Paraxial Protocadherin Research Articles

Cell–cell contact landscapes in Xenopus gastrula tissues

Molecular and structural facets of cell-cell adhesion have been extensively studied in monolayered epithelia. Here, we perform a comprehensive analysis of cell-cell contacts in a series of multilayered tissues in the Xenopus gastrula model. We show that intercellular contact distances range from 10 to 1,000 nm. The contact width frequencies define tissue-specific contact spectra, and knockdown of adhesion factors modifies these spectra. This allows us to reconstruct the emergence of contact types from complex interactions of the factors. We find that the membrane proteoglycan Syndecan-4 plays a dominant role in all contacts, including narrow C-cadherin-mediated junctions. Glypican-4, hyaluronic acid, paraxial protocadherin, and fibronectin also control contact widths, and unexpectedly, C-cadherin functions in wide contacts. Using lanthanum staining, we identified three morphologically distinct forms of glycocalyx in contacts of the Xenopus gastrula, which are linked to the adhesion factors examined and mediate cell-cell attachment. Our study delineates a systematic approach to examine the varied contributions of adhesion factors individually or in combinations to nondiscrete and seemingly amorphous intercellular contacts.

Serotonin induces Arcadlin in hippocampal neurons

The monoamine hypothesis does not fully explain the delayed onset of recovery after antidepressant treatment or the mechanisms of recovery after electroconvulsive therapy (ECT). The common mechanism that operates both in ECT and monoaminergic treatment presumably involves molecules induced in both of these conditions. A spine density modulator, Arcadlin (Acad), the rat orthologue of human Protocadherin-8 (PCDH8) and of Xenopus and zebrafish Paraxial protocadherin (PAPC), is induced by both electroconvulsive seizure (ECS) and antidepressants; however, its cellular mechanism remains elusive. Here we confirm induction of Arcadlin upon stimulation of an N-methyl-d-aspartate (NMDA) receptor in cultured hippocampal neurons. Stimulation of an NMDA receptor also induced acute (20 min) and delayed (2 h) phosphorylation of the p38 mitogen-activated protein (MAP) kinase; the delayed phosphorylation was not obvious in Acad–/– neurons, suggesting that it depends on Arcadlin induction. Exposure of highly mature cultured hippocampal neurons to 1–10 μM serotonin for 4 h resulted in Arcadlin induction and p38 MAP kinase phosphorylation. Co-application of the NMDA receptor antagonist d-(-)-2-amino-5-phosphonopentanoic acid (APV) completely blocked Arcadlin induction and p38 MAP kinase phosphorylation. Finally, administration of antidepressant fluoxetine in mice for 16 days induced Arcadlin expression in the hippocampus. Our data indicate that the Arcadlin-p38 MAP kinase pathway is a candidate neural network modulator that is activated in hippocampal neurons under the dual regulation of serotonin and glutamate and, hence, may play a role in antidepressant therapies.

PAPC couples the segmentation clock to somite morphogenesis by regulating N-cadherin-dependent adhesion

Vertebrate segmentation is characterized by the periodic formation of epithelial somites from the mesenchymal presomitic mesoderm (PSM). How the rhythmic signaling pulse delivered by the Segmentation Clock is translated into the periodic morphogenesis of somites remains poorly understood. Here, we focused on the role of Paraxial protocadherin (PAPC/Pcdh8) in this process. We show that in chicken and mouse embryos, PAPC expression is tightly regulated by the Clock and Wavefront system in the posterior PSM. We observed that PAPC exhibits a striking complementary pattern to N-Cadherin (CDH2), marking the interface of the future somite boundary in the anterior PSM. Gain and loss of function of PAPC in chicken embryos disrupt somite segmentation by altering the CDH2-dependent epithelialization of PSM cells. Our data suggest that clathrin-mediated endocytosis is increased in PAPC expressing cells, subsequently affecting CDH2 internalization in the anterior compartment of the future somite. This in turn generates a differential adhesion interface, allowing formation of the acellular fissure that defines the somite boundary. Thus periodic expression of PAPC downstream of the Segmentation Clock triggers rhythmic endocytosis of CDH2, allowing for segmental de-adhesion and individualization of somites.

PAPC couples the segmentation clock to somite morphogenesis by regulating N-cadherin-dependent adhesion.

