Alexandra Schambony

Paraxial protocadherin coordinates cell polarity during convergent extension via Rho A and JNK

Convergent extension movements occur ubiquitously in animal development. This special type of cell movement is controlled by the Wnt/planar cell polarity (PCP) pathway. Here we show that Xenopus paraxial protocadherin (XPAPC) functionally interacts with the Wnt/PCP pathway in the control of convergence and extension (CE) movements in Xenopus laevis. XPAPC functions as a signalling molecule that coordinates cell polarity of the involuting mesoderm in mediolateral orientation and thus selectively promotes convergence in CE movements. XPAPC signals through the small GTPases Rho A and Rac 1 and c‐jun N‐terminal kinase (JNK). Loss of XPAPC function blocks Rho A‐mediated JNK activation. Despite common downstream components, XPAPC and Wnt/PCP signalling are not redundant, and the activity of both, XPAPC and PCP signalling, is required to coordinate CE movements.

Beta-arrestin is a necessary component of Wnt/beta-catenin signaling in vitro and in vivo.

The Wnt/β-catenin signaling pathway is crucial for proper embryonic development and tissue homeostasis. The phosphoprotein dishevelled (Dvl) is an integral part of Wnt signaling and has recently been shown to interact with the multifunctional scaffolding protein β-arrestin. Using Dvl deletion constructs, we found that β-arrestin binds a region N-terminal of the PDZ domain of Dvl, which contains casein kinase 1 (CK1) phosphorylation sites. Inhibition of Wnt signaling by CK1 inhibitors reduced the binding of β-arrestin to Dvl. Moreover, mouse embryonic fibroblasts lacking β-arrestins were able to phosphorylate LRP6 in response to Wnt-3a but decreased the activation of Dvl and blocked β-catenin signaling. In addition, we found that β-arrestin can bind axin and forms a trimeric complex with axin and Dvl. Furthermore, treatment of Xenopus laevis embryos with β-arrestin morpholinos reduced the activation of endogenous β-catenin, decreased the expression of the β-catenin target gene, Xnr3, and blocked axis duplication induced by X-Wnt-8, CK1ε, or DshΔDEP, but not by β-catenin. Thus, our results identify β-arrestin as a necessary component for Wnt/β-catenin signaling, linking Dvl and axin, and open a vast array of signaling avenues and possibilities for cross-talk with other β-arrestin-dependent signaling pathways.

Wnt/β-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled

Wnt binding to members of the seven-span transmembrane Frizzled (Fz) receptor family controls essential cell fate decisions and tissue polarity during development and in adulthood. The Fz-mediated membrane recruitment of the cytoplasmic effector Dishevelled (Dvl) is a critical step in Wnt/β-catenin signaling initiation, but how Fz and Dvl act together to drive downstream signaling events remains largely undefined. Here, we use an Fz peptide-based microarray to uncover a mechanistically important role of the bipartite Dvl DEP domain and C terminal region (DEP-C) in binding a three-segmented discontinuous motif in Fz. We show that cooperative use of two conserved motifs in the third intracellular loop and the classic C-terminal motif of Fz is required for DEP-C binding and Wnt-induced β-catenin activation in cultured cells and Xenopus embryos. Within the complex, the Dvl DEP domain mainly binds the Fz C-terminal tail, whereas a short region at the Dvl C-terminal end is required to bind the Fz third loop and stabilize the Fz-Dvl interaction. We conclude that Dvl DEP-C binding to Fz is a key event in Wnt-mediated signaling relay to β-catenin. The discontinuous nature of the Fz-Dvl interface may allow for precise regulation of the interaction in the control of Wnt-dependent cellular responses.

The Extracellular Domain of Lrp5/6 Inhibits Noncanonical Wnt Signaling In Vivo

Lrp5/6 are crucial coreceptors for Wnt/beta-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/beta-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6-/- mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.

Equine CRISP-3: primary structure and expression in the male genital tract

Although originally described in the male rodent genital tract, cysteine-rich secretory proteins (CRISPs) are expressed in a variety of mammalian tissue and cell types. The proteins of the male genital tract have been observed associated to spermatozoa and are believed to play a role in mammalian fertilization. Here we describe the identification and primary structure of the first equine member of the CRISP family. Equine CRISP-3 is transcribed and expressed in the stallion salivary gland, in the ampulla and the seminal vesicle. It displays all 16 conserved cysteine residues and shows 82% homology to human and 78% to guinea pig CRISP-2 (AA1, TPX 1) and 77% to human CRISP-3. In contrast to other mammalia, in the horse CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle, thus allowing the isolation of equine CRISP-3 in amounts suitable for biochemical, physiological and structural studies from stallion seminal plasma.

Negative regulation of Wnt signaling mediated by CK1-phosphorylated Dishevelled via Ror2.

