Plant species richness–productivity relationships in a low-productive boreal region

Local species richness–productivity (SR–P) relationship is usually reported as unimodal if long productivity gradients are sampled. However, it tends to be monotonically increasing in low-productive environments due to the decreasing part of the SR–P curve being truncated. Previous work indicated that this can hold true for forest herb layers, because of an upper bound on productivity caused mainly by canopy shading. Here, we ask whether the same pattern exists in a region with an upper bound on productivity caused by a harsh climate. We sampled herbaceous vegetation of boreal forests and grasslands in a low-productive region of central Yakutia (NE Siberia) with dry and winter-cool continental climate. We collected data on species composition, herb-layer productivity (aboveground herbaceous biomass), soil chemistry and light availability. We applied regression models to discriminate between monotonically increasing, decreasing and unimodal responses of herb-layer species richness to measured variables and analysed trends in the species-pool size and beta diversity along the productivity gradient. Our expectation of the monotonically increasing SR–P relationship was confirmed for neither forest herb layers nor grasslands. In the forest herb layers, no relationship was detected. In grasslands, the relationship was unimodal with species richness decline starting at much lower productivity levels than in more productive temperate grasslands. Potential causes for this decline are either limitation of local species richness by the species pool, which contains few species adapted to more productive habitats, or competitive exclusion, which can become an important control of species richness under lower levels of productivity than is the case in temperate grasslands.     

Novel Systems for Dynamically Assessing Insulin Action in Live Cells Reveals Heterogeneity in the Insulin Response

Regulated GLUT4 trafficking is a key action of insulin. Quantitative stepwise analysis of this process provides a powerful tool for pinpointing regulatory nodes that contribute to insulin regulation and insulin resistance. We describe a novel GLUT4 construct and workflow for the streamlined dissection of GLUT4 trafficking; from simple high throughput screens to high resolution analyses of individual vesicles. We reveal single cell heterogeneity in insulin action highlighting the utility of this approach – each cell displayed a unique and highly reproducible insulin response, implying that each cell is hard‐wired to produce a specific output in response to a given stimulus. These data highlight that the response of a cell population to insulin is underpinned by extensive heterogeneity at the single cell level. This heterogeneity is pre‐programmed within each cell and is not the result of intracellular stochastic events.     

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM .     

Importin‐7 Mediates Nuclear Trafficking of DNA in Mammalian Cells

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy‐dependent way. The imp7‐dependent pathway was specifically competed by excess DNA but not by excess of maltose‐binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly‐l ‐lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non‐redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response .     

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 .      

Aquaporin Trafficking in Plant Cells: An Emerging Membrane‐Protein Model

Aquaporins (AQPs) are channel proteins that facilitate the transport of water and small solutes across biological membranes. In plants, AQPs exhibit a high multiplicity of isoforms in relation to a high diversity of sub‐cellular localizations, at the plasma membrane (PM) and in various intracellular compartments. Some members also exhibit a dual localization in distinct cell compartments, whereas others show polarized or domain‐specific expression at the PM or tonoplast, respectively. A diversity of mechanisms controlling the routing of newly synthesized AQPs towards their destination membranes and involving diacidic motifs, phosphorylation or tetramer assembly is being uncovered. Recent approaches using single particle tracking, fluorescence correlation spectroscopy and fluorescence recovery after photobleaching have, in combination with pharmacological interference, stressed the peculiarities of AQP sub‐cellular dynamics in environmentally challenging conditions. A role for clathrin and sterol‐rich domains in cell surface dynamics and endocytosis of PM AQPs was uncovered. These recent advances provide deep insights into the cellular mechanisms of water transport regulation in plants. They also point to AQPs as an emerging model for studying the sub‐cellular dynamics of plant membrane proteins .     

Trafficking of the IKs‐Complex in MDCK Cells: Site of Subunit Assembly and Determinants of Polarized Localization

The voltage‐gated potassium channel K_V7.1 is regulated by non‐pore forming regulatory KCNE β‐subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current I_Ks. However, where the subunits assemble and which of the subunits determines localization of the I_Ks‐complex has not been unequivocally resolved yet. We employed trafficking‐deficient K_V7.1 and KCNE1 mutants to investigate I_Ks trafficking using the polarized Madin‐Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post‐endoplasmic reticulum compartment. We demonstrate that K_V7.1 targets the I_Ks‐complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical K_V7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that K_V7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of K_V7.1/KCNE complexes .     

Cryo‐Immunoelectron Microscopy of Adherent Cells Improved by the Use of Electrospun Cell Culture Substrates

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non‐disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute‐long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze‐substitution, sample rehydration and cryosection‐immunolabelling or with freeze‐fracture replica‐immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry.     

A trs20 Mutation That Mimics an SEDT‐Causing Mutation Blocks Selective and Non‐Selective Autophagy: A Model for TRAPP III Organization

TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT‐causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III‐specific subunit), Trs120p and Trs130p (TRAPP II‐specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol‐to‐vacuole (cvt) pathway as well as non‐selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed .