Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

The adaptor protein-2 (AP-2) complex is a heterotetramer involved in clathrin-mediated endocytosis of cargo proteins from the plasma membrane in animal cells. The homologous genes of AP-2 subunits are present in the genomes of plants; however, their identities and roles in endocytic pathways are not clearly defined in plants. Here, we reveal the molecular composition of the AP-2 complex of Arabidopsis thaliana and its dynamics on the plasma membrane. We identified all of the α-, β-, σ-, and μ-subunits of the AP-2 complex and detected a weak interaction of the AP-2 complex with clathrin heavy chain. The μ-subunit protein fused to green fluorescent protein (AP2M-GFP) was localized to the plasma membrane and to the cytoplasm. Live-cell imaging using a variable-angle epifluorescence microscope revealed that AP2M-GFP transiently forms punctate structures on the plasma membrane. Homozygous ap2m mutant plants exhibited abnormal floral structures, including reduced stamen elongation and delayed anther dehiscence, which led to a failure of pollination and a subsequent reduction of fertility. Our study provides a molecular basis for understanding AP-2–dependent endocytic pathways in plants and their roles in floral organ development and plant reproduction.     

The More the Merrier: Recent Hybridization and Polyploidy in Cardamine

This article describes the use of cytogenomic and molecular approaches to explore the origin and evolution of Cardamine schulzii, a textbook example of a recent allopolyploid, in its ∼110-year history of human-induced hybridization and allopolyploidy in the Swiss Alps. Triploids are typically viewed as bridges between diploids and tetraploids but rarely as parental genomes of high-level hybrids and polyploids. The genome of the triploid semifertile hybrid Cardamine × insueta (2n = 24, RRA) was shown to combine the parental genomes of two diploid (2n = 2x = 16) species, Cardamine amara (AA) and Cardamine rivularis (RR). These parental genomes have remained structurally stable within the triploid genome over the >100 years since its origin. Furthermore, we provide compelling evidence that the alleged recent polyploid C. schulzii is not an autohexaploid derivative of C. × insueta. Instead, at least two hybridization events involving C. × insueta and the hypotetraploid Cardamine pratensis (PPPP, 2n = 4x−2 = 30) have resulted in the origin of the trigenomic hypopentaploid (2n = 5x−2 = 38, PPRRA) and hypohexaploid (2n = 6x−2 = 46, PPPPRA). These data show that the semifertile triploid hybrid can promote a merger of three different genomes and demonstrate how important it is to reexamine the routinely repeated textbook examples using modern techniques.     

Naemacyclus culmigenus,a newly reported potential pathogen to Miscanthus sinensis,new to Japan

Since the summer of 2010, a discomycete with erumpent apothecia associated with a leaf blight of Miscanthus leaves, were often collected. The morphological characteristics of the fungus suggested it was a member of the Helotiales rather than the Rhytismatales and this was supported by a phylogenetic analysis. Based on a morphological comparison with the type specimen of Naemacyclus culmigenus, currently known from Poaceae (Andropogon and Panicum), it was identified as N. culmigenus, new to Japan. The molecular phylogenetic analysis showed that the generic delimitation of Naemacyclus and related species requires clarification, as does their higher classification within the Leotiomycetes.     

Pressure-Induced Endocytic Degradation of the Saccharomyces cerevisiae Low-Affinity Tryptophan Permease Tat1 Is Mediated by Rsp5 Ubiquitin Ligase and Functionally Redundant PPxY Motif Proteins

Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1^K29R-K31R-GFP remained. The HPG1-1 (Rsp5^P514T) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.     

Urinary Fetuin-A Is a Novel Marker for Diabetic Nephropathy in Type 2 Diabetes Identified by Lectin Microarray

We analyzed the urine samples of patients with type 2 diabetes at various stages of diabetic nephropathy by lectin microarray to identify a biomarker to predict the progression of diabetic nephropathy. Japanese patients with type 2 diabetes at various stages of nephropathy were enrolled and we performed lectin microarray analyses (n = 17) and measured urinary excretion of fetuin-A (n = 85). The increased signals of urine samples were observed in Siaα2-6Gal/GalNAc-binding lectins (SNA, SSA, TJA-I) during the progression of diabetic nephropathy. We next isolated sialylated glycoproteins by using SSA-lectin affinity chromatography and identified fetuin-A by liquid chromatography–tandem mass spectrometer. Urinary excretion of fetuin-A significantly increased during the progression of albuminuria (A1, 0.40±0.43; A2, 0.60±0.53; A3 1.57±1.13 ng/gCr; p = 7.29×10⁻⁸) and of GFR stages (G1, 0.39±0.39; G2, 0.49±0.45; G3, 1.25±1.18; G4, 1.34±0.80 ng/gCr; p = 3.89×10⁻⁴). Multivariate logistic regression analysis was employed to assess fetuin-A as a risk for diabetic nephropathy with microalbuminuria or GFR<60 mL/min. Fetuin-A is demonstrated as a risk factor for both microalbuminuria and reduction of GFR in diabetic nephropathy with the odds ratio of 4.721 (1.881–11.844) and 3.739 (1.785–7.841), respectively. Collectively, the glycan profiling analysis is useful method to identify the urine biomarkers and fetuin-A is a candidate to predict the progression of diabetic nephropathy.     

Measuring Airway Surface Liquid Depth in Ex Vivo Mouse Airways by X-Ray Imaging for the Assessment of Cystic Fibrosis Airway Therapies

In the airways of those with cystic fibrosis (CF), the leading pathophysiological hypothesis is that an ion channel defect results in a relative decrease in airway surface liquid (ASL) volume, producing thick and sticky mucus that facilitates the establishment and progression of early fatal lung disease. This hypothesis predicts that any successful CF airway treatment for this fundamental channel defect should increase the ASL volume, but up until now there has been no method of measuring this volume that would be compatible with in vivo monitoring. In order to accurately monitor the volume of the ASL, we have developed a new x-ray phase contrast imaging method that utilizes a highly attenuating reference grid. In this study we used this imaging method to examine the effect of a current clinical CF treatment, aerosolized hypertonic saline, on ASL depth in ex vivo normal mouse tracheas, as the first step towards non-invasive in vivo ASL imaging. The ex vivo tracheas were treated with hypertonic saline, isotonic saline or no treatment using a nebuliser integrated within a small animal ventilator circuit. Those tracheas exposed to hypertonic saline showed a transient increase in the ASL depth, which continued for nine minutes post-treatment, before returning to baseline by twelve minutes. These findings are consistent with existing measurements on epithelial cell cultures, and therefore suggest promise for the future development of in vivo testing of treatments. Our grid-based imaging technique measures the ASL depth with micron resolution, and can directly observe the effect of treatments expected to increase ASL depth, prior to any changes in overall lung health. The ability to non-invasively observe micron changes in the airway surface, particularly if achieved in an in vivo setting, may have potential in pre-clinical research designed to bring new treatments for CF and other airway diseases to clinical trials.     

Spatial covariation of fish population vital rates in a stream network

Animal populations are spatially structured in heterogeneous landscapes, in which local patches with differing vital rates are connected by dispersal of individuals to varying degrees. Although there is evidence that vital rates differ among local populations, much less is understood about how vital rates covary among local patches in spatially heterogeneous landscapes. In this study, we conducted a nine-year annual mark–recapture survey to characterize spatial covariation of survival and growth for two Japanese native salmonids, white-spotted charr Salvelinus leucomaenis japonicus and red-spotted masu salmon Oncorhynchus masou ishikawae, in a headwater stream network composed of distinctly different tributary and mainstem habitats. Spatial structure of survival and growth differed by species and age class, but results provided support for negative covariation between vital rates, where survival was higher in the tributary habitat but growth was higher in the mainstem habitat. Thus, neither habitat was apparently more important than the other, and local habitats with complementary vital rates may make this spatially structured population less vulnerable to environmental change (i.e. portfolio effect). Despite the spatial structure of vital rates and possibilities that fish can exploit spatially distributed resources, movement of fish was limited due partly to a series of low-head dams that prevented upstream movement of fish in the study area. This study shows that spatial structure of vital rates can be complex and depend on species and age class, and this knowledge is likely paramount to elucidating dynamics of spatially structured populations.     

P2X7 receptors regulate engulfing activity of non-stimulated resting astrocytes

We previously demonstrated that P2X7 receptors (P2X7Rs) expressed by cultured mouse astrocytes were activated without any exogenous stimuli, but its roles in non-stimulated resting astrocytes remained unknown. It has been reported that astrocytes exhibit engulfing activity, and that the basal activity of P2X7Rs regulates the phagocytic activity of macrophages. In this study, therefore, we investigated whether P2X7Rs regulate the engulfing activity of mouse astrocytes. Uptake of non-opsonized beads by resting astrocytes derived from ddY-mouse cortex time-dependently increased, and the uptaken beads were detected in the intracellular space. The bead uptake was inhibited by cytochalasin D (CytD), an F-actin polymerization inhibitor, and agonists and antagonists of P2X7Rs apparently decreased the uptake. Spontaneous YO-PRO-1 uptake by ddY-mouse astrocytes was reduced by the agonists and antagonists of P2X7Rs, but not by CytD. Down-regulation of P2X7Rs using siRNA decreased the bead uptake by ddY-mouse astrocytes. In addition, compared to in the case of ddY-mouse astrocytes, SJL-mouse astrocytes exhibited higher YO-PRO-1 uptake activity, and their bead uptake was significantly greater. These findings suggest that resting astrocytes exhibit engulfing activity and that the activity is regulated, at least in part, by their P2X7Rs.     

Population genomic footprints of selection and associations with climate in natural populations of Arabidopsis halleri from the Alps

Natural genetic variation is essential for the adaptation of organisms to their local environment and to changing environmental conditions. Here, we examine genomewide patterns of nucleotide variation in natural populations of the outcrossing herb Arabidopsis halleri and associations with climatic variation among populations in the Alps. Using a pooled population sequencing (Pool‐Seq) approach, we discovered more than two million SNPs in five natural populations and identified highly differentiated genomic regions and SNPs using F_ST‐based analyses. We tested only the most strongly differentiated SNPs for associations with a nonredundant set of environmental factors using partial Mantel tests to identify topo‐climatic factors that may underlie the observed footprints of selection. Possible functions of genes showing signatures of selection were identified by Gene Ontology analysis. We found 175 genes to be highly associated with one or more of the five tested topo‐climatic factors. Of these, 23.4% had unknown functions. Genetic variation in four candidate genes was strongly associated with site water balance and solar radiation, and functional annotations were congruent with these environmental factors. Our results provide a genomewide perspective on the distribution of adaptive genetic variation in natural plant populations from a highly diverse and heterogeneous alpine environment.