Dynamic Meso-Scale Anchorage of GPI-Anchored Receptors in the Plasma Membrane: Prion Protein vs. Thy1.

The central mechanism for the transmission of the prion protein misfolding is the structural conversion of the normal cellular prion protein to the pathogenic misfolded prion protein, by the interaction with misfolded prion protein. This process might be enhanced due to the homo-dimerization/oligomerization of normal prion protein. However, the behaviors of normal prion protein in the plasma membrane have remained largely unknown. Here, using single fluorescent-molecule imaging, we found that both prion protein and Thy1, a control glycosylphosphatidylinositol-anchored protein, exhibited very similar intermittent transient immobilizations lasting for a few seconds within an area of 24.2 and 3.5 nm in diameter in CHO-K1 and hippocampal neurons cultured for 1- and 2-weeks, respectively. Prion protein molecules were immobile during 72% of the time, approximately 1.4× more than Thy1, due to prion protein's higher immobilization frequency. When mobile, prion protein diffused 1.7× slower than Thy1. Prion protein's slower diffusion might be caused by its transient interaction with other prion protein molecules, whereas its brief immobilization might be due to temporary association with prion protein clusters. Prion protein molecules might be newly recruited to prion protein clusters all the time, and simultaneously, prion protein molecules in the cluster might be departing continuously. Such dynamic interactions of normal prion protein molecules would strongly enhance the spreading of misfolded prion protein.

Groucho-associated transcriptional repressor ripply1 is required for proper transition from the presomitic mesoderm to somites.

Concomitant with the transition from the presomitic mesoderm (PSM) to somites, the periodical gene expression characteristic of the PSM is drastically changed and translated into the segmental structure. However, the molecular mechanism underlying this transition has remained obscure. Here, we show that ripply1, encoding a nuclear protein associated with the transcriptional corepressor Groucho, is required for this transition. Zebrafish ripply1 is expressed in the anterior PSM and in several newly formed somites. Ripply1 represses mesp-b expression in the PSM through a Groucho-interacting motif. In ripply1-deficient embryos, somite boundaries do not form, the characteristic gene expression in the PSM is not properly terminated, and the initially established rostrocaudal polarity in the segmental unit is not maintained, whereas paraxial mesoderm cells become differentiated. Thus, ripply1 plays dual roles in the transition from the PSM to somites: termination of the segmentation program in the PSM and maintenance of the rostrocaudal polarity.

Analysis of combinatorial effects of Wnts and Frizzleds on beta-catenin/armadillo stabilization and Dishevelled phosphorylation.

Both Wnt ligands and Frizzled (Fz) receptors each constitute a large family in vertebrates, but the receptor specificity of each Wnt has remained largely unknown. Here, we examined the receptor specificity of two typical Wnts, Wnt-3a and Wnt-5a, in signal transmission. To investigate systematically the combinatorial effects of these Wnts, various Fzs on canonical Wnt/beta-catenin signaling, we analyzed the ability of these Wnt proteins to increase stability of armadillo/beta-catenin proteins in Drosophila S2 cells expressing vertebrate Fzs. Wnt-3a increases the amount of armadillo proteins in cells expressing Fzs 4, 5 and 8, but not Fzs 3 and 6; whereas Wnt-5a does not increase it in any cell line. In contrast, both Wnt-3a and Wnt-5a increase the phosphorylation of Dsh in combination with most of the Fzs. This Dsh phosphorylation is abrogated by decreasing the levels of casein kinase I alpha by double-stranded RNA-mediated translational interference. These observations indicate that both Wnt proteins can interact with the majority of Fz receptors and elicit signaling reactions exemplified by Dsh phosphorylation but that the stabilization of beta-catenin/armadillo proteins in the Wnt/beta-catenin signaling occurs only when specific combinations of Wnt and Fz meet.

Zebrafish hairy/enhancer of split protein links FGF signaling to cyclic gene expression in the periodic segmentation of somites.

Notch and fibroblast growth factor (FGF) signaling pathways have been implicated in the establishment of proper periodicity of vertebrate somites. Here, we show evidence that a Hes6-related hairy/Enhancer of split-related gene, her13.2, links FGF signaling to the Notch-regulated oscillation machinery in zebrafish. Expression of her13.2 is induced by FGF-soaked beads and decreased by an FGF signaling inhibitor. her13.2 is required for periodic repression of the Notch-regulated genes her1 and her7, and for proper somite segmentation. Furthermore, Her13.2 augments autorepression of her1 in association with Her1 protein. Therefore, FGF signaling appears to maintain the oscillation machinery by supplying a binding partner, Her13.2, for Her1.

Separate domains of AID are required for somatic hypermutation and class-switch recombination.

Activation-induced cytidine deaminase (AID) is essential for class-switch recombination (CSR) and somatic hypermutation (SHM). Mutants with changes in the C-terminal region of AID retain SHM but lose CSR activity. Here we describe five mutants with alterations in the N-terminal region of AID that caused selective deficiency in SHM but retained CSR, suggesting that the CSR and SHM activities of AID may dissociate via interaction of CSR- or SHM-specific cofactors with different domains of AID. Unlike cells expressing C-terminal AID mutants, B cells expressing N-terminal AID mutants had mutations in the switch micro region, indicating that such mutations are generated by reactions involved in CSR but not SHM. Thus, we propose that separate domains of AID interact with specific cofactors to regulate these two distinct genetic events in a target-specific way.