Subcellular localization and signaling properties of dishevelled in developing vertebrate embryos

The Dishevelled protein mediates several diverse biological processes. Intriguingly, within the same tissues where Xenopus Dishevelled (Xdsh) controls cell fate via canonical Wnt signaling, it also controls cell polarity via the vertebrate planar cell polarity (PCP) cascade [1, 2, 3, 4, 5, 6, 7, 8 and 9]. The relationship between subcellular localization of Dishevelled and its signaling activities remains unclear; conflicting results have been reported depending upon the organism and cell types examined [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20]. We have approached this issue by developing new reagents to sequester wild-type Dishevelled protein either at the cell membrane or away from the cell membrane. Removal of Dishevelled from the cell membrane disrupts convergent extension by preventing Rho/Rac activation and mediolateral cell polarization. By manipulating the subcellular localization of K-->M (dsh1), we show that this mutation inhibits Dishevelled activation of Rac, regardless of its subcellular localization. These data demonstrate that membrane localization of Dishevelled is a prerequisite for vertebrate PCP signaling. However, both membrane-targeted and cytoplasm-targeted Dishevelled can potently activate canonical Wnt signaling, suggesting that local concentration of Dishevelled protein, but not its spatial localization, is central to canonical Wnt signaling. These results suggest that in vertebrate embryos, subcellular localization is insufficient to account for the pathway specificity of Dishevelled in the canonical Wnt versus PCP signaling cascades.     

Distinct PAR-1 proteins function in different branches of Wnt signaling during vertebrate development

The kinase PAR-1 plays conserved roles in cell polarity. PAR-1 has also been implicated in axis establishment in C. elegans and Drosophila and in Wnt signaling, but its role in vertebrate development is unclear. Here we report that PAR-1 has two distinct and essential roles in axial development in Xenopus mediated by different PAR-1 isoforms. Depletion of PAR-1A or PAR-1BX causes dorsoanterior deficits, reduced Spemann organizer gene expression, and inhibition of canonical Wnt-beta-catenin signaling. By contrast, PAR-1BY depletion inhibits cell movements and localization of Dishevelled protein to the cell cortex, processes associated with noncanonical Wnt signaling. PAR-1 phosphorylation sites in Dishevelled are required for this translocation, but not for canonical Wnt signaling. We conclude that PAR-1BY is required in the PCP branch and mediates Dsh membrane localization while PAR-1A and PAR-1BX are essential for canonical signaling to beta-catenin, possibly via targets other than Dishevelled.     

PDZ domain from Dishevelled -- a specificity study

Intracellular signaling cascades induced by Wnt proteins play a key role in developmental processes and are implicated in cancerogenesis. It is still unclear how the cell determines which of the three possible Wnt response mechanisms should be activated, but the decision process is most likely dependent on Dishevelled proteins. Dishevelled family members interact with many diverse targets, however, molecular mechanisms underlying these binding events have not been comprehensively described so far. Here, we investigated the specificity of the PDZ domain from human Dishevelled-2 using C-terminal phage display, which led us to identification of a leucine-rich binding motif strongly resembling the consensus sequence of a nuclear export signal. PDZ interactions with several peptide and protein motifs (including the nuclear export signal sequence from Dishevelled-2 protein) were investigated in detail using fluorescence spectroscopy, mutational analysis and immunoenzymatic assays. The experiments showed that the PDZ domain can bind the nuclear export signal sequence of the Dishevelled-2 protein. Since the intracellular localization of Dishevelled is governed by nuclear localization and nuclear export signal sequences, it is possible that the intramolecular interaction between PDZ domain and the export signal could modulate the balance between nuclear and cytoplasmic pool of the Dishevelled protein. Such a regulatory mechanism would be of utmost importance for the differential activation of Wnt signaling cascades, leading to selective promotion of the nucleus-dependent Wnt β-catenin pathway at the expense of non-canonical Wnt signaling.     

