Dishevelled-associated Activator Of Morphogenesis Research Articles

Wnt5a/ROR1 activates DAAM1 and promotes the migration in osteosarcoma cells.

Receptor tyrosine kinase like orphan receptor 2 (ROR2) regulates Wnt5a-induced cell migration by phosphorylating PI3K/Akt and activating RhoA in osteosarcoma. However, the role of Wnt5a signaling and its corresponding receptors in the regulation of osteosarcoma metastasis remains poorly understood. ROR1 monoclonal antibody (mAb) and short hairpin (sh)RNA targeting ROR2 markedly inhibited the activity of dishevelled associated activator of morphogenesis 1 (DAAM1) and RhoA and retarded cell migration in osteosarcoma. ROR1 mAb and ROR2 shRNA destroyed the microfilament formation of osteosarcoma cells. Silencing of DAAM1 (with DAAM1 shRNA) downregulated RhoA activity and inhibited cell migration. The decrease of cell migration caused by DAAM1 shRNA was rescued by wild-type DAAM1 overexpression. DAAM1 and PI3Kα/Akt were parallel signaling pathways mediating osteosarcoma cell migration in response to Wnt5a. It was concluded that Wnt5a promotes osteosarcoma cell migration via ROR1/2 receptors, and then activates DAAM1 and RhoA.

Overexpressed DAAM1 correlates with metastasis and predicts poor prognosis in breast cancer

Recent studies have reported that dishevelled-associated activator of morphogenesis 1 (DAAM1) is remarkably essential for mediating cell migration and invasion in breast cancer (BrCa). Nonetheless, the definite expression profile of DAAM1 in BrCa patients and the impact on metastasis of BrCa in vivo have not been explored up to now. The differential expression of DAAM1 in BrCa and adjacent tissues was assessed via immunohistochemistry (IHC) staining. The metastatic capacities of BrCa SUM-1315 cells were examined in BALB/c nude mice. Besides, the prognostic values of DAAM1 mRNA in BrCa were explored based on Kaplan-Meier (KM) plotter. The expression of DAAM1 protein was notably overexpressed in BrCa tissues compared with that in paired normal breast tissues. The high expression of DAAM1 in BrCa tissues was significantly associated with lymph-node metastasis. Furthermore, DAAM1 overexpression promoted the invasive capacity of BrCa cells and stimulated lung metastatic extent in vivo. We also found that overexpressed DAAM1 mRNA was significantly associated with poor relapse-free survival (RFS), overall survival (OS), distance-metastasis-free survival (DMFS), and post-progression survival (PPS). Our findings reveal that DAAM1 might be a novel therapeutic target to manage the deteriorated metastasis of BrCa and identified DAAM1 as a promising biomarker for unfavorable prognosis in BrCa patients.

PTPN3 suppresses lung cancer cell invasiveness by counteracting Src-mediated DAAM1 activation and actin polymerization

Cancer cell migration plays a crucial role during the metastatic process. Reversible tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) have been implicated in the regulation of cancer cell migration and invasion. However, the underlying mechanisms have not been fully elucidated. Here, we show that depletion of the FERM and PDZ domain-containing protein tyrosine phosphatase PTPN3 enhances lung cancer cell migration/invasion and metastasis by promoting actin filament assembly and focal adhesion dynamics. We further identified Src and DAAM1 (dishevelled associated activator of morphogenesis 1) as interactors of PTPN3. DAAM1 is a formin-like protein involved in the regulation of actin cytoskeletal remodeling. PTPN3 inhibits Src activity and Src-mediated phosphorylation of Tyr652 on DAAM1. The tyrosine phosphorylation of DAAM1 is essential for DAAM1 homodimer formation and actin polymerization. Ectopic expression of a DAAM1 phosphodeficient mutant inhibited F-actin assembly and suppressed lung cancer cell migration and invasion. Our findings reveal a novel mechanism by which reversible tyrosine phosphorylation of DAAM1 by Src and PTPN3 regulates actin dynamics and lung cancer invasiveness.

