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The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/β-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with β-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or β-catenin–induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of β-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling. Mol Cancer Ther; 13(4); 812–22. ©2014 AACR.

PDF file - 515KB, Supplementary Materials and Methods. Supplementary Figure S1. Densitometric analysis of western blots shown in Figs. 2D (A), 3C (B) and 4D (C). Supplementary Figure S2. Monensin suppresses Wnt signaling activated by TALEN-induced disruption of the APC gene. Supplementary Figure S3. Monensin does not interfere with Dvl phoshorylation. Supplementary Figure S4. The inhibitory effect of monensin on Wnt signaling is not restricted to the vesicular acidity blockade. Supplementary Figure S5. Monensin inhibits aberrant Wnt signaling in CRC cells COLO320 and LS174T. Supplementary Figure S6. ROS concentration is not increased by monensin. Supplementary Figure S7. Membranous localization of beta-catenin in HCT116 cells. Supplementary Figure S8. Monensin does not inhibit beta-catenin acetylation. Supplementary Figure S9. Monensin does not change the fraction of proliferating cells in intestinal tumors. Supplementary Table S1. Sequences of primers used for qRT-PCR analyses. Supplementary Table S2. Monensin does not inhibit enzymatic activity of any of the tested protein kinases. Supplementary Table S3. qRT-PCR analysis of the TCF/beta-catenin target gene expression in CRC cells. Supplementary Table S4. Cell cycle analysis of SW480 and HCT116 cells. Supplementary Table S5. Analysis of phosphorylated amino acid residues in beta-catenin.

PDF file - 160K, Determination of IC50-values in HEK293 cells (ST-Luc) and biochemical assays (TNKS1/2 and PARP) using XLfit

PDF file - 65K, The distribution and steady-state level of b-catenin as visualized by immunofluorescence

PDF file - Human liver microsome (HLM) test of JW55 (A), weight measurement of ApcCKO/CKOLgr5-CreERT2+ mice treated with JW55 or vehicle (B) and an example on quantification of tumor number and area using Ellipse software (C).

Supplementary Figure 6 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

Supplementary Figures 1-4 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 178K, Pictures of the ileum (DMSO treated small intestine) that show delineated flat adenomas (A), with a few goblet cells (arrow)(B), a transition between normal epithelium and neoplastic glands (arrow) (C) and aberrant crypt foci in areas with no obvious adenoma formation (arrows) (D).

Supplementary Figure 7 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

Supplementary Figure Legends 1-8, Table Legend 1 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 171K, Superposition of JW55 (docking) and IWR-1 (X-ray, PDB code 3UA9) in the PARP domain of TNKS2 show similar binding characteristics.

Supplementary Figure 5 from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 216K

Supplementary Methods from Novel Synthetic Antagonists of Canonical Wnt Signaling Inhibit Colorectal Cancer Cell Growth

PDF file - 77K, The figure shows full-length and uncropped Western blots of analyzed lysates from SW480 cells exposed to different concentrations of JW55 for 24 hours

XLS file - 574K, The table shows the results from an Illumina expression analysis that examined mRNA from SW480 CRC cells exposed to JW55 for 72 hours.

The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/β-catenin pathway stimulates expression of β-catenin-Lef/Tcf regulated Wnt target genes and a regulatory network giving rise to the formation of the Spemann organizer. Non-canonical pathways, by contrast, mainly regulate cell polarization and migration, in particular convergent extension movements of the trunk mesoderm during gastrulation. By transcriptome analyses, we found caveolin1, caveolin3 and cavin1 to be regulated by Lef1 in the involuting mesoderm of Xenopus embryos at gastrula stages. We show that caveolins and caveolin dependent endocytosis are necessary for proper gastrulation, most likely by interfering with Wnt5a/Ror2 signaling. Wnt5a regulates the subcellular localization of receptor complexes, including Ror2 homodimers, Ror2/Fzd7 and Ror2/dsh heterodimers in an endocytosis dependent manner. Live-cell imaging revealed endocytosis of Ror2/caveolin1 complexes. In Xenopus explants, in the presence of Wnt5a, these receptor clusters remain stable exclusively at the basolateral side, suggesting that endocytosis of non-canonical Wnt/receptor complexes preferentially takes place at the apical membrane. In support of this blocking endocytosis with inhibitors prevents the effects of Wnt5a. Thus, target genes of Lef1 interfere with Wnt5a/Ror2 signaling to coordinate gastrulation movements.

