Analysis of Wnt signaling β-catenin spatial dynamics in HEK293T cells

Background Wnt/β-catenin signaling is involved in different stages of mammalian development and implicated in various cancers (e.g. colorectal cancer). Recent experimental and computational studies have revealed characteristics of the pathway, however a cell-specific spatial perspective is lacking. In this study, a novel 3D confocal quantitation protocol is developed to acquire spatial (two cellular compartments: nucleus and cytosol-membrane) and temporal quantitative data on target protein (e.g. β-catenin) concentrations in Human Epithelial Kidney cells (HEK293T) during perturbation (with either cycloheximide or Wnt3A). Computational models of the Wnt pathway are constructed and interrogated based on this data. Results A single compartment Wnt pathway model is compared with a simple β-catenin two compartment model to investigate Wnt3A signaling in HEK293T cells. When protein synthesis is inhibited, β-catenin decreases at the same rate in both cellular compartments, suggesting diffusional transport is fast compared to β-catenin degradation in the cytosol. With Wnt3A stimulation, the total amount of β-catenin rises throughout the cell, however the increase is initially (~first hour) faster in the nuclear compartment. While both models were able to reproduce the whole cell changes in β-catenin, only the compartment model reproduced the Wnt3A induced changes in β-catenin distribution and it was also the best fit for the data obtained when active transport was included alongside passive diffusion transport. Conclusions This integrated 3D quantitation imaging protocol and computational modeling approach allowed cell-specific compartment models of the signaling pathways to be constructed and analyzed. The Wnt models constructed in this study are the first for HEK293T and have suggested potential roles of inter-compartment transport to the dynamics of signaling

Wnt/β-catenin signaling stimulates the expression and synaptic clustering of the autism-associated Neuroligin 3 gene

Indexación: Scopus.Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/β-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/β-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous β-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co-localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/β-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.https://www.nature.com/articles/s41398-018-0093-y.pd

Elucidating Mechanisms of Canonical Wnt - ephrin-B Crosstalk

Throughout development, canonical Wnt signaling contributes to the formation and maintenance of a wide array of cells, tissues, and organs. Dys-regulated Wnt signaling during embryonic development is implicated in developmental defects known as neurochristopathies, including craniofacial and heart defects, as well as defects in neural development. Due to its roles in stem cell maintenance and self-renewal, tissue homeostasis, and regeneration, aberrant Wnt signaling in adult tissues can result in various forms of cancer, including colorectal cancer, breast cancer, lung cancer, and gastro-intestinal cancer, among others. Dys-regulated Wnt signaling is also implicated in other pathologies including bone disease, and metabolic diseases, such as Type II diabetes. Our lab has previously identified a novel crosstalk between canonical Wnt signaling and ephrin signaling. Ephrin signaling occurs through the interaction of ephrin ligands and Eph receptor tyrosine kinases, and is bidirectional. Due to the roles of ephrin signaling in tissue development and maintenance, aberrant ephrin signaling is implicated in many diseases including bone remodeling diseases, diabetes, and cancer. The molecular mechanism of the crosstalk between canonical Wnt signaling and ephrin-B signaling remains unknown. beta-catenin is a key intracellular effector of canonical Wnt signaling that transduces the signal to the nucleus, where beta-catenin interacts with the TCF/LEF transcription factors and activates transcription of target genes. Due to its central role in transducing the canonical Wnt signal to the nucleus, we predict that ephrin-B signaling antagonizes canonical Wnt signaling by affecting the stability and/or sub-cellular localization of beta-catenin, or the interaction between beta-catenin and TCF/LEF transcription factors. By employing mouse ephrin-B constructs in human cell lines, we show that the canonical Wnt - ephrin-B crosstalk is conserved between frogs and mammals. We also found that ephrin-B antagonism of canonical Wnt signaling is likely independent of ubiquitin proteasome system (UPS)-mediated degradation of beta-catenin. Furthermore, confocal immunofluorescence microscopy revealed that overexpression of ephrin-B in HEK293T cells treated with lithium chloride (LiCl) seems to promote membrane localization of beta-catenin, particularly at the apical Z sections. These results suggests that re-localization of beta-catenin to the cell membrane may contribute to the ephrin-B antagonism of canonical Wnt signaling

Tyrosine-Based Signals Regulate the Assembly of Daple⋅PARD3 Complex at Cell-Cell Junctions.

