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The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer. Details concerning selectivity and differences in relative binding affinities of 19 mammalian WNTs to the cysteine-rich domain (CRD) of their receptors���the ten mammalian Frizzleds (FZDs)���remain unclear. Here, we used eGFP-tagged mouse WNT-3A for a systematic analysis of WNT interaction with every human FZD paralogue in HEK293A cells. Employing HiBiT-tagged full-length FZDs, we studied eGFP-WNT-3A binding kinetics, saturation binding, and competition binding with commercially available WNTs in live HEK293A cells using a NanoBiT/BRET-based assay. Further, we generated receptor chimeras to dissect the contribution of the transmembrane core to WNT-CRD binding. Our data pinpoint distinct WNT-FZD selectivity and shed light on the complex WNT-FZD binding mechanism. The methodological development described herein reveals yet unappreciated details of the complexity of WNT signaling and WNT-FZD interactions, providing further details with respect to WNT-FZD selectivity.

Development of non-viral gene vectors which can efficiently and safely transfect plasmid DNA and siRNA into cells is of great importance for gene therapy. Despite lots of efforts spent, it is still imperative to develop suitable gene vectors with better transfection efficiency and low cytotoxicity. To this end, we successfully designed, synthesized and screened a library of 120 polymers (via nucleophilic substitution reaction between dihalides and amines). With cell-based transfection screening assays, 120 polymers were tested to evaluate their transfection efficiency of transporting DNA and siRNA into cells. Our results indicated that hydrophobic modification could greatly enhance cationic polymers’ transfection efficiency, and polymers with long linkers usually showed better transfection performance, especially for polymers with the linker of 1, 12-dibromododecane (L3 linker). Besides, polyalkylamines exhibited better transfection efficiency with the polymer particle size around 200 nm and the zeta potential in the range of + 40 mV to +50 mV. Interestingly, polymer particles made from N15HL3 not only exhibited better DNA transfection efficiency in HEK 293T cells but also showed higher siRNA transfection efficiency in U87 Luc-GFP cells together with low cell toxicity than Lipofectamine 2000 (one of commercial transfection reagents). Therefore, it is hoped that our study here not only provides promising gene vector candidates for further evaluation in gene therapy, but also provides valuable insights for better understanding of the relationship between the chemical structures and gene transfection efficiency to rationally design better non-viral gene vectors for gene therapy in the future.

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1–10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.

International audience; p53 is one of the most important tumour suppressor proteins currently known. It is activated in response to DNA damage and this activation leads to proliferation arrest and cell death. The abundance and activity of p53 are tightly controlled and reductions in p53's activity can contribute to the development of cancer. Here, we show that Fam83F increases p53 protein levels by protein stabilisation. Fam83F interacts with p53 and decreases its ubiquitination and degradation. Fam83F is induced in response to DNA damage and its overexpression also increases p53 activity in cell culture experiments and in zebrafish embryos. Downregulation of Fam83F decreases transcription of p53 target genes in response to DNA damage and increases cell proliferation, identifying Fam83F as an important regulator of the DNA damage response. Overexpression of Fam83F also enhances migration of cells harbouring mutant p53 demonstrating that it can also activate mutant forms of p53.

Artificial multicellular systems are gaining importance in the field of tissue engineering and regenerative medicine. Reconstruction of complex tissue architectures in vitro is nevertheless challenging, and methods permitting controllable and high-throughput fabrication of complex multicellular architectures are needed. Here, a facile and high-throughput method is developed based on a tunable droplet-fusion technique, allowing programmed assembly of multiple cell spheroids into complex multicellular architectures. The droplet-fusion technique allows for construction of various multicellular architectures (double-spheroids, multi-spheroids, hetero-spheroids) in a miniaturized high-density array format. As an example of application, the propagation of Wnt signaling is investigated within hetero-spheroids formed from two fused Wnt-releasing and Wnt-reporter cell spheroids. The developed method provides an approach for miniaturized, high-throughput construction of complex 3D multicellular architectures and can be applied for studying various biological processes including cell signaling, cancer invasion, embryogenesis, and neural development.

