A proteomic snapshot of the human heat shock protein 90 interactome

Heat shock protein 90 (Hsp90) is a molecular chaperone which modulates several signalling pathways within a cell. By applying co-immunoprecipitation with endogeneous Hsp90, we were able to identify 39 novel protein interaction partners of this chaperone in human embryonic kidney cells (HEK293). Interestingly, levels of DNA-activated protein kinase catalytic subunit, an Hsp90 interaction partner found in this study, were found to be sensitive to Hsp90 inhibitor treatment only in HeLa cells but not in HEK293 cells referring to the tumorgenicity of this chaperone.     

Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis

Fluorescence recovery after photobleaching (FRAP) is a widely used imaging technique for measuring the mobility of fluorescently tagged proteins in living cells. Although FRAP presumes that high-intensity illumination causes only irreversible photobleaching, reversible photoswitching of many fluorescent molecules, including GFP, can also occur. Here, we show that this photoswitching is likely to contaminate many FRAPs of GFP, and worse, the size of its contribution can be up to 60% under different experimental conditions, making it difficult to compare FRAPs from different studies. We develop a procedure to correct FRAPs for photoswitching and apply it to FRAPs of the GFP-tagged histone H2B, which, depending on the precise photobleaching conditions exhibits apparent fast components ranging from 9-36% before correction and ～1% after correction. We demonstrate how this ～1% fast component of H2B-GFP can be used as a benchmark both to estimate the role of photoswitching in previous FRAP studies of TATA binding proteins (TBP) and also as a tool to minimize the contribution of photoswitching to tolerable levels in future FRAP experiments. In sum, we show how the impact of photoswitching on FRAP can be identified, minimized, and corrected.     

Increased microbial growth,biomass, and turnover drive soil organic carbon accumulation at higher plant diversity

Species‐rich plant communities have been shown to be more productive and to exhibit increased long‐term soil organic carbon (SOC) storage. Soil microorganisms are central to the conversion of plant organic matter into SOC, yet the relationship between plant diversity, soil microbial growth, turnover as well as carbon use efficiency (CUE) and SOC accumulation is unknown. As heterotrophic soil microbes are primarily carbon limited, it is important to understand how they respond to increased plant‐derived carbon inputs at higher plant species richness (PSR). We used the long‐term grassland biodiversity experiment in Jena, Germany, to examine how microbial physiology responds to changes in plant diversity and how this affects SOC content. The Jena Experiment considers different numbers of species (1–60), functional groups (1–4) as well as functional identity (small herbs, tall herbs, grasses, and legumes). We found that PSR accelerated microbial growth and turnover and increased microbial biomass and necromass. PSR also accelerated microbial respiration, but this effect was less strong than for microbial growth. In contrast, PSR did not affect microbial CUE or biomass‐specific respiration. Structural equation models revealed that PSR had direct positive effects on root biomass, and thereby on microbial growth and microbial biomass carbon. Finally, PSR increased SOC content via its positive influence on microbial biomass carbon. We suggest that PSR favors faster rates of microbial growth and turnover, likely due to greater plant productivity, resulting in higher amounts of microbial biomass and necromass that translate into the observed increase in SOC. We thus identify the microbial mechanism linking species‐rich plant communities to a carbon cycle process of importance to Earth's climate system.     

Environmental and developmental effects on the biosynthesis of UV-B screening pigments in Scots pine (Pinus sylvestris L.) needles

The major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris) needles are flavonol 3-o-glycosides (F3Gs) esterified with hydroxycinnamic acids at positions 3" and 6". Acylation is the last step in biosynthesis and is catalysed by position-specific hydroxycinnamoyl transferases (3" and 6"HCT). The UV-B dependence of these enzyme activities was studied in primary needles of Scots pine seedlings grown under different UV-B conditions in environmentally controlled sun simulators. 6"HCT activity was induced upon UV-B irradiation while 3"HCT activity was not induced but showed high constitutive values. To investigate the biosynthesis of diacylated F3Gs during needle development under natural conditions, the HCT activities and metabolite contents were analysed in needles of field-grown mature pine trees. Accumulation of diacylated compounds as well as of 6"HCT activity occurred transiently in the first year of needle development only. In contrast, 3"HCT activity exhibited broad maxima in two consecutive years during needle growth. The data suggest that acylated F3Gs are first formed as soluble compounds which are then translocated into the cell wall to be bound by their hydroxycinnamoyl residues.     

