InteractiveFly: GeneBrief

canoe : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

canoe(cno) has a dual character, interacting as it does with genes in both the Notch and the Ras pathways. In the Notch pathway, canoe interacts with the Notch pathway genes Notch and scabrous. Cno protein and its mammalian homolog AF-6 have also been shown to physically interact with Ras. The cno locus was defined by Jurgens (1984). Extra machrochaetes (sensory bristles) are occasionally seen in the head, notum and scutellum, and some mutants show variable but conspicous wing phenotypes such as a notched blade and the loss of a cross vein. In the eye, clones of mutant photoreceptor cells are devoid of any ommatidia, while small mutant patches have reduced numbers of photoreceptors. Ommatidia of mutant pupae often contain extra primary pigment cells. Some of these ommatidia lack a secondary pigment cell, resulting in the apparent fusion of adjacent facets (Miyamoto, 1995).

Canoe and the Notch pathway

Fused ommatidia have been reported to occur in scabrous mutants; sca mutants are also accompanied by bristle abnormalities. Although the compound eye of sca1 homozygotes contains some fused facets and others with excess or reduced numbers of photoreceptors, the majority of ommatidia have a normal complement of eight photoreceptors. When sca1 homozygotes are also heterozygous for the mutant allele cnomis1, the phenotype is dramatically enhanced: in such cases, many ommatidia are fused to one another. The eyes of double mutants for sca1 and cnomis1 are extremely rough, accompanied by giant ommatidia, formed as a result of fusion. The bristle phenotype of sca1 is also enhanced by cnomis1. Double mutant flies show supernumerary bristles. There is a genetic interaction between the two loci, also revealed by an examination of wing morphology. Double mutants have rumpled wings curved downward, with occasional shortening of the anterior cross vein. cno likewise interacts 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 cnomis1 (Miyamoto, 1995).

Additonal interactions occur between cno and shaggy, armadillo, and myospheroid. It is intriguing that the genes encoding proteins involved in adhesive cell-cell communications are shown to interact with cno. Proteins coded for by these genes mediate the adhesion of the different cell layers in a variety of developing tissues. It is conceivable that Cno is localized to junctional complexes (Miyamoto, 1995).

Canoe and the Ras pathway

Just when it seemed that Canoe was being understood in terms of its interaction with the Notch pathway, it was also becoming clear that Canoe and its mammalian homolog AF-6 physically interact with Ras (Kuriyama, 1996). Morphological studies of the fly's compound eye suggest that induction of photoreceptors, including the R7 photoreceptor, is unaffected by cno mutations. The linear alignment of the R7 photoreceptor is disrupted in the pupal retina of cno mutants, but without exception a single R7 photoreceptor is formed in each ommatidium in cno mutant retinae (Matsuo, 1997). Since Ras signaling is involved in R7 fate determination (see Sevenless), what exactly is the role of Canoe in eye development?

It has been suggested that R7 photoreceptors occupy unusual positions in pupal discs because the mutant discs fail to form a correct mesh of pigment cells, which shape ommatidia into regular hexagonal structures. A strong mutation in cno eliminates from one to three cone cells from most ommatidia. In contrast, overexpression of cno+ results in the formation of supernumerary cone cells. These results indicate that an increase in cno+ expression promotes cone cell induction and a significant decrease in cno+ expression prevents cone cell precursors from differentiating into cone cells (Matsuo, 1997).

The involvement of Ras in non-neural retinal cell formation has attracted little attention. Freeman (1996) has provided evidence that the EGF receptor is required for the development of all retinal cells, including cone cells and pigment cells. A dominant negative form of EGF receptor prevents cell differentiation, whereas a secreted form of Spitz, the EGF receptor ligand, causes differentiation of supernumerary cells when expressed in the R7 equivalence group cells (cells from which arise a single R7 photoreceptor). Expression of a constitutively active form of Ras1, an important component in the EGF receptor signal transduction, causes a strong rough-eye phenotype. The rough-eye phenotype of active Ras is dramatically enhanced in cno heterozygotes, even though cno mutation is completely recessive and flies heterozygous for cno exhibit no specific phenotype. Overexpression of cno+ in R7 equivalence group cells similarly enhances roughness of constitutively active Ras1, without increasing the number of R7 cells beyond the level found without cno+ overexpression. Since the cno mutations interfere with cone cell formation without affecting photoreceptor induction, it seems plausible that Cno interacts with Ras1 in the developing cone cells (Matsuo, 1997).

These observations raise a question: from what cells do the extra cone cells originate? Two possibilities are considered: (1) they originate from retinal precursors overproduced due to accelerated proliferation and/or (2) they are generated at the expense of pigment cells that differentiate after cone cells, and from cells that are fated to die by apoptosis. Since proliferative activity in third instar larval eye discs is indistinguishable in mutant discs, it is likely that supernumerary cone cells are recruited from a pool of retinal precursors that normally develop into pigment cells and/or from cells that would otherwise be eliminated by apoptosis. It is thought that Cno acts in oppositon to Ras1, but the exact mechanism is not known. Nevertheless, an attractive model is that direct binding of Cno to Ras1 occludes the effector domain of Ras, which otherwise provides the binding site for Raf and consequent transduction of the Ras signal. This hypothesis is based on the results of an in vitro experiment demonstrating that mammalian cRas-1 competes with AF-6 ( a mammalian homolog of Cno) in binding to the H-Ras effector domain (Kuriyama, 1996). The direct binding of Cno to Drosophila Ras1 has been confirmed to occur in yeast, by two-hybrid assay. However, the hypothesis cannot be used to satisfactorily explain why overexpression of cno+ results in enhancement, rather than inhibition, of constitutively active Ras action in cone cell formation (Matsuo, 1997).

An alternative to either of these two models is suggested. It is postulated that there are two counteracting signaling pathways in which Cno participates: one for promoting the induction of cone cells (via Ras) and another for repressing this induction. It is postulated that Cno acts as a negative regulator for both pathways. Cno predominately associates with the repressing pathway when Ras activation is minimal, where absence of Cno causes disinhibition of the repression of the cone cell formation. With maximal Ras activation, extra cone cells form because the extremely strong activation of Ras1 overrides the inhibition by Cno and the action of the repressing pathway. Under such conditions, reduction of the Cno levels results in disinhibition of the promoting pathway, leading to enhancement of extra cone cell formation (Matsuo, 1997).

It is worth noting that reduction in Notch activity results in either an increase or a decrease in the number of cone cells, depending on the timing of Notch inactivation. Thus the Notch action is likewise 'bidirectional' in cone cell formation, as found for Cno action. In this context, it is of interest to note that some components of the EGF receptor pathway display strong genetic interactions with the Notch group of genes in eye, wing and leg morphogenesis. Cno is a reasonable candidate for a molecule that mediates such crosstalk between the Notch pathway and the EGF receptor pathway (Matsuo, 1997).

The PDZ protein Canoe regulates the asymmetric division of Drosophila neuroblasts and muscle progenitors

Asymmetric cell division is a conserved mechanism to generate cellular diversity during animal development and a key process in cancer and stem cell biology. Despite the increasing number of proteins characterized, the complex network of proteins interactions established during asymmetric cell division is still poorly understood. This suggests that additional components must be contributing to orchestrate all the events underlying this tightly modulated process. The PDZ protein Canoe (Cno) and its mammalian counterparts AF-6 and Afadin are critical to regulate intracellular signaling and to organize cell junctions throughout development. Cno functions as a new effector of the apical proteins Inscuteable (Insc)-Partner of Inscuteable (Pins)-Gαi during the asymmetric division of Drosophila neuroblasts (NBs). Cno localizes apically in metaphase NBs and coimmnunoprecipitates with Pins in vivo. Furthermore, Cno functionally interacts with the apical proteins Insc, Gαi, and Mushroom body defect (Mud) to generate correct neuronal lineages. Failures in muscle and heart lineages are also detected in cno mutant embryos. These results strongly support a new function for Cno regulating key processes during asymmetric NB division: the localization of cell-fate determinants, the orientation of the mitotic spindle, and the generation of unequal-sized daughter cells (Speicher, 2008).

NBs delaminate from the neuroectoderm inheriting the apicobasal polarity of the neuroectodermal cells, in which the PDZ proteins Bazooka (Baz)/Par-3 and DmPar-6 and the kinase DaPKC localize apicolaterally. After delamination, NBs maintain the apical localization of Baz/DmPar-6/DaPKC. The cytoplasmic PDZ protein Cno localizes at the adherens junctions of some epithelial cells, and it was asked whether Cno was also present in the neuroectoderm and in the delaminated NBs. Double immunofluorescences with antibodies against Cno and Baz showed that these proteins colocalize both apicolaterally at the adherens junctions of neuroepithelial cells and apically in the delaminated metaphase NBs (mNBs). At later phases of the NB division, Cno was no longer detected (Speicher, 2008).

Apical proteins, such as Baz/Par-3, are critically involved in regulating cell-fate determinants localization and spindle orientation at metaphase. Given that Cno was detected in an apical crescent in mNBs, it was asked whether Cno was also required for modulating those events. In control embryos, the cell-fate determinant Numb was basally located in 95.4% of mNBs. In cno² zygotic mutants, Numb was uniform or undetectable or was present in nonbasal crescents in 47.9% of the mNBs analyzed. cno² has been defined as the strongest allele of cno, although the particular lesion associated is unknown. However, cno² is probably a null allele because cno² over the Df(3R)6-7 (covering the cno gene) showed a similar percent of Numb localization failures. Additionally, cno³, another strong allele of cno considered as a null displayed defects in Numb localization in comparable cases. The basal distribution of the scaffolding protein Miranda (Mira) was also altered in 16.9% of mNBs of cno² mutants. Indeed, the localization of two Mira cargo proteins, the cell-fate determinants Prospero (Pros) and Brain Tumor (Brat), was affected in mNBs. The variable penetrance of the cno² mutant phenotype observed for the different proteins analyzed may reflect, at least in part, the different sensitivity of the antibodies used (Speicher, 2008).

Intriguingly, the orientation of the mitotic spindle in mNBs of cno² mutants was randomized in 18.3% of the cases. In control embryos, the spindle is tightly aligned with the center of Numb crescents in mNBs. In cno² mutants, the spindle was uncoupled with the Numb crescent in 7.7% of the mNBs that showed these crescents (either basal or at other incorrect localizations). The maternal contribution of cno might reduce the penetrance of these phenotypes (Speicher, 2008).

The overexpression of Cno also caused Numb localization failures (45.8%) and aberrant spindle orientations (39%) in mNBs. Hence, the results showed that Cno regulates essential processes during asymmetric NB division: the basal localization of cell-fate determinants and the proper orientation of the mitotic spindle (Speicher, 2008).

Another characteristic feature of asymmetric NB division is the different cell size of the progeny. Hence, whether Cno was also regulating this process was analyzed. Control telophase NBs (tNBs) showed unequal-sized daughter cells in 100% of the cases analyzed. In cno² mutants, equal-size divisions were observed in 21.3% of tNBs. Two redundant pathways, Baz/DaPKC/Insc and Pins-Gαi, regulate cell size and mitotic-spindle asymmetry at the NB apical pole . Only when both pathways are compromised is the different size of the daughter cells affected. The data suggested that Cno functions downstream of Gαi. Thus, Cno might belong to the Pins-Gαi pathway. Indeed, when both insc and cno were eliminated, 85.2% of tNBs showed equal-sized daughter cells, a much more penetrant phenotype than those displayed by each single mutant. Moreover, ΔGαi, cno² double mutants showed a much lower percentage of equal-sized divisions (30.4%) than the insc^P49; cno² double mutants. Hence, these results strongly suggest that Cno participates within the Pins-Gαi pathway to regulate NB progeny size (Speicher, 2008).

Given the defects observed in cno² mutant embryos during NBs division, it was asked whether neuronal lineages were altered in cno² mutants. The lineage of the ganglion mother cell (GMC) 4-2a has been extensively studied. This GMC expresses the transcription factor Even-Skipped (Eve) and divides asymmetrically to give rise to two different neurons called RP2 and RP2 sibling. Both maintain the expression of Eve initially; however, at later stages of embryogenesis, only the RP2 neuron keeps expressing Eve . In control embryos, 0.9% of the segments analyzed showed defects in the number of RP2s. In cno² mutants, two or no RP2s were detected per hemisegment in 5.7% of the segments analyzed. Such a result suggested failures in the GMC 4-2a asymmetric division. This phenotype was also observed in cno^mis1 hypomorph mutants (4.6%) as well as in mutants for genes that are critical during asymmetric cell division. For example, homozygotes for DaPKC^k06403, insc^P49, ΔGαi, and mud⁴ (zygotic null mutant embryos) showed defects in the GMC 4-2a lineage in 6.4%, 13.8%, 2.5%, and 8.3% of the segments analyzed, respectively. Hence, it was next investigated whether Cno was interacting with these proteins to properly generate the GMC 4-2a neuronal lineage. Double heterozygotes DaPKC^k06403/+; cno²/+ showed defective RP2 number in 0.8% of segments. This result is consistent with a lack of functional interactions between DaPKC and Cno. However, double heterozygotes insc^P49/+; cno²/+ and ΔGαi, +/+, cno² showed an altered RP2 lineage in 14.4% and 7.6% of the segments analyzed. In addition to the analysis of double heterozygotes, it was found that the cno^mis1 phenotype was significantly enhanced in a mud⁴ zygotic null mutant background. Altogether, these results indicated that Cno functionally interacts with the apical proteins Insc, Gαi, and Mud during the asymmetric cell divisions that generate specific neuronal lineages in the CNS (Speicher, 2008).

Since Cno functionally interacts with Insc, Gαi, and Mud, the epistatic relationships between them were analyzed. To investigate whether Cno was acting upstream of the apical proteins, the localization of Baz, Insc, and Gαi was examined in cno² mutant embryos. The distribution of all these proteins was normal in cno² mutants. This result suggested that Cno acts either downstream or in parallel to Baz, Insc, and Gαi. To clarify this point, the distribution of Cno was analyzed in loss- and gain-of-function (lof and gof) mutants for several apical proteins. In insc^P49 lof mutants, Cno was untraceable or showed a wrong orientation in 78.8% of the mNBs analyzed. Insc overexpression also caused failures in Cno localization (76%); Cno was either undetectable (13/21) or present in not-apical crescents (3/21). Likewise, in Gβ13F maternal and zygotic null mutant embryos, in which Gαi is lost, Cno was mislocalized or undetectable in 94% of the mNBs. Moreover, the overexpression of Gαi caused a striking mislocalization of Cno in 100% of the mNBs analyzed. The NuMA-related protein Mud binds the apical protein Pins and functions downstream of Pins-Gαi to regulate spindle orientation. In mud mutant NBs, the spindle fails to tightly align with the basal crescent, and this failure is also shown by cno² mutant NBs. Additionally, Cno and Mud interacted genetically. Hence, it was asked whether Cno functions along with Mud to regulate spindle orientation. In control embryos, Mud localized at the apical cortex of mNBs (97%) and at the two centrosomal regions (100%). In cno² lof mutants, Mud failed to accumulate apically in 49% of mNBs, and 15% of NBs showed Mud localization in one or none of the two centrosomes. cno gof also caused failures in Mud localization (38%). Altogether, these results strongly support a function of Cno downstream of Insc and Pins-Gαi and upstream of Mud during asymmetric NB division (Speicher, 2008).

Given the functional relationships found between Cno and apical proteins during asymmetric NB division, it was asked whether Cno was physically interacting with some of these proteins. Coimmunoprecipitation experiments from Drosophila embryo extracts showed that Cno is forming a complex with Pins. Cno did not physically interact with DmPar6, Baz, DaPKC, or other apical proteins tested such as Insc, Gαi, and Mud (Speicher, 2008).

Pins also forms a complex in the delaminated metaphase NBs with the tumor-suppressor protein Discs Large (Dlg) and the kinesin Khc-73, an astral microtubule-binding protein. First, at prophase, the DmPar6/Insc pathway is required to polarize Pins/Gαi at the apical pole of the NB. Then, at metaphase, the Pins/Gαi/Dlg/Khc-73 complex forms, and it is key for tightly coupling cortical polarity with spindle orientation. Hence, it was asked whether Cno was also forming part of this complex. Experiments showed that neither Dlg nor Khc-73 coimmunoprecipitate Cno in embryo extracts. This result indicated that Cno is not forming part of the Dlg/Khc-73 complex (Speicher, 2008).

Altogether, a working network of protein interactions is proposed. Analysis of epistatic relationships between apical proteins and Cno showed that Cno is acting downstream of Insc-Pins-Gαi and upstream of Mud. Indeed, genetic analysis suggests that, at least for the control of daughter cells size asymmetry, Cno functions within the Pins-Gαi pathway, in parallel to the DaPKC-Baz-Insc pathway. Accordingly, Cno was found to form a complex with Pins in vivo. Cno did not coimmunoprecipitate with Gαi, though. One possibility is that Cno and Gαi are mutually exclusive in the complex that each of them forms with Pins. Additionally, transient or labile interactions between Cno and Gαi may occur that were not possible to detect. Another Pins interacting partner, the microtubule-binding protein Mud contributes to coordinate spindle orientation with cortical polarity. Given the functional relationships that were found between Cno and Mud, Cno could act in a complex with Pins to modulate Mud localization and, consequently, spindle orientation (Speicher, 2008).

