17 search hits
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Regulated intramembrane proteolysis in the control of the Bacillus subtilis anti-sigma factor RsiW.
(2010)
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Janine Heinrich
- The activity of the extracytoplasmic function (ECF)-sigma factor SigW of the Gram-positive soil bacterium Bacillus subtilis is modulated by a specific membrane-bound anti-sigma factor (RsiW). Initiated most likely by cell wall stress, RsiW is degraded by the mechanism of regulated intramembrane proteolysis (RIP). This process involves two site-specific proteolytic cleavage events in the extracytoplasmic part (site-1) and in the transmembrane domain (site-2) of RsiW. In consequence, SigW is released to interact with the RNApolymerase, and the transcription of SigW-controlled genes is initiated. In general, regulation of differential gene expression by RIP seems to play a major role in prokaryotic stress responses, pathogenesis and antibiosis. However, in most cases the molecular basis is not understood. The main objective of this work was to use B. subtilis SigW/ RsiW as a model to investigate the mechanism of RIP in detail. In particular, there are significant differences to the inducing stress-signal and the site-1 protease that are described for the well investigated Escherichia coli ECF sigma factor SigE-system. The basis of this work were different mutants with a defect in RIP of RsiW that were isolated in a transposon screen. First, PrsW (formerly YpdC) was identified as the site-1 protease. It belongs to a superfamily of potential membrane embedded metalloproteases (MEM) with so far unknown function in bacteria. Further characterization of PrsW in a reconstituted E. coli system showed that PrsW cleaves RsiW in a site-specific manner to form a protein truncated for 40 C-terminal extracytoplasmic amino acid residues. The tail specific protease Tsp was shown to further degrade the extracytoplasmic part of this RsiW site-1 cleavage product, which is crucial for subsequent RasP catalyzed site-2 clipping. Several other peptidases seem to be involved in trimming of RsiW downstream of PrsW and upstream of RasP in B. subtilis. Second, the transposon screen revealed that a defect of the ABC-transporter EcsAB impairs RsiW site-2 cleavage by RasP for unknown reasons. It is conceivable that an EcsAB substrate competitively inhibits RasP activity. In summary, a new model of two proteolytic modules involved in intramembrane proteolysis of RsiW could be established. Each module consists of a site-specific processing peptidase (site-1: PrsW, site-2: RasP) that subjects cleaved RsiW to degradation by unspecific proteases (site-1: Tsp-like, site-2: Clp-proteases).
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Molekulare und physiologische Charakterisierung des Energiestoffwechsels von Nitrosomonas europaea
(2010)
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Stefan Gilch
- Die lösliche Ammoniak-Monooxygenase (AMO) aus Nitrosomonas europaea ist in ungefähr der gleichen Menge wie die membrangebundene AMO vorhanden. Beide Formen sind in vivo aktiv und besitzen eine ähnliche spezifische Aktivität. Die lösliche AMO ist ein Cu-, Fe- und möglicherweise auch Zn-haltiges Enzym und besteht aus den Untereinheiten AmoA (27 kDa), AmoB (42 kDa) und Cytochrom c1 (24 kDa). Das Enzym weist dabei eine heterotrimere Alpha3-Beta3-Gamma3-Untereinheitenstrukur mit einer molekularen Masse von etwa 316 kDa auf. Die AmoB-Untereinheit der löslichen AMO besitzt dabei eine Signalsequenz von 25 Aminosäuren. Die An- beziehungsweise Abwesenheit des Signalpeptides könnte die Konformation der AMO so weitreichend beeinflussen, dass daraus sowohl ein löslicher als auch ein membrangebundener AMO-Komplex resultiert. Das gereinigte Enzym koordiniert in vitro 9,5 Cu-, 3,9 Fe- und 0,45 bis 2,6 Zn-Atome. Nach