A Sorting Nexin 17‐Binding Domain Within the LRP1 Cytoplasmic Tail Mediates Receptor Recycling Through the Basolateral Sorting Endosome

Sorting nexin 17 (SNX17) is an adaptor protein present in early endosomal antigen 1 (EEA1)‐positive sorting endosomes that promotes the efficient recycling of low‐density lipoprotein receptor‐related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1‐positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17‐binding domain, we generated chimeric proteins in which the SNX17‐binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non‐polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized Madin‐Darby canine kidney cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17‐binding receptors and the restricted function of SNX17 in the BSE .      

Beta‐diversity in temperate and tropical forests reflects dissimilar mechanisms of community assembly

Site‐to‐site variation in species composition (β‐diversity) generally increases from low‐ to high‐diversity regions. Although biogeographical differences in community assembly mechanisms may explain this pattern, random sampling effects can create this pattern through differences in regional species pools. Here, we compared assembly mechanisms between spatially extensive networks of temperate and tropical forest plots with highly divergent species pools (46 vs. 607 species). After controlling for sampling effects, β‐diversity of woody plants was similar and higher than expected by chance in both forests, reflecting strong intraspecific aggregation. However, different mechanisms appeared to explain aggregation in the two forests. In the temperate forest, aggregation reflected stronger environmental correlations, suggesting an important role for species‐sorting (e.g. environmental filtering) processes, whereas in the tropics, aggregation reflected stronger spatial correlations, more likely reflecting dispersal limitation. We suggest that biogeographical differences in the relative importance of different community assembly mechanisms contribute to these striking gradients in global biodiversity.     

The importance of metacommunity ecology for environmental assessment research in the freshwater realm

Most bioassessment programs rest on the assumption that species have different niches, and that abiotic environmental conditions and changes therein determine community structure. This assumption is thus equivalent to the species sorting perspective (i.e. that species differ in their responses to environmental variation) in metacommunity ecology. The degree to which basing bioassessment on the species sorting perspective is reasonable is likely to be related to the spatial extent of a study and the characteristics of the organism groups (e.g. dispersal ability) with which the effects of anthropogenic changes are assessed. Recent findings in metacommunity research have stressed that community structure is determined not only by local abiotic environmental conditions but also by biotic interactions and dispersal‐related effects. For example, dispersal limitation may prevent community structure recovery from the effects of a putative stressor, as organisms may not be able to disperse to all sites in a region. Mass effects (i.e. the presence of species in environmentally suboptimal sites due to high dispersal rates from environmentally suitable sites) may, in turn, obscure the effects of a stressor, as dispersal from source sites (e.g. an unaltered site) allows persistence at sink sites (e.g. an anthropogenically altered site). Better bioassessment should thus take both niche‐ and dispersal‐related processes simultaneously into consideration, which can be accomplished by explicitly modelling spatial location as a proxy for dispersal effects. Such an integrated approach should be included in bioassessment programs using general multivariate approaches, predictive modelling, and multimetric indices.     

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel''s cartilage, nasal glands, tongue, and ear.     

Sorting nexin Snx41 is essential for conidiation and mediates glutathione-based antioxidant defense during invasive growth in Magnaporthe oryzae

The sorting nexins Atg20/Snx42 and Snx41 regulate membrane traffic and endosomal protein sorting and are essential for Cvt and/or pexophagy in yeast. Previously, we showed that macroautophagy is necessary for conidiation in the rice-blast fungus Magnaporthe oryzae. Here, we analyzed the physiological function(s) of selective autophagy in Magnaporthe through targeted deletion of MGG_12832, an ortholog of yeast SNX41 and ATG20/SNX42. Loss of MGG_12832 (hereafter SNX41) abolished conidia formation and pathogenesis in M. oryzae. Snx41-GFP localized as dynamic puncta or short tubules that are partially associated with autophagosomes and/or autophagic vacuoles. PX domain, but not macroautophagy per se, was required for such localization of Snx41-GFP in Magnaporthe. Although not required for nonselective autophagy, Snx41 was essential for pexophagy in Magnaporthe. We identified Oxp1, an ATP-dependent oxoprolinase in the gamma-glutamyl cycle, as a binding partner and potential retrieval target of Snx41-dependent protein sorting. The substrate of Oxp1, 5-oxoproline, could partially restore conidiation in the snx41Δ. Exogenous glutathione, a product of the gamma-glutamyl cycle, significantly restored pathogenicity in the snx41Δ mutant, likely through counteracting the oxidative stress imposed by the host. We propose that the gamma-glutamyl cycle and glutathione biosynthesis are subject to regulation by Snx41-dependent vesicular trafficking, and mediate antioxidant defense crucial for in planta growth and pathogenic differentiation of Magnaporthe at the onset of blast disease in rice.     

