Myosin and tropomyosin stabilize the conformation of formin-nucleated actin filaments

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins, however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long-range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments, using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Forster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments, the mechanisms of the conformational transition is different for the two proteins. Heavy meromyosin stabilizes the formin- nucleated actin filaments in an apparently single-step reaction upon binding, while the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells

DAAM is required for thin filament formation and Sarcomerogenesis during muscle development in Drosophila.

During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Z-disc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin

Underground Muography with Portable Gaseous Detectors

Muography is a novel imaging technology based on particle physics instrumentation to reveal density structure of hill-sized objects. The cosmic muon flux is attenuated while penetrating into the ground, thus the differential local flux correlates with the overburden density-length. Underground muography exploits the close-to-zenith flux, while main challenges became portability, low power consumption, and robustness against the out-of-the-laboratory environment. Various fields could benefit from this non-invasive imaging, eg. speleology, mining, archeology, or industry. Portable gaseous tracking detector systems have been designed, built, and successfully used in several underground locations. This paper presents the designed portable muography systems, the main requirements, and measurement campaigns for calibration, natural caves, and cultural heritage

Age group, location or pedagogue: factors affecting parental choice of kindergartens in Hungary

Hungary has experienced significant political, economic, demographic and social changes since the end of Soviet domination in the 1990s. The gradual move towards liberal-democracy has been accompanied by growing emphasis on individualism, choice and diversity. Universal kindergarten provision for 5-6 year olds is a long established feature of the Hungarian education system, but little is known about parental choice (Török, 2004). A case study (Yin, 2004) of factors influencing parental choice and satisfaction was undertaken in one Hungarian town. This was based on a survey of 251 parents of children attending both mixed-age and same-age groups across 12 kindergartens. Parents suggested that the most important influences were geographical location and the individual pedagogue(s). Given that traditionally each pedagogue follows ‘their’ cohort from kindergarten entry to primary school, their influence appears heightened. Although generally satisfied with their chosen arrangement, parents from same-age groups expressed significantly more confidence and satisfaction, particularly in relation to cognitive development and preparation for school. Parents appear less convinced about the trend towards mixed-age groups and questions are raised about sufficiency of evidence of their benefits in a Hungarian context and the driving factors behind change

Cavity Location by Muon Tomography

Muon tomography (or muography) is one of the most effective methods for locating unknown underground voids. If the geometric conditions are favorable for the measurements, no other geophysical method can compete with it, neither when resolution nor when simplicity is considered. In the last years, thanks to the continuous R&D of our low-cost, portable muon detectors, as well as the improved data processing methods, we have completed several successful natural and artificial cavity exploration projects, demonstrating that location is possible even if the characteristic size of cavity is 5% of the rock thickness between the detector and surface. Here we present case studies carried out in Hungarian underground sites, where we could find unknown cavities and verify the method by locating known artificial shafts and adits with high precision. Reaching these unknown caves is in progress either by conventional caving exploration techniques or by drilling. Further measurements are ongoing by the new upgraded detectors. By decreasing gas consumption and supporting electric power by solar cells, we are able to measure even at remote locations without the need of any direct access, for durations of several months

Multi-Wire Detectors for Underground Muography

The use of cosmic muons in imaging large artificial or geological structures started to flourish in the last decades, with the technological advancement in particle physics instrumentations. Muography became a most effective way to locate hidden density anomalies in geological structures, which includes revealing unknown parts of natural cave systems underneath the mountains. Our group has developed a series of gaseous multi-wire particle detectors for muography applications, with targets ranging from volcanology to speleology. Advancements in durability, power consumption, portability, and acquisition system have been proven via field measurements in natural sites besides extensive laboratory testing. The poster is dedicated to give details on the main requirements, components, and solutions which are means to transform standard particle detectors to be practically applicable in underground muography. We will present the expanded scale of experimental systems, targeting upgraded high-resolution tomography, hole-fit small-scale devices, and even economical simplified versions for exploratory measurements. These muography detectors could soon become effective novel tools in geo-sciences

Glutamate Uptake Triggers Transporter-Mediated GABA Release from Astrocytes

Background: Glutamate (Glu) and c-aminobutyric acid (GABA) transporters play important roles in regulating neuronal activity. Glu is removed from the extracellular space dominantly by glial transporters. In contrast, GABA is mainly taken up by neurons. However, the glial GABA transporter subtypes share their localization with the Glu transporters and their expression is confined to the same subpopulation of astrocytes, raising the possibility of cooperation between Glu and GABA transport processes. Methodology/Principal Findings: Here we used diverse biological models both in vitro and in vivo to explore the interplay between these processes. We found that removal of Glu by astrocytic transporters triggers an elevation in the extracellular level of GABA. This coupling between excitatory and inhibitory signaling was found to be independent of Glu receptor-mediated depolarization, external presence of Ca2+ and glutamate decarboxylase activity. It was abolished in the presence of non-transportable blockers of glial Glu or GABA transporters, suggesting that the concerted action of these transporters underlies the process. Conclusions/Significance: Our results suggest that activation of Glu transporters results in GABA release through reversal of glial GABA transporters. This transporter-mediated interplay represents a direct link between inhibitory and excitatory neurotransmission and may function as a negative feedback combating intense excitation in pathological conditions such as epilepsy or ischemia

Astrocytes convert network excitation to tonic inhibition of neurons

<p>Abstract</p> <p>Background</p> <p>Glutamate and γ-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters.</p> <p>Results</p> <p>Here we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na⁺concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg²⁺] <it>in vitro </it>model of epilepsy. Under <it>in vivo </it>conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions.</p> <p>Conclusions</p> <p>The results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamat<it>ergic </it>excitation into GABA<it>ergic </it>inhibition providing an adjustable, <it>in situ </it>negative feedback on the excitability of neurons.</p

FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres

In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor–acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10–15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations