Membrane distribution of the glycine receptor α3 studied by optical super-resolution microscopy

In this study, the effect of glycine receptor (GlyR) α3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both α3K and α3L splice variants individually and together using single- and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for α3L compared to α3K (mean radius 92 ± 4 and 56 ± 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 ± 1,433 and 1,747 ± 200 μm^?2, respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 ± 6 nm). These results demonstrate that RNA splicing determines GlyR α3 membrane distribution, which has consequences for neuronal GlyR physiology and function.     

Developmental dynamics of myogenesis in the shipworm <Emphasis Type="Italic">Lyrodus pedicellatus</Emphasis> (Mollusca: Bivalvia)

Background

The shipworm Lyrodus pedicellatus is a wood-boring bivalve with an unusual vermiform body. Although its larvae are brooded, they retain the general appearance of a typical bivalve veliger-type larva. Here, we describe myogenesis of L. pedicellatus revealed by filamentous actin labelling and discuss the data in a comparative framework in order to test for homologous structures that might be part of the bivalve (larval) muscular ground pattern.

Results

Five major muscle systems were identified: a velum retractor, foot retractor, larval retractor, a distinct mantle musculature and an adductor system. For a short period of larval life, an additional ventral larval retractor is present. Early in development, a velum muscle ring and an oral velum musculature emerge. In late stages the lateral and dorsal mantle musculature, paired finger-shaped muscles, an accessory adductor and a pedal plexus are formed. Similar to other bivalve larvae, L. pedicellatus exhibits three velum retractor muscles, but in contrast to other species, one of them disappears in early stages of L. pedicellatus. The remaining two velum retractors are considerably remodelled during late larval development and are most likely incorporated into the elaborate mantle musculature of the adult.

Conclusions

To our knowledge, this is the first account of any larval retractor system that might contribute to the adult bodyplan of a (conchiferan) mollusk. A comparative analysis shows that a pedal plexus, adductors, a larval velum ring, velum retractors and a ventral larval retractor are commonly found among bivalve larvae, and thus most likely belong to the ground pattern of the bivalve larval musculature.

     

Disproportionate Contribution of Riparian Inputs to Organic Carbon Pools in Freshwater Systems

A lack of appropriate proxies has traditionally hampered our ability to distinguish riverine organic carbon (OC) sources at the landscape scale. However, the dissection of C₄ grasslands by C₃-enriched riparian vegetation, and the distinct carbon stable isotope signature (δ¹³C) of these two photosynthetic pathways, provides a unique setting to assess the relative contribution of riparian and more distant sources to riverine C pools. Here, we compared δ¹³C signatures of bulk sub-basin vegetation (δ¹³C_VEG) with those of riverine OC pools for a wide range of sites within two contrasting river basins in Madagascar. Although C₃-derived carbon dominated in the eastern Rianala catchment, consistent with the dominant vegetation, we found that in the C₄-dominated Betsiboka basin, riverine OC is disproportionately sourced from the C₃-enriched riparian fringe, irrespective of climatic season, even though δ¹³C_VEG estimates suggest as much as 96% of vegetation cover in some Betsiboka sub-basins may be accounted for by C₄ biomass. For example, δ¹³C values for river bed OC were on average 6.9 ± 2.7‰ depleted in ¹³C compared to paired estimates of δ¹³C_VEG. The disconnection of the wider C₄-dominated basin is considered the primary driver of the under-representation of C₄-derived C within riverine OC pools in the Betsiboka basin, although combustion of grassland biomass by fire is likely a subsidiary constraint on the quantity of terrestrial organic matter available for export to these streams and rivers. Our findings carry implications for the use of sedimentary δ¹³C signatures as proxies for past forest-grassland distribution and climate, as the C₄ component may be considerably underestimated due to its disconnection from riverine OC pools.     

RpoS and Indole Signaling Control the Virulence of Vibrio anguillarum towards Gnotobiotic Sea Bass (Dicentrarchus labrax) Larvae

Quorum sensing, bacterial cell-to-cell communication with small signal molecules, controls the virulence of many pathogens. In contrast to other vibrios, neither the VanI/VanR acylhomoserine lactone quorum sensing system, nor the three-channel quorum sensing system affects virulence of the economically important aquatic pathogen Vibrio anguillarum. Indole is another molecule that recently gained attention as a putative signal molecule. The data presented in this study indicate that indole signaling and the alternative sigma factor RpoS have a significant impact on the virulence of V. anguillarum. Deletion of rpoS resulted in increased expression of the indole biosynthesis gene tnaA and in increased production of indole. Both rpoS deletion and the addition of exogenous indole (50–100 µM) resulted in decreased biofilm formation, exopolysaccharide production (a phenotype that is required for pathogenicity) and expression of the exopolysaccharide synthesis gene wbfD. Further, indole inhibitors increased the virulence of the rpoS deletion mutant, suggesting that indole acts downstream of RpoS. Finally, in addition to the phenotypes found to be affected by indole, the rpoS deletion mutant also showed increased motility and decreased sensitivity to oxidative stress.     