Ferrocenyl-substituted allenylidene complexes of chromium, molybdenum and tungsten: Synthesis, structure and reactivity

Bis(ferrocenyl)-substituted allenylidene complexes, [(CO)₅MCCCFc₂] (1a-c, Fc = (C₅H₄)Fe(C₅H₅), M = Cr (a), Mo (b), W (c)) were obtained by sequential reaction of Fc₂CO with Me₃Si-CCH, KF/MeOH, n-BuLi, and [(CO)₅M(THF)]. For the synthesis of related mono(ferrocenyl)allenylidene chromium complexes, [(CO)₅CrCCC(Fc)R] (R = Ph, NMe₂), three different routes were developed: (a) reaction of the deprotonated propargylic alcohol HCCC(Fc)(Ph)OH with [(CO)₅Cr(THF)] followed by desoxygenation with Cl₂CO, (b) Lewis acid induced alcohol elimination from alkenyl(alkoxy)carbene complexes, [(CO)₅CrC(OR)CHC(NMe₂)Fc], and (c) replacement of OMe in [(CO)₅CrCCC(OMe)NMe₂] by Fc. Complex 1a was also formed when the mono(ferrocenyl)allenylidene complex [(CO)₅CrCCC(Fc)NMe₂] was treated first with Li[Fc] and the resulting adduct then with SiO₂. The replacement route (c) was also applied to the synthesis of an allenylidene complex (7a) with a CC spacer in between the ferrocenyl unit and C_γ of the allenylidene ligand, [(CO)₅CrCCC(NMe₂)-CCFc]. The related complex containing a CHCH spacer (9a) was prepared by condensation of [(CO)₅CrCCC(Me)NMe₂] with formylferrocene in the presence of NEt₃. The bis(ferrocenyl)-substituted allenylidene complexes 1a-c added HNMe₂ across the C_α-C_β bond to give alkenyl(dimethylamino)carbene complexes and reacted with diethylaminopropyne by regioselective insertion of the CC bond into the C_β-C_γ bond to afford alkenyl(diethylamino)allenylidene complexes, [(CO)₅MCCC(NEt₂)CMeCFc₂]. The structures of 5a, 7a, and 9a were established by X-ray diffraction studies.     

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in Arabidopsis thaliana

Nicotinamide adenine dinucleotide (NAD⁺) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD⁺ biosynthesis is exclusively cytosolic. Hence, NAD⁺ must be imported into organelles to support their metabolic functions. Three NAD⁺ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T‐DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD⁺ homeostasis for both metabolic and developmental processes in plants.     

Moth assemblages in Costa Rica rain forest mirror small-scale topographic heterogeneity

In many tropical lowland rain forests, topographic variation increases environmental heterogeneity, thus contributing to the extraordinary biodiversity of tropical lowland forests. While a growing number of studies have addressed effects of topographic differences on tropical insect communities at regional scales (e.g., along extensive elevational gradients), surprisingly little is known about topographic effects at smaller spatial scales. The present study investigates moth assemblages in a topographically heterogeneous lowland rain forest landscape, at distances of less than a few hundred meters, in the Golfo Dulce region (SW Costa Rica). Three moth lineages—Erebidae–Arctiinae (tiger and lichen moths), the bombycoid complex, and Geometridae (inchworm moths)—were examined by means of automatic light traps in three different forest types: creek forest, slope forest, and ridge forest. Altogether, 6,543 individuals of 419 species were observed. Moth assemblages differed significantly between the three forest types regarding species richness, total abundance, and species composition. Moth richness and abundance increased more than fourfold and eightfold from creek over slope to ridge forest sites. All three taxonomic units showed identical biodiversity patterns, notwithstanding their strong differences in multiple eco-morphological traits. An indicator species analysis revealed that most species identified as characteristic were associated either with the ridge forest alone or with ridge plus slope forests, but very few with the creek forest. Despite their mobility, local moth assemblages are highly differentially filtered from the same regional species pool. Hence, variation in environmental factors significantly affects assemblages of tropical moth species at small spatial scales.     

Complex III releases superoxide to both sides of the inner mitochondrial membrane

Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for approximately 50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining approximately 50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result.     

Lipopolysaccharide-sensitive H+ current in dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells equipped to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Ligation of Toll-like receptor 4 (TLR4), expressed on the DC surface, by lipopolysaccharides (LPS), elements of the Gram-negative bacteria outer wall, induces DC maturation. Initial steps of maturation include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production with eventual downregulation of endocytic capacity in fully matured DCs. ROS production depends on NADPH oxidase (NOX2), the activity of which requires continuous pH and charge compensation. The present study demonstrates, for the first time, the functional expression of voltage-gated proton (Hv1) channels in mouse bone marrow-derived DCs. In whole cell patch-clamp experiments, we recorded Zn(2+) (50 μM)-sensitive outwardly rectifying currents activated upon depolarization, which were highly selective for H(+), with the reversal potential shift of 38 mV per pH unit. The threshold voltage of activation (V(threshold)) was dependent on the pH gradient and was close to the empirically predicted V(threshold) for the Hv1 currents. LPS (1 μg/ml) had bimodal effects on Hv1 channels: acute LPS treatment increased Hv1 channel activity, whereas 24 h of LPS incubation significantly inhibited Hv1 currents and decreased ROS production. Activation of H(+) currents by acute application of LPS was abolished by PKC inhibitor GFX (10 nM). According to electron current measurements, acute LPS application was associated with increased NOX2 activity.