Reduktion der löslichen AMO werden im Absorptionsspektrum die für Cytochrome des c-Typs charakteristischen Absorptionsbanden ersichtlich. Elektronspinresonanz-Spektren von luftoxidierter löslicher AMO bei einer Temperatur von 50 K weisen auf die Existenz von ein oder mehreren Typ-2-Cu(II)-Zentren hin. Bei einer Temperatur von 10 K wird sowohl ein für Häm-gebundenes Eisen als auch ein für nicht Häm-gebundenes Eisen charakteristisches Signal ersichtlich. Durch Vergleich der Cu(II)-Konzentration mit dem Gesamtgehalt an Cu konnte ermittelt werden, dass die lösliche AMO etwa sechs paramagnetische Cu(II)-Atome und drei diamagnetische Cu(I)-Atome koordiniert. Der substratanaloge kompetitive Hemmstoff Acetylen wird von der löslichen AMO zu einem Keten oxidiert. Dieses reaktive Intermediat bindet in der Folge kovalent an His 191 (AmoA). Die Bindung von Keten setzt dabei ein Cu(I)-Atom pro AmoA-Untereinheit frei und führt somit zu einer irreversiblen Hemmung der AMO. His 191 aus AmoA ist dementsprechend an der Koordination eines Cu(I)-Zentrums beteiligt welches die Oxidation von Ammoniak katalysiert. Die lösliche AMO katalysiert die Disproportionierung von Hydroxylamin zu Ammoniak, Nitrit und Nitrat nach folgender Reaktionsgleichung: 3,2 NH2OH + 2 NH3 + 0,8 NO3– --> 0,4 NO2– + 3,6 H+ + 2,4 e– Die Reaktion besitzt das Temperaturoptimum zwischen 45 und 50 °C, das pH-Optimum bei einem pH-Wert von 8,5 und wird weder von Licht noch von Sauerstoff beeinflusst. Die maximale spezifische Aktivität der Disproportionierung liegt bei etwa 1.000 nmol NH2OH (mg AMO)-1 min-1 und der Km-Wert für Hydroxylamin beläuft sich auf ungefähr 140 µM. Acetylen besitzt keinen Einfluss auf die Aktivität der Disproportionierung, jedoch wirkt Hydrazin als kompetitiver Hemmstoff für die Umsetzung von Hydroxylamin durch die lösliche AMO. Eine Disproportionierung von Hydroxylamin konnte auch bei intakten Nitrosomonas-Zellen beobachtet werden, welche so einer Akkumulation von Hydroxylamin entgegenwirken. Es ist denkbar, dass die Disproportionierung von Hydroxylamin einen Mechanismus darstellt, mittels dessen sich N. europaea vor toxischen Hydroxylaminkonzentrationen schützt und dabei gleichzeitig ihr Substrat Ammoniak zurückgewinnt. Weiterhin gelang es im Zuge dieser Arbeit die Existenz eines funktionellen Ammoniaktransporters vom Rhesus-Typ (Rh1) in N. europaea zu beweisen. Der passive Rh1-Transporter ermöglicht sowohl die Aufnahme von Ammoniak als auch von Methylammonium (MA) in das Cytoplasma von N. europaea. Die Menge an transportiertem MA korreliert dabei mit der Expression von Rh1. Der Km-Wert für MA beträgt dabei 1,8 mM bei einem pH-Wert von 7,25. Die Aufnahme von MA konnte vollständig durch Ammonium gehemmt werden [Ki(NH4+) = 0,3 mM]. Ein niedriger periplasmatischer pH-Wert (pH 5 - 6) während der Ammoniakoxidation scheint eine Akkumulation von Ammonium im Periplasma zu bewirken („Säurefalle“) und Rh1 ermöglicht in der Folge einen raschen Konzentrationsausgleich von NH3 mit dem Cytoplasma. In aerob Ammoniak-oxidierenden Nitrosomonas-Zellen korrelieren die spezifische Aktivitäten der Ammoniakoxidation, der Reduktion von Nitrit, als auch die Wachstumsrate mit den Transkriptionsintensitäten der zugehörigen Gene amoA/hao/rh1, nirK/norB und cbbL/ftsZ. Die Konzentration von amoA-, hao-, rh1-, coxA-, cbbL, ftsZ- und sodB-mRNA in anaerob Ammoniak-oxidierenden Nitrosomonas-Zellen war im Bezug auf aerob Ammoniak-oxidierende Nitrosomonas-Zellen reduziert, die Konzentration an nirK- and norB-Transkript dagegen aber erhöht. Während anaerober Denitrifikation (Pyruvat dient als Substrat) stieg die Transkription der Gene nirK, norB, aceE, gltA, und odhA deutlich an. Die Konzentrationen von amoA-, hao-, rh1-, coxA-, cbbL-, ftsZ- und sodB-mRNA war im Vergleich zu aerob und anaerob Ammoniak-oxidierenden Nitrosomonas-Zellen allerdings signifikant erniedrigt.