Wnt reception at the membrane is complex and not fully understood. CD44 is a major Wnt target gene in the intestine and is essential for Wnt-induced tumor progression in colorectal cancer. Here we show that CD44 acts as a positive regulator of the Wnt receptor complex. Downregulation of CD44 expression decreases, whereas CD44 overexpression increases Wnt activity in a concentration-dependent manner. Epistasis experiments place CD44 function at the level of the Wnt receptor LRP6. Mechanistically, CD44 physically associates with LRP6 upon Wnt treatment and modulates LRP6 membrane localization. Moreover, CD44 regulates Wnt signaling in the developing brain of Xenopus laevis embryos as shown by a decreased expression of Wnt targets tcf-4 and en-2 in CD44 morphants.

In multi-cellular organisms, cell-cell communication involves a large variety of cell-surface receptors that bind specific ligands. Unraveling these cell signaling networks in depth requires quantitative information on ligand-receptor interactions within living systems. Fluorescence correlation spectroscopy (FCS) based methods are powerful approaches to obtain these data. Still, their application to living systems have proven challenging. We have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Ligand and receptor concentrations as well as their diffusion coefficients within the cell membrane can be quantified without performing additional calibration experiments. In a first application, we have employed this robust technique to study interactions between Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), and their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of HEK293T cells.

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) function as transmembrane receptors to transduce Wnt signals. A key mechanism for signalling is Wnt-induced serine/threonine phosphorylation at conserved PPPSPxS motifs in the LRP6 cytoplasmic domain, which promotes pathway activation. Conserved tyrosine residues are positioned close to all PPPSPxS motifs, which suggests they have a functional significance. Using a cell culture-based cDNA expression screen, we identified the non-receptor tyrosine kinases Src and Fer as novel LRP6 modifiers. Both Src and Fer associate with LRP6 and phosphorylate LRP6 directly. In contrast to the known PPPSPxS Ser/Thr kinases, tyrosine phosphorylation by Src and Fer negatively regulates LRP6-Wnt signalling. Epistatically, they function upstream of β-catenin to inhibit signalling and in agreement with a negative role in regulating LRP6, MEF cells lacking these kinases show enhanced Wnt signalling. Wnt3a treatment of cells enhances tyrosine phosphorylation of endogenous LRP6 and, mechanistically, Src reduces cell surface LRP6 levels and disrupts LRP6 signalosome formation. Interestingly, CK1γ inhibits Fer-induced LRP6 phosphorylation, suggesting a mechanism whereby CK1γ acts to de-represses inhibitory LRP6 tyrosine phosphorylation. We propose that LRP6 tyrosine phosphorylation by Src and Fer serves a negative regulatory function to prevent over-activation of Wnt signalling at the level of the Wnt receptor, LRP6.

Growth factor antagonists play important roles in mediating the inductive effects of the Spemann organizer in amphibian embryos and its equivalents in other vertebrates. Dual inhibition of Wnt and BMP signals has been proposed to confer head organizer activity. We tested the requirement of this coinhibition in Xenopus and mice. In Xenopus, simultaneous reduction of the BMP antagonists chordin and noggin, and the Wnt antagonist dickkopf1 (dkk1) leads to anterior truncations. In mice, compound mutants for dkk1 and noggin display severe head defects, with deletion of all head structures anterior to the mid-hindbrain boundary. These defects arise as a result of a failure in anterior specification at the gastrula stage. The results provide genetic evidence for the dual inhibition model and indicate that dkk1 and noggin functionally cooperate in the head organizer.