Vertebrate segmentation is characterized by the periodic formation of epithelial somites from the mesenchymal presomitic mesoderm (PSM). How the rhythmic signaling pulse delivered by the segmentation clock is translated into the periodic morphogenesis of somites remains poorly understood. Here, we focused on the role of paraxial protocadherin (PAPC/Pcdh8) in this process. We showed that in chicken and mouse embryos, PAPC expression is tightly regulated by the clock and wavefront system in the posterior PSM. We observed that PAPC exhibits a striking complementary pattern to N-cadherin (CDH2), marking the interface of the future somite boundary in the anterior PSM. Gain and loss of function of PAPC in chicken embryos disrupted somite segmentation by altering the CDH2-dependent epithelialization of PSM cells. Our data suggest that clathrin-mediated endocytosis is increased in PAPC-expressing cells, subsequently affecting CDH2 internalization in the anterior compartment of the future somite. This in turn generates a differential adhesion interface, allowing formation of the acellular fissure that defines the somite boundary. Thus, periodic expression of PAPC in the anterior PSM triggers rhythmic endocytosis of CDH2, allowing for segmental de-adhesion and individualization of somites.

Nemo-like kinase 1 (Nlk1) and paraxial protocadherin (PAPC) cooperatively control Xenopus gastrulation through regulation of Wnt/planar cell polarity (PCP) signaling

The Wnt/planar cell polarity (PCP) pathway directs cell migration during vertebrate gastrulation and is essential for proper embryonic development. Paraxial protocadherin (PAPC, Gene Symbol pcdh8.2) is an important activator of Wnt/PCP signaling during Xenopus gastrulation, but how PAPC activity is controlled is incompletely understood. Here we show that Nemo-like kinase 1 (Nlk1), an atypical mitogen-activated protein (MAP) kinase, physically associates with the C-terminus of PAPC. This interaction mutually stabilizes both proteins by inhibiting polyubiquitination. The Nlk1 mediated stabilization of PAPC is essential for Wnt/PCP signaling, tissue separation and gastrulation movements. We identified two conserved putative phosphorylation sites in the PAPC C-terminus that are critical for Nlk1 mediated PAPC stabilization and Wnt/PCP regulation. Intriguingly, the kinase activity of Nlk1 itself was not essential for its cooperation with PAPC, suggesting an indirect regulation for example by impeding a different kinase that promotes protein degradation. Overall these results outline a novel, kinase independent role of Nlk1, wherein Nlk1 regulates PAPC stabilization and thereby controls gastrulation movements and Wnt/PCP signaling during development.

Lack of phosphomannomutase 2 affects Xenopus laevis morphogenesis and the non-canonical Wnt5a/Ror2 signalling.

Reduced phosphomannomutase 2 activity in man leads to hypoglycosylation of glycoconjugates causing PMM2-CDG, the most common type of congenital disorders of glycosylation. Here we show that an antisense morpholino-mediated knockdown of the Xenopus laevis phosphomannomutase 2 gene provoked a general underglycosylation in frog embryos, which led to an altered phenotype and reduced glycosylation of Wnt5a as member of the non-canonical Wnt signalling. Loss of function experiments in hemi-sectioned embryos proved that due to the phosphomannomutase 2 knockdown expression of the Wnt5a/Ror2 target gene paraxial protocadherin was significantly decreased. Regarding the expression of paraxial protocadherin, a gain of function could only be achieved by injections of wnt5a and ror2 in dorsal neighbouring blastomeres, while a parallel injection of phosphomannomutase 2 morpholino led to a significant reduced level of expression. Our data show for the first time that a knockdown of phosphomannomutase 2 influences in vivo the non-canonical Wnt signalling during early embryogenesis.

PAPC mediates self/non-self-distinction during Snail1-dependent tissue separation.

Cleft-like boundaries represent a type of cell sorting boundary characterized by the presence of a physical gap between tissues. We studied the cleft-like ectoderm-mesoderm boundary in Xenopus laevis and zebrafish gastrulae. We identified the transcription factor Snail1 as being essential for tissue separation, showed that its expression in the mesoderm depends on noncanonical Wnt signaling, and demonstrated that it enables paraxial protocadherin (PAPC) to promote tissue separation through two novel functions. First, PAPC attenuates planar cell polarity signaling at the ectoderm-mesoderm boundary to lower cell adhesion and facilitate cleft formation. Second, PAPC controls formation of a distinct type of adhesive contact between mesoderm and ectoderm cells that shows properties of a cleft-like boundary at the single-cell level. It consists of short stretches of adherens junction-like contacts inserted between intermediate-sized contacts and large intercellular gaps. These roles of PAPC constitute a self/non-self-recognition mechanism that determines the site of boundary formation at the interface between PAPC-expressing and -nonexpressing cells.

