scholarly journals Single molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation

2019 ◽  
Author(s):  
Wenzhe Ma ◽  
Maorong Chen ◽  
Hong Kang ◽  
Zachary Steinhart ◽  
Stephane Angers ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Single Molecule ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
Membrane Receptors ◽  
Pathway Activation ◽  
Mean Size ◽  
Membrane Bound

AbstractDvl (Dishevelled) is one of several essential non-enzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits other components of the destruction complex leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for this process is lacking. In this study, we tagged mEGFP to C-terminus of dishevlled2 gene using CRISPR/Cas9 induced homologous recombination and observed its dynamics directly at the single molecule level with Total Internal Reflection Fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and the dynamic features of membrane-associated Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane bound Dvl2 is predominantly monomer in the absent of Wnt (mean size 1.10). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.08/μm2 to 0.34/μm2. Wnt3a also leads to increased oligomerization which raises the weighted averaged mean size of Dvl2 complexes to 1.4; with 65% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of Dvl2 at the membrane caused by increased affinity of Dvl2 for Fzd, the Dvl2 and Fzd binding is independent of LRP5/6. The oligomerized Dvl2 complexes have greatly increased dwell time, 2~3 minutes compared to less than 1 second for monomeric Dvl2. These properties make Dvl a unique scaffold dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.Significance StatementCanonical Wnt signaling is one of the most widely distributed pathways in metazoan development. Despite intense genetic and biochemical study for over 35 years, the major features of signaling across the plasma membrane are still poorly understood. Dishevelled serves as an essential bridge between the membrane receptors and downstream signaling components. Attempts to reconstruct the pathway and analyze its biochemical features in vitro have been hampered by Dishevelled’s tendency to aggregate in vitro and to form large aggregates of dubious significance in vivo. To obtain a molecular understanding of the role of Dvl in Wnt signaling, while circumventing these aggregation problems we have expressed a fluorescent tagged Dishevelled in cells at their physiological concentration and quantified the size distribution of Dishevelled before and after Wnt treatment. We found that limited oligomerization in response to the Wnt ligand is very dynamic and provides a key step of signal transduction.

scholarly journals Single-molecule dynamics of Dishevelled at the plasma membrane and Wnt pathway activation

2020 ◽  
Vol 117 (28) ◽  
pp. 16690-16701 ◽  
Author(s):  
Wenzhe Ma ◽  
Maorong Chen ◽  
Hong Kang ◽  
Zachary Steinhart ◽  
Stephane Angers ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Single Molecule ◽  
Wnt Pathway ◽  
Total Concentration ◽  
Wnt Signaling Pathway ◽  
Dynamic Features ◽  
Single Molecule Level ◽  
Pathway Activation ◽  
Mean Size ◽  
Membrane Bound

Dvl (Dishevelled) is one of several essential nonenzymatic components of the Wnt signaling pathway. In most current models, Dvl forms complexes with Wnt ligand receptors, Fzd and LRP5/6 at the plasma membrane, which then recruits the destruction complex, eventually leading to inactivation of β-catenin degradation. Although this model is widespread, direct evidence for the individual steps is lacking. In this study, we tagged mEGFP to C terminus ofdishevelled2gene using CRISPR/Cas9-induced homologous recombination and observed its dynamics directly at the single-molecule level with total internal reflection fluorescence (TIRF) microscopy. We focused on two questions: 1) What is the native size and what are the dynamic features of membrane-bound Dvl complexes during Wnt pathway activation? 2) What controls the behavior of these complexes? We found that membrane-bound Dvl2 is predominantly monomer in the absence of Wnt (observed mean size 1.1). Wnt3a stimulation leads to an increase in the total concentration of membrane-bound Dvl2 from 0.12/μm2to 0.54/μm2. Wnt3a also leads to increased oligomerization which raises the weighted mean size of Dvl2 complexes to 1.5, with 56.1% of Dvl still as monomers. The driving force for Dvl2 oligomerization is the increased concentration of membrane Dvl2 caused by increased affinity of Dvl2 for Fzd, which is independent of LRP5/6. The oligomerized Dvl2 complexes have increased dwell time, 2 ∼ 3 min, compared to less than 1 s for monomeric Dvl2. These properties make Dvl a unique scaffold, dynamically changing its state of assembly and stability at the membrane in response to Wnt ligands.

