A high-throughput assay for modulators of NNT activity in permeabilized yeast cells

Nicotinamide nucleotide transhydrogenase (NNT) mutant mice show glucose intolerance with impaired insulin secretion during glucose tolerance tests. Uncoupling of the β cell mitochondrial metabolism due to such mutations makes NNT a novel target for therapeutics in the treatment of pathologies such as type 2 diabetes. The authors propose that increasing NNT activity would help reduce deleterious buildup of reactive oxygen species in the inner mitochondrial matrix. They have expressed human Nnt cDNA for the first time in Saccharomyces cerevisiae, and transhydrogenase activity in mitochondria isolated from these cells is six times greater than is seen in wild-type mitochondria. The same mitochondria have partially uncoupled respiration, and the cells have slower growth rates compared to cells that do not express NNT. The authors have used NNT's role as a redox-driven proton pump to develop a robust fluorimetric assay in permeabilized yeast. Screening in parallel a library of known pharmacologically active compounds (National Institute of Neurological Disorders and Stroke collection) against NNT ± cells, they demonstrate a robust and reproducible assay suitable for expansion into larger and more diverse compound sets. The identification of NNT activators may help in the elucidation of the role of NNT in mammalian cells and assessing its potential as a therapeutic target for insulin secretion disorders.     

Phylogenetic constraints on digesta separation: Variation in fluid throughput in the digestive tract in mammalian herbivores

The relevance of the mean retention time (MRT) of particles through the gastrointestinal tract (GIT) is well understood and MRT(particle)GIT is an important parameter in digestion models. Solute markers have been used to estimate MRT(solute)GIT (or 'fluid passage') in animals, but the relevance of this measure is less evident and is usually sought in its relation to MRT(particle)GIT. The ratio between the two measures indicates the degree of 'digesta washing', with little washing occurring at ratios of 1, aborad washing at ratios >1 (where the solute marker travels faster than the particle marker), and orad (retrograde) washing at ratios <1 (where the solute marker travels slower than the particle marker). We analysed digesta washing in a dataset of 98 mammalian species including man of different digestion types (caecum, colon and nonruminant foregut fermenters, and ruminants), controlling for phylogeny; a subset of 72 species allowed testing for the influence of food intake level. The results indicate that MRT(solute)GIT and the degree of digesta washing are related to digestion type, whereas variation in MRT(particle)GIT is influenced mainly by effects of body mass and food intake. Thus, fluid throughput and digesta washing emerge as important correlates of digestive anatomy. Most importantly, primates appear constrained to little digesta washing compared to non-primate mammalian herbivores, regardless of their digestion type. These results may help explain the absence of primates from certain herbivore niches and represent a drastic example of a physiologic limitation in a phylogenetic group. More experimental research is required to illuminate relative benefits and costs of digesta washing.     

Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division

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Highlights? A conserved protein, CdvA, recruits ESCRT-III to membranes during cell division ? Recruitment is mediated by a peptide-winged helix domain interaction ? We have determined the structure of this complex ? CdvA and a single ESCRT-III protein can drive membrane deformation in vitro     

Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to the growing cell tip (anterograde). Occasionally, EEs run up to 90 μm in one direction. The underlying MT array consists of unipolar MTs at both cell ends and antipolar bundles in the middle region of the cell. Cytoplasmic MT-organizing centers, labeled with a γ-tubulin ring complex protein, are distributed along the antipolar MTs but are absent from the unipolar regions. Dynein colocalizes with EEs for 10-20 μm after they have left the cell tip. Inactivation of temperature-sensitive dynein abolishes EE motility within the unipolar MT array, whereas long-range motility is not impaired. In contrast, kinesin-3 is continuously present, and its inactivation stops long-range EE motility. This indicates that both motors participate in EE motility, with dynein transporting the organelles through the unipolar MT array near the cell ends, and kinesin-3 taking over at the beginning of the medial antipolar MT array. The cooperation of both motors mediates EE movements over the length of the entire cell.     