Finally, it was asked whether the function of Cno during asymmetric cell division was conserved in different tissues. Since the NBs of the CNS, the Drosophila somatic muscle and heart progenitors divide asymmetrically to give rise to two different founder cells. Cno is present in the somatic mesoderm and is required for muscle and heart progenitor specification. Hence, it was aked whether Cno was also functioning during the asymmetric division of muscle and heart progenitors. For this analysis, focus was placed on two dorsal progenitors called P2 and P15 that express the transcription factor Eve and whose lineages have been characterized in detail. In this study, it was found that the transcription factor Seven-up (Svp), a characteristic marker of a subset of cardial cells, was expressed in a dorsal founder cell of unknown identity until now, which is here named founder of Svp cardial cells (FSvpCs). With all these markers, specific for individual derivatives, whether dorsal muscle and cardial lineages were altered in cno² mutants was analyzed. It was found that at late stages (stage 14), 3.1% of hemisegments (n = 96) showed simultaneously either loss of EPCs and gain of DO2 muscle or gain of EPCs and loss of the DO2 muscle (P2 lineage). In control embryos, this phenotype was not observed in any of the hemisegments analyzed. Indeed, Numb localization, which was basal in 100% of the metaphase P2s analyzed in control embryos, was altered in 93% of metaphase P2s in cno² mutants. Hemisegments showing duplication of DA1 muscle and loss of SvpCs or DA1 muscle loss and gain of SvpCs (P15 lineage) were also detected in cno² mutants. Hence, Cno was required for the asymmetric division of progenitor cells both in the CNS and in the mesoderm (Speicher, 2008).

In conclusion, the discovery of new modulators of asymmetric cell division, as described in this study, for the PDZ protein Cno, is key to complete understanding of this intricate process. Especially challenging in the future will be unraveling the complete network of connections between all the players required for an accurate asymmetric cell division (Speicher, 2008).

Rap1 acts via multiple mechanisms to position Canoe and adherens junctions and mediate apical-basal polarity establishment

Epithelial apical-basal polarity drives assembly and function of most animal tissues. Polarity initiation requires cell-cell adherens junction assembly at the apical-basolateral boundary. Defining the mechanisms underlying polarity establishment remains a key issue. Drosophila embryos provide an ideal model, as 6000 polarized cells assemble simultaneously. Current data place the actin-junctional linker Canoe (fly homolog of Afadin) at the top of the polarity hierarchy, where it directs Bazooka/Par3 and adherens junction positioning. This study defines mechanisms regulating Canoe localization/function. Spatial organization of Canoe is multifaceted, involving membrane localization, recruitment to nascent junctions and macromolecular assembly at tricellular junctions. The data suggest apical activation of the small GTPase Rap1 regulates all three events, but support multiple modes of regulation. The Rap1GEF Dizzy (PDZ-GEF) is crucial for Canoe tricellular junction enrichment but not apical retention. The Rap1-interacting RA domains of Canoe mediate adherens junction and tricellular junction recruitment but are dispensable for membrane localization. Our data also support a role for Canoe multimerization. These multifactorial inputs shape Canoe localization, correct Bazooka and adherens junction positioning, and thus apical-basal polarity. This study integrates the existing data into a new polarity establishment model (Bonello, 2018).

Apical-basal polarity establishment is a key step in animal development, driving the assembly of epithelial tissues and organs and creating the architecture that enables morphogenetic movements. Early fly embryos provide a premier model of polarity establishment. Work from many labs has defined the assembly and apical positioning of cell-cell AJs as the key initial step. Efforts then focused on defining molecular and cellular mechanisms underlying this, revealing key roles for Baz/Par3 and, more recently, the junctional actin crosslinker Cno and its upstream activator Rap1. This refocused attention on the next level of mechanistic analysis: how does Rap1 regulate Cno localization and do Rap1-independent cues also play a role? Apical Rap1 activity promotes Cno junctional recruitment at the top of the polarity hierarchy (Bonello, 2018).

The current model of apical-basal polarity establishment during fly embryogenesis suggests the key upstream step is positioning Cno at the site where AJs will form. The small GTPase Rap1 and F-actin play roles in Cno positioning. However, the mechanism by which Rap1 acts remained unclear, as Rap1 localizes all along the invaginating membrane. The new data support a model in which apical Rap1 activity plays a key role. The ability of GTP-locked constitutively active Rap1 to recruit Cno all along the basolateral membrane and the loss of Cno from the membrane induced by GDP-locked Rap1 are both consistent with this model. In the future, it would be valuable to design a Rap1 activity sensor to confirm this hypothesis. As is discussed below, it was intriguing that although the Cno RA domain plays a role in apical Cno recruitment/retention, Rap1 also influences localization of a cno mutant lacking the RA domain. Finally, it was of interest that both the GDP- and GTP-locked Rap1 mutants altered Baz localization, raising the possibility that Baz localization requires cycling of Rap1 between the active and inactive states (Bonello, 2018).

The next task was to define Rap1 activation mechanisms. Rap1 has many GEFs, including C3G, Epac, CalDAGGEF1 and PDZ-GEF. Dzy (PDZ-GEF) was the most likely candidate as it has a role 30 min later in mesoderm apical constriction, which also requires Rap1 and Cno. Strikingly, although the analyses suggest Dzy regulates Cno in later morphogenetic events such as germband extension, junctional planar polarization and segmental groove retraction, maternal/zygotic Dzy loss did not fully mimic effects of Rap1 loss on polarity establishment. Dzy loss replicated only a subset of these effects, as cortical Cno recruitment and apical restriction were unaffected. Instead, Dzy loss specifically affected Cno enrichment at TCJs, and also led to defects in columnar cell shape like those caused by Rap1 loss. Based on this, it is hypothesized that multiple GEFs regulate Rap1 activity, each directing specific aspects of cellularization and polarity establishment. Each might create temporally or spatially restricted pools of active Rap1, with different Rap1 pools mediating different effects on Cno localization. In the accompanying paper, Schmidt (2018) describes a role for the unconventional GEF ELMO-Sponge complex in regulating initial apical positioning of Cno, consistent with this hypothesis. In the future, it will be important to investigate other Rap1GEFs, such as C3G. The cortical and apical localization of Dzy that was observed during cellularization and early gastrulation are consistent with the idea that localized Dzy provides a direct input into Rap1 activation. The data also suggest that in later events in embryogenesis, Dzy and Cno work together in many events with Rap1, but that Rap1 is also likely to regulate events where it has other activators and effectors. Another alternative is apical restriction of Rap1 activation via basolateral Rap1GAPs, as occurs at other times, and this will be important to explore. The RA domain plays important roles in Cno localization and activity but CnoΔRA retains significant function (Bonello, 2018).

The N-terminus of Cno carries two RA domains that bind Rap1. Given the essential role of Rap1 in Cno localization and function, it is suspected that the RA domains would be similarly essential. Two hypotheses were tested for the mechanism by which Rap1 could act via the RA domains to regulate Cno function during polarity establishment: (1) Rap1-GTP binding to the RA domain opens a closed, autoinhibited conformation; or (2) Rap1-GTP binding to the RA domain physically recruits Cno to sites where AJs will be assembled. The data essentially rules out the first hypothesis, as it predicted that CnoΔRA recruitment to nascent AJs would be Rap1 independent. The data are largely consistent with the second hypothesis, although the effect of Rap1 on CnoΔRA localization revealed that the Rap1-RA domain interaction is not the only means of regulation (Bonello, 2018).

The data also suggest that after gastrulation onset Rap1-independent mechanisms of Cno localization come into play, as cortical localization of both wild-type Cno and CnoΔRA begin to be restored at that stage in Rap1 knockdown embryos. Although some residual Rap1 might remain, this is thought to be unlikely as it was not detected by immunoblotting at stages when Cno localization is restored. Cno can localize to the cortex during dorsal closure in embryos expressing GDP-locked Rap1, also supporting Rap1-independent mechanisms. One potential mechanism of post-gastrulation Cno recruitment is via its known interactions with AJ proteins. Before AJs assemble, Rap1 might be essential for cortical Cno recruitment, but once AJs reappear after gastrulation onset then interactions between Cno and α-Catenin or DE-cadherin might restore Cno recruitment to AJs. Both CnoWT and CnoΔRA also retained significant function in Rap1 knockdown embryos, suggesting that Cno has Rap1-independent activity, at least when expressed at elevated levels. CnoΔRA was significantly less effective than CnoWT, confirming and extending earlier work during dorsal closure. However, CnoΔRA retained significant function in Cno knockdown embryos, suggesting that the RA domain is not absolutely essential. It might facilitate some Cno/Afadin activities; for example, the RA domain of mammalian Afadin regulates interactions with p120-catenin and modulates E-cadherin endocytosis. Cno may act as a coincidence detector, with multiple simultaneous inputs regulating its positioning and that of nascent AJs (Bonello, 2018).

Several observations support the hypothesis that Cno localization responds to multiple inputs. Rap1 activity plays a key role, as during cellularization Cno cannot localize to the membrane in the absence of Rap1 or when its ability to load GTP is compromised. It seems likely that direct interactions between Rap1 and the Cno RA domain help regulate Cno localization. However, since CnoΔRA still requires Rap1 to localize correctly, this suggests additional complexity. A second Rap1 interaction site might exist in Cno outside the RA domain. Alternately, other Rap1 effectors might regulate Cno localization by different mechanisms. During cellularization, Rap1 has Cno-independent effects on apical contractility and thus columnar cell shape. Perhaps this postulated effector alters the actomyosin cytoskeleton in a way that promotes Cno binding, since intact actin is required for Cno cortical localization. However, Cno does not simply colocalize with actin or myosin, as both are most enriched at the leading edge of the invaginating membrane. Perhaps there is an apical pool of actin in a particular conformation or with particular binding partners that allow Cno to 'choose' the correct localization. Consistent with an important role for the Cno C-terminal actin-binding domain and/or intrinsically disordered linker in localization, CnoFHA-PDZ did not localize cortically in wild-type embryos, unlike CnoΔRA. Together, these data suggest a multifactorial recruitment mechanism (Bonello, 2018).

Drosophila embryos afford unmatched temporal resolution, allowing AJ morphogenesis to be followed both in the context of apical-basal polarity establishment and later in polarity maintenance. This complex process begins with the appearance of cadherin-catenin clusters, which arise independently of a Baz polarization cue. It continues through formation of mature SAJs, which coordinate with the contractile cytoskeleton to enable the first morphogenetic movements of gastrulation. These events are now viewed as involving two independent but interlocked processes: apical restriction of Cno and AJ proteins, and their assembly into higher-order multiprotein complexes. Integrating these data with previous analyses prompts the following model. Baz helps ensure that small cadherin-catenin complexes present before cellularization are recruited/retained at an apicolateral position and assembled into larger complexes containing over 1000 cadherin molecules. Cno plays an important role, helping retain Baz at the apicolateral site of SAJ assembly. The new data suggest that Rap1 activity guides two important aspects of AJ morphogenesis -- AJ protein recruitment/retention at the apicolateral interface and the specialized assembly of larger macromolecular complexes at TCJs. These data further suggest that these two events require at least two spatial cues directed by active Rap1, one acting via the Cno RA domains and one independent of that. The results with the Rap1GEF Dzy suggest these two events have different modes of regulation. Finally, the data are consistent with a Cno-driven, self-reinforcing feedback loop in which correct Cno localization can recruit more Cno to the membrane. Cno clustering is particularly prominent at TCJs. Intriguingly, recent work in mammalian cells suggests that actomyosin cables anchor end-on at TCJs and Afadin acts there to maintain trapezoidal cell shapes. Whether Cno oligomerization is intrinsic to Cno itself or is mediated by other partners is an important question for future work. These data also have potential implications for the roles of mammalian Afadin in epithelial polarity in the kidney and intestine, another avenue for further research (Bonello, 2018).

Rap1, canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor

In Drosophila epithelial cells, apical exclusion of Bazooka/Par3 defines the position of the Zonula Adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. This study shows that the small GTPase Rap1, its effector AF6/Canoe (Cno) and the Cdc42-effector Pak4/Mushroom bodies tiny (Mbt), converge in regulating epithelial E-Cadherin, and Bazooka retention at the ZA. Furthermore, the results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating their functions during ZA morphogenesis are interlinked. In this context, the Rap1-GEF Dizzy was found to be enriched at the ZA and the results suggest it promotes Rap1 activity during ZA morphogenesis. Altogether, it is proposed the Dizzy, Rap1/Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis (Walther, 2018).

In the pupal photoreceptor, ZA morphogenesis is orchestrated by a conserved protein network that includes Cdc42, Par6, aPKC, Baz, Crb and its binding partner Sdt, and Par1. In turn, AJ material is an essential part of the regulatory network that orchestrates polarity. Previous work has shown that Mbt regulates pupal photoreceptor development by promoting ZA morphogenesis. During this process Mbt contributes in preventing Baz from spreading to the lateral membrane, a regulation that this study found to depend in part on the phosphorylation of Arm by Mbt at S561 and S688. It is proposed that Mbt regulates photoreceptor polarity by promoting the retention of Baz at the developing ZA. Failure in ZA retention leads to Baz spreading to the lateral membrane where it is eliminated through Par1-mediated displacement. In these cells, failure to retain AJ material, including Baz, at the ZA leads to its shortening along the apical basal axis and can impact on the polarization program of the photoreceptor (Walther, 2018).

This study shows that Mbt function is linked to that of Dzy, Rap1 and Cno. First, Cno and Mbt accumulation at the ZA is interdependent, reflecting a tight coupling between the Rap1 and Cno pathway and Mbt. Second, it was found that Cno promotes Baz retention at the ZA, as cnoIR leads to shorter ZAs that can be depleted of Arm and Baz. This phenotype resembles that of mbt mutant cells and is also seen when overexpressing a version of Arm that cannot be phosphorylated by Mbt. These observations prompted a test or the hypothesis that Rap1, Cno and Mbt might function as part of a linear pathway promoting Baz retention at the ZA. In this pathway, it was reasoned that Mbt could mediate Rap1 function through Arm phosphorylation. In testing this hypothesis, it was found that this is not the case. Instead, the observation that expressing a version of Arm that mimics its constitutive phosphorylation by Mbt does not ameliorate the cnoIR phenotype suggests that Rap1, Cno, and Mbt converge in promoting Baz retention at the ZA, and cannot compensate for each other during this process. This conclusion is well supported by the finding that overexpressing cno in mbt mutant cells does not lead to an amelioration of the mbt phenotype. Third, it was found that Mbt influences the distribution of Rap1 along the apical-basal axis of the cell in that Rap1::GFP no longer accumulates preferentially at the ZA. This correlates with a loss of Dzy::GFP at the plasma membrane, raising the possibility that Mbt might regulate Rap1 through Dzy. However, the dzy phenotype is milder than that seen with Rap1 or cno, in that loss of dzy does not lead to cell delamination from the retina. This suggests that, as has been reported in the cellularizing embryo, other GEFs regulate Rap1 during epithelial morphogenesis (Walther, 2018).

An interesting aspect of the cnoIR phenotype is the defects in apical accumulation of aPKC and Crb. These defects are not observed in the dzy mutant or Rap1IR cells, indicating that Cno might function independently of Rap1 during this process. However, it is noted that while Cno was not detected at the ZA of cnoIR cells, it can still be detected in Rap1IR cells. It is therefore hypothesized that residual Cno in Rap1IR cells supports optimum aPKC and Crb accumulation at the apical membrane. In this model, Dzy, Rap1 and Cno function as part of the same pathway, which includes a function in promoting optimum apical accumulation of Crb and aPKC. Baz is required for Par complex assembly and associated aPKC and Crb recruitment at photoreceptor apical membrane. It is hypothesized that the defects in Crb and aPKC that were detect in cnoIR cells are linked to the failure in retaining Baz at the ZA, which leads to its elimination from the lateral membrane by Par1. More work will be required to understand how exactly AJ material and ZA retention of Baz influences apical membrane specification (Walther, 2018).

Rap1 and cno have been shown to regulate apical-basal polarity in the cellularizing embryo. In this model system, Rap1 and Cno regulate the apical localization of Baz and Arm, which precedes the apical recruitment of Crb. In turn, Baz influences the localization of Cno. This work indicates that similar complex regulations are at play in the pupal photoreceptor. However, unlike in the early embryo, AJ material (Arm) is absolutely required for Baz (and Par6-aPKC) accumulation or retention at the cell cortex in the developing pupal photoreceptor. A model is therefore favored whereby Mbt, Rap1 and Cno influence ZA morphogenesis primarily through regulating the interface between E-Cad or Arm, Baz and the F-actin cytoskeleton. In this model, Mbt regulates this interface both through Arm phosphorylation and cofilin-dependent regulation of F-actin, and Cno contributes to this process, at least in part, through its ability to bind to F-actin (Walther, 2018).