Polarized distribution of organelles and molecules inside a cell is vital for a range of cellular processes and its loss is frequently encountered in disease. Polarization during planar cell migration is a special condition in which cellular orientation is triggered by cell-cell contact. We demonstrate that the protein Daple (CCDC88C) is a component of cell junctions in epithelial cells which serves like a cellular "compass" for establishing and maintaining contact-triggered planar polarity. Furthermore, these processes may be mediated through interaction with the polarity regulator PARD3. This interaction, mediated by Daple's PDZ-binding motif (PBM) and the third PDZ domain of PARD3, is fine-tuned by tyrosine phosphorylation on Daple's PBM by receptor and non-receptor tyrosine kinases, such as Src. Hypophosphorylation strengthens the interaction, whereas hyperphosphorylation disrupts it, thereby revealing an unexpected role of Daple as a platform for signal integration and gradient sensing for tyrosine-based signals within the planar cell polarity pathway

Wnt/PCP controls spreading of Wnt/β-catenin signals by cytonemes in vertebrates

This is the author accepted manuscript.The final version is available from eLife Sciences Publications via the DOI in this record.Signaling filopodia, termed cytonemes, are dynamic actin-based membrane structures that regulate the exchange of signaling molecules and their receptors within tissues. However, how cytoneme formation is regulated remains unclear. Here, we show that Wnt/PCP autocrine signaling controls the emergence of cytonemes, and that cytonemes subsequently control paracrine Wnt/β-catenin signal activation. Upon binding of the Wnt family member Wnt8a, the receptor tyrosine kinase Ror2 gets activated. Ror2/PCP signaling leads to induction of cytonemes, which mediate transport of Wnt8a to neighboring cells. In the Wnt receiving cells, Wnt8a on cytonemes triggers Wnt/β-catenin-dependent gene transcription and proliferation. We show that cytoneme-based Wnt transport operates in diverse processes, including zebrafish development, the murine intestinal crypt, and human cancer organoids, demonstrating that Wnt transport by cytonemes and its control via the Ror2 pathway is highly conserved in vertebrates.This project was funded by the Living Systems Institute, the University of Exeter and the Boehringer Ingelheim Foundation to SS. Studies in the DMV lab are supported by the National Research Foundation of Singapore and National Medical Research Council under its STAR Award Program. JR and AS were supported by the Impuls- und Vernetzungsfond of the Helmholtz Association. GUN was funded by the Deutsche Forschungsgemeinschaft (SFB 1324, projects A6 and Z2, GRK2039) and Helmholtz Association Program STN

