Gene name - polychaetoid
Synonyms - tamou, Drosophila ZO-1
Cytological map position - 85B9--85B9
Function - scaffolding protein
Symbol - pyd
FlyBase ID: FBgn0003177
Genetic map position - 3-48.1
Classification - MAGUK, PDZ domain, Guanylate kinase domain, Src homology 3 (SH3) domain protein
Cellular location - cell surface
|Recent literature||Shimizu, H., Wilkin, M. B., Woodcock, S. A., Bonfini, A., Hung, Y., Mazaleyrat, S. and Baron, M. (2017). The Drosophila ZO-1 protein Polychaetoid suppresses Deltex-regulated Notch activity to modulate germline stem cell niche formation. Open Biol 7(4). PubMed ID: 28424321
The developmental signalling protein Notch can be proteolytically activated following ligand-interaction at the cell surface, or can be activated independently of its ligands, following Deltex (Dx)-induced Notch endocytosis and trafficking to the lysosomal membrane. The means by which different pools of Notch are directed towards these alternative outcomes remains poorly understood. This study found that the Drosophila ZO-1 protein Polychaetoid (Pyd) suppresses specifically the Dx-induced form of Notch activation both in vivo and in cell culture assays. The physiological relevance and direction of the Pyd/Dx interaction was confirmed by showing that the expanded ovary stem cell niche phenotypes of pyd mutants require the presence of functional Dx and other components that are specific to the Dx-induced Notch activation mechanism. In S2 cells Pyd can form a complex with Dx and Notch at the cell surface and reduce Dx-induced Notch endocytosis. Similar to other known activities of ZO-1 family proteins, the action of Pyd on Dx-induced endocytosis and signalling was found to be cell density dependent. Thus, together, these results suggest an alternative means by which external cues can tune Notch signalling through Pyd regulation of Dx-induced Notch trafficking.
As its name implies, a polychaetoid (pyd) mutation (also called Tamou, which is Japanese for 'hairy') results in too many chaetae, or innervated hairs. Pyd is a PDZ domain protein whose sequence is more closely related to mammalian Zona occludens proteins ZO-1, ZO-2 and ZO-3 than to Drosophila Discs large (Dlg). Functionally, Pyd resembles the mammalian ZO proteins, thus clearly delineating a subfamily within the large PDZ domain protein family. Nevertheless, both Pyd and Dlg are junctional proteins: Polychaetoid is associated with both septate and adherens junctions; Discs large is associated only with septate junctions. While a Discs large mutation causes the dismantling of the septate junction and loss of growth control, a pyd mutation has a neurogenic effect (Takahisa, 1996 and Chen, 1996). The genetic interaction between pyd and canoe mutations results in a dorsal closure phenotype (Takahashi, 1998). Thus, while pyd and dlg belong to the same protein family, and both genes code for junctional proteins, mutations in the two genes cause different phenotypic effects, implying that the two proteins are involved in different signaling networks.
In Drosophila sensory organ development, a proneural cluster, defined as a population of cells competent for neural development, is the result of the overall positive and negative balance of activity that occurs between proneural genes and neurogenic genes. The polychaetoid mutation reduces the transcription of a repressor gene, extramacrochaetae, and causes enlargement of a proneural cluster where supernumerary precursor cells emerge, resulting in extra mechanosensory organs in the fly. Homozygotes of tam1, a mutation caused by a P-element insertion, are fully viable and fertile but ectopic macrochaetae are conspicuous on the dorsal side of the head and thorax of adults. The ectopic macrochaetae are not uniformly distributed on the notum since almost all of them emerge near the positions of the dorsocentral, postalar and/or scutellar bristles, and rarely form near the postions of the supraalar or presutural bristles. In the tam1 mutant, epithelial cells are always observed between the bristles of the notum. Therefore, the ectopic bristle (shaft and socket) do not seem to be generated by the transformation of daughter cells, which normally give rise to sheath cell and neuron, leading to the prediction that the additional bristle in the pydtam1 mutant appears through formation of additional SOPs (Takahisa, 1996 and Chen, 1996). In pyd mutant discs, the proneural cluster pattern of Achaete expression appears unchanged from the wild-type pattern: ectopic expression of Achaete is not observed. However, the subsequent refinement of Achaete expression is altered as additional cells express the high levels of Achaete characteristic of SOPs. In many pyd mutants, SOPs within a single cluster appear to be at different stages, as reflected in the normal transition from Achaete to Asense expression (Chen, 1996).
