptsächlich die Induktion von DSBs. Seit 2007 wird Tr in der Zweitlinientherapie zur Behandlung refraktärer STS, sowie bei Patienten eingesetzt, bei denen die Erstlinientherapie (Ifosfamid und/oder Doxorubicin) nicht angewendet werden kann. In Anbetracht der hitzeinduzierten Inaktivierung von BRCA2 und den DNA schädigenden Eigenschaften von Tr wurde in dieser Arbeit untersucht, ob und wie die Hyperthermie zu einer Wirkungsverstärkung der zytotoxischen Effekte von Tr beitragen kann. Tr bewirkt in vitro bei Zelllinien unterschiedlicher Sarkomentitäten (U2Os, SW872, SW982) eine dosisabhängige Reduktion des klonogenen Überlebens, das durch einen HS zusätzlich verstärkt wird. Die erhöhte antiproliferative Aktivität von Tr nach einem HS wird als thermale Chemosenitivierung definiert. Zudem konnte durch die Analyse der DNA-Verteilung bei U2Os und SW872 Zellen eine Intensivierung und Verlängerung der Tr-induzierten G2/M-Blockade nachgewiesen werden. Darüber hinaus wurden Zelllinien-spezifische Unterschiede bezüglich einer behandlungsinduzierten Apoptoseinduktion oder Senseszenzantwort identifiziert. SW872 Zellen weisen einen dosis- und temperaturabhängigen Anstieg des Anteiles apoptotischer Zellen auf, der mit einer starken Aktivierung der Effektorcaspasen 3 und 7 einhergeht. Dem entgegen gehen U2Os Zellen in eine ausgeprägte behandlungsinduzierte zelluläre Seneszenz über. Anhand der quantitativen Analyse Tr-induzierter H2AX Foci hat sich ein relevanter Anstieg an DSBs durch eine zusätzliche Hitzeexposition herausgestellt, der eine Beeinträchtigung der BRCA2-vermittelten vollständigen Assemblierung der DNA-Reparaturfoci vermuten lässt. Die Hypothese einer thermalen Chemosensitivierung gegenüber Tr durch eine hitzeinduzierte HR-Defizienz – insbesondere im Rahmen der hitzeinduzierten BRCA2 Degradierung – wurde zudem durch das Ausbleiben der hitzebedingten Verstärkung der Tr-induzierten Zytotoxizität bei BRCA2-defizienten Zellen bekräftigt. Darüber hinaus wurde durch Hochdurchsatzanalysen bestätigt, dass eine hitzevermittelte, erhöhte antiproliferative Aktivität von Tr nach einem Knockdown zahlreicher HR-spezifischer Komponenten ausbleibt. Durch Hochdurchsatzanalysen sowie durch anschließende Validierungsexperimente wurden Proteine identifiziert, die sich als relevant für weitere präklinische und klinische Untersuchungen herausgestellt haben. Die Proteine BRCA1, PARP1 und CHEK1 stellen dabei potentielle molekulare Marker für ein Tumoransprechen auf die Kombinationstherapie von Tr und Hyperthermie dar. Deren Inhibition erwies sich zudem als eine weitere Strategie, um die Effektivität der ursprünglichen Behandlung zusätzlich zu erhöhen. Darüber hinaus wurde die Funktion von FANCD2 als prädiktiver Marker und von ERCC1 als Resistenzmarker für das Therapieansprechen einer alleinigen Tr-Behandlung in vitro bestätigt. Die herausgearbeitete thermale Chemosensitivierung gegenüber Tr mit Hyperthermie durch die induzierte HR-Defizienz mittels passagerer BRCA2 Degradierung (induzierte synthetische Letalität) sowie die Identifizierung weiterer Proteine, deren medikamentöse Inhibition die Effektivität der Kombinationsbehandlung zusätzlich erhöhen könnte, eröffnen neue Möglichkeiten in der Therapie solider Tumoren.