<p>Wnt5a induces ROR1 and ROR2 to activate RhoA in esophageal squamous cell carcinoma cells</p>

Background: Wnt5a is a nontransforming Wnt family member and identified as an oncogenic role on cell motility of breast cancer and glioblastoma. However, Wnt5a signaling in esophageal squamous cell carcinoma (ESCC) progression remains poorly defined.Materials and methods: Immunohistochemistry assays were used to measure the Wnt5a expression in ESCC sections. We evaluated the role of receptor tyrosine kinase-like orphan receptor (ROR)1/2 and RhoA on the invasion of ESCC cells by using cell invasion assay, immunoprecipitation, immunofluorescence, and Rho activation assay.Results: Wnt5a was highly expressed in invasive ESCC tissues compared with that in noninvasive and nonmalignant tissues. In vitro assay showed that sfrp2 (Wnt5a antagonist) largely blocked the invasion but not the colony formation of KYSE410 and KYSE520 ESCC cells. Anti-ROR1 mAb and ROR2-shRNA markedly inhibited the disheveled-associated activator of morphogenesis 1 (DAAM1) activity, RhoA activity, microfilament formation and the invasion of ESCC cells. Fluorescent phalloidin staining experiment showed ROR1/ROR2, receptors of Wnt5a signaling, and regulated the reassembly of actin filaments in ESCC cells. Further experiments showed that ROR1 was strongly associated with ROR2 in KYSE410 cells. The activation of RhoA, not Rac1 or Rac2, was involved in ROR1/ROR2 signaling pathway. By using DAAM1 shRNA, we found that RhoA was downstream of DAAM1, which could be rescued by the overexpression of wild-type DAAM1. This could be further proved by a RhoA inhibitor CCG-1423 which could inhibit the invasion of ESCC cells but not DAAM1 activity.Conclusions: Wnt5a promotes ESCC cell invasion via ROR1 and ROR2 receptors and DAAM1/RhoA signaling pathway.

Proteins of the Wnt signaling pathway as targets for the regulation of CD133+ cancer stem cells in glioblastoma.

Glioblastoma multiforme (GBM) is one of the most aggressive types of brain tumor and is highly resistant to therapy. The median survival time for patients with GBM is 15 months. GBM resistance to treatment is associated with cancer stem cells (CSCs). CD133 membrane glycoprotein is the best‑known marker of GBM CSCs. The Wnt signaling pathway plays an important role in the proliferation of all stem cells. To the best of our knowledge, the present study was the first to examine the expression levels of proteins associated with the Wnt signaling pathway in СD133+ CSCs of human GBM. Furthermore, potential targets that may regulate СD133+ CSCs in human GBM were investigated. The human GBM U‑87MG cell line was cultured in neurobasal medium supplemented with B27, fibroblast growth factor, epidermal growth factor and no serum. Immunohistochemical characteristics of glioma spheres were investigated based on the expression of key markers of CSCs. CD133+ cells were extracted from glioma spheres by cell sorting and then lysed. High‑performance liquid chromatography‑mass spectrometry was used for proteome analysis. Lysates of CD133‑ cells in GBM were used for comparison. The present study was the first to describe the conceptual proteome differences between GBM and CD133+ CSCs of the common pool. Major differences were identified in the glycolysis/gluconeogenesis, focal adhesion, tight junction and Wnt signaling pathways. This study aimed to analyze the crucial role that proteins of the Wnt signaling pathway play in stem cell proliferation. The identified proteins were analyzed for their association with the Wnt signaling pathway using the international open databases PubMed, Protein Analysis Through Evolutionary Relationships, Gene Ontology, Kyoto Encyclopedia of Genes and Genomes and Search Tool for the Retrieval of Interacting Genes/Proteins. An increased expression of 12 proteins associated with the Wnt signaling pathway were identified in GBM CD133+ CSCs, which included catenin β‑1, disheveled associated activator of morphogenesis 1, RAC family small GTPase 2 and RAS homolog gene family member A, a number of which are also associated with adherens junctions. The Wnt signaling pathway is not upregulated in CSCs; however, the high expression levels of adenomatous polyposis coli, β‑catenin, C‑terminal binding protein (CtBP) and RuvB‑like AAA ATPase 1 (RUVBL1 or Pontin52) proteins suggest the possibility of alternative activation of specific genes in the nuclei of these cells. Calcyclin‑binding protein, casein kinase II α, casein kinase II β, CtBP1, CtBP2, CUL1 and RUVBL1 proteins may be used as targets for the pharmaceutical regulation of CSCs in complex GBM treatment.