Der Sudafrikanische Krallenfrosch Xenopus laevis ist einer der Klassiker in der Reihe tierischer Modellorganismen. Die grose Anzahl an sich auserhalb des Muttertiers entwickelnden Nachkommen gepaart mit der Grose der Eizellen erlaubte zahlreiche fundamentale Entwicklungsbiologische Fragen aufzuklaren. Daruber hinaus wurden mit diesem Modellorganismus auch Nobelpreise in der klassischen Physiologie (Funktion der Aquaporine) und der Stammzellforschung gewonnen.

A complex network of transcription factors regulates specification of neural crest cells at early neurula stage by stabilizing neural crest identity and activating neural crest effector genes so that distinct subpopulations evolve. In this network, c-myc acts on top of the gene hierarchy controlling snail2, AP2 and prohibitin1 (phb1) expression. While snail2 and AP2 are well studied neural crest specifier genes little is known about the role of phb1 in this process. To identify phb1 regulated genes we analyzed the transcriptome of neural crest explants of phb1 morphant Xenopus embryos. Among 147 phb1 regulated genes we identified the membrane-associated protein-tyrosine phosphatase PRP4A3 (prl3) and the atypical cadherin and Wnt-PCP component van gogh like1 (vangl1). Gain of function, loss of function and epistasis experiments allowed us to allocate both genes in the neural crest specification network between phb1 and twist. Interestingly, both, vangl1 and prl3 regulate only a small subset of neural crest marker genes. The identification of two membrane-associated proteins as novel neural crest specifiers indicates that in addition to gene regulation by combinatory effects of transcription factors also post-translational modifications (prl3) and cell-cell adhesion and/or regulation of cell-polarity (vangl1) specify the identity of neural crest cell populations. genesis 53:627-639, 2015. © 2015 Wiley Periodicals, Inc.

Die Autoren dieses Titels vermitteln einen Überblick über Prüfungswissen im Fach Tierphysiologie. Dabei wird Wert darauf gelegt, Themengebiete der Tierphysiologie so zu verknüpfen, dass das Denken in Schubladen aufgebrochen und Transferwissen aufgebaut wird. Mit spezifischen Fragestellungen werden beispielhaft unterschiedliche Aspekte aus verschiedenen Bereichen der Physiologie zusammengeführt. Zusätzliche Übungsklausurfragen sollen eigene Überlegungen anregen. Mit diesem Buch können Studierende insbesondere der Biologie und verwandter Studiengänge der Lebenswissenschaften das im Studium erworbene Wissen aus unterschiedlichen Blickwinkeln abfragen und Verständnisprobleme erkennen. Ideal geeignet als zusätzliche Lernhilfe zu den klassischen Lehrbüchern und zur erfolgreichen Prüfungsvorbereitung!

Summary Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration. genesis 52:120–126. © 2013 Wiley Periodicals, Inc.

The double Friedel–Crafts acylation of readily accessible biaryls with oxalyl chloride delivers the respective phenanthrene-9,10-diones, providing an alternative to the traditional methods, which require harsh oxidizing conditions and multistep sequences. This simple method allows the synthesis of various symmetrical and non-symmetrical targets, and is even effective for the synthesis of the parent ring system from (unactivated) biphenyl.

