Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping

Background

Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis.

Methods

Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme.

Results

Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5 μm in diameter.

Conclusions

Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before.

General significance

μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.     

Free mRNA in excess upon polysome dissociation is a scaffold for protein multimerization to form stress granules

The sequence of events leading to stress granule assembly in stressed cells remains elusive. We show here, using isotope labeling and ion microprobe, that proportionally more RNA than proteins are present in stress granules than in surrounding cytoplasm. We further demonstrate that the delivery of single strand polynucleotides, mRNA and ssDNA, to the cytoplasm can trigger stress granule assembly. On the other hand, increasing the cytoplasmic level of mRNA-binding proteins like YB-1 can directly prevent the aggregation of mRNA by forming isolated mRNPs, as evidenced by atomic force microscopy. Interestingly, we also discovered that enucleated cells do form stress granules, demonstrating that the translocation to the cytoplasm of nuclear prion-like RNA-binding proteins like TIA-1 is dispensable for stress granule assembly. The results lead to an alternative view on stress granule formation based on the following sequence of events: after the massive dissociation of polysomes during stress, mRNA-stabilizing proteins like YB-1 are outnumbered by the burst of nonpolysomal mRNA. mRNA freed of ribosomes thus becomes accessible to mRNA-binding aggregation-prone proteins or misfolded proteins, which induces stress granule formation. Within the frame of this model, the shuttling of nuclear mRNA-stabilizing proteins to the cytoplasm could dissociate stress granules or prevent their assembly.     

Early life expenditure in sexual competition is associated with increased reproductive senescence in male red deer

The evolutionary theories of senescence predict that investment in reproduction in early life should come at the cost of reduced somatic maintenance, and thus earlier or more rapid senescence. There is now growing support for such trade-offs in wild vertebrates, but these exclusively come from females. Here, we test this prediction in male red deer (Cervus elaphus) using detailed longitudinal data collected over a 40-year field study. We show that males which had larger harems and thereby allocated more resources to reproduction during early adulthood experienced higher rates of senescence in both harem size and rut duration. Males that carried antlers with more points during early life did not show more pronounced declines in reproductive traits in later life. Overall, we demonstrate that sexual competition shapes male reproductive senescence in wild red deer populations and provide rare empirical support for the disposable soma theory of ageing in males of polygynous vertebrate species.     

Thermal Fluctuations in Amphipol A8-35 Particles: A Neutron Scattering and Molecular Dynamics Study

Amphipols are a class of polymeric surfactants that can stabilize membrane proteins in aqueous solutions as compared to detergents. A8-35, the best-characterized amphipol to date, is composed of a polyacrylate backbone with ~35 % of the carboxylates free, ~25 % grafted with octyl side-chains, and ~40 % with isopropyl ones. In aqueous solutions, A8-35 self-organizes into globular particles with a molecular mass of ~40 kDa. The thermal dynamics of A8-35 particles was measured by neutron scattering in the 10-picosecond, 18-picosecond, and 1-nanosecond time-scales on natural abundance and deuterium-labeled molecules, which permitted to separate backbone and side-chain motions. A parallel analysis was performed on molecular dynamics trajectories (Perlmutter et al., Langmuir 27:10523–10537, 2011). Experimental results and simulations converge, from their respective time-scales, to show that A8-35 particles feature a more fluid hydrophobic core, predominantly containing the octyl chains, and a more rigid solvent-exposed surface, made up predominantly of the hydrophilic polymer backbone. The fluidity of the core is comparable to that of the lipid environment around proteins in the center of biological membranes, as also measured by neutron scattering. The biological activity of proteins depends sensitively on molecular dynamics, which itself is strongly dependent on the immediate macromolecular environment. In this context, the characterization of A8-35 particle dynamics constitutes a step toward understanding the effect of amphipols on membrane protein stability and function.     