Dishevelled (Dvl) is a multifunctional effector of different Wnt cascades. Both canonical Wnt3a and noncanonical Wnt5a stimulate casein‐kinase‐1 (CK1) ‐mediated phosphorylation of Dvl, visualized as electrophoretic mobility shift [phosphorylated and shifted Dvl (ps‐Dvl)]. However, the role of this phosphorylation remains obscure. Here we report the functional interaction of ps‐Dvl with the receptor tyrosine kinase Ror2, which is an alternative Wnt receptor and is able to inhibit canonical Wnt signaling. We demonstrate interaction between Ror2 and ps‐Dvl at the cell membrane after Wnt3a or Wnt5a stimulus dependent on CK1. Ps‐Dvl interacts with the C‐terminal proline‐serine‐threonine‐rich domain of Ror2, which is required for efficient inhibition of canonical Wnt signaling. We further show that the Dvl C terminus, which seems to be exposed in ps‐Dvl and efficiently binds Ror2, is an intrinsic negative regulator of the canonical Wnt pathway downstream of ß‐catenin. The Dvl C terminus is necessary and sufficient to inhibit canonical Wnt/ß‐catenin signaling, which is dependent on the presence of Ror2. Furthermore, both the Dvl C terminus and CK1E can inhibit the Wnt5a/Ror2/ATF2 pathway in mammalian cells and Xenopus explant cultures. This suggests that phosphorylation of Dvl triggers negative feedback regulation for different branches of Wnt signaling in a Ror2‐dependent manner.—Witte, F., Bernatik, O., Kirchner, K., Masek, J., Mahl, A., Krejci, P., Mundlos, S., Schambony, A, Bryja, V., Stricker, S. Negative regulation of Wnt signaling mediated by CK1‐phosphorylated Dishevelled via Ror2. FASEB J. 24, 2417–2426 (2010). www.fasebj.org

β-Arrestin and casein kinase 1/2 define distinct branches of non-canonical WNT signalling pathways

Recent advances in understanding β‐catenin‐independent WNT (non‐canonical) signalling suggest an increasing complexity, raising the question of how individual non‐canonical pathways are induced and regulated. Here, we examine whether intracellular signalling components such as β‐arrestin (β‐arr) and casein kinases 1 and 2 (CK1 and CK2) can contribute to determining signalling specificity in β‐catenin‐independent WNT signalling to the small GTPase RAC‐1. Our findings indicate that β‐arr is sufficient and required for WNT/RAC‐1 signalling, and that casein kinases act as a switch that prevents the activation of RAC‐1 and promotes other non‐canonical WNT pathways through the phosphorylation of dishevelled (DVL, xDSH in Xenopus). Thus, our results indicate that the balance between β‐arr and CK1/2 determines whether WNT/RAC‐1 or other non‐canonical WNT pathways are activated.

Waif1/5T4 Inhibits Wnt/β-Catenin Signaling and Activates Noncanonical Wnt Pathways by Modifying LRP6 Subcellular Localization

Wnt proteins can activate distinct signaling pathways, but little is known about the mechanisms regulating pathway selection. Here we show that the metastasis-associated transmembrane protein Wnt-activated inhibitory factor 1 (Waif1/5T4) interferes with Wnt/β-catenin signaling and concomitantly activates noncanonical Wnt pathways. Waif1 inhibits β-catenin signaling in zebrafish and Xenopus embryos as well as in mammalian cells, and zebrafish waif1a acts as a direct feedback inhibitor of wnt8-mediated mesoderm and neuroectoderm patterning during zebrafish gastrulation. Waif1a binds to the Wnt coreceptor LRP6 and inhibits Wnt-induced LRP6 internalization into endocytic vesicles, a process that is required for pathway activation. Thus, Waif1a modifies Wnt/β-catenin signaling by regulating LRP6 subcellular localization. In addition, Waif1a enhances β-catenin-independent Wnt signaling in zebrafish embryos and Xenopus explants by promoting a noncanonical function of Dickkopf1. These results suggest that Waif1 modulates pathway selection in Wnt-receiving cells.

Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns

With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast, one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDS-OH peptide. We successfully applied our coupling method to functionalize surface patterns created by microcontact printing (microCP) and chemical etching. Cells recognize areas selectively coated with RGD-containing peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide - even for the preparation of peptide-functionalized small surface areas.

Molecular characterization of the equine testis-specific protein 1 (TPX1) and acidic epididymal glycoprotein 2 (AEG2) genes encoding members of the cysteine-rich secretory protein (CRISP) family

The cysteine-rich secretory protein (CRISP) family consists of three members called acidic epididymal glycoprotein 1 (AEG1), AEG2, and testis-specific protein 1 (TPX1), which share 16 conserved cysteine residues at their C-termini. The CRISP proteins are primarily expressed in different sections of the male genital tract and are thought to mediate cell-cell interactions of male germ cells with other cells during sperm maturation or during fertilization. Therefore, their genes are of interest as candidate genes for inherited male fertility dysfunctions and as putative quantitative trait loci for male fertility traits. In this report, the cloning and DNA sequence of 137 kb of horse genomic DNA from equine chromosome 20q22 containing the closely linked equine TPX1 and AEG2 genes are described. The equine TPX1 gene consists of ten exons spanning 18 kb while the AEG2 gene consists of eight exons that are spread over 24 kb. The expression of these two genes was investigated in several tissues by reverse transcription polymerase chain reaction analysis and Western blotting. Comparative genome analysis between horse, human, and mouse indicates that all three CRISP genes are clustered on one chromosomal location, which shows conserved synteny between these species.