A DAAM1 3'-UTR Variant Promotes Breast Cancer Metastasis Via Reducing microRNA-208a-5p Binding Affinity

Background: Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a commonly expressed member of diaphanous-related formins and plays a key role in controlling the migration and haptotaxis of breast cancer cells. However, the genotype and pathological type of DAAM1 in breast cancer patients remains largely unknown. Methods: We utilized a large patient cohort and breast cancer cells to determine the role of functional SNP in microRNA-208a-5p binding site of DAAM1 3'-UTR and associated mechanism in breast cancer metastasis. Findings: The expression and activation of DAAM1 increased markedly in lymphnode metastatic tissues. A genetic variant (rs79036859 A/G) was present in the miR-208a-5p binding site of DAAM1 3'-UTR. Compared with the AA genotype, G was a risk genotype for the metastasis of breast cancer by reducing binding affinity of miR-208a-5p for the DAAM1 3'-UTR. Furthermore, the miR-208a-5p expression level was significantly suppressed in lymphnode metastatic tissues. Overexpression of miR-208a-5p inhibited DAAM1/RhoA signaling pathway and thus leaded to decreasing of the migratory ability. Interpretation: We imply that the rs79036859 G variant of DAAM1 3'-UTR contributes to the likelihood of breast cancer metastasis via a novel mechanism of subtle gene regulation through miR-208a-5p binding capacity. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (81472703) to Yichao Zhu, the Natural Science Foundation of Jiangsu Province (Grant No. BK20181367) to Yichao Zhu, and the Science and Technology Foundation of Nanjing Medical University (2017NJMU001) to Ting Yan. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: Ethical approval for the study was obtained from the Clinical Research Ethics Committee, Nanjing Medical University. Pathologic staging was determined by a pathologist based on AJCC Cancer Staging Manual 8th classification criteria. All the participants voluntarily joined this study with informed constents.

Integrin αvβ3–associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells

The formin protein dishevelled-associated activator of morphogenesis 1 (DAAM1) polymerizes straight actin filaments and mediates migration of cancer cells. However, how DAAM1 governs cell haptotaxis in response to collagen remains unexplored in breast cancer cells. We hypothesized that DAAM1 mediates invadopodia extension and cell haptotaxis in response to type IV collagen in association with integrin receptors. Using Boyden chamber membranes coated with type IV collagen, we show here that type IV collagen activates both DAAM1 and Ras homolog family member A (RHOA) and promotes haptotaxis of MDA-MB-231 and MDA-MB-453 breast cancer cells, a process abolished by treatment with the integrin αvβ3 inhibitor cyclo(-RGDfK). shRNA-mediated knockdown of DAAM1 or a dominant-negative DAAM1 mutation (N-DAAM1) significantly decreased collagen-induced RHOA activity and the assembly of stress fibers, invadopodia extension, and cell haptotaxis. Immunoprecipitation and pulldown assays revealed that integrin αvβ3 is associated with, but only indirectly binds to, the C-terminal DAD domain of DAAM1 in mammalian cells. Blockade of RHOA activation with a specific inhibitor (CCG-1423) or via a dominant-negative RHOA mutation (RHOA-N19) suppressed collagen-induced invadopodia extension and haptotaxis of the MDA-MB-231 and MDA-MB-453 cells. Immunoblotting and immunofluorescence assays indicated high DAAM1 and RHOA expression in invadopodia, which was abolished by cyclo(-RGDfK) treatment or DAAM1 knockdown. These findings have uncovered an integrin αvβ3/DAAM1/RHOA signaling pathway for type IV collagen-induced invadopodia extension and haptotaxis in breast cancer cells. Targeting this pathway may be a means for reducing invasiveness and metastasis of breast cancer.

