Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

Elevated CO2 concentration and temperature effects on the partitioning of chemical components along juvenile Scots pine stems (Pinus sylvestris L.)

The effects of doubled ambient [CO₂] and different temperature levels on young Pinus sylvestris growing in phytotron chambers were studied. Five chambers were supplied with ~380 (‘ambient air’) and five with ~700 μmol mol⁻¹ CO₂ (‘elevated [CO₂]’). Temperature levels in the chambers ranged in increment steps of 2°C from −4°C to +4°C relative to the long-term monthly (day and night) average air temperature levels in Berlin–Dahlem. Substrate was medium fertile; soil moisture and air humidity were kept constant. After three vegetation periods twigs and stems were harvested, weighed, homogenized, and analyzed chemically. There was no significant temperature effect on wood mass accumulation, clearest positive [CO₂] effect occurred in the youngest twigs. In total, wood mass increased by 28.5% at doubled ambient [CO₂]. N-contents (percentage) decreased at elevated [CO₂] in the uppermost stem sections and not in twig wood causing wider C/N ratios in total. In response to elevated temperature, N-contents decreased slightly in twigs (~0.3%). Traces of free glucose, fructose and sucrose, which decreased from the top to the bottom, were found in stem wood, in contrast to traces of starch that increased from the top to the bottom. In response to elevated [CO₂] only a little more (0.05%) was accumulated in the top shoot and in tendency; glucose, fructose, and sucrose contents were lower at the bottom of stems as compared to the control. There was no obvious response of these non-structural carbohydrates to elevated temperature except for starch that decreased to half of the content from the lowest to the highest temperature level. Among the hemicellulose compounds, rhamnose and arabinose declined from the top shoot to the bottom of stem, whereas 4-O-methyl-d-glucuronic-acid, mannose, and xylose increased. Contents (percentage) of galactose remained approximately stable along the stem. The clearest positive effect of elevated [CO₂] along the whole stem was found for mannose with differences of 0.6–0.3%. In contrast to rhamnose and arabinose that showed a negative response to elevated [CO₂], mannose was reduced towards the uppermost stem sections. The 4-O-methyl-d-glucuronic-acid was slightly lowered at the bottom, and galactose and xylose showed no [CO₂] response. The only hemicellulose compound which reacted to temperature elevation was galactose. It increased slightly (~0.1% per 1°C). Cellulose and lignin (Klason) behaved oppositely: cellulose increased and lignin decreased from the top to the bottom. These structural components behaved reversely also in response to elevated [CO₂]. In stem parts above the bottom section, cellulose content was slightly higher at elevated [CO₂], and lignin content was slightly lower at the bottom. Lignin reacted to temperature elevation by a very slight increase on the average (~0.1% per one 1°C). Cellulose, however, decreased by ~0.2% per 1°C temperature elevation. The importance of persistent sinks of carbon in woody plant parts is discussed in respect to the greenhouse effect.     

Floral isolation is the main reproductive barrier among closely related sexually deceptive orchids

Floral isolation is an important component of pollinator-driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators' mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F(1) hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination.     

Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease

Marble brain disease (MBD) also known as Guibaud-Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded beta-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619-633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.     

The crystal structure of three site-directed mutants of Escherichia coli dihydrodipicolinate synthase: further evidence for a catalytic triad

Dihydrodipicolinate synthase (DHDPS, EC 4.2.1.52) catalyses the branchpoint reaction of lysine biosynthesis in plants and microbes: the condensation of (S)-aspartate-beta-semialdehyde and pyruvate. The crystal structure of wild-type DHDPS has been published to 2.5A, revealing a tetrameric molecule comprised of four identical (beta/alpha)(8)-barrels, each containing one active site. Previous workers have hypothesised that the catalytic mechanism of the enzyme involves a catalytic triad of amino acid residues, Tyr133, Thr44 and Tyr107, which provide a proton shuttle to transport protons from the active site to solvent. We have tested this hypothesis using site-directed mutagenesis to produce three mutant enzymes: DHDPS-Y133F, DHDPS-T44V and DHDPS-Y107F. Each of these mutants has substantially reduced activity, consistent with the catalytic triad hypothesis. We have determined each mutant crystal structure to at least 2.35A resolution and compared the structures to the wild-type enzyme. All mutant enzymes crystallised in the same space group as the wild-type form and only minor differences in structure are observed. These results suggest that the catalytic triad is indeed in operation in wild-type DHDPS.     