Distinct Amino Acids in the C-Linker Domain of the Arabidopsis K+ Channel KAT2 Determine Its Subcellular Localization and Activity at the Plasma Membrane

Shaker K⁺ channels form the major K⁺ conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K⁺ channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K⁺ rectifying channel1 (AtKC1). These two subunits contribute to K⁺ transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch ₃₁₂TVRAASEFA₃₂₀ that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K⁺) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function.In plants, potassium channels from the Shaker family dominate the plasma membrane (PM) conductance to K⁺ in most cell types and play crucial roles in sustained K⁺ transport (Blatt et al., 2012; Hedrich, 2012; Sharma et al., 2013). Plant Shaker channels, like their homologs in animals (Craven and Zagotta, 2006; Wahl-Schott and Biel, 2009), belong to the six transmembrane-one pore (6TM-1P) cation channel superfamily. Functional channels are tetrameric proteins arranged around a central pore (Daram et al., 1997; Urbach et al., 2000; Dreyer et al., 2004). These channels can result from the assembly of Shaker subunits encoded by the same gene (homotetramers) or by different Shaker genes (heterotetramers). Heterotetramerization has been extensively reported within the inwardly rectifying Shaker channel group (five members in Arabidopsis [Arabidopsis thaliana]) and increased channel functional diversity (Jeanguenin et al., 2008; Lebaudy et al., 2008a).Based on in silico sequence analyses, plant Shaker subunits display a short cytosolic N-terminal domain, followed by the 6TM-1P hydrophobic core, and a long C-terminal cytosolic region in which several domains can be identified. The first one, named C-linker (about 80 amino acids in length), is followed by a cyclic nucleotide-binding domain (CNBD), an ankyrin domain (involved in protein-protein interaction; Lee et al., 2007, Grefen and Blatt, 2012), and a domain named K_HA (Ehrhardt et al., 1997) rich in hydrophobic and acidic residues. Sequence analysis of plant Shaker channels indicates that, among these cytosolic domains, the highest levels of similarity are displayed by the C-linker and the CNBD domains. Interestingly, both domains are also highly conserved in some members from the animal K⁺ channel superfamily, like Hyperpolarization-activated, cyclic nucleotide-gated K⁺ channel (HCN), K⁺ voltage-gated channel, subfamily H (KCNH), and Cyclic-nucleotide-gated ion channel (CNGC). In these animal 6TM-1P channels, the roles of C-linker and CNBD domains have been extensively investigated via crystal structure analyses (Zagotta et al., 2003; Brelidze et al., 2012), whereas plant Shaker channels are still poorly characterized at the structural level (Dreyer et al., 2004; Gajdanowicz et al., 2009; Naso et al., 2009; Garcia-Mata et al., 2010).Aiming at investigating the structure-function relationship of plant Shaker channels, we have used the Arabidopsis Shaker subunit K⁺ channel in Arabidopsis thaliana2 (KAT2) as a model. We developed a subdomain-swapping strategy between KAT2 and another Shaker subunit displaying distinctive features, Arabidopsis thaliana K⁺ rectifying channel1 (AtKC1). The KAT2 subunit can form homomeric or heteromeric inwardly rectifying K⁺ channels at the PM and has been shown to be strongly expressed in guard cells, where it provides a major contribution to the membrane conductance to K⁺ (Pilot et al., 2001; Lebaudy et al., 2008b). In contrast, the behavior of AtKC1 is more complex. In planta, this subunit is coexpressed with other inwardly rectifying Shaker subunits, including KAT2, in different plant tissues (Jeanguenin et al., 2011), and in roots, direct evidence has been obtained that AtKC1 is involved in functional heterotetrameric channel formation with AKT1 (Reintanz et al., 2002; Honsbein et al., 2009). However, experiments performed in tobacco (Nicotiana tabacum) mesophyll protoplasts have revealed that when expressed alone, AtKC1 is entrapped in the endoplasmic reticulum (ER). However, in tobacco protoplasts and Xenopus laevis oocytes, coexpression of AtKC1 with KAT2 or other inwardly rectifying Shaker subunits (AKT1, KAT1, or AKT2) gives rise to functional heteromeric channels (Duby et al., 2008; Jeanguenin et al., 2011). In Arabidopsis, it is interesting that evidence of the AtKC1 retention in the ER compartment, in the absence of other Shaker subunits, is lacking, since in the native tissues, AtKC1 is always expressed with its inward partners, with which it is able to form heteromeric channels.Here, we took advantage of the unique behavior of AtKC1 when expressed in heterologous systems to investigate the structure-function relationship of the C-linker of KAT2 by sequence exchange between these two channel subunits and by site-directed mutagenesis. The C-linker domain, which, to our knowledge, had never been studied as such in plant Shaker channels before, could be predicted to play crucial roles in channel properties due to its strategic location between the channel transmembrane core and the cytoplasmic CNBD domain. The resulting KAT2-AtKC1 chimeras were expressed in tobacco mesophyll protoplasts and in X. laevis oocytes for investigating their subcellular localization and measuring their activity at the cell membrane. Here, we show that two amino acids present in the C-linker are important for channel subcellular location and that a stretch of nine amino acids forming a short helix just below the membrane, downstream of the sixth transmembrane segment of the channel hydrophobic core, is involved in channel gating. The obtained experimental results are discussed in relation with a KAT2 C-linker/CNBD three-dimensional (3D) model based on animal HCN channels as structure templates.     