The phylotypic stage, as part of the embryonic period, is the stage where embryos of different species of a phylum show a high degree of similarity. Johann Friedrich Meckel, Karl Ernst von Baer and Ernst Haeckel already described it for vertebrates in the 19th century. They observed that vertebrate embryos pass through a period of morphological similarity. Since then, scientists have researched the field of the phylotypic stage and it was subject of many controversial discussions. The name “phylotypic stage” was coined by Klaus Sander in 1983 and describes not only the stage of the highest similarity but also the stage, typical (characteristic) for a phylum. The following study examines the phylotypic stage of zebrafish (Danio rerio). Looking at different conserving mechanisms like internal constrains and stabilizing selection, different hypothesis and concepts by several researchers were tested. To test if the phylotypic stage is accessible to selection (although it generally is considered a conserved evolutionary stage) I have studied patterns of variation during embryogenesis. I have looked at the phenotypic variance and the number of significant correlations among embryonic traits and described the phylotypic stage as a period characterized by a high number of internal correlations and declining phenotypic variance. Then, I tested if changes in the raising conditions could elicit phenotypic changes. Therefore, zebrafish embryos have been raised under different experimental conditions to see if developmental plasticity can be induced during the early developmental period and if clearly defined modules can be identified. Eggs of zebrafish were raised in: (1) different temperatures; (2) different salinities; and (3) different levels of oxygen concentration. Up to 14 characters of individual embryos were measured during early development, encompassing the phylotypic stage. In particular I found a considerable degree of heterochrony and modularity. Embryos grew slower at lower temperatures and lower oxygen levels. Plasticity was detected in the overall size of the embryo and the size of somites in the oxygen and temperature experiment. The development of the eye and otic vesicle was shifted to a later x stage under severe hypoxia. Thus, eye and otic vesicle could be identified as modules, which can be dissociated from other characters of the developing embryo (heterochrony). Changes in raising condition affect early development of the zebrafish on three levels: (1) developmental rate (2) size and shape, and (3) dissociation of modules. Thus, plasticity and modularity are effective during early embryonic development. Finally I studied the heritability of embryonic traits to examine how inheritance contributes to the stabilization of the phylotypic stage in variable environments. Following the heritabilities of certain traits reveals that the phylotypic stage is not characterized by a certain pattern of decreased heritability and thus decreased additive genetic variance. The results suggest that the phylotypic stage of zebrafish is constrained by multiple internal correlations when embryos are developing in standard conditions. However, under marginal developmental conditions so far ineffective modules become effective and buffer the embryo against disruptive effects of the environment. Patterns of family resemblance are present, indicating an inherited genetic portion of the phylotypic stage. However, under strong environmental influence it is dominated by variation associated with phenotypic plasticity. My general conclusion is that the phylotypic stage is not established because additive genetic variance is exhausted during the early period of vertebrate development but that it is under environmental and genetic influence, thus is accessible to selection. Internal constraints could be identified to stabilize morphology during the phylotypic stage, but a certain degree of phenotypic variation can be observed.

Labile characters, like behaviors, are phenotypes that are expressed repeatedly in the life of an individual. These types of characters allow individuals to adjust their phenotype to various levels of environmental variation, and therefore play a key role in the evolutionary process. Labile phenotypes are distinct because of their multi-level nature; individuals can differ in their average phenotypic expression (causing among-individual variation), but they can also vary their phenotype in each expression (causing within-individual variation). In order to understand the role of labile characters in the evolutionary process it is necessary to acknowledge that variation at each level is caused by different processes. Variation at the among-individual level is caused by genetic or environmental differences having a permanent effect on an individual’s phenotype, whereas variation at the within-individual level is caused by an individual’s adjustment of its phenotype to a changing environment. The implications of these multi layered effects in the expression of labile characters have been acknowledged by different fields of evolutionary ecology, but major areas of evolutionary research do not fully incorporated this idea. The general aim of my thesis was to fully integrate this multi-level nature in the study of the adaptive causes and evolutionary consequences of variation in labile characters. My thesis is composed of five chapters: the first three are conceptual and methodological works aimed at integrating the multi-level nature of labile characters into already existing evolutionary frameworks. The last two chapters describe, as a worked example, how the different levels of variation and covariation between (labile) fertilization related traits affect the evolution of the alternative reproductive strategies in a wild passerine bird (the great tit). The first chapter is a conceptual work focusing on how to define and statistically characterize behavioral characters. We argue that behavioral characters can be studied using the “evolutionary character concept”. This framework was developed to study characters that only vary among individuals (i.e. “fixed characters”); therefore we extended this framework to include characters that also vary within-individuals. The second chapter of the thesis is a methodological work where we proposed a way to quantify multi-level variation in reaction norms, which allows the estimation of repeatability of plasticity. Behavioral ecologists have recently developed theory predicting the ecological conditions where repeatable vs. non-repeatable variation in phenotypic plasticity should evolve. However, there was no methodological framework to estimate repeatability of plasticity. Therefore, we proposed a study design and mixed effect model structure to estimate repeatability of plasticity. To help researchers use the proposed methodology, we developed an R simulation package to estimate bias, precision and accuracy for different sampling designs. The third chapter is an opinion paper that urges researchers to combine theory and methods developed in behavioral ecology and quantitative genetics to study phenotypic variation in a social context. Quantitative geneticists have developed a framework to study social evolution aimed at predicting the evolutionary response to selection of traits affected by the phenotypes of other individuals (the “social environment”). Phenotypes expressed in a social context, also called interactive phenotypes, exhibit a particular evolutionary dynamic because their environmental component is composed of genes and can thus evolve. Despite that fact that the effects of the social environment are commonly mediated by labile characters, this social evolution framework has not fully considered the multi-level nature of labile characters. Therefore, for chapter three we integrated the multi-level nature of labile characters in this social evolution framework. The final two chapters focus, as a worked example, on within-pair and extra-pair reproductive behavior in great tits. For these chapters, we utilized the theoretical and methodological developments of the previous chapters to study the sources of evolutionary constraints on alternative fertilization routes in male great tits. One of the chapters has a more evolutionary perspective, while the other applies a more behavioral ecology view point. In chapter four we studied male extra-pair and within-pair reproduction as interactive phenotypes that are affected by the phenotypes of both the male and the female member of great tit breeding pairs. We showed that male fertilization strategies depend heavily on the phenotype of their female. This social environment effect should influence the evolutionary response to selection of male fertilization strategies, and could partly explain evolutionary stasis, observed in natural populations, in traits so closely linked to fitness. In chapter four we also studied whether trade-offs among- or within-individuals can constrain the phenotypic evolution of male alternative reproductive strategies. We showed that among-male trade-offs between within-pair and extra-pair reproduction could also be a source of evolutionary constrain. In chapter five, we corroborated the existence of trade-offs between alternative reproductive routes by studying whether within-pair and extra-pair fertilizations are obtained at the same time, allowing for the possibility of a trade-off between the two. We found that a male's extra-pair fertilization success is actually higher when it constrains his ability to secure within-pair fertilizations. This result is consistent with our finding that there is indeed a trade-off between extra-pair and within-pair reproduction in this species. The empirical works in this thesis highlight the importance of the social environment as a source of phenotypic variation in the expression of labile traits. But more generally, from the works in this thesis, we can conclude that to fully understand the role of labile characters in the evolutionary process it is necessary to acknowledge their multi-level nature.