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Control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda
(2010)
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Digali Lwalaba
- This thesis investigates control of the release of digestive enzymes in the cricket Gryllus bimaculatus and the fall armyworm, Spodoptera frugiperda. 1. Control of enzyme release in the cricket G. bimaculatus. Using flat-sheet preparations of the caecum, digestive enzyme release was investigated. More trypsin, aminopeptidase and amylase are secreted in the caecum of fed crickets than in unfed crickets, but basal levels of certain enzymes are released continuously even in unfed animals. A variable ratio of nutrients in ingested food leads to a different ratio of digestive enzyme release, but a high nutrient component in the food does not necessarily induce a high digestive enzyme release for that component. Maltose and glucose elevate amylase release from the tissues into the incubation medium, but starch does not. Bovine serum albumin (BSA), peptone and a mixture of amino acids have almost no effect on the release of aminopeptidase, and only low concentrations of peptone increase trypsin release. In crickets, the continuous release of proteases is sufficient to meet the needs for growth, and only moderate stimulation of trypsin results from feeding. Carbohydrates are used for energy, and the release of amylase is adjusted to the amount of food ingested. The neuropeptide allatostatin Grybi-AS 5 elevates the release of amylase in fed females, but not of trypsin or aminopeptidase, however, both amylase and trypsin release are stimulated by AS 5 in unfed crickets. Fed crickets have sufficient trypsin to obtain needed amino acids, but unfed do not, therefore the AS stimulation of trypsin release in unfed crickets makes sense. 2. Control of enzyme release in the larvae of S. frugiperda. A flat-sheet preparation of the ventriculus was used to test the release of amylase, trypsin and aminopeptidase in response to specific nutrients in the food and to specific neuropeptides. The epithelial secretion rate of amylase and trypsin was about 20% of the amount of enzyme present in the ventricular lumen, which, considering the efficient counter-current recycling of enzymes, suggests that the secretion rate is adequate to replace egested enzymes. Dietary carbohydrates are used for energy, and larvae adjust amylase activity to carbohydrate ingestion. Amylase activity is 5-times higher in fed compared to unfed larvae, and sugars in the incubation medium induces more than a 300 % increase in amylase release. Plants contain a low level of protein, but larvae need proteins for growth, thus the larvae can not afford to lose proteins by egestion. Therefore, trypsin activity remains high even in unfed larvae. As a result, proteins and amino acids have little or no effect on trypsin or aminopeptidase release in incubated tissues. The control of enzymes release in response to food is most likely mediated through neurohormones. Indeed, an allatostatin (Spofr-AS A5) inhibits amylase and trypsin release, and allatotropin (Manse- AT) stimulates amylase and trypsin release. Spofr-AS A5 also inhibits ileum myoactivity and Manse- AT stimulates myoactivity. 3. Inhibition of enzyme release in the larvae of S. frugiperda. Exogenous inhibitors are produced by plants, and are ingested by the insect. Endogenous inhibitors are produced by the gut epithelial cells themselves. A dose-dependent inhibition of endogenous enzymes occur in the lumen after feeding L6 larvae with the exogenous serine protease inhibitor from soybean (SBTI), the specific trypsin inhibitor TLCK, an aminopeptidase inhibitor (bestatin), and an amylase inhibitor from wheat. Inhibition in tissue extracts is seen only with higher doses of SBTI and TLCK. Inhibition of enzyme release into the incubation medium is apparent only with very high doses of SBTI. Inhibition in the tissues and inhibition of release indicate a direct cellular response to an inhibitor present in the lumen. The elevation of aminopeptidase activity in response to ingested trypsin inhibitors indicates a cellular synthesis in response to the product of a digestive enzyme. The enzymes investigated are irreversibly inactivated by 10 min at 90°C, but the corresponding inhibitors are not, therefore endogenous inhibitors could be identified. Endogenous inhibitors are present in the ventricular cells and in the lumen. We suggest that the endogenous protease inhibitors may protect the epithelium by inactivation of the trypsin in underfed larvae. This is the first explanation of how insects are able to secrete an active trypsin.
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Molecular Characterization of Sugarcane Yellow Leaf Virus (SCYLV) and its Effect on Sucrose Transporters in Sugarcane Saccharum spp. hybrids
(2010)
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Abdelaleim Ismail Ibrahim ElSayed
- Sugarcane is an important crop plant and has served as a source of sugar for hundreds of years, recently it is used to produce bioethanol, a renewable bio-fuel energy source. Sugarcane yellow leaf virus (SCYLV) was detected in the late 1990s first in Hawaii as a causal agent of a sugarcane disease which leads to sugarcane yellow leaf syndrome and reduced sugar yield. The presence of Sugarcane yellow leaf virus was determined by RT-PCR in several sugarcane cultivars, mostly from Hawaii. Interesting was the comparison of so-called susceptible versus resistant cultivars. As expected, the susceptible Hawaiian cultivars H73-6110 and H87-4094 showed strong PCR amplification products of SCYLV, while the virus-free line H87-4094, produced by tissue culture, showed no PCR product. The three resistant cultivars H87-4319, H78-4153 and H78-7750 showed quite different amplification patterns. While H78-4153 and H78-7750 expressed a weak but specific band of the correct size, unexpectedly H87-4319 showed strong amplification product. Three Cuban cultivars (C1051-73, JA-605 and CP52-43) showed low titer of SCYLV. No PCR amplificate was obtained with the moderately susceptible cultivar H65-7052. Aphids feeding on cv. H87-4094 contained sufficient virus to yield a SCYLV-signal similar in strength as from preparations from resistant cultivars. Northern blot analysis supported the results obtained from RT-PCR. The presence of SCYLV in the cultivars with low amount of virus titer (H87-4319, H78-7750 and H78-4153) indicated that they should better be called tolerant for the virus in the sense that they allow a low replication