To probe Rap1 and Cno function during photoreceptor ZA morphogenesis, the effect of decreasing Rap1 expression on E-Cad stability was assessed. Consistent with the notion that the function of mbt and Rap1 are linked during ZA morphogenesis, it as found that, as it is the case for Mbt , Rap1 is required to stabilize E-Cad::GFP at the photoreceptor ZA. However, the mobile fraction estimated for E-Cad is much higher in Rap1IR cells than in mbt^P1 null cells (evaluated at ~70% for Rap1IR and 45% for mbt^P1). Together with the finding that Mbt accumulation at the ZA is decreased in Rap1IR cells, FRAP data are therefore compatible with Mbt mediating part of the function of Rap1 in promoting E-Cad stability. However, the much larger mobile fraction were estimated in the Rap1IR genotype when compared to mbt^P1 photoreceptors indicates that Rap1 must also regulate E-Cad stability independently of Mbt. The longer time scale for E-Cad::GFP to recover in Rap1IR cells when compared to mbt^P1 mutant cells is compatible with Rap1 functioning, in part, through promoting E-Cad delivery (Walther, 2018).

Tissue tension and not interphase cell shape determines cell division orientation in the Drosophila follicular epithelium

This study investigated the cell behaviors that drive morphogenesis of the Drosophila follicular epithelium during expansion and elongation of early-stage egg chambers. Cell division was found not to be required for elongation of the early follicular epithelium, but drives the tissue toward optimal geometric packing. The orientation of cell divisions with respect to the planar tissue axis was examined, and a bias toward the primary direction of tissue expansion was found. However, interphase cell shapes demonstrate the opposite bias. Hertwig's rule, which holds that cell elongation determines division orientation, is therefore broken in this tissue. This observation cannot be explained by the anisotropic activity of the conserved Pins/Mud spindle-orienting machinery, which controls division orientation in the apical-basal axis and planar division orientation in other epithelial tissues. Rather, cortical tension at the apical surface translates into planar division orientation in a manner dependent on Canoe/Afadin, which links actomyosin-to-adherens junctions. These findings demonstrate that division orientation in different axes-apical-basal and planar-is controlled by distinct, independent mechanisms in a proliferating epithelium (Finegan, 2018).

The results demonstrate that planar cell division orientation in the FE is independent of Pins/Mud. Instead, it relies on tissue tension and Canoe, which links actomyosin-based cortical tension to the cell. These findings show that division orientation occurs independently in the apical-basal and planar tissue axes. Although this study has not investigated the effects of tension modulation or Cno disruption in the embryonic ectoderm, the data suggest that bimodal spindle orientation is common to immature epithelia (Finegan, 2018).

It is proposed that on the tissue scale, tension is oriented toward the elongation axis, but local anisotropies in the cortex, which could be stochastic or the consequence of cell behaviors, cause local differences in the tension exerted on individual cells. This is consistent with a model whereby division orientation is not dictated by an intrinsic genetic or developmental program, but is instead mediated by extrinsic physical factors. This bias in division orientation is a granular property of the epithelium; the division angle of any one cell cannot be predicted based on tissue-level tension, but a population bias exists such that cells will have bias to divide toward the axis of tissue elongation (Finegan, 2018).

This study raises the question of whether biphasic spindle orientation is special to the FE, or a broad feature of immature epithelia. In other tissues, cell shape reflects tissue-level tension and cell shape cues direct the orientation of the spindle to establish the cell division angle. It was previously observed that dividing cells can move apically, detaching from the basement membrane. A potential explanation for this movement is that it allows dividing cells to escape the migration force that drives cell shape. This raises the question of how, if not through cell shape, apical tension is communicated to the spindle-orienting machinery in the FE. This question will form the basis of future work (Finegan, 2018).

Mutations in the Drosophila tricellular junction protein M6 synergize with Ras(V12) to induce apical cell delamination and invasion

Complications from metastasis are responsible for the majority of cancer-related deaths. Despite the outsized medical impact of metastasis, remarkably little is known about one of the key early steps of metastasis: departure of a tumor cell from its originating tissue. It is well documented that cellular delamination in the basal direction can induce invasive behaviors, but it remains unknown if apical cell delamination can induce migration and invasion in a cancer context. To explore this feature of cancer progression, a genetic screen was performed in Drosophila, and mutations in the protein M6 were found to synergize with oncogenic Ras to drive invasion following apical delamination without crossing a basement membrane. Mechanistically, it was observed that M6-deficient Ras(V12) clones delaminate as a result of alterations in a Canoe-RhoA-myosin II axis that is necessary for both the delamination and invasion phenotypes. To uncover the cellular roles of M6, this study showed that it localizes to tricellular junctions in epithelial tissues where it is necessary for the structural integrity of multicellular contacts. This work provides evidence that apical delamination can precede invasion and highlights the important role that tricellular junction integrity can play in this process (Dunn, 2018).

Cells are known to delaminate from their tissues in both the apical and basal directions during development and in disease conditions. Importantly, cell delamination plays a vital role in cancer progression as it is one way that a cancer cell can escape its originating tissue before spreading to more distant sites. During tumor progression, different models have revealed that cell delamination in the apical direction can lead either to the elimination of the delaminated cells or to the overgrowth of those cells. However, invasive behaviors have not been observed to follow apical delamination but instead have been shown to occur only through basal delamination (Dunn, 2018).

During basal delamination-induced invasion, basement membrane degradation and cell invasion into the underlying tissue can be observed in fixed tissues. On the other hand, if cancer cells leave the tissue by migrating and invading following apical delamination, the invasion would not leave such a histologically visible trail as this invasion could occur without crossing the basement membrane but instead through migration along connected tissues. As such, alternate methods in a suitable system would be needed to recognize if apical delamination is able to induce invasion. Thus, although previous work has documented direct basal delamination and invasion during metastasis in animal models and human patients, it does not preclude the possibility that invasion can be initiated by apical delamination as well. Drosophila cancer models are well suited to address the role of apical delamination in inducing invasive behaviors due to their simple tissue architecture that allows for the easy identification of an apical delamination event, as well as established techniques to image intact living tissues over time to follow the fates of apically delaminated cells. This study documents that cell migration and invasion can be induced via apical delamination through the characterization of a tumor suppressor, M6, in Drosophila (Dunn, 2018).

While bicellular junctions have been well studied for their roles in tissue integrity and signaling, the importance of TCJs has been gradually coming to light in recent years as they have been shown to be key players in ionic barrier formation and maintenance, pathogen spread, and orientation of cell division. This study demonstrates that inactivation of a TCJ protein, M6, disrupts the structural integrity of multicellular contacts and induces apical delamination and invasion of otherwise benign RasV12 tumors in a manner dependent upon a Cno-RhoA-MyoII axis. This study thus provides a causative role for TCJ mutations in driving delamination and invasion in vivo, highlighting the importance of these junctions in tissue integrity and cancer biology (Dunn, 2018).

This study demonstrates a functional link between tricellular junctions and RhoA, which is a known cytoskeletal regulator. This finding adds to recent work that has begun suggesting that TCJs act as centers for cytoskeletal organization. It will be interesting to further learn the mechanisms and consequences of functionally linking RhoA and cytoskeletal components to TCJs. Additionally, RhoA is known to affect a variety of proteins and cellular processes in addition to Sqh. As such, it is highly possible that RhoA is inducing apical delamination and invasion through multiple routes in addition to its effects on sqh. Also, since Cno localizes at the adherens junctions, which are apical to M6, it is plausible that M6 only indirectly affects Cno, and thus RhoA, through alterations in epithelial integrity rather than through direct means (Dunn, 2018).

Finally, invasion of cancer cells into surrounding tissues was previously thought to occur only through direct basal delamination and subsequent invasion. This work shows that apical delamination can also precede migration and invasion to distant tissues. Furthermore, since no basement membrane degradation was observed in invasive RasV12; M6-/- clones, the invasion most likely occurs along connected tissues rather than through an apical delamination to the basal penetration route, but further experiments are needed to confirm this hypothesis. Although mammalian anatomy differs markedly from that of the simple architecture of the Drosophila imaginal discs, it will be interesting to learn if apical delamination, such as is observed in early stage human breast cancer, can also precede invasion in mammalian models. Further investigation into this paradigm of apical delamination-induced invasion could aid in understanding of the mechanisms underlying cancer progression and metastasis (Dunn, 2018).

Abl and Canoe/Afadin mediate mechanotransduction at tricellular junctions

Epithelial structure is generated by the dynamic reorganization of cells in response to mechanical forces. Adherens junctions transmit forces between cells, but how cells sense and respond to these forces in vivo is not well understood. This study identified a mechanotransduction pathway involving the Abl tyrosine kinase and Canoe/Afadin that stabilizes cell adhesion under tension at tricellular junctions in the Drosophila embryo. Canoe is recruited to tricellular junctions in response to actomyosin contractility, and this mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine in the Canoe actin-binding domain. Preventing Canoe tyrosine phosphorylation destabilizes tricellular adhesion, and anchoring Canoe at tricellular junctions independently of mechanical inputs aberrantly stabilizes adhesion, arresting cell rearrangement. These results identify a force-responsive mechanism that stabilizes tricellular adhesion under tension during epithelial remodeling (Yu, 2020).

This study shows that Canoe and Abl function in a mechanotransduction pathway that regulates dynamic changes in cell adhesion at tricellular junctions during epithelial remodeling. Canoe localization to tricellular junctions is acutely disrupted by laser ablation, demonstrating that Canoe localization is rapidly modulated by mechanical perturbation. Canoe mechanosensitivity requires Abl-dependent phosphorylation of a conserved tyrosine (Y1987) in the Canoe actin-binding domain, and Canoe localization to tricellular junctions is required to stabilize tricellular adhesion. Conversely, constitutively anchoring Canoe at tricellular junctions arrests cell rearrangement, indicating that Canoe levels can tune the rate of junctional remodeling. These results demonstrate that the mechanosensitivity of this critical junctional regulator is modulated by phosphorylation of a single tyrosine residue and reveal an essential role of Canoe in coupling tricellular adhesion with mechanical forces during epithelial remodeling (Yu, 2020).

Mechanical forces trigger a cascade of molecular events in cells that translate biophysical signals into altered cellular behaviors. However, the mechanosensors that directly change conformation under tension in vivo are not well defined. Cell surface receptors are well positioned to detect forces generated by neighboring cells, but Canoe localization and function at tricellular junctions do not require the PDZ domain that mediates its interaction with known receptors. One possibility is that the Canoe protein itself could act as a mechanosensor. Canoe is predicted to be anchored to the membrane through interactions with Rap1 and the actin cytoskeleton. Thus, cytoskeletal tension could stretch the Canoe protein and expose tyrosine 1987 to phosphorylation by Abl. Alternatively, Abl or its upstream activators could be regulated by tension during this process, because the activity of Abl and other tyrosine kinases such as Src and FAK has been shown to be regulated by tension in vitro. In a third possibility, the regulation of Canoe by Abl could allow Canoe to detect force-induced changes in other molecules at tricellular junctions. Because tyrosine 1987 is in the Canoe actin-binding domain, phosphorylation at this site could allow Canoe to recognize distinct actin structures at tricellular junctions, force-induced conformational changes in the cadherin-catenin complex, or other specialized features of tricellular junction composition or geometry. Once recruited to tricellular junctions, Canoe could stabilize adhesion at these sites by reinforcing the connection between adherens junctions and the actomyosin cytoskeleton (Yu, 2020).

Abl tyrosine kinases influence many structural changes that are driven by mechanical forces in tissues, including epithelial remodeling, tissue invagination, axon guidance, and cell migration. Abl has been shown to regulate a number of proteins that act at tension-bearing structures in cells in addition to Canoe/Afadin recruitment to tricellular junctions, including vinculin localization and β-catenin recycling at bicellular junctions and regulation of membrane curvature by BAR-domain proteins. Together, these results raise the possibility that Abl could transduce mechanical forces into a wide range of structural changes within cells. For example, during cell rearrangement, Abl could simultaneously stabilize tricellular adhesion by recruiting Canoe and destabilize bicellular adhesion by enhancing β-catenin turnover, which could allow bicellular junctions to complete contraction before tricellular junctions are remodeled. Tricellular junctions serve many important roles in epithelial development and homeostasis, including modulating cell rearrangement, orienting mitotic spindles, balancing stem cell proliferation and differentiation, and maintaining epithelial barrier function. An understanding of how mechanical inputs affect the conformation, localization, activity, and interactions of proteins at tricellular junctions will provide insight into how these structures sense and integrate mechanical forces in epithelial tissues (Yu, 2020).

REGULATION

Protein Interactions

A Ras-interacting protein with molecular mass of about 180 kDa (p180) was partially purified from bovine brain membrane extract by Ha-Ras (Drosophila homolog Ras85D) affinity column chromatography. This protein binds to a GTP Ha-Ras affinity column, but not to a column containing GDP-Ha-Ras. or to a column made with Ras that contains a mutation in the effector domain (Ha-RasA38). The amino acid sequences of the peptides derived from p180 are almost identical to those of human AF-6; AF-6 has been identified as the fusion partner of the ALL-1 protein. The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity to Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interact with GTP Ha-Ras. The known Ras target c-Raf-1 inhibits the interaction of AF-6 with GTP gamma Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras (Kuriyama, 1996). The direct interaction of Raf, phosphatidylinositol-3-OH kinase, Ral GDS and Rin1 with activated Ras has been demonstrated. There is no obvious homology with AF-6 or Canoe among the Ras-interacting interfaces of these proteins, indicating that activated Ras can recognize a variety of target interfaces (Kuriyama, 1996 and references). The interaction between AF-6 and Ras was also noted by Van Aelst (1994).

canoe and polychaetoid 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. cno^mis1 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 cno^mis1, polychaetoid was identified as an enhancer of cno phenotypes. Flies doubly homozygous for pyd^tam1 and cno^mis1 die as embryos; this represents a synthetic lethal combination. Examination of embryonic cuticles demonstrates that the cno^mis1;pyd^tam1 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. cno^mis1;pyd^tam1 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 Drac1^V12, a constitutively active form of Drac1. If Drac1 is upstream of cno, then the effect of Drac1^V12 on puc-lacZ transcription should be blocked by the loss of cno function. Targeted expression of Drac1^V12 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. Pyd and mammalin ZO-1 also interact with Drosophila Cortactin and mammalian cortactin respectively. 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. 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. It would thus seem that Rac signaling is tied to actin dynamics and Polychaetoid/ZO-1 function both in Drosophila and mammals (Takahashi, 1998 and references).

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 (Takahashi, 1998).

The AF-6 homolog Canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo

Rap1 belongs to the highly conserved Ras subfamily of small GTPases. In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown. Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, has been shown to act as an effector of Rap1 in vivo. Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets. Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact. Rap1 acts upstream of Cno, but Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process (Boettner, 2003).

Rap1 cycles between an inactive GDP-bound and an active GTP-bound state, eliciting distinct downstream responses in the active state. Mammalian Rap proteins were originally identified as antagonists of oncogenic Ras, but more recent studies suggest that the function of Rap1 is largely independent of Ras. While Ras is mainly localized at the plasma membrane, Rap1 has been found in different membrane compartments, depending on the cell type. Further, Rap1 activation appears to be stimulated by numerous exchange factors that do not act on the prototypic Ras GTPases. Rap1 has been shown to act in a Ras-independent manner in the production of superoxide, in cAMP-induced neurite outgrowth, and, most recently, in the regulation of integrin-mediated cell adhesion and AMPA receptor trafficking during synaptic plasticity (Boettner, 2003 and references therein).

Perhaps the most important insights into the function of Rap1 are emerging from studies in Drosophila. Loss-of-function (lof) mutations in Drosophila Rap1 cause severe morphogenetic abnormalities during embryonic development, while cell proliferation and cell fate determination, processes that rely heavily on regulation by Ras, appear to be unaffected. Specifically, the ventral invagination and migration of mesodermal precursors in the embryo are severely impaired, as are head involution, dorsal closure, and the migration of gonadal precursors (Asha, 1999). More recently, Rap1 has been shown to play a role in cell adhesion, specifically in the positioning of adherens junctions in proliferating epithelial cells (Knox, 2002). These findings strongly suggest that Rap1 plays a largely Ras-independent role in cell migration and morphogenesis (Boettner, 2003 and references therein).

Little is currently known about the signaling pathways mediating the downstream effects of Rap1 in vertebrates or Drosophila. A number of molecules that were originally identified in vertebrates as Ras-interacting proteins, including B-Raf, members of the RalGEF family, and AF-6, were subsequently shown to associate with Rap1 as well. However, the relevance of these interactions for Rap1 function in vivo remains largely unknown; to date, none of these molecules have been shown to act as Rap1 targets in an in vivo context (Boettner, 2003 and references therein).

This study reports that Canoe (Cno), the Drosophila ortholog of AF-6, acts as an effector of Rap1 during dorsal closure (DC) of the Drosophila embryo. DC is a morphogenetic process that occurs during midembryogenesis and involves the dorsalward movement of the lateral ectoderm over the amnioserosa, a transient structure that covers the dorsal aspect of the embryo, to enclose the embryo. This process relies entirely on the migration and elongation of ectodermal cells, without cell recruitment or proliferation, and is akin to the epithelial cell sheet movements that occur during wound healing. Among the genes identified as necessary for normal DC are proteins associated with the cytoskeleton and/or cell junctions and components of the Drosophila Jun N-terminal kinase (JNK) and Decapentaplegic (Dpp) pathways. cno is required for DC; its protein is localized to the adherens junction and feeds into the JNK pathway by an unknown mechanism. Apart from the fact that it interacts with the ZO-1 homolog Tamou, nothing is known about the regulation of Cno activity at the adherens junction (Boettner, 2003).

