Remorin proteins in Arabidopsis thaliana

Die Plasmamembran lebender Zellen stellt die Hauptbarriere für alle Arten von extrazellulären Signalen dar. Viele davon werden ins Innere der Zelle weitergeleitet, hier lösen sie im Kern transkiptionelle Veränderungen und damit die Anpassung der Zelle auf Proteinebene aus. Andere wiederum werden direkt erkannt und in unmittelbare molekulare Antworten umgewandelt, wie zum Beispiel die Sekretion von gespeicherten Stoffen oder Konformations-änderungen von Proteinen. Besonders in Pflanzen, welche durch ihre sesshafte Lebensweise auf die rechtzeitige und spezifische Erkennung von Umweltveränderungen angewiesen sind, hat sich ein höchst diverses Rezeptorsystem entwickelt. In der Ackerschmalwand Arabidopsis thaliana, der in dieser Arbeit verwendeten Modellpflanze, wurden 610 verschiedene Rezeptorproteine identifiziert, welche wiederum von zahlreichen interagierenden, und bis jetzt weitestgehend unerforschten Proteinen reguliert werden. Als entscheidendes Prinzip, dieses Aufgebot an membran-gebundenen Komponenten von Signalkaskaden zu organisieren, gilt inzwischen die zeitliche und lokale Kompartimentierung der Plasmamembran. Durch Akkumulation relevanter Bestandteile von biologischen Prozessen in sogenannten Membrandomänen werden kurze Reaktionszeiten und die unmittelbare Signalweiterleitung garantiert. Besonders wichtig bei solchen Prozessen sind sogenannte Gerüstproteine, welche als Adaptoren zwischen anderen Komponenten fungieren. In dieser Arbeit wurden Remorine, eine Familie pflanzenspezifische Proteinen ohne bisher definierte Funktion, aufgrund ihrer Eigenschaft Membrandomänen zu markieren und ihrer mutmaßlichen Beteiligung an Pflanzen-Pathogen-Interaktionen, genauer untersucht. Eine systematische Expression von Remorinen als Fluorophor-Fusionen mit anschließender hochauflösender mikroskopischer und quantitativer Untersuchung offenbarte, dass die meisten Remorine sich in deutlich unterschiedlichen Mustern an der Membran verteilen. Untersucht wurden dabei Parameter wie die Größe der erkennbaren Domänen, die Form, die Helligkeit, aus welcher auf die Proteinkonzentration rückgeschlossen werden kann, sowie die Domänendichte an der Membran. Diese Ergebnisse wurden von Kolokalisationsanalysen unterstützt, welche die Lokalisation in unterschiedlichen, koexistierenden Membrankompartimenten erkennen ließen. Ferner wurden die Eigenschaften der von Remorinen markierten Membrandomänen, wie zum Beispiel der Austausch an Proteinen mit der umgebenden Membran, sowie lokale und zeitliche Dynamik und Stabilität untersucht. Dabei konnte eine hohe Fluktuation einzelner Proteine zwischen Domäne und umliegender Membran, jedoch eine klare laterale Immobilität der gesamten Domäne nachgewiesen werden. Zusätzlich zeichneten sich die untersuchten Domänen teilweise durch eine außerordentlich große zeitliche Stabilität aus, andere wiederum scheinen abhängig von bestimmten Stimuli zu entstehen. Weitergehende Arbeiten dienten der Identifizierung der Funktion einzelner Bereiche der Proteine. Hierbei konnte die entscheidende Rolle des äußersten C-terminalen Bereichs, des so- genannten RemCAs (Perraki et al., 2012; Konrad et al., 2014) als Membrananker bestätigt werden. Zusätzlich wurden mit Hilfe eines Hefe-2-Hybrid Ansatzes zahlreiche neue Interaktoren für eine Auswahl von Remorinen identifiziert. Dabei wurde ein essentieller Rezeptor der basalen Immunantwort, BAK1 als Interaktor für Remorin 6.4 gefunden. Zuletzt wurden einige wenige Remorine mit Hilfe von Mutantenlinien in einer genetischen Studie phänotypischen Analysen bezüglich ihrer Funktion bei Pflanzen-Pathogen Interaktionen unterzogen. Remorin 6.4 spielt hiernach eine Rolle bei der Immunantwort nach Befall mit virulenten Bakterien. Die grundlegende Erkenntnis, dass in lebenden Zellen zahlreiche klar unterscheidbare Arten an Membrandomänen koexistieren, ist ein Meilenstein auf dem Weg zur Anerkennung einer neuen Vorstellung vom Aufbau der Zytoplasmamembran. Diese wird häufig noch als undifferenzierte zweidimensionale Flüssigkeit beschrieben, in welcher stellenweise sogenannte Lipidflöße, festere Strukturen aus Cholesterin und Sphingolipiden, die auch bestimmte Proteine beherbergen können, auftreten. Anhand der in dieser Arbeit gewonnen Ergebnisse, sowie ähnlicher Studien in Hefe lässt sich nun folgendes Bild zeichnen: Es ist davon auszugehen, dass unterschiedliche Proteine, welche im selben biologischen Prozess involviert sind, in unmittelbarer Nachbarschaft oder sogar im selben Proteinkomplex in der Membran organisiert sind. Die Lipidzusammensetzung in der unmittelbaren Umgebung wird von diesen Proteinen bestimmt, bietet jedoch auch die Grundlage für die Bildung der Domäne, indem sie die Lokalisation der Komponenten in diesem Bereich fördert. Die zahlreichen an der Zellmembran gleichzeitig ablaufenden, unterschiedlichen Prozesse erfordern eine hochkomplexe, zeitlich und räumlich stark regulierte Kompartimentierung der Membran. Es kann vermutet werden, dass Remorine eine Rolle als Gerüstproteine bei der Ausbildung einer Auswahl dieser Domänen bilden. Im Fall von Remorin 6.4 ist das Protein für den Prozess der Flagellin-Erkennung und die unmittelbaren Abwehrantworten, welche nachweislich eine Präformierung der beteiligten Proteinkomplexe voraussetzen, notwendig

