Polar auxin transport: an early invention

In higher plants, cell-to-cell polar auxin transport (PAT) of the phytohormone auxin, indole-3-acetic acid (IAA), generates maxima and minima that direct growth and development. Although IAA is present in all plant phyla, PAT has only been detected in land plants, the earliest being the Bryophytes. Charophyta, a group of freshwater green algae, are among the first multicellular algae with a land plant-like phenotype and are ancestors to land plants. IAA has been detected in members of Charophyta, but its developmental role and the occurrence of PAT are unknown. We show that naphthylphthalamic acid (NPA)-sensitive PAT occurs in internodal cells of Chara corallina. The relatively high velocity (at least 4-5 cm/h) of auxin transport through the giant (3-5 cm) Chara cells does not occur by simple diffusion and is not sensitive to a specific cytoplasmic streaming inhibitor. The results demonstrate that PAT evolved early in multicellular plant life. The giant Chara cells provide a unique new model system to study PAT, as Chara allows the combining of real-time measurements and mathematical modelling with molecular, developmental, cellular, and electrophysiological studies.     

Molecular mechanism of 14-3-3 protein-mediated inhibition of plant nitrate reductase

14-3-3 proteins regulate key processes in eukaryotic cells including nitrogen assimilation in plants by tuning the activity of nitrate reductase (NR), the first and rate-limiting enzyme in this pathway. The homodimeric NR harbors three cofactors, each of which is bound to separate domains, thus forming an electron transfer chain. 14-3-3 proteins inhibit NR by binding to a conserved phosphorylation site localized in the linker between the heme and molybdenum cofactor-containing domains. Here, we have investigated the molecular mechanism of 14-3-3-mediated NR inhibition using a fragment of the enzyme lacking the third domain, allowing us to analyze electron transfer from the heme cofactor via the molybdenum center to nitrate. The kinetic behavior of the inhibited Mo-heme fragment indicates that the principal point at which 14-3-3 acts is the electron transfer from the heme to the molybdenum cofactor. We demonstrate that this is not due to a perturbation of the reduction potentials of either the heme or the molybdenum center and conclude that 14-3-3 most likely inhibits nitrate reductase by inducing a conformational change that significantly increases the distance between the two redox-active sites.     

Regulation of voltage-gated potassium channels by PI(4,5)P2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) regulates activities of numerous ion channels including inwardly rectifying potassium (K_ir) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K_V) channels might be regulated by PI(4,5)P₂. Wide expression of K_V channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K_V channels by PI(4,5)P₂, we have coexpressed several of them in tsA-201 cells with a G protein–coupled receptor (M₁R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P₂ with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P₂ at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K_V7.1, K_V7.2/7.3, and K_ir2.1 channel current by 90–95%. Activation of M₁R inhibited K_V7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P₂ regulation of activity of K_V1.1/K_Vβ1.1, K_V1.3, K_V1.4, and K_V1.5/K_Vβ1.3, K_V2.1, K_V3.4, K_V4.2, K_V4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K_V1.1/K_Vβ1.1 and K_V3.4, resulting in up-regulation of current density upon activation of M₁R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P₂ at the plasma membrane by enzymes does not seem to influence activity of most tested K_V channels, whereas it does strongly inhibit members of the K_V7 and K_ir families.     

SNP-based association mapping of the polled gene in divergent cattle breeds

Naturally, hornless cattle are called polled. Although the POLL locus could be assigned to a c. 1.36‐Mb interval in the centromeric region of BTA1, the underlying genetic basis for the polled trait is still unknown. Here, an association mapping design was set up to refine the candidate region of the polled trait for subsequent high‐throughput sequencing. The case group comprised 101 homozygous polled animals from nine divergent cattle breeds, the majority represented by Galloway, Angus, Fleckvieh and Holstein Friesian. Additionally, this group included some polled individuals of Blonde d’Aquitaine, Charolais, Hereford, Jersey and Limousin breeds. The control group comprised horned Belgian Blue, Fleckvieh, Holstein Friesian and Illyrian Bu?a cattle. A genome‐wide scan using 49 163 SNPs was performed, which revealed one shared homozygous haplotype block consisting of nine neighbouring SNPs in all polled animals. This segment defines a 381‐kb interval on BTA1 that we consider to be the most likely location of the POLL mutation. Our results further demonstrate that the polled‐associated haplotype is also frequent in horned animals included in this study, and thus the haplotype as such cannot be used for population‐wide genetic testing. The actual trait‐associated haplotype may be revealed by using higher‐density SNP arrays. For the final identification of the causal mutation, we suggest high‐throughput sequencing of the entire candidate region, because the identification of functional candidate genes is difficult owing to the lack of a comparable model.     

Identification of human proteins that modify misfolding and proteotoxicity of pathogenic ataxin-1

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1-interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.     

Acetal linked oligoethylenimines for use as pH-sensitive gene carriers

Two types of acid-degradable nonviral gene carriers, OEI-MK and OEI-BAA, were synthesized by polymerizing oligoethylenimine of 800 Da (OEI800) with the pH-sensitive acetone ketal cross-linker 2,2-bis(N-maleimidoethyloxy) propane (MK) or the 4-methoxybenzaldehyde bisacrylate acetal cross-linker 1,1-bis-(2-acryloyloxy ethoxy)-[4-methoxy-phenyl]methane) (BAA). Corresponding acid-insensitive counterparts (OEI-BM and LT-OEI-HD) were synthesized as well, representing control polymers. Kinetics of hydrolysis were measured and confirmed the pH-dependent degradation profile of the acetal functions, with short half-lives of 3 min at pH 5.0, and 5 h (OEI-MK) or 3.5 h (OEI-BAA) at physiological pH 7.4 and 37 degrees C. DNA polyplexes of a luciferase expression plasmid were tested for gene transfer efficiency and biocompatibility in two cell lines (B16F10 and Neuro2A). Polyplexes with acid-labile polymers showed an improved toxicity profile compared to those made with acid-stable polymer analogues. At low cation/plasmid (c/p) w/w ratios the transfection efficiency of pH-sensitive polymers was slightly reduced, but it became similar or superior to the efficiency of acid-stable polymers at higher c/p ratios. An improved in vivo biocompatibility of the acid-degradable polymers over the stable control polymers was confirmed by liver histology after systemic administration of polymers in Balb/c mice.     

The human cerebrospinal fluid metabolome

With continuing improvements in analytical technology and an increased interest in comprehensive metabolic profiling of biofluids and tissues, there is a growing need to develop comprehensive reference resources for certain clinically important biofluids, such as blood, urine and cerebrospinal fluid (CSF). As part of our effort to systematically characterize the human metabolome we have chosen to characterize CSF as the first biofluid to be intensively scrutinized. In doing so, we combined comprehensive NMR, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography (LC) Fourier transform-mass spectrometry (FTMS) methods with computer-aided literature mining to identify and quantify essentially all of the metabolites that can be commonly detected (with today's technology) in the human CSF metabolome. Tables containing the compounds, concentrations, spectra, protocols and links to disease associations that we have found for the human CSF metabolome are freely available at http://www.csfmetabolome.ca.