First evidence of DAAM1 localization in mouse seminal vesicles and its possible involvement during regulated exocytosis

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a protein belonging to the formin family, which regulates, together with the small GTPase RhoA, the nucleation and the assembly of actin fibres through Wnt-Dishevelled PCP pathway. Its role has been investigated in essential biological processes, such as cell polarity, movement and adhesion during morphogenesis and organogenesis. In this work, we studied the expression of DAAM1 mRNA and protein by PCR and Western blot analyses and its co-localization with actin in adult mouse seminal vesicles by immunofluorescence. We show that both proteins are cytoplasmic: actin is evident at cell–cell junctions and at cell cortex; DAAM1 had a more diffused localization, but is also prominent at the apical plasmatic membrane of epithelial cells. These findings support our hypothesis of a role of DAAM1 in cytoskeletal rearrangement that occurs during the exocytosis of secretory vesicles, and in particular concerning actin filaments. We were also able to detect DAAM1 and actin association in the smooth muscle cells that surround the epithelium too. In this case, we could only speculate the possible involvement of this formin in muscular cells in the maintenance and the regulation of the contractile structures. The present results strongly suggest that DAAM1 could have a pivotal role in vesicle exocytosis and in the physiology of mouse seminal vesicles.

MiR-613 inhibits cell migration and invasion by downregulating Daam1 in triple-negative breast cancer

Dishevelled-associated activator of morphogenesis 1 (Daam1) is a formin protein and participates in regulating cell migration of triple-negative breast cancer (TNBC) cells. The specific miRNA targeting Daam1 and mediating cell migration and invasion remains obscure. This experiment investigated the suppressive role of miR-613 in TNBC cells. The luciferase activity of Daam1 3′-untranslated region (3′-UTR) based reporters constructed in HEK-293T and MCF-7 cells suggested that Daam1 was the target gene of miR-613. Overexpressed miR-613 reduced the protein level of Daam1, weakened RhoA activity, and retarded the cell migration, cell invasion and colony formation of TNBC cells. Overexpression of Daam1 or RhoA rescued cell migration and invasion in miR-613-overexpressed TNBC cells, but failed to reverse colony formation. MiR-613 was significantly downregulated in breast cancer tissues compared with that in adjacent normal tissues. This downregulation in TNBC tissues and lymphnode metastatic breast cancer tissues was more obvious than that in non-TNBC tissues and non-metastatic cancer tissues, respectively. MiR-613 weakens the resistance of TNBC cells against paclitaxel rather than adriamycin, cyclophosphamide, docetaxel, and kaempferol. Taken together, miR-613 is involved in cell migration and invasion of TNBC cells via targeting Daam1/RhoA signaling pathway.

The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones

Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin-microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in Drosophilamelanogaster primary neurons. In vitro, DAAM binds to F-actin as well as to microtubules and has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of DAAM affects growth cone and microtubule morphology, and several aspects of microtubule dynamics; and biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together, these data suggest that, besides operating as an actin assembly factor, DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth.

The activities of the C-terminal regions of the formin protein disheveled-associated activator of morphogenesis (DAAM) in actin dynamics

Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of Drosophila DAAM in its interaction with actin with in vitro biochemical and in vivo genetic approaches. We found that the DAD-CT region binds actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context.

Identification of Different Binding Partners of the F‐BAR Proteins Cdc42 Interacting Protein 4 (CIP4) and Formin Binding Protein 17 (FBP17) in Cortical Neurons

During neuronal development, neurons perform many dynamic processes, including migration and differentiation, which require the coordination of movements between both the plasma membrane and the underlying cytoskeleton. The Cdc42 Interacting Protein 4 (CIP4) subfamily of F‐BAR proteins contains an N‐terminal F‐BAR domain that can interact with the plasma membrane and C‐terminal HR1 and SH3 domains that can interact with Rho GTPases and actin associated proteins, respectively. F‐BAR proteins, which function in detecting and inducing membrane curvature, can effectively link the plasma membrane and the cytoskeleton. Previous studies have shown that CIP4 and another family member, Formin Binding Protein 17 (FBP17), exhibit distinct functions in neurons. CIP4 tends to accumulate in the extending peripheral membrane, inhibiting neurite protrusion, while FBP17 tends to localize in membrane tubules, function in endocytosis, and assist in neurite/axon outgrowth. In this work, we used Western blot and immunoprecipitation assays to show that CIP4 and FBP17 interact with different subsets of proteins to produce their distinctive functions. We have shown previously that CIP4 colocalizes with actin‐associated proteins such as WASP Family Verprolin‐Homologous Protein 1 (WAVE1) and Disheveled Associated Activator of Morphogenesis 1 (DAAM 1). Here we will determine if WAVE1 and DAAM1 form a complex with CIP4 and if FBP17 interacts with a different set of proteins than CIP4 in cortical neurons, including N‐WASP (Neural Wiskott‐Aldrich syndrome protein) and dynamin. Results from these studies will help determine how two very similar proteins function in entirely different processes in developing neurons.