rate for SCYLV. Virus preparations from 3 Hawaiian cultivars (two susceptible and one resistant) were fully sequenced. Quantitative analysis for four different genome regions of SCYLV covering the 6 ORFs has been performed for these 3 cultivars using the GeXP analysis system. The transcript levels of the different regions of SCYLV in these cultivars were present at very different quantities, for example ORF0-1 transcripts were up to 10 times more frequent than transcripts of ORF3-4. The SCYLV-sequences from the 3 Hawaiian cultivars were aligned to published full and partial sequences. The phylograms corroborated previous findings that the so-called YLS-segment coding for the coat protein shows the least genetic diversity, whereas the other sequence fragments A-D, representing the ORFs 0-5, expressed a twofold higher diversity. The phylograms of partial sequences and of the whole genome placed the Hawaiian SCYLV-strains next to the Peru strain, apart from the BRA-strains and well apart from the REU-strains. It is proposed that the Hawaiian SCYLV is considered as own group together with the Peru strain as HAW-PER. The sequences from the two susceptible cultivars had a deletion of 48 to 54 nt in ORF1, which codes for the gene silencing suppressor/RNA-dependent RNA-polymerase complex. It is speculated that this deletion is important for the proliferation rate of the virus in the plant. Sucrose is the main product of sugarcane, which accumulates in the stalk internodes in excess of 50 % of the dry weight. To gain an overview of the physiological status of SCYLV-infected sugarcane compared to virus-free plants, gene expression. Sucrose increased rapidly between internodes 3 and 7, reaching a maximum in internodes 7. Sugars content in leaves, seedling and internodes were increased as effect of the SCYLV-infection. Sucrose phosphate synthase (SPSII) transcript levels were approximately the same in sink, source and internodes with a trend to be higher in the mature internodes. A sucrose transporter of Hawaiian cultivar was isolated and sequenced and classified as ShSUT1A. There is high variability among the SUT1 subfamily with identities of 70-97%. The identity between ShSUT1A and ShSUT1 was 97.4%. It is expressed in sink, source and storage tissues. The ShSUT1A was expressed at approximately similar extent in SCYLV-infected and virus-free sugarcane. In addition a partial sequence of a sucrose transporter belonging to the SUT4 family was first obtained in sugarcane and its transcript levels in plant organs were measured. Quantitative analysis for sucrose transporters (ShSUT1 and ShSUT4) using the GeXP analysis system showed that sucrose transporter ShSUT1 was at a higher transcript expression than ShSUT4 in sink and source leaves, but not in mature internodes. In conclusion, - SCYLV from Hawaiian cultivars was characterized as belonging to an own subgroup (HAW- PER), - A deletion of 48-54 nt was detected in the SCYLV-sequence from susceptible cultivars, which may be correlated to virus proliferation, and - large differences in transcript levels of the viral ORFs were found. - Sucrose transporter transcripts and SPSII transcripts were not strictly correlated to SCYLV- infection and do not explain the pathological effect of SCYLV on sugarcane.
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Tropical bracken, a powerful invader of pastures in South Ecuador: Species composition, ecology, control measures, and pasture restoration
(2010)
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Kristin Roos
- Bracken (Pteridium spec.) is one of the most wide-spread weeds, especially where fire has been used for forest clearing or maintenance of agricultural areas. Taxonomically, it is considered an aggregate that separates into a northern hemispherical and a southern, tropical complex. Different from the extensively studied northern bracken, the knowledge of ecology and control measures of the tropical species is still fragmentary. The main aims of my thesis were (1) identification and population structure of bracken, (2) ecology of tropical bracken with particular emphasis on its survival of bushfires, and (3) development of an effective bracken control strategy and subsequent re-pasturisation of abandoned areas. The bracken vegetation of the study area consists of mixed stands of Pteridium arachnoideum (KAULF.) MAXON and P. caudatum (L.) MAXON with a proportion of 3:2, and of a hybrid (ca. 2%). Identification was by leaf morphology, allozyme analysis, comparison of particular chloroplastic DNA sequences, and analysis of four genomic and one plastidic microsatellites. Dominance of P. arachnoideum was explained by the fact that P. caudatum, as a lowland species, reaches its upper altitudinal limit in the research area. Analysis of heterozygosity indicated a higher genetic stability of the diploid P. arachnoideum population as compared to the allotetraploid P. caudatum population. Spatial extension of the individual clones is much smaller than reported for the northern bracken, indicating higher significance of sexual reproduction for the tropical fern in comparison to vegetative propagation by rhizome fragmentation. Four weeks after burning the natural rain forest, vigorously sprouting bracken sporophytes were observed. These developed from gametophytes, which germinated from the wind dispersed spores. Fast growth of the young sporophytes established the fern in the areas. After planting pasture grass, bracken was supported by repeated burning of the areas. In the long run, the grass was outcompeted by the fern possibly due to weakening of its vitality by burning and grazing, and the areas have been abandoned. The density of bracken fronds in a settled bracken area of our research site remained constant over years with small deviations caused by particular weather situations. Since this balance holds also for patchy fern canopies, it is assumed that this is due to nutrient shortage of the soil. Most probably, a new leaf can only develop from the nutrients remobilized from a senescing old leaf. Two to three months after a fire, an explosive emergence of new leaves was observed at rates, which substantially exceeded those under undisturbed growth. The newly formed leaves showed an extended life-span, which was attributed to a better nutrient supply from the ash. Subsequent self-thinning reduced the density of the leaves to a stable level within two years. In a laboratory experiment, the effects of heat pulse by a simulated bushfire on the bracken rhizomes were investigated. Separated long and short