Among Xenopus Lef/Tcfs, XTcf-4 has an outstanding role. In early development it is located exclusively in the midbrain where it is essential for midbrain and isthmus development. In order to identify transcription factors responsible for the restriction of XTcf-4 expression we isolated a 3.8 kb fragment of the XTcf-4 promoter. We found that this promoter fragment is sufficient to mimic endogenous XTcf-4 expression in the midbrain. Characterization of putative binding sites for en2 and pax2/5 revealed that en2, but not pax2/5 directly represses XTcf-4 promoter activity. Gain-of-function experiments in Xenopus embryos confirmed this en2-mediated repression. Loss-of-function experiments demonstrate that both en2 and pax2/5 are essential for endogenous XTcf-4 expression. The primary effect of pax2/5 depletion thereby appears to be a reduced en2 expression at neurula stages. Because en2 can compensate for the depletion of pax2/5, we assume a hierarchical regulation of gene expression in the midbrain/isthmus region with pax2/5 acting upstream of en2. Furthermore, since the XTcf-4 expression domain does not overlap with the expression domains of the isthmus marker genes en2 and pax2/5, we conclude that the knock-down of en2 and pax2/5 results in a downregulation of a paracrine growth factor regulating XTcf-4 expression. We found that the growth factor for this non-cell-autonomous effect of en2 and pax2/5 is wnt-1 acting on the − 1437 Lef/Tcf binding site on the XTcf-4 promoter. We provide evidence that the main nuclear wnt transducer for the autoregulation of XTcf-4 is XTcf-1.

The restricted expression of XTcf-4 in the anterior midbrain is regulated via an active wnt/beta-catenin pathway (Kunz et al.,2004, Dev Biol 273:390-401). The molecular mechanism of this autoregulatory loop, however, remained elusive. Here we show that the activity of a 1,775 bp promoter fragment containing a consensus Lef/Tcf binding site at position -1,437 to -1,428 is upregulated by activating transcription factors of the Lef/Tcf family. Furthermore, chromatin immunoprecipitation revealed that endogenous beta-catenin is bound to the Lef/Tcf site on the promoter. Thus, regulation of XTcf-4 by canonical wnt-signaling is directly controlled by binding to and activating a consensus Lef/Tcf binding site within its own promoter.

Nitrilase1 was classified as a tumour suppressor in association with the fragile histidine-triad protein Fhit. However, knowledge about nitrilase1 and its tumour suppressor function is still limited. Whereas nitrilase1 and Fhit are discrete proteins in mammals, they are merged in Drosophila melanogaster and Caenorhabditis elegans. According to the Rosetta-Stone hypothesis, proteins encoded as fusion proteins in one organism and as separate proteins in another organism may act in the same signalling pathway. Although a direct interaction of human nitrilase1 and Fhit has not been shown, our previous finding that Fhit interacts with β-catenin and represses its transcriptional activity in the canonical Wnt pathway suggested that human nitrilase1 also modulates Wnt signalling. In fact, human nitrilase1 forms a complex with β-catenin and LEF-1/TCF-4, represses β-catenin-mediated transcription and shows an additive effect together with Fhit. Knockdown of human nitrilase1 enhances Wnt target gene expression. Moreover, our experiments show that β-catenin competes away human nitrilase1 from LEF-1/TCF and thereby contributes to the activation of Wnt-target gene transcription. Inhibitory activity of human nitrilase1 on vertebrate Wnt signalling was confirmed by repression of Wnt-induced double axis formation in Xenopus embryogenesis. In line with this finding, the Drosophila fusion protein Drosophila NitFhit directly binds to Armadillo and represses the Wingless pathway in reporter gene assays. Genetic experiments confirmed the repressive activity of Drosophila NitFhit on Wingless signalling in the Drosophila wing imaginal disc. In addition, colorectal tumour microarray analysis revealed a significantly reduced expression of human nitrilase1 in poorly differentiated tumours. Taken together, repression of the canonical Wnt pathway represents a new mechanism for the human nitrilase1 tumour suppressor function.

Interactions between neoplastic and stromal cells contribute to tumor progression. Wnt genes, involved in cell migration and often deregulated in cancers, are attractive candidates to regulate these effects. We have recently shown that coculture of breast cancer cells with macrophages enhances invasiveness via matrix metalloproteases and TNF-α. Here we demonstrate that coculture of MCF-7 cells and macrophages leads to up-regulation of Wnt 5a in the latter. This was accompanied by activation ofAP-1/c-Junin MCF-7. Recombinant Wnt 5a mimicked the coculture effect. Wnt 5a was also detectable in tumor-associated macrophages in primary breast cancers. Experiments with agonists and antagonists of Wnt signaling revealed that a functional canonical pathway in the tumor cells was a necessary prerequisite; however, noncanonical signaling via Wnt 5a and the Jun-N-terminal kinase pathway was critical for invasiveness. It was also responsible for induction of matrix metalloprotease-7, known to release TNF-α. All these effects could be antagonized by dickkopf-1. Our results indicate that Wnt 5a is essential for macrophage-induced invasiveness, because it regulates tumor cell migration as well as proteolytic activity of the macrophages. The function of Wnt 5a as either a suppressor or promoter of malignant progression seems to be modulated by intercellular interactions. Wnt 5a detection in tumor-associated macrophages in breast cancer biopsies supports the assumption that similar events play a rolein vivo.