scholarly journals Replicating Adenoviruses That Target Tumors with Constitutive Activation of the wnt Signaling Pathway

2001 ◽  
Vol 75 (6) ◽  
pp. 2857-2865 ◽  
Author(s):  
Michele Brunori ◽  
Maddalena Malerba ◽  
Haruhiko Kashiwazaki ◽  
Richard Iggo
Keyword(s):  
Wnt Signaling ◽  
Cancer Cells ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
Lung Model ◽  
Colorectal Tumors ◽  
Colon Tumors ◽  
Treat All

ABSTRACT Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects. Selectively replicating viruses have a major advantage over nonreplicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumor. To permit rational targeting of colon tumors, we have developed replicating adenoviruses that express the viral E1B and E2 genes from promoters controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli and β-catenin genes in virtually all colon tumors and is constitutively repressed by Groucho and CtBP in normal tissue. The Tcf-E2 and Tcf-E1B promoters are active in many, but not all, cell lines with activation of the wnt pathway. Viruses with Tcf regulation of E2 expression replicate normally in SW480 colon cancer cells but show a 50- to 100-fold decrease in replication in H1299 lung cancer cells and WI38 normal fibroblasts. Activation of wnt signaling by transduction of a stable β-catenin mutant into normal fibroblasts renders the cells permissive for virus replication. Insertion of Tcf4 sites in the E1B promoter has only small effects on replication in vitro but significantly reduces the inflammatory response in a rodent lung model in vivo. Replicating adenoviruses with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumors, but additional changes will be required to produce a virus that can be used to treat all colon tumors.

scholarly journals Misregulation of Wnt Signaling Pathways at the Plasma Membrane in Brain and Metabolic Diseases

Membranes ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 844
Author(s):  
Mustafa Karabicici ◽  
Yagmur Azbazdar ◽  
Evin Iscan ◽  
Gunes Ozhan
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Metabolic Diseases ◽  
Wnt Signaling Pathway ◽  
Membrane Domains ◽  
Physiological Processes ◽  
Pathway Activation ◽  
Wnt Pathways ◽  
Membrane Components

Wnt signaling pathways constitute a group of signal transduction pathways that direct many physiological processes, such as development, growth, and differentiation. Dysregulation of these pathways is thus associated with many pathological processes, including neurodegenerative diseases, metabolic disorders, and cancer. At the same time, alterations are observed in plasma membrane compositions, lipid organizations, and ordered membrane domains in brain and metabolic diseases that are associated with Wnt signaling pathway activation. Here, we discuss the relationships between plasma membrane components—specifically ligands, (co) receptors, and extracellular or membrane-associated modulators—to activate Wnt pathways in several brain and metabolic diseases. Thus, the Wnt–receptor complex can be targeted based on the composition and organization of the plasma membrane, in order to develop effective targeted therapy drugs.

scholarly journals Heme Oxygenase-1 Suppresses Wnt Signaling Pathway in Nonalcoholic Steatohepatitis-Related Liver Fibrosis

2020 ◽  
Vol 2020 ◽  
pp. 1-15
Author(s):  
Jinghua Du ◽  
Weiguang Ren ◽  
Qingshan Zhang ◽  
Na Fu ◽  
Fang Han ◽  
...  
Keyword(s):  
Liver Fibrosis ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Heme Oxygenase ◽  
Nonalcoholic Steatohepatitis ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
Heme Oxygenase 1 ◽  
Inflammatory Infiltration

