Collapsin response mediator protein-4 regulates F-actin bundling

Collapsin response mediator proteins (CRMPs) form a family of cytosolic phosphoproteins which are involved in the signal transduction of semaphorin 3A leading to growth cone collapse. These proteins interact with a variety of cytosolic proteins including tubulin heterodimers. Here, we show that CRMP-4 co-localizes with F-actin in regular rib-like structures within lamellipodia of B35 neuroblastoma cells. Furthermore, depolymerization of actin fibers changed the distribution of GFP-CRMP-4 in vivo. In vitro, recombinant CRMP-4 formed homo-oligomers, bound to F-actin and organized F-actin into tight bundles. Both oligomerization and F-actin bundling depended on the C-terminal part of CRMP-4. The stoichiometry of actin and CRMP-4 in bundles was approximately 1:1 and the apparent equilibrium constant of the microfilament-CRMP-4 interaction was estimated from bundling assays as K(app) = 730 mM(-1). CRMP-4 was abundant in the cytosol of B35 neuroblastoma cells and its concentration was measured as approximately 1.7 microM. Overexpression of CRMP-4 inhibited the migration of B35 neuroblastoma cells, while knockdown of CRMP-4 enhanced cell migration and disturbed rib-like actin-structures in lamellipodia. Taken together, our data indicate that CRMP-4 promotes bundling of F-actin in vitro, that it is an important component of rib-like actin bundles in lamellipodia in vivo and that it functionally regulates the actin cytoskeleton in motile cells. These findings suggest a specific regulatory role of CRMP-4 towards the actin cytoskeleton which may by be relevant for growth cone collapse.     

Thesaurus-based disambiguation of gene symbols

Background  

Massive text mining of the biological literature holds great promise of relating disparate information and discovering new knowledge. However, disambiguation of gene symbols is a major bottleneck.     

Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Structural and functional consequences of altering a peptide MHC anchor residue

To better understand TCR discrimination of multiple ligands, we have analyzed the crystal structures of two Hb peptide/I-E(k) complexes that differ by only a single amino acid substitution at the P6 anchor position within the peptide (E73D). Detailed comparison of multiple independently determined structures at 1.9 A resolution reveals that removal of a single buried methylene group can alter a critical portion of the TCR recognition surface. Significant variance was observed in the peptide P5-P8 main chain as well as a rotamer difference at LeuP8, approximately 10 A distal from the substitution. No significant variations were observed in the conformation of the two MHC class II molecules. The ligand alteration results in two peptide/MHC complexes that generate bulk T cell responses that are distinct and essentially nonoverlapping. For the Hb-specific T cell 3.L2, substitution reduces the potency of the ligand 1000-fold. Soluble 3.L2 TCR binds the two peptide/MHC complexes with similar affinity, although with faster kinetics. These results highlight the role of subtle variations in MHC Ag presentation on T cell activation and signaling.     

Behavioural and physiological consequences of acute social defeat in growing gilts: effects of the social environment

Endocrine, behavioural and immunologic processes, together with body growth, were evaluated in gilts that were defeated at 10 weeks of age in resident-intruder tests. Immediately after defeat, gilts were either separated from or reunited with a familiar conspecific (litter-mate; always a barrow). Gilts were assigned to one of four treatments: (a) DI: defeat, followed by isolation (separation from original litter-mate; n=8); (b) I: no defeat, isolation (control group; n=9); (c) DP; defeat, followed by pair-housing (reunion with original litter-mate; n=8); and (d) P: no defeat, pair-housing (control group; n=8). The following general conclusions were derived: (1) social defeat caused pronounced short-term elevations in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal medullary activities, and of prolactin levels. Moreover, as soon as 1h after defeat, percentages of blood lymphocytes and neutrophilic granulocytes were, respectively, decreased and increased; (2) social defeat had some long-lasting influence on behaviour and physiology, but isolation predominantly determined responses in the longer term. Defeat, as well as isolation, resulted in increased cardiovascular activities compared to P controls, as observed in a novel object test (NOT: +7 days) and an aversion test (AVT: +14 days). Moreover, defeated as well as isolated gilts did not habituate to a repeated novel environment test (NET: -7, +2 and +7 days) in terms of frequencies of vocalising, whereas P controls did. Isolation, through the separation from any other pig, was responsible for the other observed long-term characteristics, which developed progressively. Isolated gilts showed high mobilities and high cortisol responses in the repeated NET (+7 days), not being habituated. This contrasted the reactions of pair-housed gilts, which were much reduced. In addition to their high cardiovascular activities in the NOT and the AVT, isolated gilts also displayed higher heart rates in the repeated NET and during human presence following the NOT, compared to pair-housed gilts. Finally, isolated gilts were more inhibited to approach a novel object (in the NOT) than pair-housed pigs; and (3) stress responses of defeated gilts were modulated by the subsequent social environment. Stimulation of the HPA-axis (plasma- and salivary cortisol) was prolonged in those defeated gilts which were isolated (observed in the first hour). Changes in leucocyte subsets were still observed after 3 days in DI, but were 'normalised' within 1 day in DP gilts. Two days after defeat, habituation to the repeated NET in terms of mobility and salivary cortisol responses occurred in control and DP gilts, but not in DI gilts. We argue that these effects of the social environment shortly after defeat were related to a stress-reducing effect of a stable social relationship, i.e. social support.     