shoots were heated for a short time either in a water bath or embedded in soil. Subsequent to this heat pulse, they were cultivated in original soil. Short shoots showed a significantly higher heat resistance (up to 80°C) than long shoots (up to 60°C). In addition, the short shoots showed elongation growth and an enhanced frond production, whereas long shoots were not stimulated by the heat pulse. In a bracken control experiment, thirteen control measures (cutting of the fronds, several herbicides, covering with plastic foil and combinations thereof) were applied over a time-period of 23 months. Each treatment was repeated six times and the effects were recorded monthly. Quarterly cutting of the leaves as well as treatment with a customary herbicide mixture (picloram and metsulforon methyl) were the most effective treatments resulting in a reduction of the standing biomass by 65%. Monthly records of the resprouting bracken was necessary to work out the minimum number of treatments required for a clear control effect. For the five most efficient treatments among two to four applications were necessary. However, complete eradication of bracken was not possible. For re-pasturisation, the common pasture grass Setaria sphacelata was planted on the treated areas within a long-term experiment. After nearly two years of observation, the system had stabilized with a cover of S. sphacelata of 75% and of bracken of below 40%. This result demonstrated that the competitive strength of S. sphacelata was sufficient to control bracken once weakened by control treatments. The long-term experiment and, in addition, an experiment in which a gradually bracken-infested area is subjected to controlled burning, are continued.
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Advanced Technologies in Education - Developing the Science Classroom of the Future
(2010)
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Sofoklis A. Sotiriou
- Information communication technology (ICT) nowadays provides innovative learning systems which although routinely available needs adjustment to real educational environments. Due to the complexity of the task an appropriate integration into everyday classrooms is an important global research challenges focusing on its utilization and effects in both, classroom and non-classroom settings. By rigorous collecting data on teaching methods, classroom characteristics and students learning effects needs analysis by concentrating on selected variables that may determine effectiveness as well as teachers characteristics such as teacherspreparation and professional development. Therefore, the aim of the four presented research papers focuses on envision the science classroom of the future, by constructing a framework for improving current educational practices and learning processes in science and mathematics through the effective implementation of advanced technological tools and applications. Overall this work presents a vision for the science classroom of the future: It will not be an island, a self-contained campus, a counter-world. The classroom of the future will be able to emit and absorb along different wavelengths, be immersed in contemporary culture, be open to the emotions, facts and news of its time. It will be permeated by society, but not unprotected: the relationship between school and society will be one of osmosis, where the pedagogical tools and applications act as a membrane and interface. For this purpose, four empirical studies were carried out in real school environments, based on the use of advanced educational systems. (i) The first system under study is the COSMOS Portal, which is an educational repository that offers access to a network of robotic telescopes across the world. At the same time it offers access to more than 85,000 educational resources. The behaviour of the teachers who are using this system was mapped through the log files of the system database for a period of one year. (ii) The second system, called Lab of Tomorrow, is a wearable device that allows high school students to use their every day life as the field where they will conduct sophisticated experiments and thus will deepen their understanding of the science concepts involved in the activities. The impact of the system on students learning and to the lesson profile was studied for a period of one school year. (iii) The third system, called CONNECT, is also a wearable device that includes an advanced visualization system that augments additional information to the optical view of the user. The system is used in the framework of educational visits in science centres and museums enriching the experiences of the visitors. The effectiveness of the system in supporting the students conceptual change was studied in this case. (iv) The fourth system, called EXPLOAR, evolved from the described CONNECT system to a much more user-friendly handheld device. Taking into account the school curriculum we have designed a series of scenarios of use of these tools. The scenarios of use include classroom activities, field trips in science centres and museums, informal learning activities, professional development opportunities and community building. In all four studies, students cognitive learning is analysed as well as selected teachers tasks on the job. By applying different assessment methods and tools (questionnaires, video captures of lessons, log files and web based data) we monitored the implementation procedure across different European countries. Our working hypothesis is that amending the traditional scientific methodology for experi¬mentation with visualization applications and model building tools will help students to articulate their mental models, make better predictions, and reflect more effectively. Additionally, working to reconcile the gaps and inconsistencies within their mental models, system models, predictions and results, will provide the learners with a powerful, explicit representation of their misconceptions and a means to repair them. Additionally our aim is to support teachers professional development. Apart from the purely technical training, in order for teachers to introduce ICT-enhanced learning methods into their everyday practice, they will have to perform a change in behaviour and to adapt a new culture and philosophy. The use of the new tools asks for systematic and detailed lesson planning procedures and use of student centred approaches. In our work we are demonstrating methods for involving teachers in this process but also tools to monitor this behavioural change.
