Establishment of a tagged variant of Lgr4 receptor suitable for functional and expression studies in the mouse

Leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4) is produced in a broad spectrum of mouse embryonic and adult tissues and its deficiency results in embryonal or perinatal lethality. The LGR4 function was mainly related to its potentiation of canonical Wnt signaling; however, several recent studies associate LGR4 with additional signaling pathways. To obtain a suitable tool for studying the signaling properties of Lgr4, we generated a tagged variant of the Lgr4 receptor using gene targeting in the mouse oocyte. The modified Lgr4 allele expresses the Lgr4 protein fused with a triple hemagglutinin (3HA) tag located at the extracellular part of the protein. The allele is fully functional, enabling tracking of Lgr4 expression in the mouse tissues. We also show that via surface labeling, the 3HA tag allows direct isolation and analysis of living Lgr4-positive cells obtained from the small intestinal crypts. Finally, the HA tag-specific antibody can be employed to characterize the biochemical features of Lgr4 and to identify possible biding partners of the protein in cells derived from various mouse tissues.     

Reconstruction, modeling & analysis of Halobacterium salinarum R-1 metabolism

We present a genome-scale metabolic reconstruction for the extreme halophile Halobacterium salinarum. The reconstruction represents a summary of the knowledge regarding the organism's metabolism, and has already led to new research directions and improved the existing annotation. We used the network for computational analysis and studied the aerobic growth of the organism using dynamic simulations in media with 15 available carbon and energy sources. Simulations resulted in predictions for the internal fluxes, which describe at the molecular level how the organism lives and grows. We found numerous indications that cells maximized energy production even at the cost of longer term concerns such as growth prospects. Simulations showed a very low carbon incorporation rate of only approximately 15%. All of the supplied nutrients were simultaneously degraded, unexpectedly including five which are essential. These initially surprising behaviors are likely adaptations of the organism to its natural environment where growth occurs in blooms. In addition, we also examined specific aspects of metabolism, including how each of the supplied carbon and energy sources is utilized. Finally, we investigated the consequences of the model assumptions and the network structure on the quality of the flux predictions.     

Characterization of mutations in crucial residues around the Q(o) binding site of the cytochrome bc complex from Paracoccus denitrificans

The protonation state of residues around the Q(o) binding site of the cytochrome bc(1) complex from Paracoccus denitrificans and their interaction with bound quinone(s) was studied by a combined electrochemical and FTIR difference spectroscopic approach. Site-directed mutations of two groups of conserved residues were investigated: (a) acidic side chains located close to the surface and thought to participate in a water chain leading up to the heme b(L) edge, and (b) residues located in the vicinity of this site. Interestingly, most of the mutants retain a high degree of catalytic activity. E295Q, E81Q and Y297F showed reduced stigmatellin affinity. On the basis of electrochemically induced FTIR difference spectra, we suggest that E295 and D278 are protonated in the oxidized form or that their mutation perturbs protonated residues. Mutations Y302, Y297, E81 and E295, directly perturb signals from the oxidized quinone and of the protein backbone. By monitoring the interaction with the inhibitor stigmatellin for the wild-type enzyme at various redox states, interactions of the bound stigmatellin with amino acid side chains such as protonated acidic residues and the backbone were observed, as well as difference signals arising from the redox active inhibitor itself and the replaced quinone. The infrared difference spectra of the above Q(o) site mutations in the presence of stigmatellin confirm the previously established role of E295 as a direct interaction partner in the enzyme from P.denitrificans as well. The protonated residue E295 is proposed to change the hydrogen-bonding environment upon stigmatellin binding in the oxidized form, and is deprotonated in the reduced form. Of the residues located close to the surface, D278 remains protonated and unperturbed in the oxidized form but its frequency shifts in the reduced form. The mechanistic implications of our observations are discussed, together with previous inhibitor binding data, and referred to the published X-ray structures.     

Feeding ecology of Xenoturbella bocki (phylum Xenoturbellida) revealed by genetic barcoding

The benthic marine worm Xenoturbella is frequently contaminated with molluscan DNA, which had earlier caused confusion resulting in a suggested bivalve relationship. In order to find the source of the contaminant, we have used molluscan sequences derived from Xenoturbella and compared them to barcodes obtained from several individuals of the nonmicroscopic molluscs sharing the same environment as Xenoturbella. Using cytochrome oxidase 1, we found the contaminating sequences to be 98% similar to the bivalve Ennucula tenuis. Using the highly variable D1-D2 region of the large ribosomal subunit in Xenoturbella, we found three distinct species of contaminating molluscs, one of which is 99% similar to the bivalve Abra nitida, one of the most abundant bivalves in the Gullmarsfjord where Xenoturbella was found, and another 99% similar to the bivalve Nucula sulcata. These data clearly show that Xenoturbella only contains molluscan DNA originating from bivalves living in the same environment, refuting former hypotheses of a bivalve relationship. In addition, these data suggest that Xenoturbella feeds specifically on bivalve prey from multiple species, possibly in the form of eggs and larvae.     