Wnt signaling enhances FGF2-triggered lens fiber cell differentiation

Wnt signaling is implicated in many developmental processes, including cell fate changes. Several members of the Wnt family, as well as other molecules involved in Wnt signaling, including Frizzled receptors, LDL-related protein co-receptors, members of the Dishevelled and Dickkopf families, are known to be expressed in the lens during embryonic or postembryonic development. However, the function of Wnt signaling in lens fiber differentiation remains unknown. Here, we show that GSK-3beta kinase is inactivated and that beta-catenin accumulates during the early stages of lens fiber cell differentiation. In an explant culture system, Wnt conditioned medium (CM) induced the accumulation of beta-crystallin, a marker of fiber cell differentiation, without changing cell shape. In contrast, epithelial cells stimulated with Wnt after priming with FGF elongated, accumulated beta-crystallin, aquaporin-0, p57kip2, and altered their expression of cadherins. Treatment with lithium, which stabilizes beta-catenin, induced the accumulation of beta-crystallin, but explants treated with lithium after FGF priming did not elongate as they did after Wnt application. These results show that Wnts promote the morphological aspects of fiber cell differentiation in a process that requires FGF signaling, but is independent of beta-catenin. Wnt signaling may play an important role in lens epithelial-to-fiber differentiation.     

The SHP—2 tyrosine phosphatase：Signaling mechanisms and biological functions

Cellular biological avtivities are tightly controlled by intracellular signaling processes initiated by extracellular signals.Protein tyrosine phosphatases,which remove phosphate groups from phosphorylated signaling molecules,play equally important tyrosine roles as protein tyrosine kinases in signal transduction.SHP-2 a cytoplasmic SH2 domain containing protein tyrosine phosphatase,is involved in the signaling pathways of a variety of growth factors and cytokines.Recent studies have clearly demonstrated that this phosphatase plays an important role in transducing signal relay from the cell surface to the nucleus,and is a critical intracellular regulator in mediating cell proliferation and differentiation.     

Dvl2 silencing in postdevelopmental cells results in aberrant cell membrane activity and actin disorganization

The upstream events by which endothelial cells perceive the necessity for migration and how this signal results in coordinated movement is unknown. The synchrony underlying these events shares parallels to events occurring during the movement of tissues in embryogenesis. While Wnt signaling is an important pathway in development, components of the cascade exist in postdevelopment endothelial cells. The objective of this study was to determine whether Dishevelled, a key modulation protein in canonical and PCP-CE Wnt signaling was present in endothelium and its potential function. Western blots of cell lysates and immunolabeling studies confirmed that Dishevelled 2 (Dvl2) is an abundant phosphoprotein in endothelial cells. Dvl2 was localized within the cytoplasm of cells as either F-actin-free or F-actin-associated. The disappearance of F-actin-free Dvl2 in vesicle-like organelles and targeting of actin filaments correlated with a loss in cell motility. Gene silencing of Dishevelled by siRNA duplexes resulted in cells with aberrant membrane activity and an inability to extend lamellipodia. Underlying these abnormalities was a disorganization of the actin filament system, including loss of actin-rich densities, indistinct stress fibers and an accompanying increase in diffuse and aggregate cytoplasmic actin. This study represents the first documentation of Dvl2 in postdevelopmental endothelial cells and its possible role in cell migration via manipulation of actin filament bundles.     

Identification of three novel Smad binding proteins involved in cell polarity

A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor beta (TGFbeta) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFbeta could alter cell polarity via a Smad-dependent signaling mechanism.     

Transient overexpression of murine dishevelled genes results in apoptotic cell death

The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are implicated in Wnt signal transduction. In mammals, the manner in which Wnt signals are transduced remains unclear. The biochemical and molecular mechanisms defining the Wnt-1 pathway are of great interest because of its important role in development and its activation in murine breast tumors. In order to elucidate Dvl's role in Wnt signaling, we attempted to overexpress Dvl in cells, but were unable to obtain stable cell lines. We show here that the overexpression of Dvl genes alters nuclear and cellular morphology of COS-1 and C57MG cells and causes cell death due to the induction of apoptosis. Deletion studies demonstrate that all three conserved domains of Dvl (DIX, PDZ, and DEP) are required for Dvl-mediated cell death. Coexpression of protein phosphatase 2Calpha, a Dvl-interacting protein identified in yeast two-hybrid studies, protects cells from the cell death observed in cells overexpressing Dvl alone. Furthermore, the adenomatous polyposis coli (APC) gene product appears to be required for Dvl-mediated cell death. The relevance of these findings to Wnt signal transduction, as well as to developmental processes and disease, are discussed.     