Cno has been identified as a protein that interacts with activated Rap1 in a yeast two-hybrid screen. To address the physiological relevance of this interaction, localization studies, a comparative phenotypic analysis, and genetic interaction experiments were undertaken for the two proteins. Rap1 and cno loci are shown to interact synergistically in DC and the physical interaction between Rap1 and Cno is required for DC. The role of Canoe in promoting JNK pathway activity is independent of Rap1 and Canoe therefore has two separate functions in DC (Boettner, 2003).

In Drosophila, embryos lacking both zygotic and maternal Rap1 display strong defects in diverse morphological aspects of embryogenesis, such as ventral invagination, migration of mesodermal precursors, head involution, and DC. A key question is which effector pathways mediate the morphogenetic functions of Rap1. The yeast two-hybrid system was used to identify Drosophila Rap1-specific effector molecules from an embryonic library and several cDNAs encoding Cno were retrieved. Both N-terminal Ras-binding domains (RA1 and RA2) of Cno possess Rap1-binding potential and they interact only with a constitutively active Rap1 mutant, Rap1^V12, but not with a dominant negative version of Rap1, Rap1^N17, suggesting that Cno may act as an effector for Rap1 (Boettner, 2003).

Several lines of evidence are provided confirming this hypothesis. Rap1 and Cno partially colocalize at the adherens junction in the two tissues that are involved in DC, the amnioserosa and the lateral ectoderm, with Rap1 being present at the entire lateral membrane and also showing vesicular expression throughout the cytoplasm. Moreover, loss of function of the two molecules leads to similar phenotypes, at both the cuticular and the cellular level. To directly address the question whether Rap1 utilizes Cno as an effector during DC, a series of genetic experiments were conducted. They demonstrate that the two molecules act in the same pathway and their physical interaction is essential for their function in DC: (1) Removal of zygotic Rap1 strongly enhances the phenotype of a weak heteroallelic cno combination; (2) removal of the RA-interaction domains and, thus, removal of the ability to bind Rap1, reduces the ability of cno transgenes to rescue the cno lof phenotype, and (3) removal of the RA-interaction domains eliminates the ability of cno to rescue Rap1^N17. Finally, the finding that activated Rap1^V12 fails to rescue the cno lof defects indicates that Rap1 acts upstream of Cno. Taken together, the yeast two-hybrid data, colocalization results, and genetic interaction experiments provide comprehensive evidence that Cno functions as a downstream effector of Rap1 in the DC process. These findings represent the first demonstration of a protein acting as a Rap1 effector in vivo (Boettner, 2003).

The events downstream of Rap1 and Cno, however, appear to be more complex. Several independent findings suggest that Cno's role in DC can be separated into Rap1-independent and Rap1-dependent functions: removal of the RA-interaction domains does not affect the ability of the remainder of the protein to localize to the adherens junction, and the mutant protein retains the capacity to partially rescue the DC defect of a cno lof mutant. Further, Cno feeds into the JNK pathway, while Rap1 does not: dpp expression levels in the LE are significantly reduced in cno lof embryos at later stages of DC, but appear unaffected in Rap1 mutants. In addition, cno lof is partially rescued by overexpressing bsk (DJNK), whereas the Rap1^N17 defect is not. Given the multidomain structure of Cno, it is not surprising that the molecule would participate in multiple pathways. Such a bifurcation of the pathway would also explain the lack of transitivity observed in rescue experiments: Rap1 lof is (partially) rescued by cno overexpression, cno lof is (partially) rescued by bsk overexpression, but Rap1 lof is not rescued by bsk overexpression. The fact that both cno^DeltaN and bsk are unable to rescue Rap1 lof demonstrates that the Rap1-independent function of Cno cannot compensate for the loss of Rap1. This leaves the reciprocal question of whether Rap1 may have a second, Cno-independent function in DC. The fact that the DC phenotype of Rap1^N17 is as severe as that of cno lof without affecting JNK pathway signaling might suggest that Rap1 has additional effectors in DC (as does the fact that the phenotype of Rap1^N17 is more severe than that of cno²; ptcGAL4 UAScno^DeltaN). However, no conclusive evidence has been found to support this idea, since the additional effectors of Rap1 identified in the yeast two-hybrid screen have not been investigated for their role in DC (Boettner, 2003).

One obstacle in investigating the function of Rap1 is its pleiotropy. A detailed analysis of DC defects, in particular, is difficult to perform in Rap1 null embryos, due to the severe disruption of multiple aspects of embryonic development prior to DC. Therefore, use was made of the dominant negative Rap1^N17 mutant. When expressed at appropriate stages in the epithelial cells that are involved in the DC process, this transgene results in robust DC defects. However, early in vitro studies appeared to show that the Rap1^N17 mutant does not compete well with normal Rap1 for the GEF C3G, calling into question whether this mutant protein can be regarded as a Rap1 dominant negative. But in vivo studies using mammalian Rap1 and now the current study clearly show that Rap1^N17 acts as a dominant negative mutant in Rap1 signaling. The successful rescue of Rap1^N17 with a concomitantly expressed Rap1^wt transgene demonstrates the specificity of the mutant. Further, dominant negative versions of Drosophila Ras1 and Ras2, the counterparts of the mammalian H, K, and N-Ras and of the R-Ras proteins, respectively, do not disrupt DC when they are examined under the same conditions. This shows that the interaction between DRas1 and Cno detected in vitro and the genetic interaction between DRas1 and Cno that influences cone cell formation in the Drosophila eye, have no role during DC (Boettner, 2003).

On which cellular processes might Rap1 and Cno act? Cno is a multidomain protein consisting of several known and putative protein-interaction domains, including the two RA domains and a PDZ domain, which targets proteins to specific cell membranes and assembles proteins into supramolecular signaling complexes, but no catalytic domain. Cno localizes to the adherens junction and may act by localizing and clustering signal transduction components at the junction or by modulating the mechanical resistance of the adherens junction, and thus, directly or indirectly, influence JNK signaling. Since Cno is found at the adherens junctions under Rap1 lof conditions as well as in the absence of its RA domains, Rap1 cannot be required for the initial localization of the Cno protein, suggesting that Rap1 influences the activity of Cno by changing its conformation. However, another possibility is suggested by a study by Knox (2002), where it was found that Rap1 function is required for evenly (re-)distributing adherens junction components in wing disc epithelial cells after mitosis. It is likely that the adherens junctions in the cells that undergo stretching in the embryonic ectoderm during DC are similarly subject to dynamic reorganization, which may in part be regulated by the Rap1/Cno complex. This idea would be consistent with the observation that in Rap1 and cno lof mutants the lateral ectoderm begins its dorsal stretching, but is then unable to complete the process. Interestingly, Rap1 in mammalian cells has been shown to be activated in cell-stretching assays. In this system, force initiation apparently results in the activation of the JNK kinase family member p38, suggesting the existence of a Rap1-dependent 'mechanosensory' pathway. The data fit this idea. Future studies using fluorescently tagged Rap1 and Cno proteins and live imaging will shed light on dynamic aspects of their localization and function during DC (Boettner, 2003).

The Rap GTPase activator Drosophila PDZ-GEF regulates cell shape in epithelial migration and morphogenesis

Epithelial morphogenesis is characterized by an exquisite control of cell shape and position. Progression through dorsal closure in Drosophila gastrulation depends on the ability of Rap1 GTPase to signal through the adherens junctional multidomain protein Canoe. This study provides genetic evidence that epithelial Rap activation and Canoe effector usage are conferred by the Drosophila PDZ-GEF (dPDZ-GEF) exchange factor. dPDZ-GEF/Rap/Canoe signaling modulates cell shape and apicolateral cell constriction in embryonic and wing disc epithelia. In dPDZ-GEF mutant embryos with strong dorsal closure defects, cells in the lateral ectoderm fail to properly elongate. Postembryonic dPDZ-GEF mutant cells generated in mosaic tissue display a striking extension of lateral cell perimeters in the proximity of junctional complexes, suggesting a loss of normal cell contractility. Furthermore, the data indicate that dPDZ-GEF signaling is linked to myosin II function. Both dPDZ-GEF and cno show strong genetic interactions with the myosin II-encoding gene, and myosin II distribution is severely perturbed in epithelia of both mutants. These findings provide the first insight into the molecular machinery targeted by Rap signaling to modulate epithelial plasticity. It is proposed that dPDZ-GEF-dependent signaling functions as a rheostat linking Rap activity to the regulation of cell shape in epithelial morphogenesis at different developmental stages (Boettner, 2007).

In developing tissues, Rap has been found to promote various morphogenetic processes, ranging from epithelial migration and invagination in embryogenesis to the maintenance of epithelial integrity in proliferating tissues at later stages. However, the mechanisms by which Rap is regulated and mediates its effects in morphogenetic episodes remain poorly understood. This report delineates a pathway in which the Drosophila GEF dPDZ-GEF links Rap activity to MyoII and the regulation of lateral contractility and cell shape in different epithelial morphogenetic episodes (Boettner, 2007).

This study identified dPDZ-GEF as a putative activator of Rap GTPases in a yeast two hybrid (YTH) screen and subsequently demonstrated that it specifically associates with Rap, but not Ras, GTPases. PDZ-GEF is highly conserved among metazoans, suggesting that it might serve common physiological roles. dPDZ-GEF was found to be highly expressed in epithelial tissues involved in embryonic dorsal closure (DC), and, importantly, the data revealed that it functions as an activator of Rap1/Cno signaling in this process. First, as in the case of Rap1 and cno, loss of zygotic dPDZ-GEF function is associated with an ectodermal failure, which is manifested by dorsal-open phenotypes. Eliminating both zygotic and maternal dPDZ-GEF elevates the frequency of late gastrulation defects. Second, the genetic analysis places dPDZ-GEF upstream of the Rap/Cno GTPase/effector complex, as both Rap1 and Cno were able to rescue the dPDZ-GEF LOF phenotype to a large extent. Third, all three proteins show an overlapping localization at AJs in ectodermal cells involved in DC. Thus, these findings demonstrate that dPDZ-GEF serves as a Rap1/Cno activator to promote late epithelial gastrulation movements. In support of a conserved role of dPDZ-GEF in epithelial morphogenesis, studies with C. elegans demonstrated that pxf-1, the dPDZ-GEF homolog, is vital for epithelial integrity. pxf-1 mutant animals often are confronted with hypodermal malfunctions; the underlying cellular basis of these defects, however, remains to be elucidated (Boettner, 2007).

Epithelial migration processes often entail striking alterations in cell shape, and much effort has been devoted to unraveling the underlying cellular and molecular mechanisms. This study highlights that dPDZ-GEF as a Rap activator adjusts cell shape to the demands of morphogenetic movements and imaginal disc morphogenesis. It was observed that dPDZ-GEF mutant embryos involved in DC often exhibit bunched regions in their leading edge and an incompetence of ectodermal cells to elongate dorsally. These phenotypes also characterize embryos that either overexpress DN Rap1 or are mutant for cno. Thus, signaling through dPDZ-GEF, Rap, and Cno (1) is vital for the organization of a coherently moving leading edge and (2) enables the typical dorsoventral stretching of lateral ectodermal cell sheets. These studies also unveiled a requirement for dPDZ-GEF for the adjustment of epithelial cell shape in the differentiation program of the wing imaginal disc. It was found that a dPDZ-GEF LOF situation generated in a clonal analysis of mosaic wing discs is associated with a decline in apicolateral contractility in the vicinity of junctional complexes. Loss of contractility, as visualized by a widening of apicolateral circumferences, is coupled to a partially compensating gain of contractility in adjacent wild-type tissue. Wild-type cells in close proximity to mutant clones display smaller apicolateral circumferences. Interestingly, overexpression of dPDZ-GEF in restricted areas of the wing disc causes contractile aberrations. When ectopically expressed in the posterior compartment, dPDZ-GEF leads to a loss of apicolateral contractility in cells lining the A/P boundary. Thus, both gain and loss of dPDZ-GEF function compromise normal contractile strength and result in aberrant adult tissue formation. These observations suggest that a finely tuned level of Rap activation is crucial for normal cellular and organismal development to occur. Tight requirements for activation of small GTPases in vivo have been documented previously, e.g., for Rho GTPases and their function in axon guidance. Importantly, this study found in genetic modification experiments that reduced or enhanced dPDZ-GEF activity in the developing wing can be rescued by ectopic Rap1 or lowered cno doses, respectively, suggesting that signaling through the dPDZ-GEF/Rap/Cno module at least partially controls disc morphogenesis. This, together with the vital cooperative roles of all three genes in embryonic cell sheet migration, corroborates the reiterative function of dPDZ-GEF/Rap/Cno signaling during epithelial development (Boettner, 2007).

What are the mechanisms that translate Rap signaling downstream of dPDZ-GEF into the modulation of cell shape? This analysis of dPDZ-GEF LOF situations during gastrulation and wing disc morphogenesis showed that junctional integrity is not corrupted. Both AJ and SJ belts around the apicolateral circumference are seamlessly maintained in dPDZ-GEF LOF tissue. However, the data support a role for the MyoII heavy chain, the product of the zip gene, as an effector. MyoII assembly and disassembly in migrating cells and tissue homeostasis are tightly balanced processes. In epithelial cells, MyoII localizes to cell-cell junctional complexes and is essential for establishing and maintaining intercellular adhesion and tension. This study found that the decline in apicolateral constriction associated with dPDZ-GEF LOF in mitotic clones in the wing disc epithelium is accompanied by a less compact MyoII localization and that adjacent constricted wild-type cells display overassembled MyoII, which concentrates in ectopic focal structures. Also, in the DC paradigm, dPDZ-GEF and cno mutant embryos that are involved in DC exhibited failures of leading-edge cells to properly assemble MyoII. The abundant MyoII localization at the leading edge that characterizes wild-type embryos during DC is significantly diminished, and the bars-on-a-string-like MyoII distribution is lost in these mutants. In particular, regions of the leading edge adjacent to the bunched segments retain only minimal amounts of assembled MyoII. These observations strongly suggest that loss of MyoII control at the leading edge is contributing to the bunching phenotype observed in dPDZ-GEF and cno mutants. Consistent with a spatiotemporal regulation of MyoII in distinct regions of the leading edge are elegant life-imaging studies undertaken with embryos undergoing DC. Dynamic cycles of MyoII-dependent contraction and relaxation occur that are limited to smaller regions within the leading edge during the migration process (Boettner, 2007).

In further support of the notion that dPDZ-GEF signaling acts on MyoII, evidence was obtained that dPDZ-GEF and cno genetically interact with zip in late gastrulation and, moreover, that cno is genetically linked to zip in wing morphogenesis. In particular, the data show a strong enhancement of dorsal-open frequencies in embryos that are double transheterozygous for hypomorphic combinations of zip and either dPDZ-GEF or cno. Combined mutations at the zip and cno loci were also found to cause a malformation of wings. Together, these findings imply that signaling through the dPDZ-GEF/Rap/Cno module is required for MyoII function at different stages of epithelial development. Future experimentation will be required to determine the precise biochemical link between this module and MyoII regulation. Of note, a recent study demonstrated that the mammalian Cno homolog, AF-6/Afadin, in a two-dimensional tissue culture system moves together with MyoII at the edge of wounds induced by laser ablation. At the onset of wound closure, a subpopulation of MyoII resides apically in the lateral membranes of cells lining the wound. However, when closure progresses into advanced stages, MyoII, together with AF-6/Afadin, migrates basalward to constrict both the wound perimeter and the apicobasal membranes facing the wound (Boettner, 2007).

These data support a model in which dPDZ-GEF, through Rap activation and MyoII regulation, contributes to the adjustment of lateral cell contractility in epithelial cells of the embryo and the developing wing. In a previous study, the analysis of Rap1 mutant clones in the wing imaginal disc revealed a direct effect of Rap1 on the reorganization of AJs at the end of cytokinesis, where resealing of their belts has to occur between daughter cells. Since the data showed that AJ integrity is unperturbed in clones comprised of dPDZ-GEF LOF cells, it is surmised that dPDZ-GEF either is not relevant for Rap1 activation in the reconstitution of a seamless AJ belt during cytokinesis or is compensated for by a still-unknown factor conferring the necessary exchange activity. In contrast, the apicolateral constriction defects detected as a consequence of clonal loss of dPDZ-GEF function so far have not been described for Rap1 mutant clones in the same scenario. It is presumed either that they have escaped scrutiny or, more likely, that Rap1 acts redundantly with its close homolog Rap2l in adjusting apicolateral constriction, while the reorganization of AJs during cytokinesis relies solely on Rap1. In fact, Rap1 and Rap2l have been shown to compensate for each other in the male stem cell niche. In this context, both Rap proteins cooperate downstream of dPDZ-GEF to anchor germ line stem cells to their niche. In future experiments, it is planned to generate Rap1 and Rap2l mitotic clones in parallel and to examine and compare their effects on cell shape and contractility (Boettner, 2007).