Live imaging at the onset of cortical neurogenesis reveals differential appearance of the neuronal phenotype in apical versus basal progenitor progeny

The neurons of the mammalian brain are generated by progenitors dividing either at the apical surface of the ventricular zone (neuroepithelial and radial glial cells, collectively referred to as apical progenitors) or at its basal side (basal progenitors, also called intermediate progenitors). For apical progenitors, the orientation of the cleavage plane relative to their apical-basal axis is thought to be of critical importance for the fate of the daughter cells. For basal progenitors, the relationship between cell polarity, cleavage plane orientation and the fate of daughter cells is unknown. Here, we have investigated these issues at the very onset of cortical neurogenesis. To directly observe the generation of neurons from apical and basal progenitors, we established a novel transgenic mouse line in which membrane GFP is expressed from the beta-III-tubulin promoter, an early pan-neuronal marker, and crossed this line with a previously described knock-in line in which nuclear GFP is expressed from the Tis21 promoter, a pan-neurogenic progenitor marker. Mitotic Tis21-positive basal progenitors nearly always divided symmetrically, generating two neurons, but, in contrast to symmetrically dividing apical progenitors, lacked apical-basal polarity and showed a nearly randomized cleavage plane orientation. Moreover, the appearance of beta-III-tubulin-driven GFP fluorescence in basal progenitor-derived neurons, in contrast to that in apical progenitor-derived neurons, was so rapid that it suggested the initiation of the neuronal phenotype already in the progenitor. Our observations imply that (i) the loss of apical-basal polarity restricts neuronal progenitors to the symmetric mode of cell division, and that (ii) basal progenitors initiate the expression of neuronal phenotype already before mitosis, in contrast to apical progenitors.     

Molecular dynamics and mutational analysis of a channelopathy mutation in the IIS6 helix of Ca V 1.2

A channelopathy mutation in segment IIS6 of Ca(V)1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarized voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in Ca(V)1.2 (midpoint of activation curve (V(0.5)) shifted to -37.7 +/- 1.2 mV). We show here that wild-type gating can partially be restored by a helix stabilizing rescue mutation N785A. V(0.5) of I781T/N785A (V(0.5) = -21.5 +/- 0.6 mV) was shifted back towards wild-type (V(0.5) = -9.9 +/- 1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform Molecular Dynamics-simulations (MD-simulations) on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187-F1194) and in helix IIS6 (N785-L786) were studied. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild-type Ca(V)1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.     

Pore stability and gating in voltage-activated calcium channels

Calcium channel family members activate at different membrane potentials, which enables tissue specific calcium entry. Pore mutations affecting this voltage dependence are associated with channelopathies. In this review we analyze the link between voltage sensitivity and corresponding kinetic phenotypes of calcium channel activation. Systematic changes in hydrophobicity in the lower third of S6 segments gradually shift the activation curve thereby determining the voltage sensitivity. Homology modeling suggests that hydrophobic residues that are located in all four S6 segments close to the inner channel mouth might form adhesion points stabilizing the closed gate. Simulation studies support a scenario where voltage sensors and the pore are essentially independent structural units. We speculate that evolution designed the voltage sensing machinery as robust "all-or-non" device while the varietys of voltage sensitivities of different channel types was accomplished by shaping pore stability.     

Beta-AMYLASE4, a noncatalytic protein required for starch breakdown, acts upstream of three active beta-amylases in Arabidopsis chloroplasts

This work investigated the roles of beta-amylases in the breakdown of leaf starch. Of the nine beta-amylase (BAM)-like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable beta-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized beta-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active beta-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total beta-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that beta-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.     

Spliceosomal small nuclear ribonucleoprotein particles repeatedly cycle through Cajal bodies

The Cajal body (CB) is a nuclear structure closely associated with import and biogenesis of small nuclear ribonucleoprotein particles (snRNPs). Here, we tested whether CBs also contain mature snRNPs and whether CB integrity depends on the ongoing snRNP splicing cycle. Sm proteins tagged with photoactivatable and color-maturing variants of fluorescent proteins were used to monitor snRNP behavior in living cells over time; mature snRNPs accumulated in CBs, traveled from one CB to another, and they were not preferentially replaced by newly imported snRNPs. To test whether CB integrity depends on the snRNP splicing cycle, two human orthologues of yeast proteins involved in distinct steps in spliceosome disassembly after splicing, hPrp22 and hNtr1, were depleted by small interfering RNA treatment. Surprisingly, depletion of either protein led to the accumulation of U4/U6 snRNPs in CBs, suggesting that reassembly of the U4/U6.U5 tri-snRNP was delayed. Accordingly, a relative decrease in U5 snRNPs compared with U4/U6 snRNPs was observed in CBs, as well as in nuclear extracts of treated cells. Together, the data show that particular phases of the spliceosome cycle are compartmentalized in living cells, with reassembly of the tri-snRNP occurring in CBs.     

The temperature sensitivity of soil: microbial biodiversity,growth, and carbon mineralization

Microorganisms drive soil carbon mineralization and changes in their activity with increased temperature could feedback to climate change. Variation in microbial biodiversity and the temperature sensitivities (Q₁₀) of individual taxa may explain differences in the Q₁₀ of soil respiration, a possibility not previously examined due to methodological limitations. Here, we show phylogenetic and taxonomic variation in the Q₁₀ of growth (5–35 °C) among soil bacteria from four sites, one from each of Arctic, boreal, temperate, and tropical biomes. Differences in the temperature sensitivities of taxa and the taxonomic composition of communities determined community-assembled bacterial growth Q₁₀, which was strongly predictive of soil respiration Q₁₀ within and across biomes. Our results suggest community-assembled traits of microbial taxa may enable enhanced prediction of carbon cycling feedbacks to climate change in ecosystems across the globe.Subject terms: Biogeochemistry, Ecosystem ecology, Soil microbiology