Myometrial cyclic AMP function

Background Uncovering the processes that drive labour onset is essential to reduce the adverse consequences of dysfunctional labour. Myometrial cAMP signalling is upregulated during pregnancy promoting uterine quiescence. Changes in its components and effectors have been identified at the onset of term labour. Preterm labour (PTL) treatments targeting this pathway have limited effectiveness and serious maternal effects. In this study, real-time FRET imaging was used to investigate compartmentalised cAMP signals at distinct cellular sites. Methods Myometrial biopsies were obtained from women at term or in distinct causes of PTL. Tissues were processed for mRNA and protein extraction or cell isolation. Primary myometrial cells (HPMCs) and an hTERT-HM cell line expressed either a cytosolic (EPAC-SH187) or plasmalemma (AKAP79-CUTie) genetically encoded FRET sensor. The florescence emission changes were monitored following isoproterenol and PGE2 treatment to determine intracellular cAMP concentrations. Results Differences in cAMP signalling components were detected in PTL compared to term with variations in effector predominance and an associated increase in OTR expression in twin-PTL. Stimulus-specific subcellular compartmentalisation of cAMP was identified in both cell types with differential regulation by phosphodiesterases (PDEs). Significant disparities were detected in the amplitude, kinetics, and regulation of cAMP signals between the two cell types. For the HPMCs, a prolonged time in culture was associated with a reduction in PDE activity and altered cell phenotype. Conclusion The cAMP signalling system is influential in the final pathway of labour, primarily regulating OTR expression. This study established the technique of FRET imaging in human myometrial cells, determining the cell model of choice and culture conditions to explore localised cAMP signalling. The findings provide new insights into the spatial and temporal dynamics of cAMP in the human myometrium and pave the way for unravelling the details of how this fundamental pathway operates and its role in pregnancy and labour.Open Acces

Loss of the SNARE protein Sec22p selectively represses caesium accumulation in yeast and plants

The rare alkali ion caesium (Cs+) is assimilated by eukaryotes, even though it is not an essential nutrient. It poses an environmental concern through the anthropogenic release of its radioisotopes, 134Cs and 137Cs. Bioavailability and long half-lives favour its uptake and accumulation in plants, via which radiocaesium can be introduced to the food chain. Cs+ ions are taken up via potassium-(K+)-related pathways due to the biophysical similarity of these cations. This makes it difficult to solely manipulate Cs+ accumulation in plants without disturbing the homeostasis of essential ions at the same time. This work shows that the soluble N-ethylmaleimide sensitive factor attachment receptor (SNARE) Sec22p, previously described as a member of the protein sorting machinery, specifically affects Cs+ accumulation in yeast by regulating the selectivity of vacuolar deposition. A similar phenotype became apparent for a homologous plant protein, SEC22. The loss of Saccharomyces cerevisiae Sec22p reduces Cs+ uptake by more than half, while at the same time leaving essential cations undisturbed. Mathematical modelling of wild-type and mutant Cs+ uptake kinetics proposes that sec22Δ is defective in vacuolar compartmentalisation of Cs+, which is proven by biochemical fractionation. Morphological alterations were not produced by the loss of Sec22p, only a Cs+-dependent vacuolar fragmentation can be observed. These results indicate a so far undescribed function of Sec22p in assuring a non-selective ion deposition to the vacuole, which is necessary in ion detoxification, while its loss induces discrimination against vacuolar Cs+ deposition. A developmentally controlled loss-of-function mutant of the orthologous gene SEC22 (At1g11890) in A. thaliana displays a similar phenotype, having specifically reduced Cs+ enrichment without detrimental growth defects, thereby translating the yeast findings to a multicellular context. Furthermore, a functional complementation of the yeast mutant Cs+ phenotype by the plant gene transcript was possible. Selective reduction of Cs+ accumulation in plants by loss of a single gene product represents a new route to limit radiocaesium input to the food chain without disturbing basic plant nutrition and growth