A picture is emerging in which specialized GEFs activate Rap GTPases and selective effectors in different morphogenetic scenarios and cellular processes. For example, Rap1 signaling has been implicated in cell/extracellular matrix-dependent force transduction at focal adhesion sites of cultured cells. In this scenario, Rap1 is regulated by an Src/p130Cas/C3G-triggered mechanism. Also, apical constriction during neural tube closure in the Xenopus blastula has been demonstrated to depend on Rap1 function downstream of the Shroom protein; however, the relevant GEF in this scenario remains to be identified. The notion that Rap activation in distinct developmental processes is specified by dedicated GEFs also suggests that Rap effectors are selected in order to fulfill pathway requirements. In light of this, dPDZ-GEF and Rap1 have been implicated in the regulation of mitogen-activated protein kinase activity during differentiation of the Drosophila compound eye, and another reported that D-Raf relays a signal from Rap to mitogen-activated protein kinase in Torso-receptor-dependent terminal differentiation of the early Drosophila embryo. Together, these findings suggest the possibility that dPDZ-GEF could trigger the activation of the Rap/D-Raf pathway to regulate certain differentiation processes. The data reveal a novel function for dPDZ-GEF as an activator of Rap in the implementation of epithelial cell shape changes required for sheet migration and homeostatic cell shape maintenance in the genesis of the wing imaginal disc epithelium. Evidence is provided that Cno functions as a relevant effector of Rap downstream of dPDZ-GEF in these events and that the dPDZ-GEF/Rap/Cno module is connected to the regulation of MyoII and the generation and modulation of appropriate lateral cell contractility. Thus, these findings have unveiled a pathway linking the Rap activator dPDZ-GEF to MyoII and the regulation of lateral contractility and cell shape in epithelium migration and homeostasis. Further elucidation of dPDZ-GEF-interacting proteins and the molecular underpinnings of MyoII regulation downstream of this module in epithelial cells will be key to understanding these aspects of tissue morphogenesis (Boettner, 2007).

Echinoid is a component of adherens junctions that cooperates with DE-Cadherin to mediate cell adhesion

Echinoid is an immunoglobulin domain-containing transmembrane protein that modulates cell-cell signaling by Notch and the EGF receptors. In the Drosophila wing disc epithelium, Echinoid is a component of adherens junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and β-catenin (a DE-Cadherin interacting protein) each possess a C-terminal PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly position Bazooka to adherens junctions. Echinoid also links to actin filaments by binding to Canoe/AF-6/afadin. Moreover, interfaces between Echinoid^- and Echinoid⁺ cells, like those between DE-Cadherin^- and DE-Cadherin⁺ cells, are deficient in adherens junctions and form actin cables. These characteristics probably facilitate the strong sorting behavior of cells that lack either of these cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin accumulate a high density of the reciprocal protein, further suggesting that Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion (Wei, 2005).

Several observations prompted the study of Ed as a canonical CAM in the monolayered wing imaginal disc. Thus, mitotic recombination clones of cells mutant for the null allele ed^1x5 exhibit rounded and smooth contours, in contrast to clones of wild-type cells that show wiggly shapes. This indicated that ed^{- /-} cells have distinct adhesive properties and assort with themselves rather than with the surrounding ed^+/- M^+/- cells. (ed^1x5 clones were M⁺, since without a growth advantage they hardly survive). It was also observed that Ed was absent from the membrane of the heterozygous cells that contacted the mutant cells, a finding consistent with the observation that Ed forms homophilic interactions and that these are required to incorporate/stabilize Ed at the cell membrane. Finally, Ed was found to localize basally to the apical marker Crb and apically to the basolateral marker Dlg. In fact, Ed colocalizes with both DE-Cad and Arm, and, therefore, it might be part of AJs. AJs are structures important for cell-cell contact and recognition. So, these results suggested that Ed plays a role in cell-cell adhesion (Wei, 2005).

Whether Ed affects components of AJs was examined by analyzing the localization of Arm within ed mutant clones. Arm strongly accumulates at the apical membranes of ed^{- /-} cells, and these cells have a reduced apical surface. Both effects are clear in small clones, but cells within larger clones (over hundreds of cells) had both the density of Arm and the apical surface more similar to those of the wild-type cells. Similar observations were made with DE-Cad and Actin. It is suggested that the increased concentration of these molecules in small clones most probably results from the apical constriction as supported by the accumulation of nonmuscle myosin II, without a net per cell increment of these proteins. Alternatively, it could result from increased stability of these proteins. The apical constriction continued through the SJs and ended at the planes just below the GJs as revealed by an Innexin antibody. Hence, these ed^{- /-} cells adopt a bottle shape. In contrast, the apposed ed^{- /-} and ed^+/- cells that form the border of the clone enlarge and adopte a rectangular shape. At this interface, the ed^{- /-} cells often contacted the heterozygous cells by their long sides, as if in an attempt to minimize the number of cells that formed the interface (Wei, 2005).

Interestingly, Arm and DE-Cad, but not Actin, are depleted at the interface membrane of both small and large clones. This suggests that ed^{- /-} and ed heterozygous cells discriminate one another and that AJs do not form properly in between them (Wei, 2005).

ed clones are surrounded by an Actin 'cable'. High-magnification images suggest that the cable is contained within the ed heterozygous cells surrounding the clone and that it is therefore generated by these cells. Several observations suggest that this Actin cable exerts a force. The cells surrounding an ed clone elongate toward the clone and accumulate nonmuscle myosin II at the interface membrane, as if attempting to cover the space exposed by the apically constricted ed^{- /-} cells. This effect is reminiscent of the stretching of the leading-edge cells that will cover the underlying amnioserosa during dorsal closure of the embryos. In the wing disc, the boundary that separates the dorsal (D) and ventral (V) regions of the wing pouch has the shape of a smooth arc and contains an actin 'fence'. After the second instar, this boundary corresponds to a compartment border that imposes absolute restrictions to cell lineages. Large ed^{- /-} clones close to or touching this boundary displace it toward the clones. In contrast, ed clones that straddle the boundary do not overtly distorted it, although the boundary could be less smooth within the clone. (Straddling clones might be originated before the compartment border was established or might be formed of D and V clones that fuse together). Moreover, the Actin cable surrounding the clones fuse with the Actin fence at the D/V boundary, suggesting that the distortion of this boundary is effected through this Actin linkage. Control ed⁺ M⁺ clones do not induce such distortions. These observations suggest that the Actin cable may contribute to the roundish shape of the ed clones and help confine their cells (Wei, 2005).

DE-Cad is a classical homophilic cell adhesion molecule of AJs. It interacts with β-catenin/Arm, which in turn binds α-catenin. Through the association between α-catenin and F-Actin, DE-Cad establishes links between cells that connect to the Actin cytoskeleton. This study shows that Ed is another CAM that, at the resolution of confocal microscopy, is also located at the AJs of imaginal disc cells. While cells in clones mutant for ed still seem to form normal AJs, the cells at the border of the clone seem impaired in forming them. It is hypothesized that this may help them segregate from surrounding ed^+/- cells. Ed was identified as a binding partner for PDZ proteins that, similarly to Arm, helps localize Baz to AJs. Moreover, it was found that through the binding of Cno, Ed, like DE-Cad/β-catenin, may link to F-Actin. Hence, Ed has functions in cell-cell adhesion similar to those of DE-Cad (Wei, 2005).

The differential adhesion hypothesis proposes that cell sorting may be driven by differences in the quantity and/or quality of adhesive molecules displayed on the surface of cells. In keeping with this hypothesis, it was found that ed^{- /-} cells sort out from ed^+/- cells, as shown by the remarkably round shapes and smooth contours of the ed clones. Moreover, their differential adhesiveness is also manifest by the fusion of different ed clones to yield composite but still roundish clones. It is suggested that contraction of the apically enriched Actin network and of the actin cable surrounding the clone, possibly by interaction with nonmuscle myosin II also present there, may contribute to the the apical constriction of the ed^{- /-} cells. It was also observed that the interface between ed^+/- and ed^{- /-} cells is depleted of DE-Cad, Arm and Baz, besides completely lacking Ed. This strongly suggests that this interface is deficient in AJs and probably helps to insulate ed^{- /-} cells from the surrounding ed heterozygous cells. It is hypothesized that this deficiency of AJs, which may reduce adhesion between ed^+/- and ed^{- /-} cells, and the inward-pulling force generated by apical constriction and the actin cable may help create the smooth and rounded contour of the clones at the level of AJs. At the plane of SJs, the clonal boundary is not as smooth. This may be due to the presence of normal levels of SJs, since seemingly wild-type amounts of Dlg were detected at the interface membrane. Normal levels of SJs may allow the clones to remain integrated in the epithelium. It is stressed that when ed clones grow large, the apical constriction disappears, suggesting that the forces responsible for this constriction become insufficient or no longer operate. If the force is exerted, at least in part, by the Actin cable surrounding the clone, as in a purse-string mechanism, it would make sense that this force becomes ineffectual as the number of cells within the clone increases. Remarkably, these differences of apical cell constriction observed in small and large ed clones have a correlate on the adult wing blade: small clones display an increased density of trichomes, implying that their cells are small or more tightly packed, whereas large clones have cells of normal size. This indicates that the apical constriction is retained through imaginal disc eversion, when the disc epithelium changes from columnar to planar (Wei, 2005).

In the embryonic epithelium, Baz, localized to both AJs and the marginal zone, is the initial apical regulator. How is Baz recruited to the apical domain? In the follicular epithelium, Baz is localized to this domain through lateral exclusion mediated by PAR-1/14-3-3 and apical anchoring by Crb/Sdt/Patj. The data support an additional mechanism to localize Baz to the apical domain. Both Ed and Arm can bind Baz through their C-terminal PDZ binding motif and therefore they may redundantly localize Baz to AJs. Indeed, the localization of Baz to AJs is relatively normal in the absence of either one. Most Baz is lost only when both Arm and Ed are depleted, as occurs at the interface membrane of ed clones or in large shg clones where Ed gradually breaks down. In the latter case, there is good colocalization between Baz and the sites maintaining residual Ed. It is suggested that in the epithelium of the wing disc, Baz localizes to AJs by the combined effects of its binding to Ed/Arm and the lateral exclusion of PAR-1/14-3-3. Additionally, apical anchoring of Baz may be mediated by direct association between the Baz and Crb apical complexes. During early embyogenesis, Ed is also present at pseudocleavage furrows. This observation, together with the ability of Ed to localize Baz to AJs, may explain the finding that during cellularization, Baz can accumulate apically in the absence of Arm. Ed also binds to the PDZ domain of Cno and mediates its localization to AJs, where Cno interacts with F-Actin either directly or indirectly through the association with Polychaetoid/ZO-1. Interestingly, the evolutionally conserved EIIV domain of Ed binds Baz and Cno in a mutually exclusive manner. Thus, the concentrations of and differential affinities between Ed, Baz, and Cno should determine their dynamic equilibrium at AJs (Wei, 2005).

Although Baz is critical to form AJs in the blastoderm and in the follicular epithelium, removal of Baz (or Par-6) from cells of the wing disc does not affect the localization of DE-Cad or Ed to AJs. This is consistent with the report that in imaginal discs, Baz does not affect the localization of DE-Cad and Dlg but is required for the asymmetric localization of cell fate determinants. Together, these results suggest that in wing discs, the Baz complex is not critical for the formation of AJs, and that the effect of the loss of Ed on AJs formation/maintenance is not due to Baz depletion (Wei, 2005).

Several similarities between the roles of DE-Cad and Ed in the wing disc epithelium are worth noting. Both Ed and DE-Cad are CAMs that establish homophilic interactions and localize to AJs. The absence of either Ed or of DE-Cad in cells of small clones causes their apical constriction and strong segregation from wild-type cells, giving rise to smooth round borders. In both cases, the mutant cells are impaired in forming AJs with neighboring wild-type or heterozygous cells and are surrounded by an Actin cable. Ed interacts with Cno, and DE-Cad with Arm, and both Cno and Arm directly or indirectly associate with F-Actin. Thus, Ed and DE-Cad represent two distinct classes of CAMs, with widely different chemical compositions, that connect to F-Actin, contribute to cell adhesion in the wing disc, and seem to have partially overlapping functions (Wei, 2005).

In contrast, DE-Cad and Ed differ in their ability to regulate the apical/basal cell polarity. Ed affects components of AJs, but not those of the apical Crb and the basolateral Dlg complexes. In contrast, DE-Cadherin is necessary for Crb localization, but similarly to Ed, it is not required for Dlg localization. Furthermore, the maintenance of Ed at AJs requires DE-Cad. In contrast, localization of DE-Cad to AJs is independent of Ed. Interestingly, the DE-Cad/Arm complex is not essential for the formation of the follicular epithelium, but upon removal of this complex, the integrity of the epithelium is lost slowly over the period of several days. This suggests that other molecules may be maintaining the epithelial structure. During stages 1 to 10 of oogenesis Ed is mainly expressed in the follicle cells, and these cells, if mutant for ed, show at low frequency a multilayered structure with disrupted expression of some polarity markers. Thus, it will be of interest to elucidate whether, in this epithelium, Ed and DE-Cad/Arm also play partially redundant roles in cell adhesion and apical/basal polarity. While both Ed and DE-Cad contribute to cell adhesion and recognition, it is unclear whether each molecule imparts specific recognition properties to cells, so that the final cell-cell affinity results from the sum of distinct affinities mediated by these different CAMs. More specifically, can an increased level (density) of DE-Cad replace the absence of Ed? The results showing that ed^{- /-} cells, with either normal levels (in large clones) or high density (in small clones) of DE-Cad, do not intermix with wild-type cells suggests that the binding specificity provided by a given CAM is not overruled by a higher level (density) of a different CAM. Moreover, the cell sorting properties conferred by Ed cannot account for the separation of cells at both sides of the A/P compartment boundary of the wing disc because A and P cells do not intermingle within composite ed, smo double mutant clones. (Similarly, DE-Cad is not responsible for the sorting out of A and P cells. Hence, cell-cell adhesion in the wing disc appears to depend on multiple CAMs (Ed, DE-Cad, etc.), each imparting specific cell recognition properties. Although Ed and its C-terminal EIIV motif are conserved in invertebrates, no clear vertebrate homolog with 7 Ig domains and a PDZ domain binding motif has been found. Nectin1-4 comprises a family of 3 Ig domain-containing CAM that have several differentially spliced forms and localize to AJs. Most spliced forms share a conserved C-terminal E/A-X-Y-V that binds the PDZ domain of Afadin. Moreover, this motif also interacts with Par-3, the vertebrate homolog of Baz. In spite of these similarities, overexpression of either nectin 1-α or 3-α does not rescue the remarkable clonal phenotype of ed (Wei, 2005).

The Drosophila afadin homologue Canoe regulates linkage of the actin cytoskeleton to adherens junctions during apical constriction

Cadherin-based adherens junctions (AJs) mediate cell adhesion and regulate cell shape change. The nectin-afadin complex also localizes to AJs and links to the cytoskeleton. Mammalian afadin has been suggested to be essential for adhesion and polarity establishment, but its mechanism of action is unclear. In contrast, Drosophila's afadin homologue Canoe (Cno) has suggested roles in signal transduction during morphogenesis. Cno as completely removed from embryos, testing these hypotheses. Surprisingly, Cno is not essential for AJ assembly or for AJ maintenance in many tissues. However, morphogenesis is impaired from the start. Apical constriction of mesodermal cells initiates but is not completed. The actomyosin cytoskeleton disconnects from AJs, uncoupling actomyosin constriction and cell shape change. Cno has multiple direct interactions with AJ proteins, but is not a core part of the cadherin-catenin complex. Instead, Cno localizes to AJs by a Rap1- and actin-dependent mechanism. These data suggest that Cno regulates linkage between AJs and the actin cytoskeleton during morphogenesis (Sawyer, 2009; full text of article).

Canoe functions at the CNS midline glia in a complex with Shotgun and Wrapper-Nrx-IV during neuron-glia interactions

Vertebrates and insects alike use glial cells as intermediate targets to guide growing axons. Similar to vertebrate oligodendrocytes, Drosophila midline glia (MG) ensheath and separate axonal commissures. Neuron-glia interactions are crucial during these events, although the proteins involved remain largely unknown. This study shows that Canoe (Cno), the Drosophila ortholog of AF-6, and the DE-cadherin Shotgun (Shg) are highly restricted to the interface between midline glia and commissural axons. cno mutant analysis, genetic interactions and co-immunoprecipitation assays unveil Cno function as a novel regulator of neuron-glia interactions, forming a complex with Shg, Wrapper and Neurexin IV, the homolog of vertebrate Caspr/paranodin. These results also support additional functions of Cno, independent of adherens junctions, as a regulator of adhesion and signaling events in non-epithelial tissues (Slováková, 2011).

The midline constitutes a key boundary of bilateral organisms. In vertebrates, it is the floorplate and the functionally equivalent structure in Drosophila is the mesectoderm, which gives rise to all midline cells, neurons and glia, in the most ventral part of the embryo. MG are of great relevance at the midline as an intermediate target during axonal pathfinding, providing both attractive and repulsive guidance cues. These signals allow contralateral axons to cross the midline but never to recross, and they also keep ipsilateral axons away from the midline. In addition to this early function in guiding commissural axons towards the midline, MG are also fundamental later on to separate the commissures by enwrapping and subdividing them. This study shows that the PDZ protein Cno and the DE-cadherin Shg participate in, and contribute to, the regulation of these later stage neural differentiation events, in which neuron-glia interactions play a central role (Slováková, 2011).

