Cerebrospinal fluid (CSF) is the liquid that fills the brain ventricles. CSF represents not only a mechanical brain protection but also a rich source of signalling factors modulating diverse ...processes during brain development and adulthood. The choroid plexus (CP) is a major source of CSF and as such it has recently emerged as an important mediator of extracellular signalling within the brain. Growing interest in the CP revealed its capacity to release a broad variety of bioactive molecules that, via CSF, regulate processes across the whole central nervous system (CNS). Moreover, CP has been also recognized as a sensor, responding to altered composition of CSF associated with changes in the patterns of CNS activity. In this review, we summarize the recent advances in our understanding of the CP as a signalling centre that mediates long-range communication in the CNS. By providing a detailed account of the CP secretory repertoire, we describe how the CP contributes to the regulation of the extracellular environment-in the context of both the embryonal as well as the adult CNS. We highlight the role of the CP as an important regulator of CNS function that acts via CSF-mediated signalling. Further studies of CP-CSF signalling hold the potential to provide key insights into the biology of the CNS, with implications for better understanding and treatment of neuropathological conditions.
Wnt signalling is known to generate cellular asymmetry via Wnt/planar cell polarity pathway (Wnt/PCP). Wnt/PCP acts locally (i) to orient membrane polarity and asymmetric establishment of ...intercellular junctions via conserved set of PCP proteins most specifically represented by Vangl and Prickle, and (ii) to asymmetrically rearrange cytoskeletal structures via downstream effectors of Dishevelled (Dvl). This process is best described on stable phenotypes of epithelial cells. Here, however, we review the activity of Wnt signalling in migratory cells which experience the extensive rearrangements of cytoskeleton and consequently dynamic asymmetry, making the localised effects of Wnt signalling easier to distinguish. Firstly, we focused on migration of neuronal axons, which allows to study how the pre-existent cellular asymmetry can influence Wnt signalling outcome. Then, we reviewed the role of Wnt signalling in models of mesenchymal migration including neural crest, melanoma, and breast cancer cells. Last, we collected evidence for local Wnt signalling in amoeboid cells, especially lymphocytes. As the outcome of this review, we identify blank spots in our current understanding of this topic, propose models that synthesise the current observations and allow formulation of testable hypotheses for the future research.
Wnt signaling cascade has developed together with multicellularity to orchestrate the development and homeostasis of complex structures. Wnt pathway components - such as β-catenin, Dishevelled (DVL), ...Lrp6, and Axin-- are often dedicated proteins that emerged in evolution together with the Wnt signaling cascade and are believed to function primarily in the Wnt cascade. It is interesting to see that in recent literature many of these proteins are connected with cellular functions that are more ancient and not limited to multicellular organisms - such as cell cycle regulation, centrosome biology, or cell division. In this review, we summarize the recent literature describing this crosstalk. Specifically, we attempt to find the answers to the following questions: Is the response to Wnt ligands regulated by the cell cycle? Is the centrosome and/or cilium required to activate the Wnt pathway? How do Wnt pathway components regulate the centrosomal cycle and cilia formation and function? We critically review the evidence that describes how these connections are regulated and how they help to integrate cell-to-cell communication with the cell and the centrosomal cycle in order to achieve a fine-tuned, physiological response.
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BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
In this review, we discuss the intricate roles of the Wnt signalling network in the development and progression of mature B‐cell‐derived haematological malignancies, with a focus on chronic ...lymphocytic leukaemia (CLL) and related B‐cell lymphomas. We review the current literature and highlight the differences between the β‐catenin‐dependent and ‐independent branches of Wnt signalling. Special attention is paid to the role of the non‐canonical Wnt/planar cell polarity (PCP) pathway, mediated by the Wnt‐5–receptor tyrosine kinase‐like orphan receptor (ROR1)–Dishevelled signalling axis in CLL. This is mainly because the Wnt/PCP co‐receptor ROR1 was found to be overexpressed in CLL and the Wnt/PCP pathway contributes to numerous aspects of CLL pathogenesis. We also discuss the possibilities of therapeutically targeting the Wnt signalling pathways as an approach to disrupt the crucial interaction between malignant cells and their micro‐environment. We also advocate the need for research in this direction for other lymphomas, namely, diffuse large B‐cell lymphoma, Hodgkin lymphoma, mantle cell lymphoma, Burkitt lymphoma and follicular lymphoma where the Wnt signalling pathway probably plays a similar role.