Activation of Wnt/beta-catenin target genes is regulated by a heterodimer of beta-catenin and the high mobility group box transcription factors of the lymphoid enhancer factor (LEF)/T-cell factor (TCF) family. In vertebrates, four LEF/TCF family members have been identified. They all contain a conserved beta-catenin-binding motif at the N terminus and a highly conserved high mobility group box for DNA binding. The core sequence between these motifs is less conserved and contributes to the specific properties of the individual family members. To identify interacting proteins that allocate specific functions to the individual LEF/TCF transcription factors, we performed a yeast two-hybrid screen using the less conserved core sequence as bait. We isolated the murine LIM protein HIC-5 (hydrogen peroxide-induced clone 5; also termed ARA-55 (androgen receptor activator of 55 kDa)) and cloned the highly conserved Xenopus homolog. In addition, we report that the LIM domain-containing C-terminal half of HIC-5 binds to a conserved alternatively spliced exon in LEF/TCF transcription factors. Our functional analyses revealed that HIC-5 acts as negative regulator of a subset of LEF/TCF family members, which have been characterized as activators in reporter gene analyses and in the Xenopus axis induction assay. In addition, we observed a repressive interference of LEF/TCF family members with HIC-5-mediated activation of glucocorticoid-driven transcription, which again could be allocated to specific LEF/TCF subtypes. With the characterization of HIC-5 as a binding partner of the alternatively spliced exon in LEF/TCF transcription factors, we identified a novel molecular mechanism in the dialog of steroid and canonical Wnt signaling that is LEF/TCF subtype-dependent.

Pontin (Tip49) and Reptin (Tip48) are highly conserved components of multimeric protein complexes important for chromatin remodelling and transcription. They interact with many different proteins including TATA box binding protein (TBP), beta-catenin and c-Myc and thus, potentially modulate different pathways. As antagonistic regulators of Wnt-signalling, they control wing development in Drosophila and heart growth in zebrafish. Here we show that the Xenopus xPontin and xReptin in conjunction with c-Myc regulate cell proliferation in early development. Overexpression of xPontin or xReptin results in increased mitoses and bending of embryos, which is mimicked by c-Myc overexpression. Furthermore, the knockdown of either xPontin or xReptin resulted in embryonic lethality at late gastrula stage, which is abrogated by the injection of c-Myc-RNA. The N-termini of xPontin and xReptin, which mediate the mitogenic effect were mapped to contain c-Myc interaction domains. c-Myc protein promotes cell cycle progression either by transcriptional activation through the c-Myc/Max complex or by repression of cyclin dependent kinase inhibitors (p21, p15) through c-Myc/Miz-1 interaction. Importantly, xPontin and xReptin exert their mitogenic effect through the c-Myc/Miz-1 pathway as dominant negative Miz-1 and wild-type c-Myc but not a c-Myc mutant deficient in Miz-1 binding could rescue embryonic lethality. Finally, promoter reporter studies revealed that xPontin and xReptin but not the N-terminal deletion mutants enhance p21 repression by c-Myc. We conclude that xPontin and xReptin are essential genes regulating cell proliferation in early Xenopus embryogenesis through interaction with c-Myc. We propose a novel function of xPontin and xReptin as co-repressors in the c-Myc/Miz-1 pathway.

The complex cross-regulation between Wnt signaling, cell-cell adhesion, and cell-matrix adhesion has revealed a number of regulatory components important in development and cancer progression. In the following, we would like to highlight and summarize some of the steps where pathways converge or diverge in regulating Wnt activity, matrix-induced pathways, and cell adhesion. We would like to focus on the involvement of heparan sulfate proteoglycan-rich proteins (HSPGs), integrin-mediated outside-in signaling, and cadherin-mediated cell-cell adhesion on Wnt pathways and the transcriptional regulation of extracellular matrix components and cell adhesion molecules by Wnt signaling.