Proton transfer in and polarizability of hydrogen bonds in proteins. Tyrosine-lysine and glutamic acid-lysine hydrogen bonds — IR investigations

The OH N O^– H⁺N hydrogen bonds formed between tyrosine and lysine, and between glutamic acid and lysine residues are studied by infrared spectroscopy considering the following systems: (l-lys)_n + phenol, copoly (l-lys, l-tyr)_n, (l-lys)_n + (l-tyr)_n and (l-lys)_n + (l-glu)_n. The phenol-lysine hydrogen bonds are largely symmetrical in the average if the pK_a of the protonated lysine is 2.2 units larger than that of the phenols. In the case of the hydrogen bonds between tyrosine and lysine residues in copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n, the weight of the proton limiting structure OH N is 80–90%, and that of the polar O^– H⁺N structure 10–20%. Double minimum proton potentials occur but the proton is preferentially present at the tyrosine residues. In the (l-lys)_n + (l-glu)_n system, the protons are present at the lysine residues. Thus, these hydrogen bonds have very large dipole moments (about 10 D). With the lysine-phenole hydrogen bonds, hydration shifts the proton transfer equilibrium a little in favour of the polar proton limiting structure O^– H⁺N. These hydrogen bonds are broken to a large extent, however, when only about 3 water molecules are present per lysine residue. When less water is present, as in the copoly (l-lys, l-tyr)_n and (l-lys)_n + (l-tyr)_n systems, these hydrogen bonds are, however, formed quantitatively. Thus — as discussed in this paper — the tyrosine-lysine hydrogen bonds can participate in proton conducting hydrogen bonded systems — as, for instance, present in bacteriorhodopsin — performing the proton transport through hydrophobic regions of biological membranes.     

A century of tree line changes in sub‐Arctic Sweden shows local and regional variability and only a minor influence of 20th century climate warming

Aim Models project that climate warming will cause the tree line to move to higher elevations in alpine areas and more northerly latitudes in Arctic environments. We aimed to document changes or stability of the tree line in a sub‐Arctic model area at different temporal and spatial scales, and particularly to clarify the ambiguity that currently exists about tree line dynamics and their causes. Location The study was conducted in the Torneträsk area in northern Sweden where climate warmed by 2.5 °C between 1913 and 2006. Mountain birch (Betula pubescens ssp. czerepanovii) sets the alpine tree line. Methods We used repeat photography, dendrochronological analysis, field observations along elevational transects and historical documents to study tree line dynamics. Results Since 1912, only four out of eight tree line sites had advanced: on average the tree line had shifted 24 m upslope (+0.2 m year^?1 assuming linear shifts). Maximum tree line advance was +145 m (+1.5 m year^?1 in elevation and +2.7 m year^?1 in actual distance), whereas maximum retreat was 120 m downslope. Counter‐intuitively, tree line advance was most pronounced during the cooler late 1960s and 1970s. Tree establishment and tree line advance were significantly correlated with periods of low reindeer (Rangifer tarandus) population numbers. A decreased anthropozoogenic impact since the early 20th century was found to be the main factor shaping the current tree line ecotone and its dynamics. In addition, episodic disturbances by moth outbreaks and geomorphological processes resulted in descent and long‐term stability of the tree line position, respectively. Main conclusions In contrast to what is generally stated in the literature, this study shows that in a period of climate warming, disturbance may not only determine when tree line advance will occur but if tree line advance will occur at all. In the case of non‐climatic climax tree lines, such as those in our study area, both climate‐driven model projections of future tree line positions and the use of the tree line position for bioclimatic monitoring should be used with caution.     

Purification of TAXI-like Endoxylanase Inhibitors from Wheat (Triticum Aestivum L.) Whole Meal Reveals a Family of Iso-forms

An affinity chromatography method has been developed for purification of endoxylanase inhibitors concentrated by cation exchange chromatography from wheat whole meal and is based on immobilisation of a Bacillus subtilis family 11 endoxylanase on N -hydroxysuccinimide activated Sepharose 4 Fast Flow. When followed by high-resolution cation exchange chromatography, the purification of seven TAXIs, Triticum aestivum L. endoxylanase inhibitors was achieved so extending the number of such proteins known to date (TAXI I and II). Based on their inhibition activities against a B. subtilis family 11 and an Aspergillus niger family 11 endoxylanase, six TAXI I- and only one TAXI II-like inhibitor could be distinguished. The first type of endoxylanase inhibitor is active against both endoxylanases and the second type only has significant activity against the B. subtilis endoxylanase.     

Zur Flora der Petzenalpe in Kärnthen

Ohne Zusammenfassung     

Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding

TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.     

Structural insight into unique cardiac myosin-binding protein-C motif: a partially folded domain

The structural role of the unique myosin-binding motif (m-domain) of cardiac myosin-binding protein-C remains unclear. Functionally, the m-domain is thought to directly interact with myosin, whereas phosphorylation of the m-domain has been shown to modulate interactions between myosin and actin. Here we utilized NMR to analyze the structure and dynamics of the m-domain in solution. Our studies reveal that the m-domain is composed of two subdomains, a largely disordered N-terminal portion containing three known phosphorylation sites and a more ordered and folded C-terminal portion. Chemical shift analyses, d(NN)(i, i + 1) NOEs, and (15)N{(1)H} heteronuclear NOE values show that the C-terminal subdomain (residues 315-351) is structured with three well defined helices spanning residues 317-322, 327-335, and 341-348. The tertiary structure was calculated with CS-Rosetta using complete (13)C(α), (13)C(β), (13)C', (15)N, (1)H(α), and (1)H(N) chemical shifts. An ensemble of 20 acceptable structures was selected to represent the C-terminal subdomain that exhibits a novel three-helix bundle fold. The solvent-exposed face of the third helix was found to contain the basic actin-binding motif LK(R/K)XK. In contrast, (15)N{(1)H} heteronuclear NOE values for the N-terminal subdomain are consistent with a more conformationally flexible region. Secondary structure propensity scores indicate two transient helices spanning residues 265-268 and 293-295. The presence of both transient helices is supported by weak sequential d(NN)(i, i + 1) NOEs. Thus, the m-domain consists of an N-terminal subdomain that is flexible and largely disordered and a C-terminal subdomain having a three-helix bundle fold, potentially providing an actin-binding platform.