The people smugglers’ business model

While discussions continue on how best to go about breaking the people smugglers’ business model, those participating in the debate have not publicly articulated the business model to which they refer. This has significant policy implications, given that the first step in developing a response is defining the problem to be addressed. This paper examines first some of the more recent international research, and second, relevant Australian case law and the more limited body of Australian research on people smuggling, to determine whether this business model can be identified. It does not seek to evaluate the appropriateness or efficacy of specific anti-people smuggling policies that have been or are currently being pursued by Australian Governments. Key points: Since the late 1990s, people smuggling has been a key focus of political debate on irregular migration to Australia. Most recently, attention has turned to how to ‘break the people smugglers’ business model’ . While there is continuing debate about how best to achieve this objec tive, the business model being referred to remains largely unarticulated , at least publicly . E xamination of recent open source research and Australian case law reveals there is no single ‘people smugglers’ business model’ that explains how people smugglers operate , either internationally or to Australia . However, certain themes are evident, including the predominance of fluid networks over more hierarchical organisations and the flexibility, adaptability and resilience of those involved. It appears that a v ariety of business models are employed (either explicitly or implicitly) and that they are c onstantly evolving. Some basic characteristics of maritime people smuggling from Indonesia to Australia can be discerned from open source information that can be us ed to outline some of the basic components of a common business model that seems to operate. However, there appear to be many variations on different components of the model. Further, the Indonesia to Australia leg is only part of a much longer journey, an d Indonesia is not the only departure point for boats to Australia organised by people smugglers. Finally, while p olitical and popular attention has focused largely on irregular maritime arrivals, people smuggling to Australia also occurs by air. The use o f the singular terms ‘the people smugglers’ business model’ or ‘the people smuggling business model’ gives the impression of a homogeneous market for which a single measure or ‘one size fits all’ solution might exist. The reality of a variety of business m odels operating at different stages of the supply chain between source countries and destination countries, including Australia, points to the need for a more tailored and considered approach. The point s at which to intervene in order to ‘break’ a certain business model , and the most appropriate mode s of intervention , will depend on the particular characteristics of that model

Investigating Socio-critical Discourses in Assessment of Senior Physical Education in New Zealand

There has been a growing interest in and, to some extent, adoption of, ‘socio-critical’ discourses in Health and Physical Education (HPE) in New Zealand and in physical education internationally. Evidence of a paradigm shift involving ‘socio-critical’ discourses is reflected in curriculum documents, course developments and assessment. With a gap in the research that addresses assessment practice in senior physical education in New Zealand, this study explored the selection, interpretation and application of National Certificate of Educational Achievement (NCEA) Level 1-3 standards. A case study methodology was used to generate in-depth insights into the factors influencing patterns of selection and non- selection of those standards explicitly linked to socio-critical discourses. NZQA data on national standard selection in NCEA Level 1-3 physical education from 2006 to 2010 was analysed to inform the case study selection and inquiry. Four teachers at two schools were involved in the research. Data collection methods included document reading, archival records, semi-structured interviews (individual), and a reflective diary. Data analysis was based on Miles and Huberman’s (1994) data management and analysis methods. The key findings showed that factors influencing selection / non-selection of socio-critical standards are complex and decision-making about selection can involve dichotomous thinking. The latter was associated with socio-critical and biophysical discourses, theoretical and practical knowledge and learning, body and mind, and physical education and health as separate knowledge bases. The data provided insight into the impact that issues associated with standard selection and interpretation can have in relation to teachers’ design of teaching and learning programmes, students’ pedagogical experiences and assessment associated with NCEA physical education. Furthermore, teachers’ own habitus, beliefs, value orientation, language and pedagogical practice were shown to have a strong influence on understandings and application of standards. Issues of alignment of curriculum, assessment and pedagogy were also explored. The study highlights the importance of teachers’ understanding of the tensions, knowledge structures and power relations at play between curriculum, assessment and pedagogy. Data revealed important ways in which these matters inform and limit understandings of what constitutes legitimate and valued practice and learning in senior physical education. Implications of this inquiry are explored for educational policy developers, senior secondary HPE teachers, all HPE teachers, HPE departments, pre-service teacher educators, senior secondary teachers working in other subject/learning areas and research. An extensive list of recommendations has been made. Several areas are identified as requiring further research. Further exploration of teachers’ habitus, beliefs and values and the influence these have on the alignment of curriculum, assessment and pedagogy would be useful. In addition research into ‘holistic’ physical education ‘in’, ‘through’ and ‘about’ movement, in the context of NCEA, would facilitate more accurate and meaningful conclusions about teaching and learning and assessment experiences for secondary school students in NCEA physical education