After the primary anterior–posterior patterning of the neural plate, a subset of wnt signaling molecules including Xwnt-1, Xwnt-2b, Xwnt-3A, Xwnt-8b are still expressed in the developing brain in a region spanning from the posterior part of the diencephalon to the mesencephalon/metencephalon boundary. In this expression field, they are colocalized with the HMG-box transcription factor XTcf-4. Using antisense morpholino loss-of-function strategies, we demonstrate that the expression of this transcription factor depends on Xwnt-2b, which itself is under the control of XTcf-4. Marker gene analyses reveal that this autoregulatory loop is important for proper development of the midbrain and the isthmus. Staining for NCAM reveals a lack of dorsal neural tissue in this area. This reduction is caused by a reduced proliferation rate as shown by staining for PhosphoH3 positive nuclei. In rescue experiments, we demonstrate that individual isoforms of XTcf-4 control the development of different parts of the brain. XTcf-4A restored the expression of the mesencephalon marker genes pax-6 and wnt-2b but not the isthmus marker gene en-2. XTcf-4C, in contrast, restored en-2, but had only weak effects on pax-6 and wnt-2b. Thus, autoregulation of canonical Wnt signaling and alternative expression of different isoforms of XTcf-4 is essential for specifying the developing CNS.

Given its fundamental role in development and cancer, the Wnt-β-catenin signaling pathway is tightly controlled at multiple levels. RING finger protein 43 (RNF43) is an E3 ubiquitin ligase originally found in stem cells and proposed to inhibit Wnt signaling by interacting with the Wnt receptors of the Frizzled family. We detected endogenous RNF43 in the nucleus of human intestinal crypt and colon cancer cells. We found that RNF43 physically interacted with T cell factor 4 (TCF4) in cells and tethered TCF4 to the nuclear membrane, thus silencing TCF4 transcriptional activity even in the presence of constitutively activemutants of β-catenin. This inhibitorymechanismwas disrupted by the expression ofRNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of theWnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate thatRNF43 inhibits theWnt pathway downstreamof oncogenicmutations that activate the pathway. Mimicking or enhancing this inhibitory activity of RNF43 may be useful to treat cancers arising from aberrant activation of the Wnt pathway., This work was supported by a grant from Deutsche Forschungsgemeinschaft (GE 2042/2-1) to M. Gerhard.

The Wnt signaling pathway is required during embryonic development and for the maintenance of homeostasis in adult tissues. However, aberrant activation of the pathway is implicated in a number of human disorders, including cancer of the gastrointestinal tract, breast, liver, melanoma, and hematologic malignancies. In this study, we identified monensin, a polyether ionophore antibiotic, as a potent inhibitor of Wnt signaling. The inhibitory effect of monensin on the Wnt/β-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with β-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or β-catenin–induced formation of secondary body axis in Xenopus embryos. In Wnt3a-activated HEK293 cells, monensin blocked the phoshorylation of Wnt coreceptor low-density lipoprotein receptor related protein 6 and promoted its degradation. In human colorectal carcinoma cells displaying deregulated Wnt signaling, monensin reduced the intracellular levels of β-catenin. The reduction attenuated the expression of Wnt signaling target genes such as cyclin D1 and SP5 and decreased the cell proliferation rate. In multiple intestinal neoplasia (Min) mice, daily administration of monensin suppressed progression of the intestinal tumors without any sign of toxicity on normal mucosa. Our data suggest monensin as a prospective anticancer drug for therapy of neoplasia with deregulated Wnt signaling. Mol Cancer Ther; 13(4); 812–22. ©2014 AACR.