A gradient of Wnt/β-catenin signalling formed by posteriorising Wnts and anteriorising Wnt antagonists regulates anteroposterior (AP) patterning of the central nervous system (CNS) during Xenopus gastrulation. In this process, the secreted Wnt antagonist Dkk1 functions in the Spemann organiser and its anterior derivatives by blocking Wnt receptors of the lipoprotein receptor-related protein (LRP) 5 and 6 class. In addition to LRP6, Dkk1 interacts with another recently identified receptor class, the transmembrane proteins Kremen1 (Krm1) and Kremen2 (Krm2) to synergistically inhibit LRP6. We have investigated the role of Krm1 and Krm2 during early Xenopusembryogenesis. Consistent with a role in zygotic Wnt inhibition, overexpressed Krm anteriorises embryos and rescues embryos posteriorised by Wnt8. Antisense morpholino oligonucleotide (Mo) knockdown of Krm1 and Krm2 leads to deficiency of anterior neural development. In this process, Krm proteins functionally interact with Dkk1: (1) in axis duplication assays krm2 synergises with dkk1 in inhibiting Wnt/LRP6 signalling; (2) krm2rescues microcephalic embryos induced by injection of inhibitory anti-Dkk1 antibodies; and (3) injection of krm1/2 antisense Mo enhances microcephaly induced by inhibitory anti-Dkk1 antibodies. The results indicate that Krm proteins function in a Wnt inhibition pathway regulating early AP patterning of the CNS.

Background The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins. Methods Expression screening was based on co-transfection of H1299 cells with pools of cDNA’s from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins. Results More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2. Conclusions Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway. Electronic supplementary material The online version of this article (doi:10.1186/s12896-015-0208-y) contains supplementary material, which is available to authorized users.

P53 is an important tumor suppressor that, upon activation, induces growth arrest and cell death. Control of p53 is thus of prime importance for proliferating cells, but also for cancer therapy, where p53 activity contributes to the eradication of tumors. Mdm2 functionally inhibits p53 and targets the tumor suppressor protein for degradation. In a genetic screen, we identified TRIM25 as a novel regulator of p53 and Mdm2. TRIM25 increased p53 and Mdm2 abundance by inhibiting their ubiquitination and degradation in 26 S proteasomes. TRIM25 co-precipitated with p53 and Mdm2 and interfered with the association of p300 and Mdm2, a critical step for p53 polyubiquitination. Despite the increase in p53 levels, p53 activity was inhibited in the presence of TRIM25. Downregulation of TRIM25 resulted in an increased acetylation of p53 and p53-dependent cell death in HCT116 cells. Upon genotoxic insults, TRIM25 dampened the p53-dependent DNA damage response. The downregulation of TRIM25 furthermore resulted in massive apoptosis during early embryogenesis of medaka, which was rescued by the concomitant downregulation of p53, demonstrating the functional relevance of the regulation of p53 by TRIM25 in an organismal context.

The expression of the melatonin receptor is positively regulated by cAMP and negatively regulated by melatonin in the ovine pars tuberalis (PT). Furthermore, when PT cells are dispersed in primary culture, both messenger RNA (mRNA) and protein levels spontaneously increase through a process that can be blocked by melatonin, but does not involve cAMP. This suggests that other second messengers may be regulated by melatonin, which, in turn, regulates melatonin receptor mRNA and protein levels. In this study using ribonuclease protection assays, ligand binding, protein kinase C (PKC), and cAMP analysis, we demonstrate that the levels of Mel 1a mRNA and protein expression in ovine PT are reduced by phorbol 12-myristate 13-acetate in a cAMP-independent process. This is indicative of an inhibitory role for PKC in receptor regulation. Melatonin, however, does not act through PKC activation to reduce Mel 1a mRNA or protein levels. Basal PKC activity in PT cells can be inhibited by the PKC inhibitor Ro 31-8220, and this suggests that basal PKC activity may suppress Mel 1a receptor expression. Paradoxically, however, Ro 31-8220 also inhibits melatonin receptor mRNA and protein levels in PT cells by a cAMP-independent mechanism. This suggests that other undefined pathways must play an important role in the physiological self-regulation of Mel 1a receptor expression by melatonin.