Implication of overexpression of dishevelled-associated activator of morphogenesis 1 (Daam-1) for the pathogenesis of human Idiopathic Pulmonary Arterial Hypertension (IPAH)

BackgroundIdiopathic pulmonary arterial hypertension (IPAH) is a rare, fatal disease of unknown pathogenesis. Evidence from our recent study suggests that IPAH pathogenesis is related to upregulation of the Wnt/planar cell polarity (Wnt/PCP) pathway. We used microscopic observation and immunohistochemical techniques to identify expression patterns of cascading proteins—namely Wnt-11, dishevelled-2 (Dvl-2), and dishevelled-associated activator of morphogenesis 1 (Daam-1)—in pulmonary arteries.MethodsWe analyzed sections of formalin-fixed and paraffin-embedded autopsied lung tissues obtained from 9 IPAH cases, 7 associated pulmonary arterial hypertension cases, and 16 age-matched controls without pulmonary arterial abnormalities. Results of microscopic observation were analyzed in relation to the cellular components and size of pulmonary arteries.ResultsVarying rates of positive reactivity to Dvl-2 and Daam-1 were confirmed in all cellular components of pulmonary arteries, namely, endothelial cells, myofibroblasts, and medial smooth muscle cells. In contrast, none of these components was reactive to Wnt-11. No specific expression patterns were observed for endothelial cells or myofibroblasts under any experimental conditions. However, marked expression of Dvl-2 and Daam-1 was confirmed in smooth muscle cells. In addition, Dvl-2 was depleted while Daam-1 expression was elevated in IPAH, in contrast with specimens from associated pulmonary arterial hypertension cases and controls.ConclusionsHigh Daam-1 expression may upregulate the Wnt/PCP pathway and cause IPAH.

The core planar cell polarity gene, Vangl2, directs adult corneal epithelial cell alignment and migration.

This study shows that the core planar cell polarity (PCP) genes direct the aligned cell migration in the adult corneal epithelium, a stratified squamous epithelium on the outer surface of the vertebrate eye. Expression of multiple core PCP genes was demonstrated in the adult corneal epithelium. PCP components were manipulated genetically and pharmacologically in human and mouse corneal epithelial cells in vivo and in vitro. Knockdown of VANGL2 reduced the directional component of migration of human corneal epithelial (HCE) cells without affecting speed. It was shown that signalling through PCP mediators, dishevelled, dishevelled-associated activator of morphogenesis and Rho-associated protein kinase directs the alignment of HCE cells by affecting cytoskeletal reorganization. Cells in which VANGL2 was disrupted tended to misalign on grooved surfaces and migrate across, rather than parallel to the grooves. Adult corneal epithelial cells in which Vangl2 had been conditionally deleted showed a reduced rate of wound-healing migration. Conditional deletion of Vangl2 in the mouse corneal epithelium ablated the normal highly stereotyped patterns of centripetal cell migration in vivo from the periphery (limbus) to the centre of the cornea. Corneal opacity owing to chronic wounding is a major cause of degenerative blindness across the world, and this study shows that Vangl2 activity is required for directional corneal epithelial migration.

First Evidence of DAAM1 Localization During the Post-Natal Development of Rat Testis and in Mammalian Sperm.

Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a formin-family protein involved in nucleation of unbranched actin filaments and in cytoskeletal organization through Wnt-Dishevelled PCP pathway, which participates in essential biological processes, such as cell polarity, movement, and adhesion during morphogenesis and organogenesis. While its role has been investigated during development and in somatic cells, its potential association with the germinal compartment and reproduction is still unexplored. In this work, we assessed the possible association of DAAM1 with the morphogenesis of rat testis. We studied its expression and profiled its localization versus actin and tubulin, during the first wave of spermatogenesis and in the adult gonad (from 7 to 60 dpp). We show that, in mitotic phases, DAAM1 shares its localization with actin in Sertoli cells, gonocytes, and spermatogonia. Later, during meiosis, both proteins are found in spermatocytes, while only actin is detectable at the forming blood-testis barrier. DAAM1, then, follows the development of the acrosome system throughout spermiogenesis, and it is finally retained inside the cytoplasmic droplet in mature gametes, as corroborated by additional immunolocalization data on both rat and human sperm. Unlike the DAAM1, actin keeps its localization in Sertoli cells, and tubulin is associated with their protruding cytoplasm during the process. Our data support, for the first time, the hypothesis of a role for DAAM1 in cytoskeletal organization during Mammalian testis morphogenesis and gamete progression, while also hinting at its possible investigation as a morphological marker of germ cell and sperm physiology. J. Cell. Physiol. 231: 2172-2184, 2016. © 2016 Wiley Periodicals, Inc.

