Myosin 5a controls insulin granule recruitment during late-phase secretion

We have examined the importance of the actin-based molecular motor myosin 5a for insulin granule transport and insulin secretion. Expression of myosin 5a was downregulated in clonal INS-1E cells using RNAinterference. Stimulated hormone secretion was reduced by 46% and single-cell exocytosis, measured by capacitance recordings, was inhibited by 42% after silencing. Silencing of Slac-2c/MYRIP, which links insulin granules to myosin 5a, resulted in similar inhibition of single-cell exocytosis. Antibody inhibition of the myosin 5a-Slac-2c/MYRIP interaction significantly reduced the recruitment of insulin granules for release. The pool of releasable granules independent of myosin 5a activity was estimated to approximately 550 granules. Total internal reflection microscopy was then applied to directly investigate granule recruitment to the plasma membrane. Silencing of myosin 5a inhibited granule recruitment during late phase of insulin secretion. In conclusion, we propose a model where insulin granules are transported through the actin network via both myosin 5a-mediated transport and via passive diffusion, with the former playing the major role during stimulatory conditions.     

Characterization of phase separation in film forming biopolymer mixtures

Enhanced, tailor-made films can be achieved by combining the good gas barrier of the hydrophilic high amylose maize starch (hylon) with the water resistance of the hydrophobic protein zein. Two polymers are not always miscible in solution, and the phase separation behavior of the mixture is therefore important for the final film structure and its properties. Phase separation of a mixture of these two biopolymers was induced either by cooling, which was observed as growing droplets of the hylon phase which in some cases also formed small aggregates, or by solvent evaporation and studied in real-time in a confocal laser scanning microscope. Solvent evaporation had a much stronger effect on phase separation. During the early stage of phase separation, hylon formed large aggregates and subsequently smaller droplets coalesced with other droplets or large hylon aggregates. The later part of the separation seemed to take place through spinodal decomposition.     

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.     

Biomass partitioning and leaf N,P - stoichiometry: comparisons between tree and herbaceous current-year shoots

We compare the biomass partitioning patterns and the nitrogen/phosphorus (N,P) stoichiometry of the current-year shoots of tree and herbaceous species and ask whether they scale in the same ways. Our analyses indicate that few statistically significant differences exist between the shoot biomass partitioning patterns of the two functional species-groups. In contrast, statistically significant N,P - stoichiometric differences exist between the two functional groups. Across all species, dry leaf mass scales nearly as the square of basal stem diameter and isometrically with respect to dry stem mass. However, total leaf N scales as the 1.37-power and as the 1.09-power of total leaf P across herbaceous and tree shoots, respectively. Therefore, tree shoots can be viewed as populations of herbs elevated by their older, woody herbaceous cohorts. However, tree leaf stoichiometry cannot be modelled in terms of herbaceous N,P - leaf stoichiometry.     

Higher level phylogeny of Satyrinae butterflies (Lepidoptera: Nymphalidae) based on DNA sequence data

We have inferred the first empirically supported hypothesis of relationships for the cosmopolitan butterfly subfamily Satyrinae. We used 3090 base pairs of DNA from the mitochondrial gene COI and the nuclear genes EF-1alpha and wingless for 165 Satyrinae taxa representing 4 tribes and 15 subtribes, and 26 outgroups, in order to test the monophyly of the subfamily and elucidate phylogenetic relationships of its major lineages. In a combined analysis, the three gene regions supported an almost fully resolved topology, which recovered Satyrinae as polyphyletic, and revealed that the current classification of suprageneric taxa within the subfamily is comprised almost completely of unnatural assemblages. The most noteworthy findings are that Manataria is closely related to Melanitini; Palaeonympha belongs to Euptychiina; Oressinoma, Orsotriaena and Coenonympha group with the Hypocystina; Miller's (1968). Parargina is polyphyletic and its components group with multiple distantly related lineages; and the subtribes Elymniina and Zetherina fall outside the Satyrinae. The three gene regions used in a combined analysis prove to be very effective in resolving relationships of Satyrinae at the subtribal and tribal levels. Further sampling of the taxa closely related to Satyrinae, as well as more extensive sampling of genera within the tribes and subtribes for this group will be critical to test the monophyly of the subfamily and establish a stronger basis for future biogeographical and evolutionary studies.     

Dual color photoactivation localization microscopy of cardiomyopathy-associated desmin mutants

Mutations in the DES gene coding for the intermediate filament protein desmin may cause skeletal and cardiac myopathies, which are frequently characterized by cytoplasmic aggregates of desmin and associated proteins at the cellular level. By atomic force microscopy, we demonstrated filament formation defects of desmin mutants, associated with arrhythmogenic right ventricular cardiomyopathy. To understand the pathogenesis of this disease, it is essential to analyze desmin filament structures under conditions in which both healthy and mutant desmin are expressed at equimolar levels mimicking an in vivo situation. Here, we applied dual color photoactivation localization microscopy using photoactivatable fluorescent proteins genetically fused to desmin and characterized the heterozygous status in living cells lacking endogenous desmin. In addition, we applied fluorescence resonance energy transfer to unravel short distance structural patterns of desmin mutants in filaments. For the first time, we present consistent high resolution data on the structural effects of five heterozygous desmin mutations on filament formation in vitro and in living cells. Our results may contribute to the molecular understanding of the pathological filament formation defects of heterozygous DES mutations in cardiomyopathies.     

Primary metabolism in the new human cell line AGE1.HN at various substrate levels: increased metabolic efficiency and α1-antitrypsin production at reduced pyruvate load

Metabolic responses of the new neuronal human cell line AGE1.HN to various substrate levels were analyzed in this study showing that reduced substrate and especially pyruvate load improves metabolic efficiency, leading to improved growth and α₁-antitrypsin (A1AT) production. The adaptation of the metabolism to different pyruvate and glutamine concentrations was analyzed in detail using a full factorial design. The most important finding was an increasingly inefficient use of substrates as well as the reduction of cell proliferation with increasing pyruvate concentrations in the medium. Cultivations with different feeding profiles showed that the highest viable cell density and A1AT concentration (167% of batch) was reached in the culture with the lowest glucose level and without pyruvate feeding. Analysis of metabolic fluxes in the differently fed cultures revealed a more efficient metabolic phenotype in the cultures without pyruvate feeding. The measured in vitro enzyme activities of the selected enzymes involved in pyruvate metabolism were lower in AGE1.HN compared with CHO cells, which might explain the higher sensitivity and different adaptation of AGE1.HN to increased pyruvate concentrations. The results indicate on the one hand that increasing the connectivity between glycolysis and the TCA cycle might improve substrate use and, finally, the production of A1AT. On the other hand, a better balanced substrate uptake promises a reduction of energy spilling which is increased with increasing substrate levels in this cell line. Overall, the results of this study provide important insights into the regulation of primary metabolism and into the adaptation of AGE1.HN to different substrate levels, providing guidance for further optimization of production cell lines and applied process conditions.     

Reconstitution of water channel function and 2D-crystallization of human aquaporin 8

Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3?.