Individualistic species limitations of climate‐induced range expansions generated by meso‐scale dispersal barriers

Aim Evidence indicates that species are responding to climate change through distributional range shifts that track suitable climatic conditions. We aim to elucidate the role of meso‐scale dispersal barriers in climate‐tracking responses. Location South coast of England (the English Channel). Methods Historical distributional data of four intertidal invertebrate species were logistically regressed against sea surface temperature (SST) to determine a climate envelope. This envelope was used to estimate the expected climate‐tracking response since 1990 along the coast, which was compared with observed range expansions. A hydrodynamic modelling approach was used to identify dispersal barriers and explore disparities between expected and observed climate tracking. Results Range shifts detected by field survey over the past 20 years were less than those predicted by the changes that have occurred in SST. Hydrodynamic model simulations indicated that physical barriers produced by complex tidal currents have variably restricted dispersal of pelagic larvae amongst the four species. Main conclusions We provide the first evidence that meso‐scale hydrodynamic barriers have limited climate‐induced range shifts and demonstrate that life history traits affect the ability of species to overcome such barriers. This suggests that current forecasts may be flawed, both by overestimating range shifts and by underestimating climatic tolerances of species. This has implications for our understanding of climate change impacts on global biodiversity.     

Transportation of nonindigenous species via soil on international aircraft passengers’ footwear

The potential for transported soil to harbour and spread nonindigenous species (NIS) is widely recognised and many National Plant Protection Organisations (NPPOs) restrict or prohibit its movement. However, surprisingly few studies have surveyed soil while it is in transit to provide direct support for its role in accidental introductions of NIS. Moreover, there are few border interception records for soil organisms because they are neither easily detected nor routinely isolated and identified. Better data would improve evaluations of risks from soil transported via different pathways, enable targeting of management resources at the riskiest pathways, and support development of new risk management methods. We surveyed organisms present in soil that had been removed from footwear being carried in the baggage of international aircraft passengers arriving in New Zealand and recorded high incidences, counts and diversities of viable bacteria, fungi, nematodes and seeds, as well as several live arthropods. These included taxa that have not been recorded in New Zealand and were therefore almost certainly nonindigenous to this country. In each gram of soil, there was an estimated 52–84% incidence of genera that contain species regulated by New Zealand’s NPPO, which suggests many were potentially harmful. Variation in the incidences and counts of soil organisms with sample weight, footwear type and season at the port of departure indicated it may be possible to develop methods for targeting management resources at the riskiest footwear. Comparisons with previously published data supported the hypothesis that survival of soil organisms is greater when they are transported in protected (e.g. in luggage) rather than unprotected environments (e.g. external surfaces of sea containers); this offers opportunities to develop methods for targeting management resources at the most hazardous soil pathways.     

Identification and characterisation of novel Mss4-binding Rab GTPases

The Mss4 (mammalian suppressor of yeast Sec4) is an evolutionarily highly conserved protein and is expressed in all mammalian tissues. Although its precise biological function is still elusive, it has been shown to associate with a subset of secretory Rab proteins (Rab1b, Rab3a, Rab8a, Rab10) and to possess a rather low guanine nucleotide exchange factor (GEF) activity towards them in vitro (Rab1, Rab3a and Rab8a). By screening a human placenta cDNA library with Mss4 as bait, we identified several Rab GTPases (Rab12, Rab13 and Rab18) as novel Mss4-binding Rab proteins. Only exocytic but no endocytic Rab GTPases were found in our search. The binding of Mss4 to Rab proteins was confirmed by direct yeast two-hybrid interaction, by co-immunoprecipitation from lysates of mammalian cells, by immunofluorescence colocalisation as well as by direct in vitro binding studies. Analysis of Mss4 catalytic activity towards different Rab substrates confirmed that it is a somewhat inefficient GEF. These data, together with our mutational analysis of Mss4-Rab binding capacity, support the already proposed idea that Mss4 functions rather as a chaperone for exocytic Rab GTPases than as a GEF.