Csk differentially regulates Src64 during distinct morphological events in Drosophila germ cells

The Src family protein tyrosine kinases (SFKs) are crucial regulators of cellular morphology. In Drosophila, Src64 controls complex morphological events that occur during oogenesis. Recent studies have identified key Src64-dependent mechanisms that regulate actin cytoskeletal dynamics during the growth of actin-rich ring canals, which act as intercellular bridges between germ cells. By contrast, the molecular mechanisms that regulate Src64 activity levels and potential roles for Src64 in additional morphological events in the ovary have not been defined. In this report, we demonstrate that regulation of Src64 by Drosophila C-terminal-Src Kinase (Csk) contributes to the packaging of germline cysts by overlying somatic follicle cells during egg chamber formation. These results uncover novel roles for both Csk and Src64 in a dynamic event that involves adhesion, communication between cell types and control of cell motility. Strikingly, Src64 and Csk function in the germline to control packaging, not in migrating follicle cells, suggesting novel functions for this signaling cassette in regulating dynamic adhesion. In contrast to the role played by Csk in the regulation of Src64 activity during packaging, Csk is dispensable for ring canal growth control, indicating that distinct mechanisms control Src64 activity during different morphological events.     

A network model of early events in epidermal growth factor receptor signaling that accounts for combinatorial complexity

We consider a model of early events in signaling by the epidermal growth factor (EGF) receptor (EGFR). The model includes EGF, EGFR, the adapter proteins Grb2 and Shc, and the guanine nucleotide exchange factor Sos, which is activated through EGF-induced formation of EGFR-Grb2-Sos and EGFR-Shc-Grb2-Sos assemblies at the plasma membrane. The protein interactions involved in signaling can potentially generate a diversity of protein complexes and phosphoforms; however, this diversity has been largely ignored in models of EGFR signaling. Here, we develop a model that accounts more fully for potential molecular diversity by specifying rules for protein interactions and then using these rules to generate a reaction network that includes all chemical species and reactions implied by the protein interactions. We obtain a model that predicts the dynamics of 356 molecular species, which are connected through 3749 unidirectional reactions. This network model is compared with a previously developed model that includes only 18 chemical species but incorporates the same scope of protein interactions. The predictions of this model are reproduced by the network model, which also yields new predictions. For example, the network model predicts distinct temporal patterns of autophosphorylation for different tyrosine residues of EGFR. A comparison of the two models suggests experiments that could lead to mechanistic insights about competition among adapter proteins for EGFR binding sites and the role of EGFR monomers in signal transduction.     

Investigation of the Role of a Conserved Glycine Motif in the Saccharomyces cerevisiae Xylose Reductase

All yeast xylose reductases, with the exception of that from Schizosaccharomyces pombe, possess the catalytic and coenzyme-binding elements from both the aldo–keto reductase and short-chain dehydrogenase–reductase (SDR) enzyme families in their primary sequences. In the Saccharomyces cerevisiae xylose reductase (XR), the SDR-like coenzyme-binding GXXXGXG motif (Gly motif) is located between residues 128 and 134, with the third Gly residue being replaced by an Asp. We used site-directed mutagenesis to study the role of this SDR-like Gly motif in the S. cerevisiae xylose reductase. Site-directed mutagenesis of the individual conserved Gly residue positions (G128A, G132A, D134G, and D134A) did not significantly affect the specific activity, kinetic constants (K_m, K_cat, and K_cat/K_m), or dissociation constants (K_d) in any of the variants compared with the wild type. Deletion of the entire Gly motif produced an unstable protein that could not be purified. These results indicate that the SDR-like Gly motif likely provides support to the overall structure of the enzyme, but it does not contribute directly to coenzyme binding in this XR.     

Probing single-cell micromechanics in vivo: the microrheology of C. elegans developing embryos

Cells are not directly accessible in vivo and therefore their mechanical properties cannot be measured by methods that require a direct contact between probe and cell. Here, we introduce a novel in vivo assay based on particle tracking microrheology whereby the extent and time-lag dependence of the mean squared displacements of thermally excited nanoparticles embedded within the cytoplasm of developing embryos reflect local viscoelastic properties. As a proof of principle, we probe local viscoelastic properties of the cytoplasm of developing Caenorhabditis elegans embryos. Our results indicate that unlike differentiated cells, the cytoplasm of these embryos does not exhibit measurable elasticity, but is highly viscous. Furthermore, the viscosity of the cytoplasm does not vary along the anterior-posterior axis of the embryo during the first cell division. These results support the hypothesis that the asymmetric positioning of the mitotic spindle stems from an asymmetric distribution of elementary force generators as opposed to asymmetric viscosity of the cytoplasm.     