Genome mining of Streptomyces ambofaciens

Since the discovery of the streptomycin produced by Streptomyces griseus in the middle of the last century, members of this bacterial genus have been largely exploited for the production of secondary metabolites with wide uses in medicine and in agriculture. They have even been recognized as one of the most prolific producers of natural products among microorganisms. With the onset of the genomic era, it became evident that these microorganisms still represent a major source for the discovery of novel secondary metabolites. This was highlighted with the complete genome sequencing of Streptomyces coelicolor A3(2) which revealed an unexpected potential of this organism to synthesize natural products undetected until then by classical screening methods. Since then, analysis of sequenced genomes from numerous Streptomyces species has shown that a single species can carry more than 30 secondary metabolite gene clusters, reinforcing the idea that the biosynthetic potential of this bacterial genus is far from being fully exploited. This review highlights our knowledge on the potential of Streptomyces ambofaciens ATCC 23877 to synthesize natural products. This industrial strain was known for decades to only produce the drug spiramycin and another antibacterial compound, congocidine. Mining of its genome allowed the identification of 23 clusters potentially involved in the production of other secondary metabolites. Studies of some of these clusters resulted in the characterization of novel compounds and of previously known compounds but never characterized in this Streptomyces species. In addition, genome mining revealed that secondary metabolite gene clusters of phylogenetically closely related Streptomyces are mainly species-specific.     

Positional cloning of the rice male sterility gene ms-IR36, widely used in the inter-crossing phase of recurrent selection schemes

The monogenetic recessive male-sterile gene ms-IR36 is widely used to facilitate the inter-crossing phase of recurrent selection in rice (Oryza sativa), but its segregation within the progeny disturbs other breeding phases. Marker-assisted early identification of msms and Msms seedlings would help overcome this drawback. Using successively bulked segregant analysis and large F2 populations, we mapped the ms-IR36 gene to a 33-kb region on the short arm of chromosome 2 that includes 10 candidate genes. Sequencing of these candidates together with checking rice genome annotations and expression databases allowed the target to be narrowed down to one candidate gene already isolated and characterized as the tapetum degeneration retardation (TDR) gene and reported to be involved in tapetal programmed cell death. Comparison of the sequence of the TDR gene between male-sterile (MS) and male-fertile (MF) IR36 plants detected one non-synonymous nucleotide substitution affecting the active domain of the encoded protein. Perfect co-segregation was observed between polymorphism at this nucleotide (SNP) and the MS/MF phenotype of 946 F2 plants. Spatial modelling of the active domain of the candidate protein reinforced the candidate status of the only SNP identified. Histological characterization of anther development in MS IR36 revealed defects identical to the ones observed in mutants used for the isolation and characterization of the TDR gene: delayed/non-degradation of tapetum tissue and collapse of the haploid microspores. We concluded that ms-IR36 corresponded to the TDR gene with a different mutation from the earlier one described in the same gene. No significant linkage drag was associated with ms-IR36. A SNP-based marker that enables simple early identification of MS plants and MF plants with the Msms genotype was designed.     