Correlation Between Daam2 Expression Changes and Demyelination in Guillain-Barre Syndrome.

Wnt signaling has been implicated in developmental and regenerative myelination of the CNS and PNS. The present translational investigation was undertaken to assess whether a soluble factor like Wnt may be responsible for the critically important skeletal muscle neuromuscular junction-Schwann cell communication. Specifically, three key aspects were examined: (a) whether the expression of Daam2, disheveled-associated activator of morphogenesis, a key Wnt signaling downstream effector, and PIP5K is changed in the demyelinating conditions and under different stages of progress of clinical recovery of patients with Guillain-Barre syndrome; (b) whether critical protein interactions of Daam2 with disheveled and Arf6 are changed; and (c) whether expression of c-Jun/Krox, a key negative regulator of remyelination, is changed. Daam2 was elevated in acute presentation in GB syndrome. Reduction occurred with clinical improvement of the patients. With progressive clinical improvement, c-Jun/Krox expression significantly reduced with time. Wnt signaling likely causes immediate early gene activation and transcriptional shutdown of factors critical for formation and maintenance of myelination. Whether the findings of the present study are specific to pathophysiology of demyelination in acute infectious polyradiculopathy and multiple sclerosis or a generalized aspect of demyelinating diseases merits to be examined in future studies.

The effect of maternal diabetes on the Wnt-PCP pathway during embryogenesis as reflected in the developing mouse eye.

Embryopathies that develop as a consequence of maternal diabetes have been studied intensely in both experimental and clinical scenarios. Accordingly, hyperglycaemia has been shown to downregulate the expression of elements in the non-canonical Wnt-PCP pathway, such as the Dishevelled-associated activator of morphogenesis 1 (Daam1) and Vangl2. Daam1 is a formin that is essential for actin polymerization and for cytoskeletal reorganization, and it is expressed strongly in certain organs during mouse development, including the eye, neural tube and heart. Daam1gt/gt and Daam1gt/+ embryos develop ocular defects (anophthalmia or microphthalmia) that are similar to those detected as a result of hyperglycaemia. Indeed, studying the effects of maternal diabetes on the Wnt-PCP pathway demonstrated that there was strong association with the Daam1 genotype, whereby the embryopathy observed in Daam1gt/+ mutant embryos of diabetic dams was more severe. There was evidence that embryonic exposure to glucose in vitro diminishes the expression of genes in the Wnt-PCP pathway, leading to altered cytoskeletal organization, cell shape and cell polarity in the optic vesicle. Hence, the Wnt-PCP pathway appears to influence cell morphology and cell polarity, events that drive the cellular movements required for optic vesicle formation and that, in turn, are required to maintain the fate determination. Here, we demonstrate that the Wnt-PCP pathway is involved in the early stages of mouse eye development and that it is altered by diabetes, provoking the ocular phenotype observed in the affected embryos.

Interleukin-1β-induced Wnt5a enhances human corneal endothelial cell migration through regulation of Cdc42 and RhoA.

Wnt5a can activate β-catenin-independent pathways for regulation of various cellular functions, such as migration, that play critical roles in wound repair. Investigation of Wnt5a signaling may help identify therapeutic targets for enhancing corneal endothelial wound healing that could provide an alternative to corneal transplantation in patients with blindness from endothelial dysfunction. However, Wnt5a signaling in corneal endothelial cells (CECs) has not been well characterized. In this study, we show transient induction of Wnt5a by interleukin-1β (IL-1β) stimulation proceeds through NF-κB in human CECs. This leads to binding of Fzd5 to Ror2, resulting in activation of disheveled protein (Dvl) and subsequently disheveled-associated activator of morphogenesis 1 (DAAM1). This leads to activation of Cdc42 and subsequent inhibition of RhoA. Inhibition of RhoA leads to parallel dephosphorylation and inactivation of LIM domain kinase 2 along with dephosphorylation and activation of slingshot 1, resulting in dephosphorylation and activation of cofilin and leading to enhanced cell migration. These findings suggest that Wnt5a enhances cell migration through activation of Cdc42 and inactivation of RhoA in human CECs.