Membrane remodeling is a dynamic process that occurs in bacterial cells to facilitate substrate transport and to provide protection to bacteria during environmental stress. In eukaryotic cells, membrane remodeling is carried out by dynamin-like proteins (DLPs). These proteins are involved in diverse membrane-associated functions such as cargo transport via vesicles, cytokinesis, division of cell organelles and resistance to pathogens. DLPs are also conserved in bacterial species; however, their function is still not clearly understood. The genome of B. subtilis contains a gene dynA (ypbR), which encodes a large DLP (136 KDa),DynA, that can tether membranes and induce membrane fusion in vitro. Deletion of dynA in B. subtilis strain 168 fails to produce any observable growth phenotype under standard laboratory conditions. B. subtilis is a soil bacterium and prey to several environmental stress factors to which laboratory strains are normally not exposed. Hence, it was conceivable that DynA might be required when bacteria are exposed to stress. To address this hypothesis, the behavior of DynA was examined under conditions causing membrane-stress, such as exposure to antibiotics and phage infection. A strain lacking dynA showed impaired growth in the presence of sublethal amounts of antibiotics that target the cell membrane and was more sensitive to phage infection compared to wild-type strains. Time-lapse microscopy and fluorescence loss in photobleaching (FLIP) experiments showed that ΔdynA cells have compromised membrane remodeling compared to wild-type strain. In conclusion, all results propose DynA to play a role in protecting the cell membrane under stress conditions. Also, for the first time, it is shown that a bacterial DLP contributes to innate immunity of bacteria. DynA not only has a unique membrane protection function but also distinctive structural features. A single DynA polypeptide contains two dynamin-like subunits, each consisting of a GTPase domain and a dynamin-like stalk region. Both subunits, D1 and D2, share strong intra-molecular cooperativity to facilitate GTPase activity. Here, a combination of mutational analysis and subsequent in vivo and in vitro investigation was applied to further characterise structural assembly and biochemical properties of DynA. Size-exclusion chromatography elucidated that DynA dimerisation requires C-terminal amino acids 591-620. In addition, in vivo localisation, in vitro lipid-binding and GTPase analysis revealed arginine at position 512 of DynA to be a key regulator of GTP hydrolysis as well as lipid-binding. Furthermore, in vivo localisation and bacterial two-hybrid experiments were employed to confirm interaction of DynA with putative interaction partners (YneK, YwpG and YmdA). YneK was found to interact with D1 and YwpG with D1 and D2 individually, whereas YmdA required a full-length DynA (D1+D2) for interaction. Taken together, the results presented here greatly expand on current knowledge regarding functional, biochemical and structural properties of a bacterial dynamin-like protein (BDLP). This thesis not only demonstrates the preserved membrane remodeling function of DLPs in bacteria but also explain their conservation from bacteria to higher-organisms.

In all cellular systems, the transmission of bulk genetic information during proliferation occurs in the form of chromosomes. The segregation of these entities upon cell division is of pivotal importance for all forms of life. Structural maintenance of chromosomes (SMC)-kleisin complexes are ubiquitous and essential factors that ensure proper organisation and segregation of the genetic material. Aim of this work was to elucidate evolutionary conserved features in the architecture of SMC-kleisin complexes, and to probe these features for functional relevance. We find that two major architectural themes have been constrained by evolution: (I) SMC-kleisin complexes form asymmetric assemblies with a ring-like topology, whereby a kleisin monomer bridges two different binding sites on a SMC dimer, (II) SMC-kleisin complexes form rod-like structures, whereby the SMC proteins of a given dimer are closely juxtaposed in a well-defined manner. Based on these findings, we propose that SMC-kleisin complexes from all domains of life act by a unifying mechanism.