Secreted Frizzled-related Protein 2 (sFRP2) Redirects Non-canonical Wnt Signaling from Fz7 to Ror2 during Vertebrate Gastrulation.

Convergent extension movements during vertebrate gastrulation require a balanced activity of non-canonical Wnt signaling pathways, but the factors regulating this interplay on the molecular level are poorly characterized. Here we show that sFRP2, a member of the secreted frizzled-related protein (sFRP) family, is required for morphogenesis and papc expression during Xenopus gastrulation. We further provide evidence that sFRP2 redirects non-canonical Wnt signaling from Frizzled 7 (Fz7) to the receptor tyrosine kinase-like orphan receptor 2 (Ror2). During this process, sFRP2 promotes Ror2 signal transduction by stabilizing Wnt5a-Ror2 complexes at the membrane, whereas it inhibits Fz7 signaling, probably by blocking Fz7 receptor endocytosis. The cysteine-rich domain of sFRP2 is sufficient for Ror2 activation, and related sFRPs can substitute for this function. Notably, direct interaction of the two receptors via their cysteine-rich domains also promotes Ror2-mediated papc expression but inhibits Fz7 signaling. We propose that sFRPs can act as a molecular switch, channeling the signal input for different non-canonical Wnt pathways during vertebrate gastrulation.

Regulation of distinct branches of the non-canonical Wnt-signaling network in Xenopus dorsal marginal zone explants.

BACKGROUND: A tight regulation of the Wnt-signaling network, activated by 19 Wnt molecules and numerous receptors and co-receptors, is required for the establishment of a complex organism. Different branches of this Wnt-signaling network, including the canonical Wnt/ß-catenin and the non-canonical Wnt/PCP, Wnt/Ror2 and Wnt/Ca(2+) pathways, are assigned to distinct developmental processes and are triggered by certain ligand/receptor complexes. The Wnt-signaling molecules are closely related and it is still on debate whether the information for activating a specific branch is encoded by specific sequence motifs within a particular Wnt protein. The model organism Xenopus offers tools to distinguish between Wnt-signaling molecules activating distinct branches of the network. RESULTS: We created chimeric Wnt8a/Wnt11 molecules and could demonstrate that the C-terminal part (containing the BS2) of Wnt8a is responsible for secondary axis formation. Chimeric Wnt11/Wnt5a molecules revealed that the N-terminus with the elements PS3-1 and PS3-2 defines Wnt11 specificity, while elements PS3-1, PS3-2 and PS3-3 are required for Wnt5a specificity. Furthermore, we used Xenopus dorsal marginal zone explants to identify non-canonical Wnt target genes regulated by the Wnt5a branch and the Wnt11 branch. We found that pbk was specifically regulated by Wnt5a and rab11fip5 by Wnt11. Overexpression of these target genes phenocopied the overexpression of their regulators, confirming the distinct roles of Wnt11 and Wnt5a triggered signaling pathways. Furthermore, knock-down of pbk was able to restore convergent extension movements in Wnt5a morphants. CONCLUSIONS: The N-terminal part of non-canonical Wnt proteins decides whether the Wnt5a or the Wnt11 branch of the Wnt-signaling network gets activated. The different non-canonical Wnt branches not only regulate cellular behavior, but, surprisingly, also regulate the expression of different target genes. One of these target genes, pbk, seems to be the relevant target gene executing Wnt5a-mediated regulation of convergent extension movements.

Neural crest specification by Prohibitin1 depends on transcriptional regulation of prl3 and vangl1.

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.

Molecular dissection of Wnt3a-Frizzled8 interaction reveals essential and modulatory determinants of Wnt signaling activity.