In Drosophila, Wrapper and Nrx-IV physically interact to promote glia-neuron intercellular adhesion at the MG. This study proposes that Cno and Shg are important components of this adhesion complex and key to its function. Both Cno and Shg are present at the MG, being highly restricted to the interface between MG and commissural axons. Cno and Shg were detected in a complex in vivo with Wrapper at the CNS MG. Nrx-IV, which is located on the surface of commissural axons, was also consistently found in a complex with Cno, although the amount of Cno protein that was co-immunoprecipitated was much lower than that present in Cno-Wrapper complexes. One plausible explanation is that whereas Cno and Wrapper are present in the same cell (MG), Cno and Nrx-IV are in different cell types (MG and neurons, respectively) and, in addition, Cno is a cytoplasmic protein that is indirectly linked to Nrx-IV through other proteins in the same complex (i.e., Shg and Wrapper). Intriguingly, stronger genetic interactions were found between Cno and Nrx-IV than between Cno and Wrapper (double heterozygote analysis). A possible explanation for this is that Nrx-IV is not only acting through Wrapper-Shg-Cno in the MG but also through other partners. In this way, when the dose of Cno and Wrapper was halved, Nrx-IV could still function fully through these other, putative partners. However, halving the dose of Cno and Nrx-IV would impair not only the Nrx-IV-Wrapper-Cno signal but also the other potential pathways. In vertebrates, the ortholog of Nrx-IV, termed contactin-associated protein (Caspr or Cntnap) or paranodin, is located at the septate-like junctions of the axonal paranodes, where it interacts in cis with contactin (at neurons) and in trans with neurofascin (at the glia). The Drosophila homologs of these Ig superfamily proteins, Contactin and Neuroglian, interact in the same way with Nrx-IV at the septate junctions. However, there are no septate junctions at the neuron-MG interface. Hence, other, as yet unknown partners of Nrx-IV might exist at this location (Slováková, 2011).

Cno and its vertebrate orthologs afadin/AF-6/Mllt4 have been shown to localize at epithelial adherens junctions (AJs), where they regulate the linkage of AJs to the actin cytoskeleton by binding both actin and Nectin family proteins. However, Cno is not exclusively present at the AJs of epithelial tissues. Indeed, it was found that Cno is also expressed in mesenchymal tissues, where it dynamically regulates three different signaling pathways required for muscle/heart progenitor specification. The asymmetric division of these muscle/heart progenitors and of CNS progenitors also requires an AJ-independent function of Cno to asymmetrically locate cell fate determinants and properly orientate the mitotic spindle. Therefore, Cno seems to act through different mechanisms depending on the cell type. This study describes a novel function of Cno during neural differentiation. In the MG, Cno, through Shg, contributes to the tight adhesion between the MG and the commissural axons and perhaps even to the regulation of some intracellular signaling within the MG. Indeed, Cno has been shown to regulate different signaling cascades during development. No AJs or septate junctions (SJs) have been described at the MG-commissural axon interface. This suggests that the function of Cno in the midline is independent of AJs. In fact, the partner of Cno at this location, the Drosophila Nectin ortholog Echinoid, is not detected at the midline. In this context, it is worth pointing out that Shg is an epithelial cadherin key at AJs. This study has shown that Shg can also be found in non-epithelial tissues with an important function independent of AJs. A similar situation occurs with Nrx-IV. Despite Nrx-IV being a very well established component of SJs, no SJs are formed in the midline and no other known components of SJs are expressed there. Thus, different modes of Cno action, either as an AJ protein or as a signaling pathway regulator, are possible and they are not mutually exclusive: it all depends on the cell type and context (Slováková, 2011).

Canoe binds RanGTP to promote Pins^TPR/Mud-mediated spindle orientation

Regulated spindle orientation maintains epithelial tissue integrity and stem cell asymmetric cell division. In Drosophila neural stem cells (neuroblasts), the scaffolding protein Canoe (Afadin/Af-6 in mammals) regulates spindle orientation, but its protein interaction partners and mechanism of action are unknown. This paper uses a recently developed induced cell polarity system to dissect the molecular mechanism of Canoe-mediated spindle orientation. A previously uncharacterized portion of Canoe was shown to directly bind the Partner of Inscuteable (Pins) tetratricopeptide repeat (TPR) domain. The Canoe-Pins^TPR interaction recruits Canoe to the cell cortex and is required for activation of the Pins(TPR)-Mud (nuclear mitotic apparatus in mammals) spindle orientation pathway. The Canoe Ras-association (RA) domains directly bind RanGTP, and both the Canoe^RA domains and RanGTP are required to recruit Mud to the cortex and activate the Pins/Mud/dynein spindle orientation pathway (Wee, 2011).

Spindle orientation is essential to maintain epithelial integrity; planar spindle orientation results in both daughter cells maintaining apical junctions and remaining part of the epithelium, whereas apical/basal spindle orientation can lead to the loss of the basal daughter cell from the epithelium. Spindle orientation is also important during asymmetric cell division of stem, progenitor, and embryonic cells; when the spindle orients along an axis of intrinsic or extrinsic polarity, it will generate two different daughter cells, but, when the spindle aligns perpendicular to the axis of polarity, it will generate two identical daughter cells. Proper spindle orientation may even be necessary to prevent tumorigenesis. Thus, it is essential to understand the molecular mechanisms that regulate spindle orientation, particularly those that use evolutionarily conserved proteins and pathways, to help direct stem cell lineages and potentially treat pathological conditions caused by aberrant spindle orientation (Wee, 2011).

Drosophila neuroblasts provide an excellent system for studying spindle orientation during asymmetric cell division. Neuroblasts have an apical/basal polarity and orient their mitotic spindle along this cortical polarity axis to generate distinct apical and basal daughter cells. The apical neuroblast inherits fate determinants responsible for neuroblast self-renewal, whereas the basal daughter cell inherits fate determinants responsible for neuronal/glial differentiation. Genetic studies have identified proteins that regulate spindle orientation during asymmetric cell division, including the apically localized proteins Inscuteable, Partner of Inscuteable (Pins; LGN/AGS-3 in mammals), Mushroom body defect (Mud; nuclear mitotic apparatus [NuMA] in mammals), Discs large (Dlg), and Ga_i. In addition, many proteins that are not asymmetrically localized are required for spindle orientation, including the dynein complex and the Aurora A and Polo kinases (Wee, 2011).

An induced cell polarity/spindle orientation system has been developed using the normally apolar S2 cell line to biochemically dissect Drosophila and vertebrate spindle orientation (Johnston, 2009; Ségalen, 2010). Using this system to characterize Drosophila spindle orientation, it was shown that cortical Pins nucleates two spindle orientation pathways: (1) the Pins^LINKER domain is phosphorylated by Aurora A, which allows recruitment of Dlg, which interacts with the kinesin Khc-73 to promote partial spindle orientation; and (2) the Pins tetratricopeptide repeat (TPR) domain (Pins^TPR) binds Mud, which promotes dynein-dynactin complex-mediated spindle orientation (Johnston, 2009). This induced cell polarity system was used to characterize Dishevelled-mediated spindle orientation in the zebrafish embryo and in Drosophila sensory organ precursor cells, identifying a Dishevelled domain that is necessary and sufficient to bind Mud and regulate spindle orientation in both cell types (Wee, 2011).

The scaffolding protein Canoe has been shown to regulate spindle orientation and cell polarity in Drosophila neuroblasts (Speicher, 2008), although the mechanisms involved remain unknown. Canoe contains two Ras-association (RA) domains, a Forkhead domain, a myosin-like Dilute domain, and a PSD-95, Dlg, and ZO-1 (PDZ) domain. In addition to regulating neuroblast cell polarity and spindle orientation, it integrates Notch, Ras, and Wnt pathways during Drosophila muscle progenitor specification and serves as a Rap1 effector within the Jun N-terminal kinase pathway during dorsal closure of the Drosophila embryo, and the mammalian orthologue Afadin links cadherins to the actin cytoskeleton at adherens junctions. This study mapped direct Pins/Canoe and Canoe/RanGTP-binding domains and used the induced cell polarity/spindle orientation system to show that Canoe/RanGTP is required for Pins to recruit Mud and activate the Pins/Mud/dynein spindle orientation pathway (Wee, 2011).

How might Canoe/RanGTP promote Mud recruitment to the Pins cortical domain? One model is that Ran sequesters importin-a/β away from the Mud NLS, thereby allowing Mud to interact with Pins. This model is based on the observation that RanGTP inhibits binding of importin-β to the NLS of NuMA (the mammalian orthologue of Mud), increasing the pool of NuMA available to promote spindle formation. The model predicts that Mud can bind importin-a/β and that this binding prevents Mud/Pins association. Consistent with the model, importin-β/Mud were coimmunoprecipitated from S2 cell lysates, and a GST:Mud fragment containing the adjacent Mud TPR-interacting peptide (TIP)-NLS domains (GST:Mud^TIP-NLS) could bind purified importin-β in the presence of importin-a. However, it was found that increasing the concentration of purified importin-a/β did not effect the amount of Pins pulled down with GST:Mud^TIP-NLS, which does not support a model in which Ran must sequester importin-a/β to allow Pins/Mud binding. Furthermore, a GFP-tagged Mud^TIP-NLS fragment localized to Ed:Pins^TPR+LINKER crescents independently of the Canoe/Ran pathway, showing that the Mud NLS is not involved in the Canoe/Ran-regulated localization mechanism. Interestingly, Canoe/RanGTP regulation is required for recruitment of full-length endogenous Mud but not for the recruitment of the smaller Mud^TIP-NLS fragment; this indicates that Canoe/RanGTP normally functions by blocking an unknown inhibitor of the Mud-Pins^TPR interaction (Wee, 2011).

In conclusion, this study has characterized the molecular mechanism by which Canoe regulates spindle orientation. A region of Canoe (amino acids 1,755-1,950) was identified that directly interacts with the Pins^TPR domain, and it was showm that these domains are necessary and sufficient for Canoe-Pins association. It was shown that the Canoe RA domains bind directly to RanGTP, that both the Canoe RA domains and Ran are necessary for the Pins^TPR/Mud spindle orientation pathway, and that Canoe/RanGTP acts by promoting Mud recruitment to the cortical Pins domain. All of the proteins in the Pins/Canoe/Ran/Mud pathway are conserved from flies to mammals, suggesting that this pathway could be widely used to regulate spindle orientation (Wee, 2011).

Serrate-Notch-Canoe complex mediates glial-neuroepithelial cell interactions essential during Drosophila optic lobe development

It is firmly established that neuron-glia interactions are fundamental across species for the correct establishment of a functional brain. This study found that the glia of the Drosophila larval brain display an essential non-autonomous role during the development of the optic lobe. The optic lobe develops from neuroepithelial cells that proliferate by dividing symmetrically until they switch to asymmetric/differentiative divisions generating neuroblasts. The proneural gene lethal of scute (l'sc) is transiently activated by the Epidermal Growth Factor Receptor (EGFR)/Ras signal transduction pathway at the leading edge of a proneural wave that sweeps from medial to lateral neuroepithelium promoting this switch. This process is tightly regulated by the tissue-autonomous function within the neuroepithelium of multiple signaling pathways, including EGFR/Ras and Notch. This study shows that the Notch ligand Serrate (Ser) is expressed in the glia and it forms a complex in vivo with Notch and Canoe, which colocalize at the adherens junctions of neuroepithelial cells. This complex is crucial for glial-neuroepithelial cell interactions during optic lobe development. Ser is tissue-autonomously required in the glia where it activates Notch to regulate its proliferation, and non-autonomously in the neuroepithelium where Ser induces Notch signaling to avoid the premature activation of the EGFR/Ras pathway and hence of L'sc. Interestingly, different Notch activity reporters showed very different expression patterns in the glia and in the neuroepithelium, suggesting the existence of tissue-specific factors that promote the expression of particular Notch target genes or/and a reporter response dependent on different thresholds of Notch signaling (Perez-Gomez, 2013).

Cno and its vertebrate homologues AF-6/Afadin localize at epithelial AJs where they regulate the linkage of AJs to the actin cytoskeleton by binding both actin and nectin family proteins. This study found that Cno colocalizes with Notch at the AJs of NE cells in the optic lobe proliferation centers. Notch also colocalizes with its ligand Ser, which was detected at the glia, highly accumulated at the interface between NE cells and the surrounding glia. Co-immunoprecipitation experiments indicate the formation of a Ser-Notch-Cno complex in vivo, and the mutant analysis shows the functional relevance of such a complex at the glia neuroepithelium interface. The data presented in this study support the hypothesis that Cno may be stabilizing Notch at the AJs of NE cells, favoring the binding of Ser present in the adjacent glial cells. Indeed, in cno lof both Notch and Ser distribution is affected; this alteration is accompanied by an abnormally advanced proneural wave, a reminiscent phenotype to that shown by Notch lof optic lobes and also a similar phenotype found in this work in Ser lof. The activation of Notch pathway is essential to maintain the integrity of the neuroepithelium and to allow the correct progression of the proneural wave. The results show that glial Ser is responsible of such activation, promoting the expression of the m7-nuclacZ reporter in NE cells. In fact, the reduction of glial Ser either by knocking down epithelial cno or by expressing DNSer in the glia led to a decrease in the expression of the m7-nuclacZ reporter in NE cells and to an ectopic activation of the Ras/PntP1 pathway and of L'sc. It is proposed that this may be ultimately the cause of the proneural wave advance observed in those genotypes. Thus, the activation of Notch in the neuroepithelium by glial Ser, in nomal conditions, would be essential to avoid a premature activation of the EGFR/Ras/PntP1 pathway and hence of L'sc. Indeed, Notch has been shown to downregulate different EGFR/Ras signaling pathway components such as Rhomboid (Rho), required for the processing of the EGFR ligand Spitz, in other developmental contexts in which both pathways are actively cross-talking. Therefore, Notch activity in NE cells could be contributing to inhibit Rho, restricting its presence to the transition zone where Rho is very locally expressed (Perez-Gomez, 2013).

It was observed that in a WT condition Ser is present in all surface glia (perineurial and subperineurial), as shown by the expression of CD8::GFP (SerGal4>>UAS-CD8::GFP), and Notch, as tested by different reporters, is active in this tissue and highly reduced in Ser lof in the glia. This makes sense with the existence of a Ser-Notch mediated intercellular communication between the glial cells that comprise both the perineurial and subperineurial glia. Intriguingly, the knockin down and overexpression of cno in NE cells also had a clear effect on Notch activity in the glia, a reduction and an increase, respectively. This is more challenging to explain. As the cno lof in the NE led to a high reduction of both neuroepithelial Notch and glial Ser, the easiest explanation is that an 'excess' of unbound glial Ser is degraded and this impinges on the general thresholds of glial Ser, therefore causing a general reduction in the Notch activity in this tissue. This is an interesting field to explore in detail and is left open for future investigation (Perez-Gomez, 2013).

The activity of Notch in the neuroepithelium and in medulla NBs seems controversial. For example, Notch has been shown to be active in the neuroepithelium at low/null levels or in a 'salt and pepper' patter. A weak/null activity of Notch has also been reported in NBs as well as a high activation. One possibility to conciliate all these results and apparently contradictory data is that different Notch target genes used as Notch activity reporters are, in fact, differentially activated in particular regions or tissues. The results support this proposal. Four different Notch reporters were used in this study. Whereas m7-nuclacZ was expressed throughout the neuroepithelium, Gbe+Su(H)lacZ was restricted to the transition zone, although both were expressed in medulla NBs along with mβ-CD2. In addition, mβ-CD2 was strongly activated in the glia, whereas the Gbe+Su(H)lacZ and the mδ-lacZ reporters were expressed at much lower levels at this location. Differential activation of Notch targets genes has been previously reported and tissue-specific factors could contribute to this differential expression. This is an intriguing scenario to analyze in the future. The in depth analysis of other Notch reporter genes in the developing optic lobe can contribute to further clarify this issue (Perez-Gomez, 2013).

At third larval instar during optic lobe development, Dl is highly restricted to 2-3 cells at the transition zone in the neuroepithelium, where Dl activates Notch. This work has found that the other ligand of Notch, Ser, is expressed in the surrounding glia at this larval stage and it is strongly accumulated at the interface with NE cells. Ser activates Notch in the neuroepithelium and this, in turn, would contribute to restrict the activation of the Ras-PntP1 pathway and L'sc to the transition zone. Intriguingly, it was observed that Ser preferentially activates the Notch target gene m7-nuclacZ in the neuroepithelium whereas Dl activates other Notch target genes, including Gbe+Su(H)lacZ, in the transition zone. For example, the overexpression of Dl in NE cells caused an ectopic activation throughout the neuroepithelium of Gbe+Su(H)lacZ, along with dpn that also behaves as a Notch target in other systems, and a repression of m7-nuclacZ . In addition, the lof of Ser in the glia caused a striking decrease in the expression of m7-nuclacZ in the neuroepithelium. One possibility to explain these observations is that the pool of Notch associated to the AJs and activated by glial Ser is subject of particular posttranslational modifications or/and is associated with other AJs proteins (including Cno) that somehow make Notch more receptive to Ser and able to activate specific target genes (i.e., m7). In this regard, it is interesting to note that Dl ectopically expressed in the glia (i.e., repoGal4>>UAS-Dl) was not detected at the interface with NE cells, where glial Ser is highly present in contact with Notch, but Dl was restricted to the outermost surface glia (perineurial glia). This result strongly indicates that Dl cannot bind or has very low affinity for this pool of Notch at the AJs, hence being actively degraded in the subperineurial glia. This low affinity of Dl by Notch at this location further suggests that this pool of Notch at the AJs must be endowed with particular characteristics that ultimately could alter the activity properties of Su(H), explaining in turn the distinct expression pattern of Notch targets genes. Another possibility, which is not necessarily exclusive, to explain the differential activation of the Notch reporters is that they respond to different Notch thresholds. For example, m7-nuclacZ would require very low levels of Notch activation whereas Gbe+Su(H)lacZ would require high amounts of Notch signaling in NE cells. All these questions remain open for further investigation (Perez-Gomez, 2013).

