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

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 E

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

pheno

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 tw