BACKGROUND: Wnt proteins are a family of secreted signaling molecules that regulate key developmental processes in metazoans. The molecular basis of Wnt binding to Frizzled and LRP5/6 co-receptors has long been unknown due to the lack of structural data on Wnt ligands. Only recently, the crystal structure of the Wnt8-Frizzled8-cysteine-rich-domain (CRD) complex was solved, but the significance of interaction sites that influence Wnt signaling has not been assessed. RESULTS: Here, we present an extensive structure-function analysis of mouse Wnt3a in vitro and in vivo. We provide evidence for the essential role of serine 209, glycine 210 (site 1) and tryptophan 333 (site 2) in Fz binding. Importantly, we discovered that valine 337 in the site 2 binding loop is critical for signaling without contributing to binding. Mutations in the presumptive second CRD binding site (site 3) partly abolished Wnt binding. Intriguingly, most site 3 mutations increased Wnt signaling, probably by inhibiting Wnt-CRD oligomerization. In accordance, increasing amounts of soluble Frizzled8-CRD protein modulated Wnt3a signaling in a biphasic manner. CONCLUSIONS: We propose a concentration-dependent switch in Wnt-CRD complex formation from an inactive aggregation state to an activated high mobility state as a possible modulatory mechanism in Wnt signaling gradients.

Protocadherin PAPC is expressed in the CNC and can compensate for the loss of PCNS.

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.

Parenchymal cues define Vegfa-driven venous angiogenesis by activating a sprouting competent venous endothelial subtype.

Formation of organo-typical vascular networks requires cross-talk between differentiating parenchymal cells and developing blood vessels. Here we identify a Vegfa driven venous sprouting process involving parenchymal to vein cross-talk regulating venous endothelial Vegfa signaling strength and subsequent formation of a specialized angiogenic cell, prefabricated with an intact lumen and pericyte coverage, termed L-Tip cell. L-Tip cell selection in the venous domain requires genetic interaction between vascular Aplnra and Kdrl in a subset of venous endothelial cells and exposure to parenchymal derived Vegfa and Apelin. Parenchymal Esm1 controls the spatial positioning of venous sprouting by fine-tuning local Vegfa availability. These findings may provide a conceptual framework for understanding how Vegfa generates organo-typical vascular networks based on the selection of competent endothelial cells, induced via spatio-temporal control of endothelial Kdrl signaling strength involving multiple parenchymal derived cues generated in a tissue dependent metabolic context.

Subfunctionalization and neofunctionalization of vertebrate Lef/Tcf transcription factors.

Invertebrates express a multitude of Wnt ligands and all Wnt/ß-catenin signaling pathways converge to only one nuclear Lef/Tcf. In vertebrates, however, four distinct Lef/Tcfs, i.e. Tcf-1, Lef, Tcf-3, and Tcf-4 fulfill this function. At present, it is largely unknown to what extent the various Lef/Tcfs are functionally similar or diversified in vertebrates. In particular, it is not known which domains are responsible for the Tcf subtype specific functions. We investigated the conserved and non-conserved functions of the various Tcfs by using Xenopus laevis as a model organism and testing Tcfs from Hydra magnipapillata, Caenorhabditis elegans and Drosophila melanogaster. In order to identify domains relevant for the individual properties we created series of chimeric constructs consisting of parts of XTcf-3, XTcf-1 and HyTcf. Rescue experiments in Xenopus morphants revealed that the three invertebrate Tcfs tested compensated the loss of distinct Xenopus Tcfs: Drosophila Tcf (Pangolin) can substitute for the loss of XTcf-1, XTcf-3 and XTcf-4. By comparison, Caenorhabditis Tcf (Pop-1) and Hydra Tcf (HyTcf) can substitute for the loss of only XTcf-3 and XTcf-4, respectively. The domain, which is responsible for subtype specific functions is the regulatory CRD domain. A phylogenetic analysis separates Tcf-1/Lef-1 from the sister group Tcf-3/4 in the vertebrate lineage. We propose that the vertebrate specific diversification of Tcfs in vertebrates resulted in subfunctionalization of a Tcf that already united most of the Lef/Tcf functions.

The vestibuloocular reflex of tadpoles (Xenopus laevis) after knock-down of the isthmus-related transcription factor XTcf-4.

Development 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 the organizing centre, suppression of isthmus maintenance should modify vestibular morphology and function. We tested this hypothesis by downregulation of the transcription factor XTcf-4. 10 pmol l(-1) XTcf-4-specific antisense morpholino oligonucleotide was injected in one blastomere of two-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. Overexpression 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, downregulation 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) Overexpression of XTcf-4A caused similar morphological and rVOR modifications as XTcf-4 depletion, while overexpression of XTcf-4C had no effect. Because XTcf-4C has been described as an essential factor for isthmus development, we postulate that the isthmus is strongly involved in vestibular development.