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Funktionale Charakterisierung neuer Komponenten der mitochondrialen Morphogenese in Saccharomyces cerevisiae
(2010)
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Miriam Hammermeister
- Die Morphologie und die Dynamik von Mitochondrien spielen eine wichtige Rolle bei der Funktion und Vererbung dieser Organellen. Zum besseren Verständnis der beteiligten Prozesse müssen alle dazugehörigen Komponenten identifiziert und charakterisiert werden. Die Erforschung von zwei Proteinen, Mdm35 und Mdm36, die die mitochondriale Morphologie und Verteilung beeinflussen, und ihre mögliche Eingliederung in den Morphogenesapparat der Mitochondrien war Gegenstand dieser Arbeit. Der erste Teilabschnitt der Arbeit beschäftigte sich mit der Charakterisierung und Ermittlung der Funktion von Mdm36 in der Gestaltgebung von Mitochondrien. Die Dynamik der Mitochondrien wird maßgeblich von den beiden antagonistischen Prozessen Fusion und Teilung bestimmt. Dnm1, eine Dynamin-verwandte GTPase, stellt die Schlüsselkomponente der mitochondrialen Teilung in Hefe dar, die in Zusammenarbeit mit weiteren Komponenten agiert. Teilungsmutanten besitzen netzartige Mitochondrien, die aufgrund der gestörten Teilung bei fortlaufender Fusion entstehen. In dieser Arbeit wurde gezeigt, dass Deletionsmutanten von MDM36 stark verzweigte mitochondriale Netzwerke aufweisen, die denen der Teilungsmutanten sehr ähneln. Doppeldeletionsstudien mit den Fusionskomponenten Fzo1 und Mdm30 lassen annehmen, dass Mdm36 der Fusion entgegengesetzt wirkt. Außerdem ist in delta-mdm36-Mutanten die durch die Depolymerisation des Aktinzytoskeletts induzierte mitochondriale Teilung blockiert und die Anzahl an teilungsaktiven Dnm1-Komplexen reduziert. Das Zellkortex-assoziierte Protein Num1 interagiert mit mitochondrial assembliertem Dnm1 und fördert über die dadurch geschaffene Verankerung der Mitochondrien am Zellkortex die mitochondriale Teilung. Der mitochondriale Phänotyp der delta-mdm36-Mutanten und delta-num1-Mutanten ist fast identisch. Beide Mutanten besitzen netzähnliche, kompakte Mitochondrien, die wenig Nähe zur Zellperipherie aufweisen und hoch dynamisch sind. Durch die Abwesenheit von Mdm36 wird die Kolokalisation von Dnm1 und Num1 aufgehoben. Diese Ergebnisse liefern weitere Einblicke in die mitochondriale Teilungsmaschinerie und legen ein Modell nahe, in dem Mdm36 für die Bildung des Num1/Dnm1-Komplexes und folglich für die Verankerung der Mitochondrien am Zellkortex benötigt wird. Über diesen wird dann die notwendige Spannung entlang des Mitochondrientubulus für die Dnm1-abhängige Teilung erzeugt. Im zweiten Teilabschnitt der vorliegenden Arbeit wurde zunächst die mitochondriale Morphologie der Mutanten der kürzlich identifizierten Twin Cx9C-Protein-Familie analysiert. Anschließend wurde das dabei und durch eine vorhergehende systematische Durchmusterung einer Hefedeletionsstammsammlung auffällig gewordene Protein Mdm35 näher untersucht. Mdm35 ist ein mitochondriales Protein des Intermembranraums, dessen Deletionsmutante zum Großteil sphärische Mitochondrien besitzt und einen Wachstumsdefekt bedingt durch den Verlust von mitochondrialer DNA aufweist. Die Mitochondrien und das instabile mitochondriale Genom der delta-mdm35-Mutante zeigen Ähnlichkeiten zu Zellen auf, denen entweder die mitochondrialen Innenmembranproteine Mdm31 bzw. Mdm32 fehlen, oder die mitochondrialen Außenmembranproteine Mmm2, Mdm10, Mdm12 bzw. das integrale Membranprotein des Endoplasmatischen Retikulums Mmm1. Die gleichzeitige Deletion von MDM35 und MDM31 bzw. MDM32 ist synthetisch letal, was ein Hinweis darauf ist, dass die Proteine an demselben zellulären Prozess beteiligt sind. Für die Deletion von MDM31 und MDM32 konnte bereits eine synthetische Letalität mit MMM1, MMM2, MDM10 und MDM12 gezeigt werden. Mmm1, Mmm2, Mdm10 und Mdm12 bilden einen Komplex aus, der eine Bindung zwischen dem Endoplasmatischen Retikulum und den Mitochondrien herstellt und vermutlich für den Austausch von Calcium und Phospholipiden zuständig ist. Dieser Komplex befindet sich außerdem in Nachbarschaft aktiv replizierender mitochondrialer DNA. Die erhaltenen Ergebnisse deuten darauf hin, dass Mdm35 möglicherweise als Protein des Intermembranraums eine Funktion bei der Vermittlung zwischen dem Mmm1/Mmm2/Mdm10/Mdm12-Proteinkomplex und den Innenmembranproteinen Mdm31/Mdm32 besitzt und so ein koordiniertes mitochondriales Wachstum ermöglicht durch Kopplung der Replikation des mitochondrialen Genoms und der Aufrechterhaltung der mitochondrialen Membranen über den Lipidtransport.