Structural properties of prion protein protofibrils and fibrils: an experimental assessment of atomic models

Decades after the prion protein was implicated in transmissible spongiform encephalopathies, the structure of its toxic isoform and its mechanism of toxicity remain unknown. By gathering available experimental data, albeit low resolution, a few pieces of the prion puzzle can be put in place. Currently, there are two fundamentally different models of a prion protofibril. One has its building blocks derived from a molecular dynamics simulation of the prion protein under amyloidogenic conditions, termed the spiral model. The other model was constructed by threading a portion of the prion sequence through a beta-helical structure from the Protein Data Bank. Here we compare and contrast these models with respect to all of the available experimental information, including electron micrographs, symmetries, secondary structure, oligomerization interfaces, enzymatic digestion, epitope exposure, and disaggregation profiles. Much of this information was not available when the two models were introduced. Overall, we find that the spiral model is consistent with all of the experimental results. In contrast, it is difficult to reconcile several of the experimental observables with the beta-helix model. While the experimental constraints are of low resolution, in bringing together the previously disconnected experiments, we have developed a clearer picture of prion aggregates. Both the improved characterization of prion aggregates and the existing atomic models can be used to devise further experiments to better elucidate the misfolding pathway and the structure of prion protofibrils.     

Structural insights into the interaction between prion protein and nucleic acid

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.     

Comparison of a Salmonella typhimurium proteome defined by shotgun proteomics directly on an LTQ-FT and by proteome pre-fractionation on an LCQ-DUO.

Shotgun proteomics is rapidly becoming one of the most efficient and popular tools to examine protein expression in cells. Numerous laboratories now have a wide array of low- and high-performance mass spectrometry instrumentation necessary to complete proteome-wide projects. Often these laboratories have time and financial constraints that prohibit all projects from being conducted on high-performance state-of-the-art mass spectrometers. Here, we compare shotgun proteomic results using a direct 'lyse, digest and analyse' approach on a high-performance mass spectrometer (i.e. the LTQ-FT) with the results from a much lower-performance instrument (i.e. the LCQ-DUO) where, for the latter, various traditional protein pre-fractionation steps and gas-phase fractionation were used to increase the proteome coverage. Our results demonstrate that shotgun proteomic analyses conducted on the lower-performance LCQ-DUO mass spectrometer could adequately characterize a PhoP constitutive strain of Salmonella typhimurium if proteome pre-fractionation steps and gas-phase fractionation were included.     

Systems genetic analysis of multivariate response to iron deficiency in mice

The aim of this study was to identify genes that influence iron regulation under varying dietary iron availability. Male and female mice from 20+ BXD recombinant inbred strains were fed iron-poor or iron-adequate diets from weaning until 4 mo of age. At death, the spleen, liver, and blood were harvested for the measurement of hemoglobin, hematocrit, total iron binding capacity, transferrin saturation, and liver, spleen and plasma iron concentration. For each measure and diet, we found large, strain-related variability. A principal-components analysis (PCA) was performed on the strain means for the seven parameters under each dietary condition for each sex, followed by quantitative trait loci (QTL) analysis on the factors. Compared with the iron-adequate diet, iron deficiency altered the factor structure of the principal components. QTL analysis, combined with PosMed (a candidate gene searching system) published gene expression data and literature citations, identified seven candidate genes, Ptprd, Mdm1, Picalm, lip1, Tcerg1, Skp2, and Frzb based on PCA factor, diet, and sex. Expression of each of these is cis-regulated, significantly correlated with the corresponding PCA factor, and previously reported to regulate iron, directly or indirectly. We propose that polymorphisms in multiple genes underlie individual differences in iron regulation, especially in response to dietary iron challenge. This research shows that iron management is a highly complex trait, influenced by multiple genes. Systems genetics analysis of iron homeostasis holds promise for developing new methods for prevention and treatment of iron deficiency anemia and related diseases.     

The extinction context enables extinction performance after a change in context

One experiment with human participants determined the extent to which recovery of extinguished responding with a context switch was due to a failure to retrieve contextually controlled learning, or some other process such as participants learning that context changes signal reversals in the meaning of stimulus-outcome relationships. In a video game, participants learned to suppress mouse clicking in the presence of a stimulus that predicted an attack. Then, that stimulus underwent extinction in a different context (environment within the game). Following extinction, suppression was recovered and then extinguished again during testing in the conditioning context. In a final test, participants that were tested in the context where extinction first took place showed less of a recovery than those tested in a neutral context, but they showed a recovery of suppression nevertheless. A change in context tended to cause a change in the meaning of the stimulus, leading to recovery in both the neutral and extinction contexts. The extinction context attenuated that recovery, perhaps by enabling retrieval of the learning that took place in extinction. Recovery outside an extinction context is due to a failure of the context to enable the learning acquired during extinction, but only in part.