Background and Aim. Heme oxygenase-1 (HO-1) has been verified to play an important role in nonalcoholic steatohepatitis (NASH), but the mechanism remains unclear. In this study, we aimed to clarify whether induction of HO-1 reverses steatofibrosis via suppression of the Wnt signaling pathway and to explore the potential mechanism of HO-1 on NASH-related liver fibrosis. Methods. Mice were fed with a methionine-choline-deficient (MCD) diet for 8 weeks to induce steatohepatitis-related liver fibrosis and were treated with HO-1 inducer Hemin and inhibitor ZnPP. Mouse sera were collected for the biochemical analysis, and livers were obtained for further histological observation and gene expression analysis. HSC-T6 cells were cultured for the in vitro study and were administrated with Hemin and si-HO-1 to induce or inhibit the expression of HO-1. qPCR and Western blot were used to assess the mRNA and protein levels of genes. Results. MCD-fed mice developed marked macrovesicular steatosis, focal necrosis, and inflammatory infiltration and pericellular fibrosis in liver sections. Administration of Hemin could significantly ameliorate the severity of steatosis, inflammation, and fibrosis and also could decrease the serum ALT and AST. We demonstrated that HO-1 induction was able to downregulate the key regulator of the canonical Wnt pathway Wnt1 and the noncanonical Wnt pathway Wnt5a. The downstream factors of the Wnt pathway β-catenin and NFAT5 were inhibited by Hemin, but GSK-3β was upregulated compared to the MCD group, which were consistent with the in vitro study. Hemin markedly inhibited the TGF-β1/Smad signaling pathway in both in vivo and in vitro studies. Conclusion. Our study demonstrated that HO-1 inhibited the activation of canonical and noncanonical Wnt signaling pathways in NASH-related liver fibrosis. Thus, these results may suggest a new therapeutic strategy for NASH-related liver fibrosis.

scholarly journals Overexpression of ICAT Inhibits the Progression of Colorectal Cancer by Binding with β-Catenin in the Cytoplasm

2021 ◽  
Vol 20 ◽  
pp. 153303382110412
Author(s):  
Jiancong Hu ◽  
Zihan Wang ◽  
Junxiong Chen ◽  
Zhaoliang Yu ◽  
Jingdan Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Proliferation ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Transcriptional Activity ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
T Cell Factor

Inhibitor of β-catenin and T-cell factor (ICAT) was first found as a polypeptide that blocks β-catenin–TCF interaction. Abundant evidence has shown that ICAT has different functions in diverse cancers’ progression. Nevertheless, the roles it plays in colorectal cancer (CRC) have not been described. Here, we documented that ICAT expression was higher in CRC tissue than in the adjacent normal tissue and that prognosis was better in high-ICAT expression patients. The overexpression of ICAT inhibited CRC cell proliferation both in vitro and in vivo. Wnt pathway transcriptional activity was suppressed in the CRC cells with ICAT overexpression, where the CCND1 and MYC expression, which occurs downstream of the Wnt signaling pathway, was inhibited. Co-immunoprecipitation experiments showed that ICAT bound with β-catenin in stable overexpression cell lines; immunofluorescence showed the co-localization of ICAT and β-catenin in the cytoplasm. Overall, our study reveals that ICAT inhibits CRC cell proliferation by binding to cytoplasm-located β-catenin, and prevents its translocation, which results in Wnt signaling pathway inactivation. It may provide a scientific foundation for focusing on ICAT in treatments for CRC.

Epigenetic regulation of the Wnt-signaling pathway in CIMP-H BRAFV600E mCRC.

2021 ◽  
Vol 39 (3_suppl) ◽  
pp. 110-110
Author(s):  
Hey Min Lee ◽  
Stefania Napolitano ◽  
Van K. Morris ◽  
Kunal Rai ◽  
John Paul Y.C. Shen ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Braf Mutation ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
Methylation Profile ◽  
Brafv600e Mutation ◽  
Wild Type ◽  
Mutation Status ◽  
Pathway Activation ◽  
Apc Mutation