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Secondary Plant Compounds as Feeding Deterrents in the African Subterranean Termite Schedorhinotermes lamanianus Sjöstedt (Isoptera: Rhinotermitidae): A Behavioural and Neurophysiological Approach
(2010)
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Stefan Gross
- In the present study, the influence of plant-derived secondary compounds on feeding behaviour in the subterranean termite Schedorhinotermes lamanianus was investigated. Furthermore, the chemosensory input system responsible for the perception of these compounds was investigated using electrophysiological methods. The obtained results provide evidence that in S. lamanianus a variety of structurally diverse secondary plant compounds (alkaloids and non-alkaloids) other than securinega-alkaloids influence feeding behaviour. These compounds evoke an avoidance of food sources or lower food consumption under choice conditions even at lower concentrations obtained for securinega-alkaloids. Furthermore, these compounds also reduce feeding under no-choice conditions. Termites seem to ingest less food even when they started feeding and no alternative food source is available. Therefore these compounds act as repellents and feeding deterrents in S. lamanianus depending on the test conditions under which they are applied. Furthermore, the present study provides strong evidence that different proximate mechanisms explain feeding inhibition in S. lamanianus: 1) Twelve structurally very diverse alkaloids, including feeding deterrent alkaloids in S. lamanianus, stimulated the taste neuron II/3 in TP II sensilla on antennae of this termite species. Non-alkaloids did not stimulate neuron II/3. Therefore, this neuron II/3 is an "alkaloid cell" negatively influencing feeding behaviour in this termite. 2) Feeding inhibition seems also to be influenced by the inhibition of phagostimulant taste neurons ("water cells") on antennae. 3) A second sensory input system for the perception feeding deterrent plant-derived secondary compounds seems to be evident as some tested compounds (alkaloids and non-alkaloids) are clear antifeedants in S. lamanianus but do not influence feeding behaviour by the former two mechanisms. Hence, in termites feeding inhibition by secondary plant compounds is a very complex process which needs further investigation. Including neurophysiological investigations of the chemosensory input system seems to be a promising approach to better understand feeding inhibition in termites which may lead to improved wood protection and termite management.
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Identification and characterization of Sgo2 interactions - Insights into dynamic chromosomal localization, mechanism of cohesin protection and putative checkpoint function
(2010)
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Bernd Mayer
- Sister chromatids are embraced and held together by a ring-shaped multiprotein complex called cohesin, from the time of their generation in S-phase until their separation in M-phase. Shugoshins protect centromeric cohesin from premature dissociation and, hence, are important regulators of genome stability. This work demonstrates that hSgo2, one of the two shugoshins in humans, first binds to kinetochores in prophase, then localizes to centromeres in prometaphase before finally traveling back to kinetochores in metaphase. It is further shown that (1) chromatin binding requires the C-terminus of hSgo2, (2) the catalytic activity of the aurora B kinase is essential to focus hSgo2 at centromeres, and (3) attachment of microtubules to kinetochores is not only necessary for the metaphase-specific re-localization of hSgo2 but also sufficient; pulling forces are not required. These newly discovered regulations allow to formulate a mechanistic model that explains shugoshin’s dynamic subcellular localization. An existing conflict in the literature concerns the relative importance of mammalian Sgo1 and Sgo2 in mitosis. Cell biological analyses now demonstrate unambiguously that, contrary to earlier claims, hSgo2 is dispensable for several aspects of mitotic cell divisions. Thus, shugoshin functions are strictly separated, being fulfilled by hSgo1 in mitosis and by hSgo2 in meiosis. Initially, it was unclear how shugoshins exert their cohesin protective function at the molecular level. Using a biochemical approach, protein phosphatase 2A (PP2A) was identified as a prominent interactor of shugoshins in this thesis. Shortly thereafter, it was reported that shugoshins protect cohesin by mediating PP2A-dependent dephosphorylation. Nevertheless, it is shown here for the first time that hSgo2 binds PP2A via its N-terminus. A Sgo2 point mutant deficient in PP2A binding is created and characterized by cell biological experiments. Importantly, biochemical assays demonstrate that hSgo2 greatly stimulates PP2A’s enzymatic activity. Shugoshin function therefore extends beyond simple provision of a linkage between cohesin and PP2A. Mitosis and meiosis are chiefly controlled by the spindle assembly checkpoint (SAC), which allows anaphase to take place only after all chromosomes have become properly attached to the mitotic spindle. Central to SAC signaling, kinetochore bound Mad1-Mad2 complex catalyzes a conformational switch of soluble Mad2, thereby allowing its inhibitory binding to the downstream effector protein Cdc20. SAC-dependent inhibition of anaphase correlates well in timing with shugoshin-dependent protection of cohesin. Here, Mad2 is identified as another novel interactor of hSgo2. Precise mapping reveal a conserved Mad2 interaction motif (MIM) in hSgo2, which is shared by the known Mad2 interactors Mad1 and Cdc20. In fact, several lines of evidence show that the Sgo2-Mad2 complex is structurally very similar to the Mad1-Mad2 and the Cdc20-Mad2 complexes. These biochemical studies challenge the current “one source (kinetochore bound Mad1) – one target (Cdc20)” dogma in the field of SAC research. Mad2 binding is conserved in the only Xenopus laevis shugoshin, xSgo1, indicating an important function of this interaction during vertebrate meiosis.