Systematic Analysis of proteoglycan modification sites in Caenorhabditis elegans by scanning mutagenesis

Proteoglycan modification is essential for development and early cell division in Caenorhabditis elegans. The specification of proteoglycan attachment sites is defined by the Golgi enzyme polypeptide xylosyltransferase. Here we evaluate the substrate specificity of this xylosyltransferase for its downstream targets by using reporter proteins containing proteoglycan modification sites from C. elegans syndecan/SDN-1. The N terminus of the SDN-1 contains a Ser-Gly proteoglycan site at Ser(71), flanked by potential mucin and N-glycosylation sites. However, Ser(71) was exclusively used as a proteoglycan site in vivo, based on mapping studies with a Ser(71) reporter protein, glycosyltransferase RNA interference, and co-expression of worm polypeptide xylosyltransferase. To elucidate the substrate requirements of this enzyme, a library of 42 point mutants of the Ser(71) reporter was expressed in tissue culture. The nematode proteoglycan modification site in SDN-1 required serine (not threonine), two flanking glycine residues (positions -1 and +1), and either one proximal acidic N-terminal amino acid (positions -4, -3, and -2) or a pair of distal N-terminal acidic amino acids (positions -6 and -5). C-terminal acidic amino acids, although present in many proteoglycan modification sites, had minimal impact on xylosylation at Ser(71). Proline inhibited glycosylation when present at -1, +1, or +2. The position of glycine, proline, and acidic amino acids allows the glycosylation machinery to discriminate between mucin and proteoglycan modification sites. The key residues that define proteoglycan modification sites also function with the Drosophila polypeptide xylosyltransferase, indicating that the specificity in the glycosylation process is evolutionarily conserved. Using a neural network method, a preliminary proteoglycan predictor has been developed.     

Thyroxine as a Mediator of Metamorphosis of Atlantic Halibut, Hippoglossus Hippoglossus

The response of morphological, histological and endocrinological development to exogenous 1-thyroxine (T₄) and to water depth during metamorphosis in Atlantic halibut, Hippoglossus hippoglossus, was investigated. Exogenous T₄ was given in daily doses of 0.1, 0.05 ppm or a control treatment to halibut larvae at 550 daydegrees (posthatch, premetamorphic) for 14 days. Water depths of 40 cm, 10 cm or 1.5 cm were used to rear halibut larvae from 590 daydegrees for 21 days. Halibut larvae given exogenous T₄ at 0.1 ppm had accelerated eye migration relative to MH in fish given 0.05 ppm and in control fish. Pigmentation was correlated with dosage after 14 days. The volume of thyroid tissue was expressed in a dose-dependent manner and exhibited a size-dependency within each treatment. However, the follicles were atypical with reduced colloid, increased lumen and low epithelial cells even in the control group. The results indicate that T₄ is a mediator in halibut metamorphosis. In the water depth experiment, only cortisol levels of larvae reared in 1.5 cm water were significantly affected after 21 days, but this was not correlated with metamorphic rate. Hormone profiles, morphological changes and size suggest the existence of a window of opportunity for metamorphosis in halibut extending from about 16 mm and tapering off about 21 mm SL. The pooled hormone profiles indicate the commencement of a hormonal cascade similar to that of other flatfishes during metamorphosis. The results indicate that growth, neural and skeletal transformation, and pigmentation are biochemically separate processes in the metamorphosis of Atlantic halibut.     

Density of Monoporeia affinis and biogeochemistry in Baltic Sea sediments

This study focused on effects from Monoporeia affinis reworking and ventilation activities on benthic fluxes and mineralization processes during a simulated bloom event. The importance of M. affinis density for benthic solute (O₂, ΣNO₂⁻ + NO₃⁻, NH₄⁺ and HPO₄²⁻) fluxes and sediment reactivity (mobilization of NH₄⁺ and HPO₄²⁻) following additions of organic material to the sediment surface was experimentally investigated using sediment-water and closed sediment (jar) incubations. Three different densities of M. affinis were used to resemble a low, medium and high density situation (1300, 2500 and 6400 ind. m^− 2, respectively) of a natural amphipod community. The degradation of phytodetritus (Tetraselmis sp., 5 g C m^− 2) added to the sediment surface was followed over a period of 20 days. Benthic solute fluxes of O₂, ΣNO₂⁻ + NO₃⁻ and NH₄⁺ were generally progressively stimulated with increasing number of M. affinis, while no such correlation was found for HPO₄²⁻. Solute fluxes were initially enhanced 1 to 2 days after the addition of phytodetritius, caused by mineralization of the most labile organic material and a food-stimulated irrigation by the amphipods. There was no effect from the activity of M. affinis on total denitrification (Dtot = Dn + Dw) or denitrification utilizing nitrate from coupled nitrification/denitrification (Dn) for any of the densities examined. Denitrification utilizing overlying water nitrate (Dw) was only about 10% of Dtot. Dw was significantly enhanced for the highest M. affinis density investigated. The reactivity of the sediment decreased progressively with increasing density of M. affinis and with time of the experiment. However, enhanced ammonium production at least 6 days after the organic addition indicated excretion of N-containing organic compounds by M. affinis. In conclusion, large spatial and temporal variations in density of M. affinis may be of significant importance for benthic solute fluxes and overall mineralization of organic material in Baltic Sea sediments.