canoe and pyd genetically interact giving rise to a more severe dorsal closure phenotype than one resulting from canoe mutation alone. canoe3 is an embryonic lethal allele of cno and displays a typical dorsal open phenotype. cnomis1 is a weak hypomorph that yields adult flies with rough eyes and subtle changes in the bristle number. In a search for mutations that interact with cnomis1, polychaetoid was identified as an enhancer of cno phenotypes. Flies doubly homozygous for pydtam1 and cnomis1 die as embryos; this represents a synthetic lethal combination. Examination of embryonic cuticles demonstrates that the cnomis1;pydtam1 double mutant remains open dorsally. Comparisons of cell shape during dorsal closure reveal that cno3 embryos exhibit insufficient elongation of cells. This is most evident in the leading edge cells, which appear square in cno3, in contrast to the oblong cells of wild-type. cnomis1;pydtam1 double mutant embryos exhibit a more extreme phenotype than single mutants: the leading edge cells elongate even less than in cno3 mutants. These results suggest that cno and pyd are required for coordinated cell shape changes in the cells of the leading edge and the lateral ectodermal cells during dorsal closure (Takahashi, 1998).
There is compelling evidence that the small GTPase Drac1 functions in dorsal closure as an upstream (early acting) element of the JNK pathway, which is composed of hemipterous, basket and puckered. To determine if cno is further upstream of Drac1, puckered-lacZ expression was examined in cno3 homozygous embryos: the leading edge of the epidermis in these embryos is driven to express Drac1V12, a constitutively active form of Drac1. If Drac1 is upstream of cno, then the effect of Drac1V12 on puc-lacZ transcription should be blocked by the loss of cno function. Targeted expression of Drac1V12 in the leading edge cells restores puc-lacZ transcription in cno3 homozygotes to a level comparable to that of wild-type. This result is compatible with the hypothesis that cno is upstream of Drac1, or that cno functions in a pathway parallel to that of Drac1 (Takahashi, 1998).
Demonstration of a physical interaction between Cno and Pyd places Pyd similarly upstream of Rac in the dorsal closure pathway. Cno and Pyd exhibit a similar tissue distribution and appear to colocalize at junctional membrane sites within the cell. ZO-1 is a component of both tight junctions and adherens junctions in mammalian cells. Mammalian ZO-1 binds to alpha-spectrin, which cross-links with actin filaments, thereby affecting cell shape (Ito, 1993). Pyd and mammalin ZO-1 also interact with Drosophila Cortactin and mammalian cortactin respectively (Katsube, 1998). Mammalian Cortactin is known to be a filamentous actin cross-linking protein and a substrate of Src protein tyrosine kinase. Cortactin is phosphorylated at tyrosine residues upon stimulation by extracellular signals. Filamentous actin cross-linking activity of cortactin is attenuated by Src (Huang, 1998). The intracellular localization of mammalian cortactin is regulated by the activation of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and this translocation is blocked by expression of dominant negative Rac1N17. Thus in mammals, cortactin is a putative target of Rac1-induced signal transduction events involved in membrane ruffling and lamellipodia formation (Weed, 1998). It would thus seem that Rac signaling is tied to actin dynamics and Polychaetoid/ZO-1 function both in Drosophila and mammals.
