A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space

Genetically encoded, ratiometric biosensors based on fluorescence resonance energy transfer (FRET) are powerful tools to study the spatiotemporal dynamics of cell signaling. However, many biosensors lack sensitivity. We present a biosensor library that contains circularly permutated mutants for both the donor and acceptor fluorophores, which alter the orientation of the dipoles and thus better accommodate structural constraints imposed by different signaling molecules while maintaining FRET efficiency. Our strategy improved the brightness and dynamic range of preexisting RhoA and extracellular signal-regulated protein kinase (ERK) biosensors. Using the improved RhoA biosensor, we found micrometer-sized zones of RhoA activity at the tip of F-actin bundles in growth cone filopodia during neurite extension, whereas RhoA was globally activated throughout collapsing growth cones. RhoA was also activated in filopodia and protruding membranes at the leading edge of motile fibroblasts. Using the improved ERK biosensor, we simultaneously measured ERK activation dynamics in multiple cells using low-magnification microscopy and performed in vivo FRET imaging in zebrafish. Thus, we provide a construction toolkit consisting of a vector set, which enables facile generation of sensitive biosensors.

Herbivores equalize the seedling height growth of three dominant tree species in an African tropical rain forest

Determining the impact of insect herbivores on forest tree seedlings and saplings is difficult without experimentation in the field. Moreover, this impact may be heterogeneous in time and space because of seasonal rainfall and canopy disturbances, or ‘gaps’, which can influence both insect abundance and plant performance. In this study we used fine netting to individually protect seedlings of Microberlinia bisulcata, Tetraberlinia bifoliolata and Tetraberlinia korupensis trees (Fabaceae = Leguminosae) from insects in 41 paired gap-understorey locations across 80 ha of primary rain forest (Korup, Cameroon). For all species, growth in height and leaf numbers was negligible in the understorey, where M. bisulcata had the lowest survival after c. 2 years. In gaps, however, all species responded positively with pronounced above-ground growth across seasons. When exposed to herbivores their seedling height growth was similar, but in the absence of herbivores, M. bisulcata significantly outgrew both Tetraberlinia species and matched their leaf numbers. This result suggests that insect herbivores might play an important role in maintaining species coexistence by mitigating sapling abundance of the more palatable M. bisulcata, which in gaps was eaten the most severely. The higher ratio in static leaf damage of control-to-caged M. bisulcata seedlings in gaps than understorey locations was consistent with the Plant Vigour Hypothesis. This result, however, did not apply to either Tetraberlinia species. For M. bisulcata and T. korupensis, but not T. bifoliolata (the most shade-tolerant species), caging improved relative seedling survival in the understory locations compared to gaps, providing restricted support for the Limiting Resource Model. Approximately 2.25 years after treatments were removed, the caged seedlings were taller and had more leaves than controls in all three species, and the effect remained strongest for M. bisulcata. We conclude that in this community the impact of leaf herbivory on seedling growth in gaps is strong for the dominant M. bisulcata, which coupled to a very low shade-tolerance contributes to limiting its regeneration. However, because gaps are common to most forests, insect herbivores may be having impacts upon functionally similar tree species that are also characterized by low sapling recruitment much more widely than currently appreciated. An implication for the restoration and management of M. bisulcata populations in forests outside of Korup is that physical protection from herbivores of new seedlings where the canopy is opened by gaps, or by harvesting, should substantially increase its subcanopy regeneration, and thus, too, its opportunities for adult recruitment.

Virtue, Law and Mercy

The attribution of responsibility in world society is increasingly a field of contestation. On the one hand, the perception of causal and moral links reaching far in space and time are ever more explicitly pronounced; on the other hand, the very complexity of these links often engenders a fragmentation of responsibility both in law (Veitch 2007) as well as in moral commitment. Moreover, those institutions of legal responsibility attempting to reflect some of these interrelations are often criticised as insufficient by those who follow alternative narratives of causation and moral community. Current institutions of responsibility in law appear to abstract from what could be called enabling contexts; they perform their cuts in the chains of enabling interactions at very brief intervals (Strathern 2001). The result is often “organised irresponsibility” (Veitch 2007; Beck 1996), producing appeals to a global community of concern in time and space without corresponding obligatory commitments. This talk explores alternative conceptualisations of responsibility, and enquires into their notion of the person, their temporal and socio-spatial dimensions, and their notion of liability.

Atomistic simulations of 2D bicomponent self-assembly: from molecular recognition to self-healing

Supramolecular two-dimensional engineering epitomizes the design of complex molecular architectures through recognition events in multicomponent self-assembly. Despite being the subject of in-depth experimental studies, such articulated phenomena have not been yet elucidated in time and space with atomic precision. Here we use atomistic molecular dynamics to simulate the recognition of complementary hydrogen-bonding modules forming 2D porous networks on graphite. We describe the transition path from the melt to the crystalline hexagonal phase and show that self-assembly proceeds through a series of intermediate states featuring a plethora of polygonal types. Finally, we design a novel bicomponent system possessing kinetically improved self-healing ability in silico, thus demonstrating that a priori engineering of 2D self-assembly is possible.

Spatio-temporal Rho GTPase signaling - where are we now?