The Mel 1a receptor gene has recently been cloned and sequenced, and its product has been found to represent a new class of the G protein-coupled receptor superfamily. A unique aspect of its endogenous hormonal ligand, melatonin, is the prolonged duration (16 h) required to initiate appropriate photoperiodic responses in the animal. This study provides the first data on the transcriptional and translational regulation of this receptor in primary cultures of its native tissue, the pars tuberalis of the ovine pituitary. Using RNAse protection assays we show Mel 1a mRNA expression is rapidly induced through changes in intracellular cAMP levels. Forskolin enhances Mel 1a mRNA expression, whereas melatonin reverses this effect. RNA stability is also a factor, as Mel 1a mRNA expression decreases rapidly in the presence of actinomycin D. Although the expression of receptor protein is sensitive to forskolin stimulation, the lag between induction of mRNA and protein suggests that other factors (e.g. RNA splicing) contribute to functional receptor expression. Furthermore we observed that both mRNA and receptor protein levels increase spontaneously during primary culture, and these rises could be blocked by melatonin. A spontaneous rise in Mel 1a mRNA can also occur in serum-depleted cultures. In the absence of corresponding changes in cAMP, these results suggest not only that the Mel 1a receptor can autoregulate its own expression by an undefined pathway, but also they provide the first evidence that melatonin can act via a cAMP-independent signal transduction pathway to repress transcription in this tissue.

The delivery of nucleic acids such as plasmid DNA and siRNA into cells is a cornerstone of biological research and is of fundamental importance for medical therapeutics. Although most gene delivery therapeutics in clinical trials are based on viral vectors, safety issues remain a major concern. Non-viral vectors, such as cationic lipids and polymers, offer safer alternatives but their gene delivery efficiencies are usually not high enough for clinical applications. Thus, there is a high demand for more efficient and safe non-viral vectors. Here, we present a facile two-step method based on thiol-yne click chemistry for parallel synthesis of libraries of new biomimetic cationic thioether lipids. A library of novel lipids was synthesized using the developed method and more than 10% of the lipids showed highly efficient transfection in different cell types, surpassing the efficiency of several popular commercial transfection reagents. One of the new lipids showed highly efficient siRNA delivery to multiple cell types and could successfully deliver DNA plasmid to difficult-to-transfect mouse embryonic stem cells (mESC). Analysis of structure–activity relationship revealed that the length of the hydrophobic alkyl groups was a key parameter for efficient cell transfection and was more important for transfection efficiency than the nature of cationic head groups. The correlation of the size and surface charge of liposomes with transfection efficiency is described.

SummaryLow-density lipoprotein receptor related proteins 5 and 6 (LRP5/6) are transmembrane receptors that initiate Wnt/β-catenin signaling. Phosphorylation of PPPSP motifs in the LRP6 cytoplasmic domain is crucial for signal transduction. Using a kinome-wide RNAi screen, we show that PPPSP phosphorylation requires the Drosophila Cyclin-dependent kinase (CDK) L63. L63 and its vertebrate homolog PFTK are regulated by the membrane tethered G2/M Cyclin, Cyclin Y, which mediates binding to and phosphorylation of LRP6. As a consequence, LRP6 phosphorylation and Wnt/β-catenin signaling are under cell cycle control and peak at G2/M phase; knockdown of the mitotic regulator CDC25/string, which results in G2/M arrest, enhances Wnt signaling in a Cyclin Y-dependent manner. In Xenopus embryos, Cyclin Y is required in vivo for LRP6 phosphorylation, maternal Wnt signaling, and Wnt-dependent anteroposterior embryonic patterning. G2/M priming of LRP6 by a Cyclin/CDK complex introduces an unexpected new layer of regulation of Wnt signaling.

Bordetella pertussis toxin (islet activating protein, IAP) has been used to investigate the G-proteins involved in mediating the action of the melatonin receptor. Melatonin inhibits iorskolin-stimulated cyclic AMP production in ovine pars tuberalis (PT) cells. In cells treated with IAP for 16 h this response is attenuated in a dose-dependent manner, but not abolished. IAP catalyses the incorporation of [(32) P-ADP]ribose into a 41 kd protein present in PT membranes, but this labelling can be reduced if PT cells are preincubated with IAP for 16 h. Treatment of crude membrane preparations with IAP (20 /ig/ml) suppresses the binding of 2-[(125) l]iodomelatonin by 20%, whereas 1 mM GTP alone reduces binding by 40%, and in combination with IAP its effect is additive (60% inhibition). Therefore, these results indicate that the melatonin receptor acts via two G-proteins, one pertussis toxin-sensitive and the other pertussis toxin-insensitive.