Linked Articles
This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc
Dishevelled (DVL) proteins are key mediators of the Wnt/β-catenin signaling pathway. All DVL proteins contain three conserved domains: DIX, PDZ, and DEP. There is a consensus in the field that the ...DIX domain is critical for Wnt/β-catenin signaling, but contradictory evidence regarding the function of the DEP domain exists. It has been difficult, until recently, to test the importance of the DEP domain rigorously because of the interference with endogenous DVL, expressed in all Wnt-responsive cell lines. In this study, we took advantage of DVL knockout (DVL1/DVL2/DVL3 triple knockout) cells fully deficient in Wnt3a-induced signaling events and performed a series of rescue experiments. Using these complementation assays, we analyzed the role of individual DVL isoforms. Further domain mapping of DVL1 showed that both the DVL1 DEP domain and especially its N-terminal region are required and sufficient for Wnt3a-induced phosphorylation of LRP6 and TopFlash reporter activation. On the contrary, multiple DEP domain mutants deficient in the planar cell polarity (PCP) pathway could fully rescue the Wnt3a response. This study provides conclusive evidence that the DVL DEP domain is essential for Wnt/β-catenin signaling in mammalian cells and establishes an experimental system suitable for further functional testing of DVL.
The Cdk12/CycK complex promotes expression of a subset of RNA polymerase II genes, including those of the DNA damage response. CDK12 is among only nine genes with recurrent somatic mutations in ...high-grade serous ovarian carcinoma. However, the influence of these mutations on the Cdk12/CycK complex and their link to cancerogenesis remain ill-defined. Here, we show that most mutations prevent formation of the Cdk12/CycK complex, rendering the kinase inactive. By examining the mutations within the Cdk12/CycK structure, we find that they likely provoke structural rearrangements detrimental to Cdk12 activation. Our mRNA expression analysis of the patient samples containing the CDK12 mutations reveals coordinated downregulation of genes critical to the homologous recombination DNA repair pathway. Moreover, we establish that the Cdk12/CycK complex occupies these genes and promotes phosphorylation of RNA polymerase II at Ser2. Accordingly, we demonstrate that the mutant Cdk12 proteins fail to stimulate the faithful DNA double strand break repair via homologous recombination. Together, we provide the molecular basis of how mutated CDK12 ceases to function in ovarian carcinoma. We propose that CDK12 is a tumor suppressor of which the loss-of-function mutations may elicit defects in multiple DNA repair pathways, leading to genomic instability underlying the genesis of the cancer.
Over the past decades an extensive effort has been made to provide a more comprehensive understanding of Wnt signaling, yet many regulatory and structural aspects remain elusive. Among these, the ...ability of Dishevelled (DVL) protein to relocalize at the plasma membrane is a crucial step in the activation of all Wnt pathways. The membrane binding of DVL was suggested to be mediated by the preferential interaction of its C-terminal DEP domain with phosphatidic acid (PA). However, due to the scarcity and fast turnover of PA, we investigated the role on the membrane association of other more abundant phospholipids. The combined results from computational simulations and experimental measurements with various model phospholipid membranes, demonstrate that the membrane binding of DEP/DVL constructs is governed by the concerted action of generic electrostatics and finely-tuned intermolecular interactions with individual lipid species. In particular, while we confirmed the strong preference for PA lipid, we also observed a weak but non-negligible affinity for phosphatidylserine, the most abundant anionic phospholipid in the plasma membrane, and phosphatidylinositol 4,5-bisphosphate. The obtained molecular insight into DEP-membrane interaction helps to elucidate the relation between changes in the local membrane composition and the spatiotemporal localization of DVL and, possibly, other DEP-containing proteins.