En2, Pax2/5 and Tcf-4 transcription factors cooperate in patterning the Xenopus brain.

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 3.8kb 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.

Near-field optical study of protein transport kinetics at a single nuclear pore.

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.

Lef1 regulates caveolin expression and caveolin dependent endocytosis, a process necessary for Wnt5a/Ror2 signaling during Xenopus gastrulation.

The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/ß-catenin pathway stimulates expression of ß-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.

Autoregulation of XTcf-4 depends on a Lef/Tcf site on the XTcf-4 promoter.

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.

Cold-inducible RNA binding protein (CIRP), a novel XTcf-3 specific target gene regulates neural development in Xenopus.

BACKGROUND: As nuclear mediators of wnt/beta-catenin signaling, Lef/Tcf transcription factors play important roles in development and disease. Although it is well established, that the four vertebrate Lef/Tcfs have unique functional properties, most studies unite Lef-1, Tcf-1, Tcf-3 and Tcf-4 and reduce their function to uniformly transduce wnt/beta-catenin signaling for activating wnt target genes. In order to discriminate target genes regulated by XTcf-3 from those regulated by XTcf-4 or Lef/Tcfs in general, we performed a subtractive screen, using neuralized Xenopus animal cap explants. RESULTS: We identified cold-inducible RNA binding protein (CIRP) as novel XTcf-3 specific target gene. Furthermore, we show that knockdown of XTcf-3 by injection of an antisense morpholino oligonucleotide results in a general broadening of the anterior neural tissue. Depletion of XCIRP by antisense morpholino oligonucleotide injection leads to a reduced stability of mRNA and an enlargement of the anterior neural plate similar to the depletion of XTcf-3. CONCLUSION: Distinct steps in neural development are differentially regulated by individual Lef/Tcfs. For proper development of the anterior brain XTcf-3 and the Tcf-subtype specific target XCIRP appear indispensable. Thus, regulation of anterior neural development, at least in part, depends on mRNA stabilization by the novel XTcf-3 target gene XCIRP.

Pontin and Reptin regulate cell proliferation in early Xenopus embryos in collaboration with c-Myc and Miz-1.

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.

A novel role for the tumour suppressor Nitrilase1 modulating the Wnt/ß-catenin signalling pathway.

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 ß-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 ß-catenin and LEF-1/TCF-4, represses ß-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 ß-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.

The E3 ligase RNF43 inhibits Wnt signaling downstream of mutated ß-catenin by sequestering TCF4 to the nuclear membrane.

Given its fundamental role in development and cancer, the Wnt-ß-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 active mutants of ß-catenin. This inhibitory mechanism was disrupted by the expression of RNF43 bearing mutations found in human gastrointestinal tumors, and transactivation of the Wnt pathway was observed in various cells and in Xenopus embryos when the RING domain of RNF43 was mutated. Our findings indicate that RNF43 inhibits the Wnt pathway downstream of oncogenic mutations 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.

Live imaging of Xwnt5A-ROR2 complexes.

Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/ß-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner.

Monensin inhibits canonical Wnt signaling in human colorectal cancer cells and suppresses tumor growth in multiple intestinal neoplasia mice.

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/ß-catenin signaling cascade was observed in mammalian cells stimulated with Wnt ligands, glycogen synthase kinase-3 inhibitors, and in cells transfected with ß-catenin expression constructs. Furthermore, monensin suppressed the Wnt-dependent tail fin regeneration in zebrafish and Wnt- or ß-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 ß-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.

Synthesis of novel inhibitors blocking Wnt signaling downstream of ß-catenin.

Large scale screening of libraries consisting of natural and small molecules led to the identification of many small molecule inhibitors repressing Wnt/ß-Catenin signaling. However, targeted synthesis of novel Wnt pathway inhibitors has been rarely described. We developed a modular and expedient way to create the aromatic ring system with an aliphatic ring in between. Our synthesis opens up the possibility, in principle, to substitute all positions at the ring system with any desired substituent. Here, we tested five different haloquinone analogs carrying methoxy- and hydroxy-groups at different positions. Bona fide Wnt activity assays in cell culture and in Xenopus embryos revealed that two of these compounds act as potent inhibitors of aberrant activated Wnt/ß-Catenin signaling.

Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells.

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.