Regulation of Wnt/β-Catenin Signaling in Cardiac Valve Development and Disease

Non-syndromic Mitral Valve Prolapse (MVP) is a common disease with associated morbidities and mortality. Affecting 2-3% of the global population, MVP has become a significant health burden in developed countries. We recently identified mutations in the cilia gene, DZIP1 in multiple families with MVP. To initially identify the function of DZIP1 in valve biology, we performed proteomics-based approaches with the goal of identifying unique binding partners for DZIP1. These studies revealed a direct interaction between DZIP1 and the β-catenin antagonist, CBY1. We hypothesized that DZIP1 suppresses the Wnt/ β-catenin pathway during mitral valve development through CBY1. Immunofluorescence staining revealed overlap between DZIP1 and CBY1 protein at the basal body of the primary cilium. Increase of activated β-catenin was observed in the Dzip1S14R/+ valves. Co-immunoprecipitation confirmed an interaction between DZIP1, CBY1 and β-catenin. Ensuing immunofluorescence staining suggested overlap between β-catenin and the basal body. DZIP1 truncation mutants identified a minimal CBY1 interaction motif within the C-terminus of DZIP1. A membrane permeant mimetic peptide against this motif was synthesized and confirmed as being able to interact with CBY1 and β-catenin. Treatment of chicken valve interstitial cells with the mimetic peptide resulted in significant decrease in activated nuclear β-catenin. To test whether this pathway was relevant in the context of the DZIP1 mutation, we assayed nuclear vs. cytoplasmic β-catenin expression in Dzip1S14R/+ MEFs. Western blot analysis showed a significant increase in nuclear β-catenin from the mutant cells. An additional family was identified with a rare DZIP1 variant within the DZIP1-CBY1 interaction motif. The mutation resulted in reduced DZIP1 and CBY1 protein stability and a peptide synthesized with the mutation resulted in an enhanced interaction between DZIP1 and β-catenin and an inhibitory effect on β-catenin signaling. Through analysis of nuclear β-catenin expression profiles during cardiac valve development, we conclude that Wnt/β-catenin signaling is temporally and spatially regulated. It is down regulated after E13.5 and undetectable in the adult. However, β-catenin signaling is significantly upregulated in human myxomatous valves and thus may be a major contributor to disease phenotype. In conclusion, DZIP1 suppresses Wnt activity to direct mitral valve development through interacting with and stabilizing CBY1. This study characterizes the β-catenin expression profile during murine cardiac valve development and reveals a molecular mechanism, by which mutations in DZIP1 alter valve development leading to increased β-catenin signaling. Altered β-catenin signaling may be an early initiating signal in the pathogenesis of MVP

Daple is a novel non-receptor GEF required for trimeric G protein activation in Wnt signaling

Peer reviewedPublisher PD

The roles of Wnt signaling during mitosis

Wnt signaling is crucial for embryonic patterning, the formation of tissues during development, and tissue maintenance in the adult organism. The canonical Wnt pathway induces the transcription of β-catenin target genes, thereby controlling cell cycle progression during G1/S phase, stem cell self-renewal, and stem cell differentiation. However, besides its transcriptional roles, it emerged that Wnt signaling also takes post-translational functions during mitosis. Accordingly, a misregulation of the pathway causes severe mitotic defects, such as chromosome misalignments and chromosome segregation errors, which can ultimately lead to aneuploidy. In this work, I aimed to identify targets of Wnt signaling, which safeguard the correct progression through mitosis, and characterize the underlying molecular mechanisms to explain the emergence of mitotic defects upon Wnt disturbance. First, I revealed that the mitotic kinesin KIF2A is recruited by the Wnt component DVL. DVL localizes KIF2A to the mitotic spindle poles, where it regulates microtubule minus-end dynamics to ensure chromosome alignment before anaphase, both in somatic cells and pluripotent stem cells. This process is supported by the phosphorylation of KIF2A at serine 100 and the interaction with PLK1, which is positively regulated by active Wnt signaling and LRP6 signalosome formation. Second, I verified an S phase-dependent mechanism of Wnt signaling, ensuring the equal segregation of chromosomes in pluripotent stem cells during anaphase. At this, I hypothesize that Wnt signaling contributes to the error-free replication of DNA in S phase, which mediates microtubule plus-end assembly in mitosis, and thereby facilitates faithful chromosome segregation. The validation of both mechanisms in pluripotent stem cells emphasizes their relevance for the understanding of developmental defects, tissue degeneration, and cancer progression, which is often characterized by chromosomal instability. Besides, KIF2A was recruited by DVL also in interphase, indicating that the Wnt-mediated regulation of KIF2A may contribute to processes beyond mitosis, namely ciliogenesis and neurogenesis. Taken together, in my work, I revealed two novel Wnt-dependent mechanisms, which function directly in mitosis or through S phase to control microtubule minus- or plus-end dynamics respectively, ensuring the faithful progression through mitosis, preservation of euploidy, and possibly further post-mitotic processes