DEVELOPMENTAL BIOLOGY

Embryonic

In stage 5 embryos before cellularization, CNO mRNA distributes uniformly at a low level throughout the embryo, except for the pole cells. At the cellular blastoderm, signals are detected along the dorsal midline, where three intensely stained domains (anterior, central, and posterior) are descernible. In additon, three ectodermal stripes reminiscent of parasegmental expression of gap genes are seen clearly in the central domain of the embryo. In stage 7-10, expression is confined to the dorsal furrows and the posterior midgut rudiment. The pattern of mRNA distribution may imply that Cno is required for delamination of presumptive endodermal cells, a process that depends on control of adhesion between midgut epithelial cells. Ectodermal expression becomes evident at stage 10. At stage 13, focal stainings are detected near the attachment site of the midgut to the foregut and hindgut (Miyamoto, 1995).

Polychaetoid protein is localized at the cell-cell junction. Observation of the presumptive wing blade region of the wing imaginal disc reveals that Pyd has a more basal distribution compared with Shotgun. Pyd colocalizes with the scaffolding protein Canoe. In the cellular blastoderm (stage 5), the Cno protein is distributed diffusely in the cytoplasm, with significant accumulation at the apical surface. The cytoplasmic staining decreases before gastrulation. In stage 13 embryos undergoing germ-band retraction, marked accumulation of Cno is observed in the amnioserosa, with persistent expression of Cno in the lateral epidermis. The intense staining of the amnioserosa and the apposed edges of the lateral epidermis continues during dorsal closure. At this stage of embryogenesis, the trachea in each segment begins to elongate laterally to form the tracheal system across the segments. In addition, Cno is localized in Malpighian tubules, hindgut and the central nervous system. The tissue localization of Pyd is remarkably similar to that of Cno. It is present in the cytoplasm in the blastoderm stage embryo. In the later stages, Pyd is exclusively localized to cell boundaries. The epidermis, amnioserosa, the margin of the closing epidermis, the tracheal system, the Malpighian tubes, the hindgut and the CNS all express Pyd at high levels (Takahashi, 1998).

Ectodermal epithelium was examined for Pyd and Cno colocalization. The two proteins partially colocalize: Pyd expression is more widespread than Cno expression. The domain of Cno expression and that of Fas III expression are mutally exclusive, whereas the distributions of Arm and Drosophila alpha-catenin coincide with that of Cno. In contrast, Pyd is expressed in areas at which Fas III is localized. Fas III distribution is known to be restricted to septate junctions, and Drosophila alpha-catenin and Armadillo are confined to adherens junctions. Cno colocalizes with Arm but not with Fas III in the embryonic epidermis. Thus the results indicate that Pyd is present at both the septate and adherens junctions while Cno exists predominantly at adherens junctions (Takahashi, 1998).

A contractile actomyosin network linked to adherens junctions by Canoe/afadin helps drive convergent extension

Integrating individual cell movements to create tissue-level shape change is essential to building an animal. This study explored mechanisms of adherens junction (AJ):cytoskeleton linkage and roles of the linkage regulator Canoe/afadin during Drosophila germband extension (GBE), a convergent-extension process elongating the body axis. Surprising parallels were found between GBE and a quite different morphogenetic movement, mesoderm apical constriction. Germband cells have an apical actomyosin network undergoing cyclical contractions. These coincide with a novel cell shape change--cell extension along the anterior-posterior (AP) axis. In Canoe's absence, GBE is disrupted. The apical actomyosin network detaches from AJs at AP cell borders, reducing coordination of actomyosin contractility and cell shape change. Normal GBE requires planar polarization of AJs and the cytoskeleton. Canoe loss subtly enhances AJ planar polarity and dramatically increases planar polarity of the apical polarity proteins Bazooka/Par3 and atypical protein kinase C. Changes in Bazooka localization parallel retraction of the actomyosin network. Globally reducing AJ function does not mimic Canoe loss, but many effects are replicated by global actin disruption. Strong dose-sensitive genetic interactions between canoe and bazooka are consistent with them affecting a common process. A model is proposed in which an actomyosin network linked at AP AJs by Canoe and coupled to apical polarity proteins regulates convergent extension (Sawyer, 2011).

The data suggest that coupling AJs to a contractile apical actomyosin cytoskeleton plays an important role in a very different cell movement: convergent extension during Drosophila GBE. A novel cell shape change, AP cell elongation, was identified that contributes to WT GBE. Furthermore, it was found that Cno is required for maintaining attachment of the apical actomyosin network AJs in a planar-polarized way. Disrupting this connection results in failure of GBE and prevents coordination of apical myosin contractility and cell shape change. These data are consistent with a model in which Cno tightly couples apical actomyosin to AP AJs and coordinates apical polarity proteins with the network, helping to integrate individual cell shape changes across the tissue (Sawyer, 2011).

Previous studies illustrated how an apical contractile actomyosin network powers apical constriction. In contrast, convergent extension during Drosophila GBE was thought to involve planar-polarized enhancement of contractile actomyosin cables, driving cell intercalation and body elongation. It was surprising to find that, in addition to junctional cables, germband cells also have an apical actomyosin network that undergoes cyclical constriction and relaxation. This coincides with and may help to drive cell shape change. The asymmetric cue of planar-polarized myosin is likely to impose asymmetry. Together, asymmetric cortical myosin and cyclical contractions may help to extend cells in one dimension instead of shrinking them in all dimensions, thus contributing to tissue elongation. While this manuscript was being revised, two other papers independently discovered and described the apical network: the Lecuit lab data further suggest that myosin condensations preferentially move toward AP borders, helping to drive cell rearrangement (Rauzi, 2010; Fernandez-Gonzalez, 2011). Both the current data on Cno and the Lecuit lab's data on β-catenin further suggest that different proteins linking this apical network to AJs are critical for the fidelity and coupling of apical myosin contraction to cell shape change (Sawyer, 2011).

Also, a novel cell shape change was identified that may help to drive AP body axis extension - AP cell elongation. Cno and presumably linkage of the apical actomyosin network to AJs are important for this cell shape change. One speculative possibility is that an asymmetric ratchet acts in germband cells, selectively preventing elongation along the DV body axis while allowing cell elongation along the AP body axis. It is also possible that outside forces, such as shape changes of the first cells to divide, help reshape ectodermal cells, but it is thought that this is less likely, as cell shapes were examined during early GBE before germband mitotic domains divide. Ratchets have also been proposed during mesoderm invagination and during dorsal closure, where amnioserosal cells apically constrict. Before dorsal closure onset, amnioserosal cells have periodic apical actomyosin contractions, but cells only retain changes in shape after a junctional actomyosin purse string appears. Disrupting the purse string disrupts dorsal closure, suggesting that a junctional actomyosin cable can act as a ratchet (Sawyer, 2011).

Studies in Xenopus suggest that the role of a dynamic, planar-polarized apical actomyosin network in convergent extension is conserved. Myosin organizes actin into dynamic foci that move within intercalating cells along their mediolateral axis. In myosin's absence, actin foci are lost and convergent extension is disrupted. Thus dynamic actomyosin foci may play a conserved role in convergent extension (Sawyer, 2011).

It will be interesting to identify regulators shaping contractile activity in different tissues. Jak/Stat signaling restricts apical constriction to the mesoderm; in its absence apical myosin accumulates in the ectoderm, and those cells inappropriately apically constrict. Thus, although both mesoderm and ectoderm share an apical contractile network, its regulation is tuned differently. Furthermore, different actin regulators regulate apical and junctional myosin, with Wasp regulating the apical pool (Sawyer, 2011).

Linking AJs to actin is key in diverse processes from adhesion itself to morphogenetic movements as different as apical constriction and collective cell migration. Cno regulates linkage during mesoderm apical constriction, but isn'n required for cell adhesion (Sawyer, 2009). Other AJ-actin linkers act in other contexts, suggesting that cells use distinct linkers in circumstances with different force regimes. The current data suggest that during GBE, Cno regulates AJ:actomyosin network connections, acting specifically along AP borders (Sawyer, 2011).

Core AJ proteins are more reduced on AP borders in cno^MZ mutants than in WT. In WT, slightly reducing AJ proteins on AP borders may facilitate shrinkage of these borders during GBE. It is tempting to speculate that Cno enhancement along AP borders provides extra support when DEcad/Arm is reduced, strengthening AJ:actomyosin linkages along AP borders yet still allowing cell shape change. In this model, when Cno is absent, AJ:actomyosin linkage is weakened at AP borders, leading to inefficient cell shape change, impairing GBE, and accentuating reduction of AJ proteins (Sawyer, 2011).

The data further suggest that Cno is not the only AJ:actomyosin linker during GBE. Although the actomyosin network detaches from AJs in cno^MZ, it does not collapse into a ball; instead, cables remain 0.2-0.5 microm distant from AJs. A second connection is also supported by the appearance of apical strands of DEcadherin stretching from the cortex to detached myosin in cno^MZ. It will be interesting to determine what proteins compose these other AJ:actomyosin links. β-Catenin regulates actin:AJ linkage just prior to this stage and also plays a role in GBE, although how β-catenin mediates linkage remains mysterious (Sawyer, 2011).

Both myosin and Baz/Par3 are important GBE regulators. One of the most surprising consequences of Cno loss was dramatic change in Baz and aPKC localization. Their strong reduction along AP borders and restricted localization along DV borders correlates well with altered localization of apical actomyosin, which detached from AP AJs and retracted along DV borders from vertices. These data suggest that coordination of the actomyosin network and Baz/aPKC facilitates efficient cell shape change. Consistent with this, an interesting recent paper demonstrated that Baz is required for reciprocal planar-polarized distribution of myosin and AJs. Baz localization, in turn, is restricted by the cytoskeletal regulator Rho-kinase (Rok), leaving Baz enriched at DV borders (Simoes Sde, 2010). This suggests a complex network of interactions (Sawyer, 2011).

In C. elegans a contractile actomyosin cytoskeleton positions apical-polarity proteins (PAR3/PAR6/aPKC) anteriorly in one-cell embryos, and this complex then alters the actomyosin network, promoting asymmetric cortical flow to maintain anterior and posterior domains. It is tempting to speculate that the germband contractile actomyosin network plays a similar role. In this model, planar polarization of the network would create a symmetry break, helping to trigger Baz/aPKC planar polarization. They, in turn, may feed back to modulate actomyosin contractility, driving GBE. Strengthening AJ:actomyosin linkages via Cno could help to ensure efficient cell shape changes that are integrated across the tissue (Sawyer, 2011).

Several mechanistic hypotheses are consistent with these data that are not mutually exclusive. First, Cno may directly affect Baz/aPKC localization during assembly or maintenance, working in parallel or in series with Rok (Simoes Sde, 2010), with actomyosin positioning and contractility then modulated by Baz/aPKC. Consistent with this, previous work revealed that Baz remains apical in the absence of AJs; residual epithelial cells retain polarized actin but have hyperconstricted apical ends. Furthermore, PAR proteins regulate actomyosin contractility during DC. Second, Cno could alter the actomyosin network, which in turn may affect proper Baz/aPKC localization. Baz apical positioning requires the actin cytoskeleton. Actin disruption and Cno loss alter Baz localization similarly, consistent with this hypothesis. Finally, Baz/aPKC may mediate Cno apical positioning, as Baz does for AJs. Of course, more complex interplay with feedback between actomyosin and Baz/aPKC seems likely, creating a network of interactions rather than a linear pathway. Teasing out the complex coordination of AJs, apical polarity protein, and the actomyosin network during morphogenesis is an exciting challenge (Sawyer, 2011).

Rap1 and Canoe/afadin are essential for establishment of apical-basal polarity in the Drosophila embryo

The establishment and maintenance of apical-basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. This study found that the small GTPase Roughened/Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. The current linear model for polarity establishment was tested. Both Bazooka and aPKC regulate Canoe localization despite being 'downstream' of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data suggest that polarity establishment is regulated by a protein network rather than a linear pathway (Choi, 2013).

Polarity is a fundamental property of all cells, from polarized cell divisions in bacteria or fungi to the elaborate polarity of neurons. Among the most intensely studied forms of polarity in animal cells is epithelial apical-basal polarity. Polarity of epithelial sheets is key to their function as barriers between body compartments, and is also critical in collective cell migration and cell shape change during morphogenesis, as cytoskeletal and apical-basal polarity often go hand in hand. Loss of apical-basal polarity is a hallmark of metastasis. Significant advances have been made in defining the machinery required for cell polarity in many settings, but fundamental questions remain unanswered (Choi, 2013).

Cadherin-catenin complexes, which assemble into adherens junctions (AJs) near the apical end of the lateral cell interface, are critical polarity landmarks that define the boundary between apical and basolateral domains. Studies in C.elegans and Drosophila identified other key regulators of apical-basal polarity. In the textbook view, the apical domain is defined by the Par3/Par6/aPKC and Crumbs/Stardust(Pals1)/ PATJ complexes, while Scribble, Dlg, Lgl, and Par1 define the basolateral membrane (Choi, 2013).

Complex cross-regulatory interactions between apical and basolateral proteins maintain these mutually exclusive membrane territories. These proteins also regulate other types of polarity during morphogenesis; e.g., fly Par3 (Bazooka; Baz), aPKC, and AJ proteins are planar-polarized during fly convergent extension, thus regulating polarized cell movements (Choi, 2013).

Polarized cytoskeletal networks also play key roles in establishing and maintaining apical-basal and planar polarity. These networks are thought to be physically linked to apical junctional complexes. The earlier model suggesting that cadherin-catenin complexes link directly to actin via α-catenin is now viewed as over-simplified. Instead, different proteins are thought to mediate this connection in different tissues and at different times (Choi, 2013).

Among the linkers is Canoe (Cno)/Afadin, an actin-binding protein that binds transmembrane nectins via its PDZ domain. While originally hypothesized to be essential for cell adhesion, subsequent work supports a model in which afadin modulates adhesive and cytoskeletal machinery during cell migration in vitro and the complex events of mouse gastrulation. Afadin has two N-terminal Ras association domains for which the small GTPase Rap1 is the major binding partner, and Afadin and Rap1 are functionally linked in both flies and mice. Rap1, Cno, and the Rap1 GEF Dizzy/PDZGEF are all essential for maintaining effective linkage between AJs and the apical actomyosin cytoskeleton during apical constriction of Drosophila mesodermal cells during fly gastrulation. Rap1 regulates Cno localization to the membrane. Cno plays a related role during convergent extension, though its role is planar polarized during this process. Cno also regulates collective cell migration, signaling, and oriented asymmetric divisions. The Rap1/Cno regulatory module is also important in disease, as Afadin and Rap1 are implicated in congenital disorders of the cardiovascular system and cancer metastasis. It remains unclear whether these diverse roles all involve junction-cytoskeletal linkage or whether some are independent functions (Choi, 2013 and references therein).

The small GTPase Rap1 plays diverse cellular roles. Mammalian Rap1 isoforms are perhaps best known for regulating integrin-based cell matrix adhesion, but Rap1 also regulates cell-cell AJs in both Drosophila and mice. In murine endothelial cells, for example, Rap1, its effector Krit1, and VE-cadherin form a complex that regulates endothelial cell junctions and stabilizes apical-basal polarity (Choi, 2013 and references therein).

In Drosophila imaginal disc cells, Rap1 regulates the symmetric distribution of DE-cadherin (DEcad) around the apical circumference of each cell. Rap1 carries out these functions via a diverse set of effector proteins, including Krit1, TIAM, RIAM, and Cno/Afadin. Thus, Rap1 and its effectors are candidate proteins for regulating interactions between AJs, polarity proteins and the cytoskeleton during polarity establishment and maintenance (Choi, 2013).

The early Drosophila embryo provides among the best models of establishing and maintaining apical-basal polarity. Flies start embryogenesis as a syncytium, with 13 rounds of nuclear division without cytokinesis. Membranes then simultaneously invaginate around each nucleus, forming ~6000 cells in a process known as cellularization. Prior to cellularization, the egg membrane is already polarized and serves as a polarity cue for underlying nuclei. This ultimately becomes the apical end of the new cells. Epithelial apical-basal polarity is initiated during cellularization. In the absence of cadherin-catenin complexes, cells form normally but then lose adhesion and polarity as gastrulation begins. These data and earlier work from cell culture suggested AJs are the initial apical cue. However, it was found that Bazooka (Baz)/Par3 acts upstream of AJs in this process. Strikingly, Baz and DEcad apically co-localize in spot AJs from cellularization onset. In the absence of Baz, DEcad loses its apical enrichment and redistributes all along the lateral membrane, while in the absence of AJ proteins, Baz remains apically localized, and a subset of cells retain residual apical-basal polarity, although cell shapes are highly abnormal. Cadherin-catenin and Baz complexes form independently before cellularization, and Baz then helps position DEcad in the apicolateral position where spot AJs will form. This placed Baz atop of the polarization network, raising the question of how it is positioned apically. Two cytoskeletal networks play important roles in initial Baz positioning (Choi, 2013).