The pineal indoleamine, melatonin, acts on specific secretory cells of the pars tuberalis of the sheep pituitary. Using pars tuberalis cells in primary culture melatonin inhibited forskolin-stimulated cyclic AMP production in both a time - and dose-dependent manner, but the nature of the melatonin response was critically dependent upon the stimulatory concentration of forskolin used. Forskolin alone stimulated dose-dependent cyclic AMP accumulation, which reached an equilibrium state after 15 min. This was maintained for up to 3 h, indicating a lack of desensitization to forskolin. Melatonin (1 muM) inhibited this response by greater than 80% at all doses. However, 100 muM forskolin reduced both the affinity and the number of the melatonin receptors, relative to untreated controls. Consistent with this, melatonin was 100 times less potent at inhibiting forskolin-stimulated cyclic AMP production, when titrated against 100 muM forskolin as compared to 1 muM forskolin. The response to 1 muM forskolin could be potentiated by 10 muM phorbol 12,13 myristate acetate, but not by calcium ionophore (A23187). This provides evidence for the interaction of the phosphatidylinositol pathway with the cyclic AMP system in these cells. Nevertheless melatonin can inhibit both the potentiated and non-potentiated response with equal effect.

The functional significance of the pars tuberalis (PT) of the mammalian adenohypophysis has remained an enigma (1, 2). One view of its function is that it acts as an auxiliary gland to support the endocrine role of the pars distalis (PD) (2), as it has been shown to contain immunocytochemically identifiable thyrotrophs and gonadotrophs (1). Many of the cells of the PT are, however, ultrastructurally unique suggesting an independent function for this tissue. Our recent demonstration that the PT of the rat is a major binding site for the ligand iodomelatonin lends further support to this idea (3). We have utilized the highly specific ligand ['251]melatonin, and have demonstrated that it binds exclusively, with very high affinity, to the PT but not the PD of the adult sheep adenohypophysis. These findings support the conclusion that the PT has a distinct role in relation to melatonin action and seasonal reproduction. The tissue specificity of ['2sII]melatonin binding to the hypothalamic-pituitary complex of the sheep brain has been revealed by in vitro autoradiography. Fig. IA and IB shows the images produced on X-ray film by sagittal sections (20 pm thick) through the sheep brain and pituitary, after incubation with either 50 pM ['251]melatonin alone (Fig. 1~) or in the presence of 1 pM melatonin (Fig. 16). The tissues underlying these images are shown in a section after staining with toluidine blue (Fig. Ic). These results reveal [I 2sI]melatonin labelling is discretely localized on the PT of the adenohypophysis, and that the specific labelling can be completely displaced by excess cold melatonin. No labelling was found in either the PD or the median eminence of the hypothalamus. This result confirms our conclusions reached for the rat (3), and provides further support for the view, that reports which ascribe ['ZSI]melatonin labelling to the median eminence (4, 5) and the anterior lobe of the pituitary (5) may have mistaken these sites for the PT. The binding characteristics of the PT for ['2sI]melatonin were examined using homogenates of ovine PT obtained from sheep slaughtered at an abattoir during their anoestrus period. In excess of 500 PT were used in these experiments. The binding of ['2sI]melatonin was found to be dependent upon membrane concentration, time and temperature of incubation (Fig. 2~, 26). The specific binding of [1251]melatonin was shown to be linearly related to membrane concentration (Fig. 2~). The time-course of binding was found to differ considerably from those reported for other species (4, 6, 7). At 37 "C binding increases rapidly over the first 2 h before reaching equilibrium between 2 to 4 h, remaining stable for up to 6 h. Binding is also reversible, as seen by the dissociation of [' 2sI]melatonin from PT membrane in the presence of excess cold melatonin (1 pM) (Fig. 26). The dissociation rate constant (k- 1) is estimated to be 0.015 min- ', and the association