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•hDVL3/DEP domain binding to membranes with different lipid composition studied by MD simulations, flow cytometry, and QCM-D•hDVL3/DEP domain-membrane association is determined by the interaction between basic amino acids and negatively charged lipids•Phosphatidic acid (PA) is preferred over more abundant phosphatidylserine (PS) and phosphatidylinositol-bisphosphate (PIP2)•PA/PIP2/PS ratio in human plasma membrane may regulate the recruitment of hDVL3 and the activation of the Wnt signaling
Phosphorylation-dependent YAP translocation is a well-known intracellular mechanism of the Hippo pathway; however, the molecular effectors governing YAP cytoplasmic translocation remains undefined. ...Recent findings indicate that oncogenic YAP paradoxically suppresses Wnt activity. Here, we show that Wnt scaffolding protein Dishevelled (DVL) is responsible for cytosolic translocation of phosphorylated YAP. Mutational inactivation of the nuclear export signal embedded in DVL leads to nuclear YAP retention, with an increase in TEAD transcriptional activity. DVL is also required for YAP subcellular localization induced by E-cadherin, α-catenin, or AMPK activation. Importantly, the nuclear-cytoplasmic trafficking is dependent on the p53-Lats2 or LKB1-AMPK tumor suppressor axes, which determine YAP phosphorylation status. In vivo and clinical data support that the loss of p53 or LKB1 relieves DVL-linked reciprocal inhibition between the Wnt and nuclear YAP activity. Our observations provide mechanistic insights into controlled proliferation coupled with epithelial polarity during development and human cancer.
Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles ...(EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80-200 nm, microvesicles: ~200-1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.
Dishevelled (DVL) proteins are key mediators of most Wnt pathways. In all vertebrates, three DVL paralogs are present (DVL1, DVL2 and DVL3) but it is poorly defined to what extent they are ...functionally redundant. Here, we generated T-REx HEK 293 cells with only one DVL paralog (i.e., DVL1-only, DVL2-only, and DVL3-only) and compared their response to Wnt-3a and Wnt-5a ligands with wild type and DVL triple knockout cells. We show that DVL is essential, in addition to the previously shown Wnt-3a-induced phosphorylation of LRP6 and transcriptional activation of TCF/LEF-dependent reporter, also for Wnt-3a-induced degradation of AXIN1 and Wnt-5a-induced phosphorylation of ROR1. We have quantified the molar ratios of DVL1:DVL2:DVL3 in our model to be approximately 4:80:16. Interestingly, DVL-only cells do not compensate for the lack of other paralogs and are still fully functional in all analyzed readouts with the exception of Wnt-3a-induced transcription assessed by TopFlash assay. In this assay, the DVL1-only cell line was the most potent; on the contrary, the DVL3-only cell line exhibited only the negligible capacity to mediate Wnt signals. Using a novel model system – complementation assays in T-REx HEK 293 with amplified Wnt signal response (RNF43/ZNRF3/DVL1/DVL2/DVL3 penta KO cells) we demonstrate that it is not the total amount of DVL but ratio of individual paralogs what decides the signal strength. In sum, this study contributes to our better understanding of the role of individual human DVL paralogs in the Wnt pathway.
•New models for studying Wnt pathway: DVL-only cell lines and supersensitive RNF43/ZNRF3 DVL1/2/3 penta KO cells•All DVL paralogs are able at endogenous levels to mediate Wnt-3a-induced phosphorylation of LRP6 and degradation of Axin1•DVL3 is less efficient than DVL1 and DVL2 in Wnt/β-catenin downstream signalling•All DVL paralogs are able at endogenous levels mediate non-canonical Wnt-5a-induced phosphorylation of ROR1
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