beta-Catenin stabilizes the cadherin cell adhesion complex but, as a component of the Wnt/Wg signaling pathway, also controls gene expression by forming a heterodimer with a transcription factor of the LEF-TCF family. We demonstrate that the substrate adhesion molecule fibronectin is a direct target of Wnt/Wg signaling. Nuclear depletion of beta-catenin following cadherin transfection in Xenopus fibroblasts resulted in downregulation of fibronectin expression which was restored by activating the Wnt/Wg signaling cascade via LiCl treatment or transfection of either Xwnt-8 or beta-catenin. We isolated the Xenopus fibronectin gene (FN) promoter and found four putative LEF-TCF binding sites. By comparing the activities of different fibronectin gene reporter constructs in fibroblasts and cadherin transfectants, the LEF-TCF site at position -368 was identified as a Wnt/Wg response element. LEF-1-related proteins were found in nuclei of the fibroblasts but were absent in a kidney epithelial cell line. Consistent with the lack of these transcription factors, the FN promoter was silent in the epithelial cells but was activated upon transfection of LEF-1. Wild-type Xenopus Tcf-3 (XTcf-3) was unable to activate FN promoter reporter constructs, while a mutant lacking the groucho binding region behaved like LEF-1. In contrast to XTcf-3, LEF-1 does not interact with groucho proteins, which turn TCFs into activators or repressors (J. Roose, M. Molenaar, J. Hurenkamp, J. Peterson, H. Brantjes, P. Moerer, M. van de Wetering, O. Destreé, and H. Clevers, Nature 395:608-612, 1998). Together these data provide evidence that expressing LEF-1 enables fibroblasts, in contrast to epithelial cells, to respond to the Wnt/Wg signal via beta-catenin in stimulating fibronectin gene transcription. Our findings further promote the idea that due to its dual function, beta-catenin regulates the balance between cell-cell and cell-substrate adhesion.

Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration.

Raster image correlation spectroscopy is a powerful tool to study fast molecular dynamics such as protein diffusion or receptor-ligand interactions inside living cells and tissues. By analysing spatio-temporal correlations of fluorescence intensity fluctuations from raster-scanned microscopy images, molecular motions can be revealed in a spatially resolved manner. Because of the diffraction-limited optical resolution, however, conventional raster image correlation spectroscopy can only distinguish larger regions of interest and requires low fluorophore concentrations in the nanomolar range. Here, to overcome these limitations, we combine raster image correlation spectroscopy with stimulated emission depletion microscopy. With imaging experiments on model membranes and live cells, we show that stimulated emission depletion-raster image correlation spectroscopy offers an enhanced multiplexing capability because of the enhanced spatial resolution as well as access to 10-100 times higher fluorophore concentrations.

Increased nuclear accumulation of β-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/β-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the β-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the β-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the β-catenin destruction complex, followed by increased degradation of β-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of β-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/β-catenin signaling through inhibiting the PARP domain of TNKS1/2. Cancer Res; 72(11); 2822–32. ©2012 AACR.

SummaryDevelopment of the amphibian vestibular organ is regulated by molecular and neuronal mechanisms and by environmental input. The molecular component includes inductive signals derived from neural tissue of the hindbrain and from the surrounding mesoderm. The integrity of hindbrain patterning, on the other hand, depends on instructive signals from the isthmus organizer of the midbrain including the transcription factor XTcf-4. If the development of the vestibular system depends on the integrity of the isthmus as organizing centre, suppression of isthmus maintenance should modify vestibular morphology and function. We tested this hypothesis by down-regulation of the transcription factor XTcf-4. 10 pMol XTcf-4-specific antisense morpholino oligonucleotide were injected in one blastomere of 2-cell stage embryos of Xenopus laevis. For reconstitution experiments, 500 pg mRNA of the repressing XTcf-4A isoform or the activating XTcf-4C isoform were co-injected. Over-expression experiments were included using the same isoforms. Otoconia formation and vestibular controlled behaviour such as the roll-induced vestibuloocular reflex (rVOR) and swimming were recorded two weeks later. In 50% of tadpoles, down-regulation of XTcf-4 induced (1) a depression of otoconia formation accompanied by a reduction of the rVOR, (2) abnormal tail development, and (3) loop swimming behaviour. (4) All effects were rescued by co-injection of XTcf-4C but not or only partially by XTcf-4A. (5) Over-expression of XTcf-4A caused similar morphological and rVOR modifications as XTcf-4 depletion while over-expression of XTcf-4C had no effect. Because XTcf-4C has been described as essential factor for isthmus development, we postulate that the isthmus is strongly involved in vestibular development.