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Exocrine glands in Erotylidae (Coleoptera, Cucujoidea): chemical ecology, morphology and evolution
(2010)
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Kai Drilling
- In most insect orders chemical defence is highly important and a multiplicity of partly spectacular defence mechanisms were described in the last years. It is well known that members of the Erotylidae show a particularly rich equipment of exocrine compound glands. However, morphology and ultrastructure as well as the chemistry of the secretions of these compound glands remain unexplored so far. The cosmopolitan Erotylidae is assigned to the superfamily Cucujoidea (Clavicornia) of the Coleoptera-Cucujiformia and comprises about 3500 described species in 258 genera. Today the family includes both the phytophagous species of the former Languriidae and the mycophagous species of the former Erotylidae s. str. (now ranked as the subfamily Erotylinae). The adult beetles, as well as their larvae, are bounded to different bracket fungi or live under the bark. Most species are striking in appearance, frequently in combination with conspicuous patters of stripes, spots or rings. The present contribution deals with species of this coleopteran family and concerns altogether five different subject areas: (1) Morphological details of the internal structure and ultrastructure of the compound glands were examined in exemplar species of the family (Tritoma bipustulata, Triplax scutellaris) for the first time (SEM, TEM). Each compound gland consists of a central excretory duct and numerous identical gland units. These gland units are composed of two different cells, whereof one forms a cuticular ductule. Thus the glands belong to class III as defined by Noirot and Quennedey (1974, 1991). Furthermore, the glands offered two structural features (lateral appendix, the spongious wall of the ductus), which were previously not known from compound glands of beetles. (2) Hitherto hardly known was the ability of reflex bleeding in these species. The phenomenon is reported, for instance, from the closely related families Coccinellidae and Endomychidae. However, the hemolymph is not, like in the mentioned taxa, released from the joints of the leg, but from the abdominal tip. The chemistry of the reflex blood as well as the discharged secretion of the pronotal glands was examined by GC-MS for the first time. Biological effects of the identified compounds of both secretions were evaluated in bioassays and agar diffusion tests. (3) Furthermore, a study on the role of emitted fungal volatile compounds in recognition of the hostfungus by mycophagous beetles was conducted (GC-EAD, EAG). Beside the two erotylid-species (Tritoma bipustulata and Dacne bipustulata), mycophagous species of the families Tenebrionidae and Ciidae were included in this study. The scents of young as well as aged fungi were tested. The results imply that the species are able to discriminate between fungi of different ages as well as the degree of colonization. (4) Due to the multiplicity of different exocrine compound glands in Erotylidae (within the angles, as well as along the lateral margin of the pronotum, on the prosternal and mesoventral intercoxal processes, anteromesal to the compound eyes, on the subgenal braces, and rarely on the mentum), a comparative analysis on the occurrence of compound glands was carried out. 47 species were included in this analysis. The results were mapped on an existing phylogeny of the family and other phylogenetic hypotheses were discussed. Several glandular characters support the monophyly of the Erotylidae, Erotylinae as well as some tribes of the latter subfamily. Also the postulated position of Languria bicolor (Languriinae) within the Erotylinae is confirmed by glandular characters. (5) Finally, it was possible to identify Brachyserphus parvulus (Proctotrupidae) as a parasitoid of T. bipustulata. Members of this group of Hymenoptera are endoparasites in larvae of numerous families of the Coleoptera, Diptera and Lepidoptera. B. parvulus was hitherto known from species of Nitidulidae, Melandryidae, Phalacridae as well as the erotylid genus Triplax.