Exploring necrotizing autoimmune myopathies with a novel immunoassay for anti-3-hydroxy-3-methyl-glutaryl-CoA reductase autoantibodies

Introduction

Necrotizing autoimmune myopathies (NAM) have recently been defined as a distinct group of severe acquired myopathies, characterized by prominent myofiber necrosis without significant muscle inflammation. Because of the lack of appropriate biomarkers, these diseases have been long misdiagnosed as atypical forms of myositis. NAM may be associated to autoantibodies directed against signal recognition particle (SRP) or 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). The objective of this work was to quantify anti-HMGCR autoantibodies in patients with suspicion of NAM through the development of a new addressable laser bead immunoassay (ALBIA).

Methods

Recombinant HMGCR C-domain was bound to fluorescent beads. After incubation with serum, autoantibodies were revealed using class- or subclass-specific anti-human immunoglobulin G (IgG) antibodies. Anti-HMGCR levels were assayed in 150 patients with suspicion of NAM, 142 controls with different inflammatory/autoimmune diseases and 100 healthy donors. Inhibition with free recombinant HMGCR and immunoprecipitation experiments confirmed test specificity. Reproducibility and repeatability were determined from sera with various levels of anti-HMGCR autoantibodies. A multiplex assay (ALBIA-NAM) was also developed to permit the simultaneous quantification of anti-HMGCR and anti-signal recognition particle autoantibodies.

Results

No controls scored positive. Of 150 patients with suspicion of NAM, 24% were positive for anti-HMGCR autoantibodies with levels ranging from 24 to 2,656 AU/mL. Anti-HMGCR positivity could be associated to a cytoplasmic pattern in immunofluorescence assay on HEp-2 cells. Anti-HMGCR-positive patients had high creatine kinase (CK) levels (mean 6,630 IU/L) and only 40% of them had been exposed to statins. Multiplex ALBIA-NAM was equally as effective as monoplex anti-HMGCR and anti-SRP ALBIA.

Conclusions

Both monoplex ALBIA-HMGCR and multiplex ALBIA-NAM reliably detect and quantify anti-HMGCR autoantibodies. A positive result allows ascribing patients with a necrotizing myopathy to an autoimmune form. Anti-HMGCR autoantibodies may be found in patients who have not taken statins.     

PCR performance of the highly thermostable proof-reading B-type DNA polymerase from Pyrococcus abyssi

DNA polymerase from the archaeon Pyrococcus abyssi strain Orsay was expressed in Escherichia coli. The recombinant DNA polymerase (Pab) was purified to homogeneity by heat treatment followed by 5 steps of chromatography and characterized for PCR applications. Buffer optimization experiments indicated that Pab PCR performance and fidelity parameters were highest in the presence of 20 mM Tris-HCl, pH 9.0, 1.5 mM MgSO4, 25 mM KCl, 10 mM (NH4)2SO4 and 40 microM of each dNTP. Under these conditions, the error rate was 0.66.10(-6) mutations/nucleotide/duplication. Pab DNA polymerase, having a half life of 5 h at 100 degrees C, was demonstrated to be highly thermostable in PCR conditions compared to commercial Taq and Pfu DNA polymerases. These characteristics enable Pab to be one of the most efficient thermostable DNA polymerases described, exhibiting very high accuracy compared to other available commercial DNA polymerases and robust thermostable activity. This new DNA polymerase is currently on the market under the name Isis DNA Polymerase (Qbiogene Molecular Biology).