FZD7 drives in vitro aggressiveness in Stem-A subtype of ovarian cancer via regulation of non-canonical Wnt/PCP pathway

Ovarian cancer (OC) can be classified into five biologically distinct molecular subgroups: epithelial-A (Epi-A), Epi-B, mesenchymal (Mes), Stem-A and Stem-B. Among them, Stem-A expresses genes relating to stemness and is correlated with poor clinical prognosis. In this study, we show that frizzled family receptor 7 (FZD7), a receptor for Wnt signalling, is overexpressed in the Stem-A subgroup. To elucidate the functional roles of FZD7, we used an RNA interference gene knockdown approach in three Stem-A cell lines: CH1, PA1 and OV-17R. Si-FZD7 OC cells showed reduced cell proliferation with an increase in the G0/G1 sub-population, with no effect on apoptosis. The cells also displayed a distinctive morphologic change by colony compaction to become more epithelial-like and polarised with smaller internuclear distances and increased z-axis height. Immunofluorescence (IF) staining patterns of pan-cadherin and β-catenin suggested an increase in cadherin-based cell–cell adhesion in si-FZD7 cells. We also observed a significant rearrangement in the actin cytoskeleton and an increase in tensile contractility in si-FZD7 OC cells, as evident by the loss of stress fibres and the redistribution of phospho-myosin light chain (pMLC) from the sites of cell–cell contacts to the periphery of cell colonies. Furthermore, there was reciprocal regulation of RhoA (Ras homolog family member A) and Rac1 (Ras-related C3 botulinum toxin substrate 1 (Rho family, small GTP-binding protein Rac1)) activities upon FZD7 knockdown, with a significant reduction in RhoA activity and a concomitant upregulation in Rac1 activity. These changes in pMLC and RhoA, as well as the increased TopFlash reporter activities in si-FZD7 cells, suggested involvement of the non-canonical Wnt/planar cell polarity (PCP) pathway. Selected PCP pathway genes (cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), prickle homolog 4 (Drosophila) (PRICKLE4), dishevelled-associated activator of morphogenesis 1 (DAAM1), profilin 2 (PFN2), protocadherin 9 (PCDH9), protocadherin α1 (PCDHA1), protocadherin β17 pseudogene (PCDHB17), protocadherin β3 (PCDHB3), sprouty homolog 1 (SPRY1) and protein tyrosine kinase 7 (PTK7)) were found to be more highly expressed in Stem-A than non Stem-A subgroup of OC. Taken together, our results suggest that FZD7 might drive aggressiveness in Stem-A OC by regulating cell proliferation, cell cycle progression, maintenance of the Mes phenotype and cell migration via casein kinase 1ɛ-mediated non-canonical Wnt/PCP pathway.

Analysis of the local organization and dynamics of cellular actin networks

Actin filaments, with the aid of multiple accessory proteins, self-assemble into a variety of network patterns. We studied the organization and dynamics of the actin network in nonadhesive regions of cells bridging fibronectin-coated adhesive strips. The network was formed by actin nodes associated with and linked by myosin II and containing the formin disheveled-associated activator of morphogenesis 1 (DAAM1) and the cross-linker filamin A (FlnA). After Latrunculin A (LatA) addition, actin nodes appeared to be more prominent and demonstrated drift-diffusion motion. Superresolution microscopy revealed that, in untreated cells, DAAM1 formed patches with a similar spatial arrangement to the actin nodes. Node movement (diffusion coefficient and velocity) in LatA-treated cells was dependent on the level and activity of myosin IIA, DAAM1, and FlnA. Based on our results, we developed a computational model of the dynamic formin-filamin-actin asters that can self-organize into a contractile actomyosin network. We suggest that such networks are critical for connecting distant parts of the cell to maintain the mechanical coherence of the cytoplasm.