Balancing cell behavior at boundaries.

The restriction of cell intermingling across boundaries is essential for the establishment of discrete tissues. Eph receptor signaling prevents intermingling at many boundaries. In this issue, Luu et al. (2015. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201409026) report a parallel pathway, mediated by Wnt signaling, Snail1, and paraxial protocadherin (PAPC). This pathway establishes a distinctive organization of cell adhesion and intercellular gaps at the interface between tissues.

Phosphorylation-dependent ubiquitination of paraxial protocadherin (PAPC) controls gastrulation cell movements.

Paraxial protocadherin (PAPC) has been shown to be involved in gastrulation cell movements during early embryogenesis. It is first expressed in the dorsal marginal zone at the early gastrula stage and subsequently restricted to the paraxial mesoderm in Xenopus and zebrafish. Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage. Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation. PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase β-TrCP. We also show that precise control of PAPC by phosphorylation/ubiquitination is essential for normal Xenopus gastrulation cell movements. Taken together, our findings unveil a novel mechanism of regulation of a cell adhesion protein and show that this system plays a crucial role in vertebrate embryogenesis.

Protocadherin PAPC is expressed in the CNC and can compensate for the loss of PCNS

Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration.

Wnt-11 and Fz7 reduce cell adhesion in convergent extension by sequestration of PAPC and C-cadherin

Wnt-11/planar cell polarity signaling polarizes mesodermal cells undergoing convergent extension during Xenopus laevis gastrulation. These shape changes associated with lateral intercalation behavior require a dynamic modulation of cell adhesion. In this paper, we report that Wnt-11/frizzled-7 (Fz7) controls cell adhesion by forming separate adhesion-modulating complexes (AMCs) with the paraxial protocadherin (PAPC; denoted as AMCP) and C-cadherin (denoted as AMCC) via distinct Fz7 interaction domains. When PAPC was part of a Wnt-11-Fz7 complex, its Dynamin1- and clathrin-dependent internalization was blocked. This membrane stabilization of AMCP (Fz7/PAPC) by Wnt-11 prevented C-cadherin clustering, resulting in reduced cell adhesion and modified cell sorting activity. Importantly, Wnt-11 did not influence C-cadherin internalization; instead, it promoted the formation of AMCC (Fz7/Cadherin), which competed with cis-dimerization of C-cadherin. Because PAPC and C-cadherin did not directly interact and did not form a joint complex with Fz7, we suggest that Wnt-11 triggers the formation of two distinct complexes, AMCC and AMCP, that act in parallel to reduce cell adhesion by hampering lateral clustering of C-cadherin.

Xenopus paraxial protocadherin inhibits Wnt/β‐catenin signalling via casein kinase 2β

Xenopus paraxial protocadherin (PAPC) regulates cadherin-mediated cell adhesion and promotes the planar cell polarity (PCP) pathway. Here we report that PAPC functions in the Xenopus gastrula as an inhibitor of the Wnt/β-catenin pathway. The intracellular domain of PAPC interacts with casein kinase 2 beta (CK2β), which is part of the CK2 holoenzyme. The CK2α/β complex stimulates Wnt/β-catenin signalling, and the physical interaction of CK2β with PAPC antagonizes this activity. By this mechanism, PAPC restricts the expression of Wnt target genes during gastrulation. These experiments identify a novel function of protocadherins as regulators of the Wnt pathway.