The physical and genetic interactions of Pyd and Canoe proteins, and the genes that code for them, respectively, are interesting in light of the genetic interaction between canoe and Notch. Cno has a DHR motif, a conserved sequence associated with protein interaction found in Discs large and Polychaetoid. The molecular structure of Cno suggests its direct association with Ras. Cno has significant homology with a mammalian Ras-binding protein AF-6 (Kuriyama, 1996). cno interacts genetically with the split allele of Notch, for eye, bristle and wing development. An interrupted wing vein in Ax1, one N allele producing an activated form of Notch protein, is dominantly suppressed by cno mutation (Miyamoto, 1995). What exactly is the biochemical pathway leading to extra bristles in polychaetoid and canoe mutation and how might this pathway intersect with the Notch pathway? What is the connection between Pyd, Cno and Rac leading to the activation of the JNK pathway during dorsal closure? These questions await future experimentation.
Adherens and tight junctions play key roles in assembling epithelia and maintaining barriers. In cell culture zonula occludens (ZO)-family proteins are important for assembly/maturation of both tight and adherens junctions (AJs). Genetic studies suggest that ZO proteins are important during normal development, but interpretation of mouse and fly studies is limited by genetic redundancy and/or a lack of null alleles. Null alleles of the single Drosophila ZO protein Polychaetoid (Pyd), have been generated. Most embryos lacking Pyd die with striking defects in morphogenesis of embryonic epithelia including the epidermis, segmental grooves, and tracheal system. Pyd loss does not dramatically affect AJ protein localization or initial localization of actin and myosin during dorsal closure. However, Pyd loss does affect several cell behaviors that drive dorsal closure. The defects, which include segmental grooves that fail to retract, a disrupted leading edge actin cable, and reduced zippering as leading edges meet, closely resemble defects in canoe zygotic null mutants and in embryos lacking the actin regulator Enabled (Ena), suggesting that these proteins act together. Canoe (Cno) and Pyd are required for proper Ena localization during dorsal closure, and strong genetic interactions suggest that Cno, Pyd, and Ena act together in regulating or anchoring the actin cytoskeleton during dorsal closure (Choi, 2011).
ZO family proteins localize to mammalian TJs and also to AJs in mammals, flies, and nematodes. Elegant work in cell culture revealed important roles for mammalian ZO family proteins in properly localizing TJ strands into a functional, apically-localized barrier. Furthermore, whereas cultured mammalian cells lacking ZO family function can assemble AJs, their maturation into smooth belt junctions, a phenotype thought to involve remodeling the linkage to the actin cytoskeleton, is impaired (Choi, 2011).
It was thus hypothesized that ZO family proteins would be essential for AJ maturation and/or maintenance during normal development. However, assembly of spot AJs into more continuous belt AJs occurred normally in pydMZ mutants, and there were no apparent defects in DE-cad levels or localization, even late in embryonic morphogenesis. Furthermore, loss of Pyd did not perturb tracheal trunk fusion, an event that requires AJ function. Finally, loss of Pyd did not perturb the junctional localization of its AJ binding partner Cno. The data also suggest that Pyd is dispensable for assembly of tracheal septate junctions -- although this is perhaps not surprising, as fly Pyd does not localize to septate junctions. The data are consistent with analysis of the nematode ZO-1 orthologue ZOO-1 (Lockwood, 2008), which is also dispensable for AJ assembly. It will be interesting to examine mouse ZO family double and triple mutants to determine the full role of these proteins in both AJs and TJs during mammalian development (Choi, 2011).
Subtle changes in levels of AJ proteins in the absence of Pyd cannot be ruled out. Djiane (2011) recently reported that although AJs remain in pyd mutant cells, cells lacking Pyd accumulate higher levels of membrane-associated DE-cad than neighboring wild-type cells. Djiane's data provides support for a model in which Pyd binds and may regulate the E3 ubiquitin ligase Su(dx), which regulates the endocytic trafficking of Notch. Perhaps Pyd plays a similar role in regulating the trafficking of AJ proteins (Choi, 2011).