Rho-family GTPases are molecular switches that transmit extracellular cues to intracellular signaling pathways. Their regulation is likely to be highly regulated in space and in time, but most of what is known about Rho-family GTPase signaling has been derived from techniques that do not resolve these dimensions. New imaging technologies now allow the visualization of Rho GTPase signaling with high spatio-temporal resolution. This has led to insights that significantly extend classic models and call for a novel conceptual framework. These approaches clearly show three things. First, Rho GTPase signaling dynamics occur on micrometer length scales and subminute timescales. Second, multiple subcellular pools of one given Rho GTPase can operate simultaneously in time and space to regulate a wide variety of morphogenetic events (e.g. leading-edge membrane protrusion, tail retraction, membrane ruffling). These different Rho GTPase subcellular pools might be described as 'spatio-temporal signaling modules' and might involve the specific interaction of one GTPase with different guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and effectors. Third, complex spatio-temporal signaling programs that involve precise crosstalk between multiple Rho GTPase signaling modules regulate specific morphogenetic events. The next challenge is to decipher the molecular circuitry underlying this complex spatio-temporal modularity to produce integrated models of Rho GTPase signaling.

Contact, Mobility and Authenticity: Language Ideologies in Koineisation and Creolisation

Part II - Christoph Neuenschwander: Language ideologies in the legitimisation of Tok Pisin as a lingua franca Pidgins and Creoles all over the world seem to share common aspects in the historical circumstances of their genesis and evolution. They all emerged in the context of colonialism, in which not only colonisers and colonised, but also the various groups of the colonised population spoke different languages. Pidgins and Creoles, quite simply, resulted from the need to communicate.¬¬ Yet, the degree to which they became accepted as a lingua franca or in fact even as a linguistic variety in its own right, strikingly differs from variety to variety. The current research project focuses on two Pacific Creoles: Tok Pisin, spoken on Papua New Guinea, and Hawai'i Creole English (HCE). Whereas Tok Pisin is a highly stabilised and legitimised variety, used as a lingua franca in one of the most linguistically diverse countries on Earth, HCE seems to be regarded as nothing more than broken English by a vast majority of the Hawai'ian population. The aim of this project is to examine the metalinguistic comments about both varieties and to analyse the public discourses, in which the status of Tok Pisin and HCE were and still are negotiated. More precisely, language ideologies shall be identified and compared in the two contexts. Ultimately, this might help us understand the mechanisms that underlie the processes of legitimisation or stigmatisation. As Laura Tresch will run a parallel research project on language ideologies on new dialects (New Zealand English and Estuary English), a comparison between the findings of both projects may produce even more insights into those mechanisms. The next months of the project will be dedicated to investigating the metalinguistic discourse in Papua New Guinea. In order to collect a wide range of manifestations of language ideologies, i.e. instances of (lay and academic) commentary on Tok Pisin, it makes sense to look at a relatively large period of time and to single out events that are likely to have stimulated such manifestations. In the history of Papua New Guinea - and in the history of Tok Pisin, in particular - several important social and political events concerning the use and the status of the language can be detected. One example might be public debates on education policy. The presentation at the CSLS Winter School 2014 will provide a brief introduction to the history of Tok Pisin and raise the methodological question of how to spot potential sites of language-ideological production.

Wetland-Archaeology in the Alpine Space. Overview and actual research issues.

First indications of prehistoric sites in lakes of Switzerland go back more than 200 years and in 1854 Ferdinand Keller (1800-1881) published his famous book The Celtic Pile Dwellings in Swiss Lakes. Since these times, large-scale rescue excavations as well as survey and research projects have extended our knowledge about Neolithic and Bronze Age settlements in lakes, bogs and rivers around the European Alps. In 2011 a representative choice of 111 sites out of nearly 1000 in six countries around the Alps (Austria, France, Germany, Italy, Slovenia and Switzerland) were recognized by the UNESCO World Heritage committee as serial World Heritage. The lecture will give a general overview on prehistoric lake dwellings around the Alps (distribution, types of lakes/bogs and environment of sites, chronology/cultural units in the time scale 5300 to 800 BC) and present examples of well-documented settlement structures. The intense use of dendrochronological dating allowed the building up of a well-fixed chronological framework. In some cases dendrochronology is the basis for year-by-year reconstructions of prehistoric village biographies and detailed insights in the life cycle of early agrarian settlements. Beside these local events the grouped repartition of lake dwelling remains on the time scale makes a more global correlation between Holocene lake levels and the preservation of archaeological layers likely.

Capturing socially motivated linguistic change. How the use of gender-fair language afffects support for social initiatives in Austria and Poland.

Gender-fair language consists of the symmetric linguistic treatment of women and men instead of using masculine forms as generics. In this study, we examine how the use of gender-fair language affects readers' support for social initiatives in Poland and Austria. While gender-fair language is relatively novel in Poland, it is well established in Austria. This difference may lead to different perceptions of gender-fair usage in these speech communities. Two studies conducted in Poland investigate whether the evaluation of social initiatives (Study 1: quotas for women on election lists; Study 2: support for women students or students from countries troubled by war) is affected by how female proponents (lawyers, psychologists, sociologists, and academics) are referred to, with masculine forms (traditional) or with feminine forms (modern, gender-fair). Study 3 replicates Study 2 in Austria. Our results indicate that in Poland, gender-fair language has negative connotations and therefore, detrimental effects particularly when used in gender-related contexts. Conversely, in Austria, where gender-fair language has been implemented and used for some time, there are no such negative effects. This pattern of results may inform the discussion about formal policies regulating the use of gender-fair language.

Organ transplantation in Switzerland: impact of the new transplant law on cold ischaemia time and organ transports

On 1 July 2007 a new transplant law came into force in Switzerland. The principal item of this new law is the change from centre-oriented allocation to patient-oriented national allocation of organs. The aim of the present study is to assess the impact on cold ischaemia time (CIT) and transport requirements.