P11. Dissecting biological functions of a Wnt/PCP pathway component Prickle

The Wnt/planar cell polarity (PCP) pathway is one of the non-canonical Wnt signalling pathways, and known to be involved in a variety of biological processes including establishment of organised epithelia in Drosophila and regulation of grouped cell migration in vertebrate development. Prickle (Pk) belongs to the ‘core’ components of the Wnt/PCP pathway. In its N-terminal portion Pk has a PET domain, function of which is not known, and three LIM domains that are thought to be involved in protein–protein interaction. Pk is also shown to localise to the proximal side of the cells to form a complex with Vang in Drosophila PCP. However the precise mechanism by which Pk controls these biological processes remains largely unknown.To dissect and elucidate the biological functions of Pk in further detail, we have constructed a series of Pk deletion variants and tested their activities. We found that expression of a variant that lacks LIM and PET domains induces excess actin-dependent filopodia-like processes in Xenopus animal caps and HEK293 cells. This version of Pk localises to these processes. On the other hand, in animal caps expressing another variant which lacks the middle portion, cells became dissociated from each other, indicating inhibited cell adhesion. This phenotype was partially rescued by co-expression of paraxial protocadherin (PAPC).These results suggest pleiotropic roles of Pk in regulation of cytoskeleton and cell adhesion. The possible molecular interactions and mechanism underlying multiple functions of Pk will be discussed in this presentation.

XGit2 and xRhoGAP 11A regulate convergent extension and tissue separation in Xenopus gastrulation

In a microarray-based screen for genes that are involved in tissue separation downstream of Paraxial Protocadherin (PAPC) and Frizzled-7 (Fz7)-mediated signaling we identified xGit2 and xRhoGAP 11A, two GTPase-activating proteins (GAP) for small GTPases. xGit2 and xRhoGAP 11A are expressed in the dorsal ectoderm, and their transcription is downregulated in the involuting dorsal mesoderm by PAPC and Fz7. Overexpression of xGit2 and xRhoGAP 11A inhibits Rho activity and impairs convergent extension movements as well as tissue separation behaviour. We propose that Rho activity in the involuting mesoderm is enhanced through inhibition of xGit2 and xRhoGAP 11A transcription by PAPC and Fz7. By this mechanism xRhoGAP 11A and xGit2 are restricted to the dorsal ectoderm, while Rho signaling is inhibited.

Movement Directionality in Collective Migration of Germ Layer Progenitors

Collective cell migration, the simultaneous movement of multiple cells that are connected by cell-cell adhesion, is ubiquitous in development, tissue repair, and tumor metastasis [1, 2]. It has been hypothesized that the directionality of cell movement during collective migration emerges as a collective property [3, 4]. Here we determine how movement directionality is established in collective mesendoderm migration during zebrafish gastrulation. By interfering with two key features of collective migration, (1) having neighboring cells and (2) adhering to them, we show that individual mesendoderm cells are capable of normal directed migration when moving as single cells but require cell-cell adhesion to participate in coordinated and directed migration when moving as part of a group. We conclude that movement directionality is not a de novo collective property of mesendoderm cells but rather a property of single mesendoderm cells that requires cell-cell adhesion during collective migration.

A Protocadherin-Cadherin-FLRT3 Complex Controls Cell Adhesion and Morphogenesis

BackgroundParaxial protocadherin (PAPC) and fibronectin leucine-rich domain transmembrane protein-3 (FLRT3) are induced by TGFβ signaling in Xenopus embryos and both regulate morphogenesis by inhibiting C-cadherin mediated cell adhesion.Principal FindingsWe have investigated the functional and physical relationships between PAPC, FLRT3, and C-cadherin. Although neither PAPC nor FLRT3 are required for each other to regulate C-cadherin adhesion, they do interact functionally and physically, and they form a complex with cadherins. By itself PAPC reduces cell adhesion physiologically to induce cell sorting, while FLRT3 disrupts adhesion excessively to cause cell dissociation. However, when expressed together PAPC limits the cell dissociating and tissue disrupting activity of FLRT3 to make it effective in physiological cell sorting. PAPC counteracts FLRT3 function by inhibiting the recruitment of the GTPase RND1 to the FLRT3 cytoplasmic domain.Conclusions/SignificancePAPC and FLRT3 form a functional complex with cadherins and PAPC functions as a molecular “governor” to maintain FLRT3 activity at the optimal level for physiological regulation of C-cadherin adhesion, cell sorting, and morphogenesis.

PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer

Carcinoma is an altered state of tissue differentiation in which epithelial cells no longer respond to cues that keep them in their proper position. A break down in these cues has disastrous consequences not only in cancer but also in embryonic development when cells of various lineages must organize into discrete entities to form a body plan. Paraxial protocadherin (PAPC) is an adhesion protein with six cadherin repeats that organizes the formation and polarity of developing cellular structures in frog, fish and mouse embryos. Here we show that protocadherin-8 (PCDH8), the human ortholog of PAPC, is inactivated through either mutation or epigenetic silencing in a high fraction of breast carcinomas. Loss of PCDH8 expression is associated with loss of heterozygosity, partial promoter methylation, and increased proliferation. Complementation of mutant tumor cell line HCC2218 with wild-type PCDH8 inhibited its growth. Two tumor mutants, E146K and R343H, were defective for inhibition of cell growth and migration. Surprisingly, the E146K mutant transformed the human mammary epithelial cell line MCF10A and sustained the expression of cyclin D1 and MYC without epidermal growth factor. We propose that loss of PCDH8 promotes oncogenesis in epithelial human cancers by disrupting cell-cell communication dedicated to tissue organization and repression of mitogenic signaling.

Xenopus Paraxial Protocadherin regulates morphogenesis by antagonizing Sprouty

Xenopus Paraxial Protocadherin (xPAPC) has signaling functions that are essential for convergent extension (CE) movements and tissue separation during gastrulation. PAPC modulates components of the planar cell polarity (PCP) pathway, but it is not clear how PAPC is connected to beta-catenin-independent Wnt-signaling. By yeast two-hybrid screen, we found that the intracellular domain of PAPC interacts with Sprouty (Spry), an inhibitor of CE movements. Upon binding to PAPC, Spry function is inhibited and PCP signaling is enhanced. Our data indicate that PAPC promotes gastrulation movements by sequestration of Spry and reveal a novel mechanism by which protocadherins modulate beta-catenin-independent Wnt-signaling.

Structural Elements Necessary for Oligomerization, Trafficking, and Cell Sorting Function of Paraxial Protocadherin

Protocadherins have been shown to regulate cell adhesion, cell migration, cell survival, and tissue morphogenesis in the embryo and the central nervous system, but little is known about the mechanism of protocadherin function. We previously showed that Xenopus paraxial protocadherin (PAPC) mediates cell sorting and morphogenesis by down-regulating the adhesion activity of a classical cadherin, C-cadherin. Classical cadherins function by forming lateral dimers that are necessary for their adhesive function. However, it is not known whether oligomerization also plays a role in protocadherin function. We show here that PAPC forms oligomers that are stabilized by disulfide bonds formed between conserved Cys residues in the extracellular domain. Disruption of these disulfide bonds by dithiothreitol or mutation of the conserved cysteines results in defects in oligomerization, post-translational modification, trafficking to the cell surface and cell sorting function of PAPC. Furthermore, none of the residues in the cytoplasmic domain of PAPC is required for its cell sorting activity, whereas both the transmembrane domain and the extracellular domain are necessary. Therefore, protein oligomerization and/or protein interactions via the extracellular and transmembrane domains of PAPC are required for its cell sorting function.

ANR5, an FGF Target Gene Product, Regulates Gastrulation in Xenopus

Gastrulation is a morphogenetic process in which tightly coordinated cell and tissue movements establish the three germ layers (ectoderm, mesoderm, and endoderm) to define the anterior-to-posterior embryonic organization [1]. To elicit this movement, cells modulate membrane protrusions and undergo dynamic cell interactions. Here we report that ankyrin repeats domain protein 5 (xANR5), a novel FGF target gene product, regulates cell-protrusion formation and tissue separation, a process that develops the boundary between the ectoderm and mesoderm [2, 3], during Xenopus gastrulation. Loss of xANR5 function by antisense morpholino oligonucleotide (MO) caused a short trunk and spina bifida without affecting mesodermal gene expressions. xANR5-MO also blocked elongation of activin-treated animal caps (ACs) and tissue separation. The dorsal cells of xANR5-MO-injected embryos exhibited markedly reduced membrane protrusions, which could be restored by coinjecting active Rho. Active Rho also rescued the xANR5-MO-inhibited tissue separation. We further demonstrated that xANR5 interacted physically and functionally with paraxial protocadherin (PAPC), which has known functions in cell-sorting behavior, tissue separation, and gastrulation cell movements [4-6], to regulate early morphogenesis. Our findings reveal for the first time that xANR5 acts through Rho to regulate gastrulation and is an important cytoplasmic partner of PAPC, whose cytoplasmic partner was previously unknown.