To exert its activity, anthrax toxin must be endocytosed and its enzymatic toxic subunits delivered to the cytoplasm. It has been proposed that, in addition to the anthrax toxin receptors (ATRs), lipoprotein-receptor-related protein 6 (LRP6), known for its role in Wnt signalling, is also required for toxin endocytosis. These findings have however been challenged. We show that LRP6 can indeed form a complex with ATRs, and that this interaction plays a role both in Wnt signalling and in anthrax toxin endocytosis. We found that ATRs control the levels of LRP6 in cells, and thus the Wnt signalling capacity. RNAi against ATRs indeed led to a drastic decrease in LRP6 levels and a subsequent drop in Wnt signalling. Conversely, LRP6 plays a role in anthrax toxin endocytosis, but is not essential. We indeed found that toxin binding triggered tyrosine phosphorylation of LRP6, induced its redistribution into detergent-resistant domains, and its subsequent endocytosis. RNAis against LRP6 strongly delayed toxin endocytosis. As the physiological role of ATRs is probably to interact with the extracellular matrix, our findings raise the interesting possibility that, through the ATR-LRP6 interaction, adhesion to the extracellular matrix could locally control Wnt signalling.

Signalling by Wnt proteins (Wingless in Drosophila) has diverse roles during embryonic development and in adults, and is implicated in human diseases, including cancer. LDL-receptor-related proteins 5 and 6 (LRP5 and LRP6; Arrow in Drosophila) are key receptors required for transmission of Wnt/beta-catenin signalling in metazoa. Although the role of these receptors in Wnt signalling is well established, their coupling with the cytoplasmic signalling apparatus remains poorly defined. Using a protein modification screen for regulators of LRP6, we describe the identification of Xenopus Casein kinase 1 gamma (CK1gamma), a membrane-bound member of the CK1 family. Gain-of-function and loss-of-function experiments show that CK1gamma is both necessary and sufficient to transduce LRP6 signalling in vertebrates and Drosophila cells. In Xenopus embryos, CK1gamma is required during anterio-posterior patterning to promote posteriorizing Wnt/beta-catenin signalling. CK1gamma is associated with LRP6, which has multiple, modular CK1 phosphorylation sites. Wnt treatment induces the rapid CK1gamma-mediated phosphorylation of these sites within LRP6, which, in turn, promotes the recruitment of the scaffold protein Axin. Our results reveal an evolutionarily conserved mechanism that couples Wnt receptor activation to the cytoplasmic signal transduction apparatus.

The Wnt family of secreted glycoproteins mediate cell cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the beta-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular beta-catenin. Wnt/beta-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. Here we show that the transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/beta-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates.

After activation by Wnt/β-Catenin ligands, a multi-protein complex assembles at the plasma membrane as membrane-bound receptors and intracellular signal transducers are clustered into the so-called Lrp6-signalosome [Corrected]. However, the mechanism of signalosome formation and dissolution is yet not clear. Our imaging studies of live zebrafish embryos show that the signalosome is a highly dynamic structure. It is continuously assembled by Dvl2-mediated recruitment of the transducer complex to the activated receptors and partially disassembled by endocytosis. We find that, after internalization, the ligand-receptor complex and the transducer complex take separate routes. The Wnt-Fz-Lrp6 complex follows a Rab-positive endocytic path. However, when still bound to the transducer complex, Dvl2 forms intracellular aggregates. We show that this endocytic process is not only essential for ligand-receptor internalization but also for signaling. The μ2-subunit of the endocytic Clathrin adaptor Ap2 interacts with Dvl2 to maintain its stability during endocytosis. Blockage of Ap2μ2 function leads to Dvl2 degradation, inhibiton of signalosome formation at the plasma membrane and, consequently, reduction of signaling. We conclude that Ap2μ2-mediated endocytosis is important to maintain Wnt/β-catenin signaling in vertebrates.