Canonical Wnt signaling is deregulated in several types of human cancer where it plays a central role in tumor cell growth and progression. Here we report the identification of 2 new small molecules that specifically inhibit canonical Wnt pathway at the level of the destruction complex. Specificity was verified in various cellular reporter systems, a Xenopus double-axis formation assay and a gene expression profile analysis. In human colorectal cancer (CRC) cells, the new compounds JW67 and JW74 rapidly reduced active β-catenin with a subsequent downregulation of Wnt target genes, including AXIN2, SP5, and NKD1. Notably, AXIN2 protein levels were strongly increased after compound exposure. Long-term treatment with JW74 inhibited the growth of tumor cells in both a mouse xenograft model of CRC and in ApcMin mice (multiple intestinal neoplasia, Min). Our findings rationalize further preclinical and clinical evaluation of these new compounds as novel modalities for cancer treatment. Cancer Res; 71(1); 197–205. ©2010 AACR.

The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.

The application of Fluorescence Correlation Spectroscopy (FCS) to Near-field Scanning Optical Microscopy on diffusing molecules in solution is presented here. The ultra small excitation volume in such a technique allows measurements with fluorophore concentrations of up to 10μM, making FCS in biological environment at nearly native conditions possible.

The kinetics of proteins passing through individual nuclear pore complexes (NPCs) of the nuclear envelope (NE) was studied using near-field scanning optical microscopy (NSOM) in combination with fluorescence correlation spectroscopy (FCS). The NSOM probe was placed over a single pore in an unsupported native NE to observe fluorescence-labeled NTF2 moving in the transport channel. A correlation analysis of the arising fluorescence fluctuations enabled us to characterize the translocation as driven by Brownian motion and to determine the related kinetic constants. Though trapped in the pore, NTF2 turned out to be highly mobile within a large axial extension. Our findings support the idea that molecules in transit interact with NPC proteins containing phenylalanine-glycine-repeat domains at the periphery of the channel. NSOM-FCS may help to understand the facilitated translocation in more detail and offers a new way to study single molecule mobility on a nanoscale.

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.

Spinal muscular atrophy (SMA) is a motoneuron disease caused by reduced levels of survival motoneuron (SMN) protein. Previous studies have assigned SMN to uridine-rich small nuclear ribonucleoprotein particle (U snRNP) assembly, splicing, transcription, and RNA localization. Here, we have used gene silencing to assess the effect of SMN protein deficiency on U snRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to levels found in SMA patients impairs U snRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less severe knock-down conditions, zebrafish embryos proceeded through development yet exhibited dramatic SMA-like motor axon degeneration. The same was observed after silencing two other essential factors in the U snRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified U snRNPs into either SMN- or Gemin2-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of U snRNPs.

Some members of the Wnt family of extracellular glycoproteins regulate target gene expression by inducing stabilization and nuclear accumulation of beta-catenin, which functions as a transcriptional activator after binding to transcription factors of the T-cell factor/lymphoid enhancer factor (TCF/LEF) family. Three different members of this family have been identified in Xenopus laevis thus far that differ in their ability to influence mesodermal differentiation and to activate expression of the Wnt target gene fibronectin. Here we report on the isolation and characterization of additional variants of XTCF-4. We show that the differential ability of these proteins and other members of the TCF family to activate target genes is neither due to preferential interaction with transcriptional cofactors of the groucho family or SMAD4 nor to different DNA binding affinities. Expression of these proteins in an epithelial cell line reveals differences in their ability to form a ternary complex with DNA and beta-catenin. Interestingly, formation of this ternary complex was not sufficient to activate target gene expression as previously thought. Our experiments identify two amino acid sequence motifs, LVPQ and SFLSS, in the central domain of XTCF-4 that regulate the formation of the DNA-TCF-beta-catenin complex or activation of target genes, respectively. Biochemical studies reveal that the phosphorylation state of these XTCF-4 variants correlates with their ability to form a ternary complex with beta-catenin and DNA but not to activate target gene expression. The described variants of XTFC-4 with their different properties in complex formation provide strong evidence that in addition to the regulation of beta-catenin stability the isoforms of TCF/LEF transcription factors and their posttranslational modifications define the cellular response of a Wnt/wingless signal.