Nuclear Dishevelled: An enigmatic role in governing cell fate and Wnt signaling

The Dishevelled gene was first identified in Drosophila mutants with disoriented hair and bristle polarity and subsequent work has now demonstrated its importance in critical and diverse aspects of biology. Since those early discoveries, Dishevelled has been shown to coordinate a plethora of developmental and cellular processes that range from controlling cell polarity during gastrulation to partnering with chromatin modifying enzymes to regulate histone methylation at genomic loci. While the role of DVL in development is well-respected and the cytosolic function of DVL has been studied more extensively, its nuclear role continues to remain murky. In this review we highlight some of the seminal discoveries that have contributed to the field, but the primary focus is to discuss recent advances with respect to the nuclear role of Dishevelled. This nuclear function of Dishevelled is a dimension which is proving to be increasingly important yet remains enigmatic.     

A simple guide to biochemical approaches for analyzing protein-lipid interactions

Eukaryotic cells contain many different membrane compartments with characteristic shapes, lipid compositions, and dynamics. A large fraction of cytoplasmic proteins associate with these membrane compartments. Such protein-lipid interactions, which regulate the subcellular localizations and activities of peripheral membrane proteins, are fundamentally important for a variety of cell biological processes ranging from cytoskeletal dynamics and membrane trafficking to intracellular signaling. Reciprocally, many membrane-associated proteins can modulate the shape, lipid composition, and dynamics of cellular membranes. Determining the exact mechanisms by which these proteins interact with membranes will be essential to understanding their biological functions. In this Technical Perspective, we provide a brief introduction to selected biochemical methods that can be applied to study protein-lipid interactions. We also discuss how important it is to choose proper lipid composition, type of model membrane, and biochemical assay to obtain reliable and informative data from the lipid-interaction mechanism of a protein of interest.     

Profilin is an effector for Daam1 in non-canonical Wnt signaling and is required for vertebrate gastrulation

Non-canonical Wnt signaling plays important roles during vertebrate embryogenesis and is required for cell motility during gastrulation. However, the molecular mechanisms of how Wnt signaling regulates modification of the actin cytoskeleton remain incompletely understood. We had previously identified the Formin homology protein Daam1 as an important link between Dishevelled and the Rho GTPase for cytoskeletal modulation. Here, we report that Profilin1 is an effector downstream of Daam1 required for cytoskeletal changes. Profilin1 interacted with the FH1 domain of Daam1 and was localized with Daam1 to actin stress fibers in response to Wnt signaling in mammalian cells. In addition, depletion of Profilin1 inhibited stress fiber formation induced by non-canonical Wnt signaling. Inhibition or depletion of Profilin1 in vivo specifically inhibited blastopore closure in Xenopus but did not affect convergent extension movements, tissue separation or neural fold closure. Our studies define a molecular pathway downstream of Daam1 that controls Wnt-mediated cytoskeletal reorganization for a specific morphogenetic process during vertebrate gastrulation.     

Epigenome-Metabolome-Epigenome signaling cascade in cell biological processes

Cell fate determination as a fundamental question in cell biology has been extensively studied at different regulatory levels for many years. However, the mechanisms of multilevel regulation of cell fate determination remain unclear. Recently, we have proposed an Epigenome-Metabolome-Epigenome (E-M-E) signaling cascade model to describe the cross-over cooperation during mouse somatic cell reprogramming. In this review, we summarize the broad roles of E-M-E signaling cascade in different cell biological processes, including cell differentiation and dedifferentiation, cell specialization, cell proliferation, and cell pathologic processes. Precise E-M-E signaling cascades are critical in these cell biological processes, and it is of worth to explore each step of E-M-E signaling cascade. E-M-E signaling cascade model sheds light on and may open a window to explore the mechanisms of multilevel regulation of cell biological processes.     