Glycosaminoglycan-binding properties and secondary structure of the C-terminus of netrin-1

Netrins are soluble neurite-outgrowth-promoting proteins related to the laminin B2 chain. Since these proteins and their receptor DCC (the "deleted in colorectal carcinoma" gene product) bind heparin, glycosaminoglycans may modulate their biological actions in a similar fashion as described for several other ligand-receptor systems. Here we show that a polypeptide encompassing the C-terminal cluster of basic amino acids of netrin-1 (i) adopts an alpha-helical conformation in water-trifluoroethanol mixtures according to circular dichroism experiments and (ii) binds electrostatically to heparin with high affinity under physiological ionic conditions (K(D) = 15 nM for the binding to immobilized heparin according to surface plasmon resonance, K(D) = 50 nM in solution as determined with isothermal titration calorimetry). These data indicate that the cluster of basic amino acids at the C-terminus of netrin-1 forms an alpha-helical structural element which can contribute to the glycosaminoglycan-binding activity of this neurotrophic guidance molecule.     

Partial leptin receptor gene deletion in transgenic mice prevents expression of the membrane-bound isoforms except for Ob-Rc

Examination of random insertional mutations in transgenic animals harbouring an abnormal phenotype contributes to the discovery of new genes and/or the understanding of already known genes. Here we describe a transgenic mouse line showing early-onset obesity as consequence of the transgene insertion. Molecular genetic analysis revealed a partial deletion of the leptin receptor (Lepr, Ob-R) gene including the coding sequences downstream of exon 17'. This defect prevents the expression of all described membrane-bound isoforms of Ob-R except for isoform Ob-Rc in the homozygous transgenic animals. Thus, this mouse model might be useful for the investigation of the function of the short Ob-R isoforms.     

B cell helper factors. II. Synergy among three helper factors in the response of T cell- and macrophage-depleted B cells

The concanavalin A- (Con A) stimulated supernatant of normal spleen cells (normal Con A SN) was shown to contain a set of helper factors sufficient to allow T cell- and macrophage- (M phi) depleted murine splenic B cells to produce a plaque-forming cell response to the antigen sheep red blood cells (SRBC). The activity of normal Con A SN could be reconstituted by a mixture of three helper factor preparations. The first was the interleukin 2- (IL 2) containing Con A SN of the T cell hybridoma, FS6-14.13. The second was a normal Con A SN depleted of IL 2 by extended culture with T cell blasts from which the 30,000 to 50,000 m.w. factors were isolated (interleukin X, IL X). The third was a SN either from the M phi tumor cell line P388D1 or from normal M phi taken from Corynebacterium parvum-immune mice. The combination of all three helper factor preparations was required to equal the activity of normal Con A SN; however, the M phi SN had the least overall effect. The M phi SN and IL 2 had to be added at the initiation of the culture period for a maximal effect, but the IL X preparation was most effective when added 24 hr after the initiation of culture. These results indicate that at least three nonspecific helper factors contribute to the helper activity in normal Con A SN.     

Plasmatubules in the pollen tubes of Nicotiana sylvestris

Ultrastructural studies of the pollen tubes of Nicotiana sylvestris grown in the pistil revealed an extensive development of plasmatubules formed by evaginations of the plasma membrane. The plasmatubules occurred as twisted tubular structures in the periplasmic space along the tube wall and, in cross section, exhibited circular profiles with an outer diameter of 28±4 nm. They were also seen in deep, pocket-like invaginations of the plasma membrane and in this case the profiles had an outer diameter of 34±8 nm. In the pocket-like invaginations they were partially branched and often closely packed to form groups with obvious patterns. The enlargement of the plasma-membrane area resulting from plasmatubules formed along the tube wall was about six-to tenfold. Pollen tubes grown in vitro exhibited poorly developed plasmatubules. It is suggested that the large extension of the plasma membrane could enhance the uptake of nutrients, and thus might be responsible for the comparatively fast growth of pollen tubes in the pistil. Moreover, it is also assumed that the turnover rate of the Golgi apparatus must be higher in pollen tubes growing in vivo than in vitro, in order to provide a sufficient amount of membrane for the formation of the plasma membrane with its tubular modifications.     

Atmospheric hydrogen peroxide and Eoarchean iron formations

It is widely accepted that photosynthetic bacteria played a crucial role in Fe(II) oxidation and the precipitation of iron formations (IF) during the Late Archean–Early Paleoproterozoic (2.7–2.4 Ga). It is less clear whether microbes similarly caused the deposition of the oldest IF at ca. 3.8 Ga, which would imply photosynthesis having already evolved by that time. Abiological alternatives, such as the direct oxidation of dissolved Fe(II) by ultraviolet radiation may have occurred, but its importance has been discounted in environments where the injection of high concentrations of dissolved iron directly into the photic zone led to chemical precipitation reactions that overwhelmed photooxidation rates. However, an outstanding possibility remains with respect to photochemical reactions occurring in the atmosphere that might generate hydrogen peroxide (H₂O₂), a recognized strong oxidant for ferrous iron. Here, we modeled the amount of H₂O₂ that could be produced in an Eoarchean atmosphere using updated solar fluxes and plausible CO₂, O₂, and CH₄ mixing ratios. Irrespective of the atmospheric simulations, the upper limit of H₂O₂ rainout was calculated to be <10⁶ molecules cm^?2 s^?1. Using conservative Fe(III) sedimentation rates predicted for submarine hydrothermal settings in the Eoarchean, we demonstrate that the flux of H₂O₂ was insufficient by several orders of magnitude to account for IF deposition (requiring ~10¹¹ H₂O₂ molecules cm^?2 s^?1). This finding further constrains the plausible Fe(II) oxidation mechanisms in Eoarchean seawater, leaving, in our opinion, anoxygenic phototrophic Fe(II)‐oxidizing micro‐organisms the most likely mechanism responsible for Earth's oldest IF.