110 Background: BRAFV600E mutation identifies mCRC patients with poor prognosis with only little benefit from standard therapy. Analysis from TCGA revealed that 89% of BRAFV600E CRC tumors were associated with a high CpG island methylator phenotype (CIMP-H), which may result in epigenetic silencing of tumor suppressor genes, while only 29% of BRAF wild-type tumors are CIMP-H. In this study, we define key pathways regulated by global DNA hypermethylation in the context of BRAFV600E mutation. Methods: We analyzed the TCGA Illumina 450k array methylation datasets and RNA-sequencing datasets for 97 CIMP-H CRC tumors (27 BRAFV600E; 69 BRAF WT) identified by five universal CIMP annotations ( p14, p16, MLH1, MINT1, MINT2, MINT31). We defined differential methylation profile according to BRAF mutation status and calculated Spearman correlation between methylation and gene expression to identify CIMP-H BRAF-associated genes. Next, pathways enriched with CIMP-H BRAFV600E tumors were defined using PANTHER pathway analysis. Additionally, β-catenin IHC were conducted on 145 MD Anderson CRC patient samples and compared by CIMP and BRAF status. Results: BRAF mutation is associated with lower rates of APC mutation as has previously been shown (32%, 82%). We identified 6,097 differentially methylated probes by BRAF mutation status (FDR = 10-4), and as expected, our data suggests a higher methylation profile in BRAFV600E mutated tumors compared to BRAF WT. Intriguingly, CIMP-H BRAF-associated genes showed enrichment in the Wnt-signaling and cadherin signaling pathways ( p< 0.0001 (FDR < 0.0001)). Despite the epigenetic Wnt-signaling, nuclear β-catenin expression (as a measure of Wnt activity) in CIMP-H and BRAF tumors remains lower than for non-CIMP, and BRAF wild-type ( p= 0.0003 for comparison of CIMP). Conclusions: Genes under methylation regulation in the BRAF-mutant context showed enrichment in Wnt-signaling pathway. Since BRAFV600E CRC tumors have a low association with APC mutation, this data suggests role of epigenetic regulation of the Wnt-pathway activation. However, as measured by nuclear β-catenin, Wnt activation in these tumors is not as high as traditional APC-mutated CRC tumors. CIMP-H tumors with BRAFV600E mutation is a unique subset of CRC tumor that have Wnt-pathway activation regulated by epigenetic modifications more than a β-catenin activation.

scholarly journals A Review of the Role of Wnt in Cancer Immunomodulation

Cancers ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 771 ◽  
Author(s):  
Whitney N. Goldsberry ◽  
Angelina Londoño ◽  
Troy D. Randall ◽  
Lyse A. Norian ◽  
Rebecca C. Arend
Keyword(s):  
Tumor Microenvironment ◽  
Wnt Signaling ◽  
Immune Suppression ◽  
Wnt Pathway ◽  
Wnt Signaling Pathway ◽  
Cancer Therapeutics ◽  
Multiple Cancer ◽  
Pathway Activation ◽  
Tumor Types

Alterations in the Wnt signaling pathway are associated with the advancement of cancers; however, the exact mechanisms responsible remain largely unknown. It has recently been established that heightened intratumoral Wnt signaling correlates with tumor immunomodulation and immune suppression, which likely contribute to the decreased efficacy of multiple cancer therapeutics. Here, we review available literature pertaining to connections between Wnt pathway activation in the tumor microenvironment and local immunomodulation. We focus specifically on preclinical and clinical data supporting the hypothesis that strategies targeting Wnt signaling could act as adjuncts for cancer therapy, either in combination with chemotherapy or immunotherapy, in a variety of tumor types.

scholarly journals Regulation of Wnt Signaling by FOX Transcription Factors in Cancer

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3446
Author(s):  
Stefan Koch
Keyword(s):  
Transcription Factors ◽  
Wnt Signaling ◽  
Wnt Pathway ◽  
Treatment Options ◽  
Wnt Signaling Pathway ◽  
Tissue Homeostasis ◽  
Fine Tuning ◽  
Dependent Manner ◽  
Forkhead Box ◽  
Signaling Activity

Aberrant activation of the oncogenic Wnt signaling pathway is a hallmark of numerous types of cancer. However, in many cases, it is unclear how a chronically high Wnt signaling tone is maintained in the absence of activating pathway mutations. Forkhead box (FOX) family transcription factors are key regulators of embryonic development and tissue homeostasis, and there is mounting evidence that they act in part by fine-tuning the Wnt signaling output in a tissue-specific and context-dependent manner. Here, I review the diverse ways in which FOX transcription factors interact with the Wnt pathway, and how the ectopic reactivation of FOX proteins may affect Wnt signaling activity in various types of cancer. Many FOX transcription factors are partially functionally redundant and exhibit a highly restricted expression pattern, especially in adults. Thus, precision targeting of individual FOX proteins may lead to safe treatment options for Wnt-dependent cancers.

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

scholarly journals Dickkopf Wnt signaling pathway inhibitor 1 regulates the differentiation of mouse embryonic stem cells in vitro and in vivo

2015 ◽  
Vol 13 (1) ◽  
pp. 720-730 ◽  
Author(s):  
LIPING OU ◽  
LIAOQIONG FANG ◽  
HEJING TANG ◽  
HAI QIAO ◽  
XIAOMEI ZHANG ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Wnt Signaling ◽  
Signaling Pathway ◽  
Wnt Signaling Pathway ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells
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