The Guanine Nucleotide Exchange Factor RIC8 Regulates Conidial Germination through Gα Proteins in Neurospora crassa

Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia) germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.     

Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements

Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (t_Thresh and d_Thresh) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems.     

Habitat-related differences in recruitment and survival of early recruits of the invasive <Emphasis Type=Italic>Sargassum muticum</Emphasis> (Phaeophyta,Sargassaceae) in northern Portugal

Habitat-related differences in recruitment and early survival of the invasive seaweed Sargassum muticum were investigated in relation to the lunar phase (gamete release trigger) and meso-grazing between mid-intertidal and low-intertidal pools in northern Portugal. In situ experiments were performed to determine recruitment and to test the effect of intertidal level (mid-intertidal and low-intertidal pools), grazing (no cage, cage and open cage), and lunar phase (full, new and each quarter moon) on recruit survival during reproductive seasons in two consecutive years. Our results show clearly that recruitment and survival were higher in the mid-intertidal than in low-intertidal environment in both study periods. Caging treatments affected recruit survival in both environments, however, differences in patterns suggesting differences in grazing pressure due to differences in grazer density and/or the assemblages. It therefore seems that grazing on early recruits of this invader might contribute to the resistance to proliferation. Mid-intertidal pools constitute a more favourable environment for recruits of S. muticum than the low-intertidal, which may explain the mainly mid-intertidal distribution of the species in Portugal.     

Truncation of a β-Barrel Scaffold Dissociates Intrinsic Stability from Its Propensity to Aggregation

Δ98Δ is a functional all-β sheet variant of intestinal fatty acid binding protein (IFABP) that was generated by controlled proteolysis. This framework is useful to study the molecular determinants related to aggregation of β-barrel proteins. Albeit displaying increased conformational plasticity, Δ98Δ exhibits a nativelike β-barrel topology and is able to support a cooperative folding behavior. Here we present a comparative study of IFABP and Δ98Δ regarding their conformational perturbation and aggregation propensity triggered by trifluoroethanol. Both proteins share a common nucleation-elongation mechanism, whereby the rate-limiting step is the formation of stable dimeric nuclei followed by the association of monomers to the growing aggregates. Despite leading to a less stable structure, the extensive truncation of IFABP yields a form exhibiting a somewhat lower tendency to aggregate. This finding appears at odds with the established notion that a perturbation of the native compact fold should necessarily favor the population of aggregation-prone species. In addition to the aggregation propensity dictated by a given amino-acid sequence, our contention holds that long-range interactions might also play a major role in determining the overall aggregation propensity.     

Ecology and conservation of grassland birds in southeastern South America: a review

ABSTRACT The grasslands of southeastern South America (SESA), comprising one of the most extensive grassland ecosystems in the Neotropics, have been negatively impacted by the development of the livestock industry, arable agriculture, and forestry. SESA grasslands have a rich avifauna that includes 22 globally threatened and near‐threatened species, and many other species have suffered local population extinctions and range reductions. In addition to habitat loss and fragmentation, grassland birds in SESA are threatened by improper use of agrochemicals, unfavorable fire management regimes, pollution, and illegal capture and hunting. Studies to date have provided information about the distribution of grassland birds, the threats populations face, and the habitat requirements of some threatened species, but more information is needed concerning dispersal and migration patterns, genetics, and factors that influence habitat use and species survival in both natural and agricultural landscapes. There are few public protected areas in the region (1% of original grasslands), and many populations of threatened grassland birds are found on private lands. Therefore, efforts to preserve grassland habitat must reconcile the interests of land owners and conservationists. Current conservation efforts include establishment of public and private reserves, promotion of agricultural activities that reconcile production with biodiversity conservation, development of multilateral conservation projects across countries, and elaboration of action plans. Measures that result in significant losses to private land owners should include economic compensation, and use of economic incentives to promote agriculture and forestry in native grassland areas should be discouraged, especially in priority areas for grassland birds. Although more studies are needed, some actions, particularly habitat protection and improved management of public and private lands, should be taken immediately to improve the conservation status of grassland birds in SESA.     

Effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis

Here we report the effects of metronidazole and tinidazole on NTPDase1 and ecto-5'-nucleotidase from intact cells of Trichomonas vaginalis. Adenosine triphosphate (ATP) and adenosine diphosphate (ADP) hydrolysis was 5- to 7-fold higher for the fresh clinical strain, when compared with the ATCC (American Type Culture Collection) strain. ATP hydrolysis was activated in the presence of metronidazole in the ATCC strain, whilst it was inhibited 33% by 50 microM tinidazole in a fresh clinical isolate. The treatment of cells in the presence of metronidazole for 2 h inhibited ATP and ADP hydrolysis, whilst treatment with tinidazole inhibited ATP and ADP hydrolysis only in the fresh clinical isolate. The drugs did not change the ecto-5'-nucleotidase activity for both strains. Our results suggest that the modulation of extracellular ATP and ADP levels during treatment with these drugs could be a parasitic defence strategy as a survival mechanism in an adverse environment.     

Ant communities,environmental characteristics and their implications for conservation in the Brazilian Pantanal

Studies about processes determining biodiversity in Pantanal are urgently needed and the use of invertebrates for this purpose may be an efficient tool. The purpose of this paper is to verify whether ants could be used to predict human impacts on Pantanal, assessing how environmental characteristics of gallery forest influence arboreal ant species richness. Specifically, we tested the hypotheses that: (i) resource availability (estimated by tree density) and (ii) diversity of environmental conditions (estimated by structural heterogeneity) positively influence arboreal ant species richness and composition. Ants were sampled in a gallery forest in the Miranda River. Ant species richness did not respond to tree density, but increased with structural heterogeneity. Species composition was affected both by tree density and structural heterogeneity. The species list produced in the present study showed a high similarity between arboreal ant faunas of Cerrado and Pantanal, which are contiguous biomes. The relationship of ant species richness with heterogeneity, as well as the observed changes of species composition with tree density and heterogeneity may be explained by the presence of a variation of environmental conditions, which create several micro-habitats in the forest. The response of the ant community can reflect human impacts on the forests of the studied region, which are usually modified to create patches for sport fishing and by pathways used in ecotourism.     

Characterization of an allelic series in the MONOPTEROS gene of arabidopsis

Patterning of numerous features of plants depends on transduction of the auxin signal. Auxin signaling is mediated by several pathways, the best understood of which relies on the function of the MONOPTEROS (MP) gene. Seven mp mutant alleles have been described in the widely used Columbia background of Arabidopsis: two extensively characterized and five only partially characterized. One of these five mp alleles appears to be extinct and thus unavailable for analysis. We show that two of the four remaining, partially characterized mp alleles reported to be in the Columbia background are in fact not in this background. We extend characterization of the remaining two Columbia alleles of mp, and we identify and characterize four new alleles of mp in the Columbia background, among which the first low‐expression allele of mp and the strongest Columbia allele of mp. These genetic resources provide the research community with new experimental opportunities for insight into the function of MP‐dependent auxin signaling in plant development. genesis 52:127–133. © 2013 Wiley Periodicals, Inc.