Dishevelled: The hub of Wnt signaling

Wnt signaling controls a variety of developmental and homeostatic events. As a key component of Wnt signaling, Dishevelled (Dvl/Dsh) protein relays Wnt signals from receptors to downstream effectors. In the canonical Wnt pathway that depends on the nuclear translocation of β-catenin, Dvl is recruited by the receptor Frizzled and prevents the constitutive destruction of cytosolic β-catenin. In the non-canonical Wnt pathways such as Wnt-Frizzled/PCP (planar cell polarity) signaling, Dvl signals via the Daam1-RhoA axis and the Rac1 axis. In addition, Dvl plays important roles in Wnt-GSK3β-microtubule signaling, Wnt-calcium signaling, Wnt-RYK signaling, Wnt-atypical PKC signaling, etc. Dvl also functions to mediate receptor endocytosis. To fulfill its multifaceted functions, it is not surprising that Dvl associates with various kinds of proteins. Its activity is also modulated dynamically by phosphorylation, ubiquitination and degradation. In this review, we summarize the current understanding of Dvl functions in Wnt signal transduction and its biological functions in mouse development, and also discuss the molecular mechanisms of its actions.     

Stage-dependent Dishevelled-1 expression during mouse spermatogenesis suggests a role in regulating spermatid morphological changes

Dishevelled (Dsh in Drosophila or DVL in mice) is a member of the highly conserved Wg/Wnt signaling pathway, which regulates important processes such as cell proliferation, polarity, and specification of cell fate. Three orthologous genes of Dishevelled (Dvl-1, Dvl-2, and Dvl-3) have been found in both humans and mice. They play pivotal roles in regulating cell morphology and a variety of changes in cell behaviors. In the present study, we show that the expression of Dvl-1 is stage-dependent during mouse spermatogenesis, although Dvl-2 and Dvl-3 show relative consistent expression. The expression of Dvl-1 mRNA first appears in pachytene spermatocytes, increases in round and elongating spermatids, and then turns to an undetectable level in mature sperm cells. Analyses of immunohistochemistry and immunofluorescence staining show that DVL-1 is present diffusely in the cytoplasm of pachytene spermatocytes and exhibits mainly a vesicular pattern and perinuclear distribution and a weak diffusely cytoplasmic signal in round and elongating spermatids. The vesicular pattern of DVL-1 has been observed by previous studies in somatic cells, and suggested to play roles in signal transduction. Immunoprecipitation experiments show that DVL-1 coimmunprecipitates with spermatogenic cells beta-actin rather than alpha-tubulin. These results indicate that DVL-1 may be involved in spermatid morphological changes during mouse spermiogenesis through mediating signal transduction and/or regulating actin cytoskeleton organization.     

Hairy is a cell context signal controlling Notch activity

Notch signaling regulates cell fate determination and many developmental processes. Here we report that lateral inhibition, a major mechanism for Notch activity, is modulated by Hairy, a bHLH-WRPW protein. In Xenopus, Notch can have from inhibitory, permissive to enhancing roles in muscle or neural differentiation. These cell context-dependent effects correlate with Hairy expression levels from high to low, respectively, in the cells. Moreover, Notch effects can be altered upon manipulation of Hairy expression. We propose that Hairy provides a cell context in which a cell can interpret Notch and other extrinsic signals by controlling responsiveness of its target genes; this mode of Hairy-Notch interaction may apply in other systems.     

Differential transmission of G1 cell cycle arrest and mating signals by Saccharomyces cerevisiae Ste5 mutants in the pheromone pathway.

Saccharomyces cerevisiae Ste5 is a scaffold protein that recruits many pheromone signaling molecules to sequester the pheromone pathway from other homologous mitogen-activated protein kinase pathways. G1 cell cycle arrest and mating are two different physiological consequences of pheromone signal transduction and Ste5 is required for both processes. However, the roles of Ste5 in G1 arrest and mating are not fully understood. To understand the roles of Ste5 better, we isolated 150 G1 cell cycle arrest defective STE5 mutants by chemical mutagenesis of the gene. Here, we found that two G1 cell cycle arrest defective STE5 mutants (ste5M(D248V) and ste5(delta-776)) retained mating capacity. When overproduced in a wild-type strain, several ste5 mutants also showed different dominant phenotypes for G1 arrest and mating. Isolation and characterization of the mutants suggested separable roles of Ste5 in G1 arrest and mating of S. cerevisiae. In addition, the roles of Asp-248 and Tyr-421, which are important for pheromone signal transduction were further characterized by site-directed mutagenesis studies.