C1188D mutation abolishes specific recognition between MLL1-CXXC domain and CpG site by inducing conformational switch of flexible N-terminal

Mixed lineage leukemia protein (MLL1 protein) recognizes the CpG site via its CXXC domain and is frequently associated with leukemia. The specific recognition is abolished by C1188D mutation, which also prevents MLL-related leukemia. In this paper, multiple molecular dynamic (MD) simulations were performed to investigate the mechanism of recognition and influences of C1188D mutation. Started from fully dissociated DNA and MLL1-CXXC domain, remarkably, the center of mass (COM) of MLL1-CXXC domain quickly concentrates on the vicinity of the CpG site in all 53 short MD simulations. Extended simulations of the wild type showed that the native complex formed in 500 ns among 4 of 53 simulations. In contrast, the C1188D mutant COM distributed broadly around the DNA and the native complex was not observed in any of the extended simulations. Simulations on the apo MLL1-CXXC domain further suggest that the wild type protein remained predominantly in an open form that closely resembles its structure in the native complex whereas C1188D mutant formed predominantly compact structures in which the N- terminal bends to D1188. This conformational switch hinders the formation of encounter complex, thus abolishes the recognition. Our study also provides clues to the study mechanism of recognition, by the CXXC domain from proteins like DNA methyltransferase and ten-eleven translocation enzymes.     

The amphipathic helices of Arfrp1 and Arl14 are sufficient to determine subcellular localizations

The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors. By promoting GTP binding, guanine nucleotide exchange factors induce conformational changes of Arf proteins exposing their N-terminal amphipathic helices, which then insert into the membranes to stabilize the membrane association process. Here, we found that the N-terminal amphipathic motifs of the Golgi-localized Arf family protein, Arfrp1, and the endosome- and plasma membrane–localized Arf family protein, Arl14, play critical roles in spatial determination. Exchanging the amphipathic helix motifs between these two Arf proteins causes the switch of their localizations. Moreover, the amphipathic helices of Arfrp1 and Arl14 are sufficient for cytosolic proteins to be localized into a specific cellular compartment. The spatial determination mediated by the Arfrp1 helix requires its binding partner Sys1. In addition, the residues that are required for the acetylation of the Arfrp1 helix and the myristoylation of the Arl14 helix are important for the specific subcellular localization. Interestingly, Arfrp1 and Arl14 are recruited to their specific cellular compartments independent of GTP binding. Our results demonstrate that the amphipathic motifs of Arfrp1 and Arl14 are sufficient for determining specific subcellular localizations in a GTP-independent manner, suggesting that the membrane association and activation of some Arf proteins are uncoupled.     

Comparison of Complex DNA Mixtures with Generic Oligonucleotide Microchips

Abstract

The reproducibility of melting curves for repeated hybridizations of target DNA with generic oligonucleotide microchips is shown experimentally to depend on the character of matching between fragments of target DNA and immobilized oligonucleotides. The reproducibility of melting curves is higher for the perfect match duplexes and decreases as the number of mismatched pairs within duplexes increases. This effect was applied to the comparative analysis of complex DNA mixtures. We developed a scheme in which we can identify and discriminate between the probe oligonucleotides responsible for the distinctions between target DNA mixtures. A scheme is illustrated by comparing DNA mixtures corresponding to VD-J genes connected with populations of mRNAs CDR3 TCR Vb (T-cell receptor beta complementarity determining region 3) from the thymus and pancreas of NOD mice. Our results demonstrate that generic microchips can be applied efficiently to the analysis of DNA mixtures.