A high-affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [(125)|]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of radioligand was found in regions of the epithalamus and hypothalamus in the brain and the pars tuberalis of the pituitary. Excitatory amino-acid lesions destroyed [(125)|]iodomelatonin binding within the brain, demonstrating that binding sites are located on neurons. Analysis of [(125)|]iodomelatonin binding to membrane homogenates of the pars tuberalis revealed a linear relationship between specific ligand binding and the amount of tissue. The time-course of specific binding at 37 degrees C reached equilibrium after 30 min and remained stable thereafter. The addition of increasing concentrations of [(125)|]iodomelatonin alone and in the presence of 1 muM melatonin showed that specific binding reached equilibrium at 80 to 100 pM. Analysis of the saturation isotherm using a one-site binding model was consistent with a single receptor site with a K(d) of 29.3 (+/-5.9 SEM) pM and B(max) of 2.54 (+/-0.19 SEM) fmol/mg protein.

The kinetics of radiochromate-labeled autologous blood lymphocytes were studied in ten cases of chronic lymphocytic leukemia (CLL). The labeled cells equilibrated with an enlarged recirculating lymphocyte pool (RLP) distributed between the blood, the spleen, and the bone marrow. The size of the RLP, estimated from dilution of the labeled cells, was better correlated with the degree of splenomegaly and marrow infiltration than was the blood lymphocyte count. The marrow fraction of the RLP turned over more slowly than the splenic pool. The half-life of the labeled cells in the RLP varied in individual cases from one to 11 days. Lymphocyte life span was not related either to the blood lymphocyte count or to the size of the RLP. The mean half-life of 3.8 days was significantly shorter than the normal lymphocyte half-life of 18 days. We conclude that CLL is characterized by overproduction of a strain of abnormally short-lived lymphocytes.

The kinetics of radiochromate-labeled autologous blood lymphocytes were studied in normal subjects. The labeled cells equilibrated within hours with a recirculating lymphocyte pool (RLP) distributed between the blood, the spleen, and the bone marrow. The RLP was found to be about twice the size of the blood lymphocyte pool contained within it and turned over through the blood about 12 times daily. The labeled cells had an average half life in the RLP of 18 days.

THERE are problems associated with the diagnosis of gonorrhea in the asymptomatic female. For many years, the sulfonamides have had little effect in inhibiting the gonococcus, and surveillance studies indicate ever-increasing resistance to penicillin, streptomycin, and oxytetracycline (1, 2). For this reason, it is expedient to obtain accurate laboratory diagnosis rapidly in primary cases and to indicate later whether treatment with antibiotics has been successful. The conventional smear stained by Gram's method is of value in early infections when typical intracellular diplococci may be present in material taken from genital orifices. These organisms are more likely to be found in the male than in the female by this direct examination. As the infection advances or in chronic cases where few organisms are present, the value of the smear decreases (3). Because the genitourinary tract harbors staphylococci, streptococci, various rod types, and JIrim,eae or other species of Neisseria which resemble the gonococcus, information obtained from smears may not be reliable. When the examination is not performed in a venereal disease or a reference laboratory, uncertainty may be compounded because many diagnostic laboratories receive relatively few requests to identify Neisseria gonorrhoeae as compared with tlhe number of such specimens submitted to specialized laboratories. In an effort to broaden tlhe examiination, many workers also culture various exudates on chocola.te agar which well supports the growth of the gonococcus. But due to overgrowtlh, the presence of numerous bacteria that show positive oxidase reactions, and variations in colonial morphology among gonococcal varieties, correct identification of organisms may be difficult (4, 5). In recent years immunofluorescent techniques for identifying gono,cocci have bee.n used successfully (6). Thayer and Martin (7) have developed a selective medium (vanicomycin, colistimethate, nystatin medium) for the isolation and cultivation of N. gonorrhoeae. This medium, hereafter referred to a.s VCN, was prepared in the Ohio Department of Health's laboratory and was used in this study. The purpose of this paper is to report our experience using VCN as a transport medium and to demonstrate the higher percentage of positive results obtained by combining immunofluorescence with culture procedures. This study began in March 1966 and ended in January 1968. The proce.dure described in the study is now being followed routinely at the Ohio Department of Health laboratories.