Pyd's role in Notch signaling during postembryonic development was not explored in this study, since that was the subject of parallel of Djiane (2011). However, the current data do not support an essential role for Pyd in embryonic Notch signaling, as Notch mutant embryos lose ventral epidermal cells and gain excess neurons, phenotypes not observed int this study. Subtler roles for Pyd in Notch or other signaling pathways in the embryo cannot be ruled out. In fact, the presence of extra terminal cells in the tracheal system may be indicative of Notch signaling defects in that tissue (Choi, 2011).
Although Pyd is not essential for assembly or maintenance of AJs, this study found that it does play important roles in embryonic morphogenesis in both the epidermis and trachea. From 40 to 70% of embryos lacking maternal and zygotic Pyd die as embryos, with characteristic defects in head involution. This was true in embryos mutant for three different deletion alleles, two of which did not remove any other coding sequences. Even for events that usually go to completion in the absence of Pyd, like dorsal closure, execution does not proceed normally. For example, loss of Pyd disrupts coordinated cell shape changes in the epidermis during dorsal closure and significantly slows this process. Pyd also plays an important role in effective zippering together of the two epidermal sheets at the canthi and in maintaining a straight leading edge. Furthermore, the tracheal defects observed are consistent with defects in intercalation, as were previously documented in weaker alleles, along with possible defects in cell fate. Thus fly Pyd, like nematode ZOO-1 (Lockwood, 2008), is an important regulator of morphogenesis. Because Pyd is a complex, multidomain protein with many binding partners, in the future, it will be of interest to explore how the different domains of ZO-1 contribute to its functions in vivo (Choi, 2011).
Of interest, zygotic cno mutants share all of the cell shape and morphogenesis defects of pydMZ mutants. This is consistent with early data demonstrating both physical and genetic interactions, thus strongly suggesting that Cno and Pyd work together in regulating coordination of adhesion and the cytoskeleton. Recent work suggests that during apical constriction and invagination of mesoderm cells, Cno is one of the linkers anchoring the actomyosin cytoskeleton at AJs (Sawyer, 2009). Consistent with this idea, an apparent rupture of the LE actomyosin cable was shown in both pydMZ and cno mutants, leading to splayed open and hyperconstricted LE cells. During dorsal closure, these data would be consistent with a model in which Cno and Pyd specifically reinforce AJ-actomyosin connections at points where tension is the greatest. It will be interesting to examine whether mammalian ZO-1/ZO-2 and afadin functionally interact in a similar way (Choi, 2011).
Another player in dorsal closure is the fly nectin-like protein Echinoid (Ed). Like the mammalian nectins, Ed is an immunoglobulin-superfamily cell adhesion molecule. Both nectins and Ed associate with afadin/Canoe. Ed plays an important role during dorsal closure (LaPlante, 2011), and Ed, like Pyd, plays a role in tracheal development (Laplante, 2010). During dorsal closure, Ed expression is lost from the amnioserosa but maintained in the epidermis (Laplante, 2011, and juxtaposition of adjacent cells that express and those that do not express Ed can lead to actin cable assembly. However, ed maternal and zygotic mutants differ from both pydMZ and cno zygotic mutants: in ed mutants the actomyosin cable fails to assemble. Furthermore, unlike Ed, Cno and Pyd continue to be expressed in the amnioserosa. The mechanistic role of Ed remains somewhat controversial, with suggestions that it works through fly myosin VI to regulate myosin contractility and suggestions that it sets up a tissue boundary, allowing proper polarization of junctional and cytoskeletal proteins in the leading edge (Laplante, 2011). It will be interesting to explore whether Ed, Cno and Pyd work together during dorsal closure (Choi, 2011).
The suite of defects during dorsal closure shared by pydMZ and cno mutants is complex, including defects in LE cell shapes, a wavy leading edge, defects in zippering at the canthi, persistent deep segmental grooves, and simultaneous disruption of head involution. This entire suite of defects was strikingly reminiscent of those previously observed in embryos in which the function of the actin regulator Ena was disrupted by genetic inactivation, sequestration to mitochondria, or expression of a constitutively active form of its negative regulator Abelson (Abl) kinase. This led to an exploration of the hypothesis that Pyd and Cno worked together with or regulated Ena (Choi, 2011).
During dorsal closure, Ena has an interesting localization pattern in epidermal cells. It localizes to AJs and is particularly enriched at tricellular junctions. It also localizes to ends of filopodia produced by LE cells. However, the most striking feature of Ena localization during this stage is its dramatic accumulation in 'LE dots', which form at the dorsal ends of the AJs between LE cells, where they overlay the amnioserosa. These overlap locations where the actomyosin cable is anchored. It was initially hypothesized that LE dots might play a role in cadherin-based cell adhesion, but this is not disrupted in ena mutants. Reducing Ena function does reduce filopodia, which is suspected to underlie defects in zippering of the epidermal sheets. The role of Ena in LE dots is less clear. It is speculated that LE dots are Ena storage places, from which it is released to modulate cell protrusions at the leading edge. Consistent with this, activation of the formin Diaphanous leads to loss of Ena from LE dots and dramatic alterations in protrusive behavior. However, the defects seen in LE cell shape in ena mutants are also consistent with the idea that Ena plays a role in anchoring or maintaining the actin cable at the leading edge (Choi, 2011).
Clear alterations were seen in Ena localization in LE cells in pydMZ or cno mutants. Enrichment of Ena in LE dots was reduced overall and became very uneven. It is tempting to speculate that the failure to effectively recruit Ena to LE dots leads to the defects in LE cell shape observed in pydMZ or cno mutants. If failure to deliver Ena to LE dots also interfered with subsequent release to the leading edge, this might alter protrusive behavior and slow zippering of the epidermal sheets at the canthi—this remains to be tested. To test the hypothesis that regulating Ena is an important part of the roles of Pyd and Cno during dorsal closure, genetic interactions were examined. Loss of zygotic Ena has only a subtle effect on epidermal morphogenesis, as the maternal Ena suffices for most events. However, reduction of maternal/zygotic Ena significantly enhanced the epidermal phenotype of zygotic cno mutants, and reduction of maternal/zygotic Cno enhanced the epidermal phenotype of zygotic ena mutants, consistent with them working together during this process; it is important to note that in both zygotic mutants maternal Ena or Cno remains, so enhancement is a plausible prediction for double mutants of genes in the same pathway. It will be interesting to further explore this mechanistic connection, probing whether Ena physically interacts with either Cno or Pyd and how they regulate Ena localization and/or activity (Choi, 2011).
In wild type, four transcripts (5.6, 6.7, 8.1 and 9.5kb) are detected in embryos but are only very slightly detected in adult flies, whereas a 10.5-kb additional transcript is also detected in third-instar larvae and pupae. In tam1 mutants, in addition to these five transcripts, two larger transcipts (11 kb and 13 kb) are observed in embryos, third-instar larvae, and pupae (Takahisa, 1996).
Bases in 5' UTR - 319
Exons - 13
Bases in 3' UTR - 439
Polychaetoid is highly homologous to the mammalian junction-associated protein ZO-1, found in mice and humans; Pyd is slightly less homologous to human ZO-2. ZO-1 and ZO-2 are members of the MAGUK family (see Drosophila Discs large). Like ZO-1, Polychaetoid has three discs-large homologous regions (DHR); an src oncogene homology region 3 (SH3) domain; a guanylate kinase (GUK) domain, and a proline-rich (17%) carboxyl terminal region of 320 amino acids. The fact that the GUK domain of Pyd, like that of ZO-1 and ZO-2, has both deletions and nonconserved substitutions in residues thought to be essential for yeast GUK to bind the substrates ATPO and GMP suggests that Pyd does not have GUK activity. The putative leucine zipper motif conserved in the GUK domains of ZO-1 and ZO-2 is also conserved in Pyd, except for the substitution of the third leucine with isoleucine. DHR1 and DHR3 are homologous to the corresponding motifs of Drosophila Discs large and other members of the MAGUK family. The DHR2, SH3 and GUK domains show significant homology with ZO-1 and ZO-2 (Takahisa, 1996).
date revised: 15 February 98
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