Disrupting dynein led to Baz spreading along the lateral membrane, suggesting polarized transport along microtubules (MTs) plays a role. Depolymerizing actin also destabilized apical Baz, as did significantly overexpressing Baz, suggesting an actin-based scaffold with a saturable number of binding sites anchors Baz apically. While both actin and MTs are required for initial Baz polarization, they are not the only cues. Mislocalized Baz is re-recruited or re-stabilized apically at gastrulation onset if either initial cue is disrupted, suggesting a third cue perhaps involving aPKC/Par6 or Par1. Thus, the current model for initial establishment of apical-basal polarity involves a relatively simple pathway in which Baz is positioned apically, and then positions other apical polarity players. However, once initial polarity is established, events become more complex, with a network of mutually reinforcing and inhibitory interactions between apical and basolateral polarity complexes leading to polarity elaboration and maintenance. These were significant advances, but the proteins directing apical accumulation of Baz remained unknown. Work on apical constriction in the fly mesoderm, convergent extension during gastrulation, establishment of anteriorposterior polarity in one cell C. elegans embryos, and on apically constricting Drosophila amnioserosal cells, suggested that a complex network of interactions link AJs, the apical polarity proteins Baz and aPKC, and the actomyosin cytoskeleton. Recent work on Canoe and Rap1's roles in mesoderm apical constriction and convergent elongation (Sawyer, 2011) suggested they also fit into this network. These data led to an exploration of whether Rap1 and Cno play roles in initial apical positioning of AJs and Baz and thus in the establishment and early maintenance of polarity (Choi, 2013).

In regulating polarity establishment, Rap1 and Cno could act by several possible mechanisms. Their role in AJ positioning may be solely due to their effects on Baz localization, or alternatively Rap1 and Cno may independently affect the localization of both Baz and AJs. In the latter case, Cno may directly link AJs to the apical actin scaffold, as it was suggested to act in apical constriction. Rap1 and Cno also clearly regulate Baz positioning. Since Baz apical positioning requires an apical actin scaffold and dynein based MT transport, whether Rap1 and Cno act indirectly by regulating cytoskeletal organization was examined. However, the data suggest this is not the case: both the MT and actomyosin cytoskeletons appear normal in mutants. Thus the most likely model is that Rap1 and Cno are required for anchoring Baz apically. Consistent with this, when Cno was ectopically localized to artificial cell-cell contacts in cultured fly cells, it was able to recruit Baz to that site. This could occur directly, for example, by Cno binding Baz, or indirectly, via unknown intermediaries. Strikingly, however, when Baz was over-expressed in cellularizing embryos, presumably saturating its apical binding sites, it accumulated basolaterally and recruited DEcad but not Cno to these ectopic sites. Thus Cno and Baz do not co-localize obligatorily. It likely that each has multiple binding partners and that when pools are limiting, as Cno may be in this latter experiment, ectopic Baz cannot recruit Cno away from a preferred binding site. Of course, it remains possible that Cno and Rap1 also regulate Baz positioning through effects on MT transport or, given Cno's apical localization, unloading at an apical docking site. It will be important to test these possibilities. As is discussed in more detail below, it will also be important to define the Cno- and Rap1-independent mechanisms that partially restore apical Baz localization after gastrulation onset (Choi, 2013).

Since Rap1 is uniformly distributed along the apical-basal axis during cellularization, the most likely hypothesis is that it is locally activated apically by a GEF. A number of Rap1GEFs exist, many of which are conserved between mammals and flies. Recent work from the Reuter lab demonstrated that, like Cno and Rap1, the Rap1 GEF Dizzy (Dzy/PDZ-GEF) plays an important role in coordinated mesodermal apical constriction, suggesting it is the GEF acting upstream of Cno and Rap1 in that process. They also suggest that Rap1 and Dzy help regulate establishment of AJs. While similar in outline, their analysis of AJs differs from this one in detail, as they see strong effects on DEcad localization without similar effects on Arm. This is surprising, since these two proteins of the cadherin-catenin complex generally localize very similarly at the cortex. However, these differences aside, their data are consistent with Dzy acting with Cno and Rap1 in AJ establishment-it will be important to examine the effects of Dzy on Baz localization. It will also be important to determine how pre-existing egg membrane polarity is translated into localized Rap1 activity (Choi, 2013).

In addition to the parallel roles of Rap1 and Cno in regulating initial apical-basal polarization, this study identified a second role for Rap1 in establishing and maintaining columnar cell shape. The data suggest that this is partially or completely Cno-independent, and thus one of the many other Rap1 effectors may play a role in this process. It will be exciting to examine embryos mutant for other Rap1 effectors, such as Krit1/Bili, TIAM/Still life, RIAM/Pico, or RhoL to see if they are required for establishing columnar cell shape. baz and aPKC mutants also had defects in establishing columnar cell architecture. It is possible that each protein provides an independent mechanistic input into this process. This is consistent with the observed differences in the details of how columnar cell shape is disrupted, with Baz and aPKC primarily regulating apical cell area, while Rap1 affects cell shape at multiple apical-basal positions. A more speculative but perhaps less likely possibility is that Rap1 uses Baz and aPKC as effectors in establishing columnar cell shape. Fly Rap1 can form a complex with aPKC and Par6, and Rap1 acts upstream of cdc42/Par3/aPKC in regulating polarity of cultured neurons (Choi, 2013 and references therein).

Having identified Rap1's direct effector(s) in regulating cell shape, it is necessary to move downstream. Based on analogies with other epithelial tissues in fly development, it is hypothesized establishing columnar cell shape involves regulating apical tension. Other small GTPases play key roles in this; e.g., Rho and cdc42 have striking and opposing roles in apical tension regulation during fly eye development. In that context, Rho acts via separate effectors to maintain AJs and apical tension-it regulates tension via Rok, Diaphanous, and ultimately myosin contractility. It will be interesting to determine whether the defects in apical cell shape in the absence of Rap1, Baz, or aPKC also reflect unbalanced contractility in different nascent cells, and which contractility regulators are involved. However, for now, this is speculative (Choi, 2013).

Previous work has suggested a linear hierarchy regulating polarity establishment, with Baz at the top, positioning AJs and aPKC. The current work extends this hierarchy, positioning Rap1 and Cno upstream of Baz in this process. However, the data further suggest that viewing polarity establishment as a linear process is significantly over-simplified. It is now known that all of the relevant players -- including the AJ proteins, Baz, Cno and aPKC -- are at the cortex in syncytial embryos, prior to cellularization and the initiation of apical-basal polarity. This places them in position to cross-regulate one another. Consistent with this, the data suggest that viewing relationships with an 'upstream-downstream' point of view misses important reciprocal interactions that occur as polarity is established. Two examples point this out most clearly. First, earlier work suggested that localization of aPKC occurs 'downstream' of Baz, as apical positioning of aPKC at gastrulation onset requires Baz function. The new data reveal that Rap1 and Cno are, in turn, 'upstream' of Baz, and thus, if things work in a strictly linear fashion, Rap1 and Cno should be 'upstream' of aPKC. However, in contrast to this simple view, this study found that precise positioning of Cno during cellularization requires aPKC - in its absence, Cno is not cleared from the apical region, and the apical-basal cables of Cno at tricellular junctions are not properly assembled. In a similar fashion, Baz, which in a linear model is 'downstream' of Cno, also regulates precise positioning of Cno during cellularization. aPKC and Baz also play important roles in Cno localization during the early polarity maintenance phase beginning at gastrulation onset. Together, these data suggest that initial positioning of proteins along the apical-basal axis involves a network of protein interactions, similar to that previously suggested to regulate polarity elaboration during the extended germband phase and beyond, as cells develop the full suite of epithelial junctions. It will now be important to define mechanisms by which aPKC and Baz act to precisely position Cno: two broad possibilities are that they act on Cno directly, or that they modulate the fine scale architecture of the actin cytoskeleton, with indirect effects on Cno. It will also be exciting to determine if other polarity determinants, like the basolateral proteins Discs Large, Scribble or Lgl, or the basolateral kinase Par1 also play roles in polarity establishment, as they do in polarity maintenance. Consistent with this possibility, recent work from the Harris lab suggests Par1 is important for the gastrulation onset rescue of Baz localization in embryos in which early cues are disrupted. Finally, it will be interesting to identify the cues that come into play at gastrulation onset, which partially restore apical Baz localization, as part of the increasingly complex network of partially redundant regulatory cues that give polarity its robustness (Choi, 2013).

Larval

CNO transcripts are expressed ubiquitously in eye-antennal discs, with higher levels of expression in the lateral edge region (Miyamoto, 1995).

Discontinuities in Rap1 activity determine epithelial cell morphology within the developing wing of Drosophila

Mechanisms that govern cell-fate specification within developing epithelia have been intensely investigated, with many of the critical intercellular signaling pathways identified, and well characterized. Much less is known, however, about downstream events that drive the morphological differentiation of these cells, once their fate has been determined. In the Drosophila wing-blade epithelium, two cell types predominate: vein and intervein. After cell proliferation is complete and adhesive cell-cell contacts have been refined, the vast majority of intervein cells adopt a hexagonal morphology. Within vein territories, however, cell-shape refinement results in trapezoids. Signaling events that differentiate between vein and intervein cell fates are well understood, but the genetic pathways underlying vein/intervein cyto-architectural differences remain largely undescribed. This study shows that the Rap1 (Roughened) GTPase plays a critical role in determining cell-type-specific morphologies within the developing wing epithelium. Rap1, together with its effector Canoe, promotes symmetric distribution of the adhesion molecule DE-cadherin about the apicolateral circumference of epithelial cells. Evidence is provided that in presumptive vein tissue Rap1/Canoe activity is down-regulated, resulting in adhesive asymmetries and non-hexagonal cell morphologies. In particular Canoe levels are reduced in vein cells as they morphologically differentiate. It was also demonstrate that over-expression of Rap1 disrupts vein formation both in the developing epithelium and the adult wing blade. Therefore, vein/intervein morphological differences result, at least in part, from the patterned regulation of Rap1 activity (O'Keefe, 2012).

During the early, proliferative phase of epithelial development each cell strives to maintain adhesive contacts with its neighbors, generating, on average, a field of hexagonal-shaped cells. This uniformity is transient, however, as multiple cell types are frequently specified within a single epithelium, each with a unique function and cyto-architecture. Mechanisms must exist, therefore, for cell-type-specific shapes to emerge as these heterogeneous epithelia begin to morphologically differentiate. This study shows that in the Drosophila wing the regulation of Rap1 activity is one means by which non-hexagonal epithelial cell shapes are generated (O'Keefe, 2012).

These studies have focused on the Drosophila wing vein. Within the wing blade, veins comprise a small subset of cells, and during pupal stages of development it was shown that vein-precursor cells adopt a unique shape (trapezoidal), compared to surrounding intervein cells (hexagonal). Presumptive vein cells are first identified by high levels of Egfr activity, and previous studies have shown that Egfr signaling up-regulates the homophilic adhesion molecule DE-cad in these cells (both transcriptionally and post-translationally) (O'Keefe, 2007). High levels of cadherin generally result in apical constriction, a prominent characteristic of the adult vein. DE-cad is only one component of this morphogenetic process, however, as increased levels of DE-cad did not result in a vein-like trapezoidal shape. It was asked, therefore, what other mechanisms might determine the non-hexagonal morphology of vein precursors (O'Keefe, 2012).

In addition to elevated levels of DE-cad, another distinguishing feature of pupal vein cells is an asymmetric distribution of DE-cad about their apicolateral circumference, a phenotype most apparent when two-cell clones of ectopic veins were examined. As loss of Rap1 leads to asymmetric DE-cad (Knox, 2002; O'Keefe, 2009), it was hypothesized that Rap1 activity is down-regulated in vein precursor cells compared to surrounding intervein precursors. Consistent with this hypothesis, Rap1 over-expression dramatically disrupted pupal vein cell shape without affecting cell fate (i.e., DSRF levels). Rap1 over-expressing vein cells had more symmetric DE-Cad distributions, and did not adopt a trapezoidal morphology. This often led to morphological vein defects in the adult wing. In addition, the localization patterns of Rap1-GFP and Canoe suggested lower levels of Rap1 activity in pupal-vein precursors (compared with surrounding intervein cells). It has been previously demonstrated that the generation of Rap1 loss-of-function clones during larval stages results in vein loss (O'Keefe, 2009). Rap1 activity, therefore, plays a dual role in wing-vein formation. First, during larval and early pupal stages, Rap1 stabilizes adhesive contacts between adjacent epithelial cells, thereby facilitating Egfr signaling and maintaining vein-cell fate. Hours later, as the wing begins to differentiate, down-regulation of Rap1 activity drives the morphological changes necessary for vein formation (O'Keefe, 2012).

How does the down-regulation of Rap1 activity specifically increase DE-cad levels at vein-vein cell contacts? Rap1 recruits Cno to adherens junctions, where Cno forms a physical link between adherens junctions and the actin cytoskeleton (Sawyer, 2009). As such, Cno primarily acts as a non-enzymatic scaffolding protein, which suggests that stoichiometry between DE-cad and Cno is important. Based on immunofluorescence analysis of apicolateral cell junctions in the wing, there is a large disparity between Cno and DE-cad levels in vein cells, as Egfr/Ras signaling both up-regulates DE-cad, and down-regulates Cno. It is inferred from these data that vein cells contain far fewer adherens junction complexes that are associated with a molecule(s) of Cno (compared to intervein cells). As Cno represents the critical Rap1 effector in this context, these Cno-free adherens junction complexes would be functionally dissociated from Rap1 signaling, and free to localize in an asymmetric fashion. Relieved from spatial constraints concerning symmetry, adherens junction complexes would accumulate at vein-vein interfaces, where chances of encountering an intercellular binding partner are highest for two reasons: 1) adjacent vein cells express higher levels of DE-cad than adjacent intervein cells, and 2) adjacent vein cells contain Cno-free adherens junction complexes, which are similarly relieved from symmetry constraints (O'Keefe, 2012).

The formation of asymmetrical adhesive contacts in presumptive vein cells is coincident with changes in apical cell shape. It was asked, therefore, how changes in DE-cad localization might affect vein-cell shape, and have proposed a simple model based on examinations of a timecourse of vein differentiation. The balance between intercellular adhesion and cortical tension is a critical determinant of cell shape. Increased adhesion expands cell contacts, and cortical tension opposes this effect. The data suggest that after ~24 h APF, vein-vein cell contacts are characterized by high levels of adhesion (i.e., DE-cad) and decreased levels of cortical tension (i.e., Cno, which links adherens junctions to the actin cytoskeleton). It is hypothesized that these factors drive the expansion of vein-vein contacts at the expense of one vein-intervein cell contact, resulting in the formation of a pentagon. Real-time imaging of vein differentiation will be used in the future to test this model of morphogenesis (O'Keefe, 2012).

The Egfr/Ras and Dpp signaling pathways act in concert to specify vein-cell fate. At 12-16 h APF, Egfr/Ras activity turns on dpp expression in presumptive vein cells. After this stage of development, Dpp is required to maintain vein identity and high levels of Egfr/Ras signaling in presumptive vein cells (creating a positive feed-back loop). In contrast, these developmental signaling pathways have very different effects on cell adhesion and epithelial cell morphology. It has been shown previously that Egfr/Ras activity up-regulates DE-cad levels in vein precursors, and that it does so in a Dpp-independent fashion (O'Keefe, 2007). Results presented in this study indicate that Egfr/Ras signaling also plays the dominant role in regulating Rap1/Cno. Two-cell clones that express Ras^V12 phenotypically resembled Rap1 loss-of-function cells (more so than Tkv^Q235D clones). In addition, Ras^V12 down-regulated the critical Rap1 effector Cno, whereas this effect was not evident in Tkv^Q235D-expressing cells. As loss of Cno disassociates actin-myosin contractility from cell shape (Sawyer, 2009), Ras^V12 two-cell clones were less apically constricted than Tkv^Q235D-expressing cells. Egfr/Ras signaling is also associated with asymmetric adhesive contacts in other developmental contexts. In the Drosophila eye, for example, Egfr/Ras signaling is required in photoreceptors. Much like vein cells, photoreceptors adhere more tightly to one another than to surrounding cells. This raises the possibility that Egfr down-regulates Rap1 activity in multiple cell types following their specification, enabling them to differentiate appropriate cell shapes. Finally, it will be interesting to determine how the Egfr/Ras and Dpp signaling pathways regulate other aspects of vein-cell morphology (e.g., constriction along the apical/basal axis to generate a lumen) (O'Keefe, 2012).

In the wing, Egfr/Ras signaling does not affect Rap1/Cno activity at every developmental stage. High levels of Egfr/Ras signaling are detected in vein cells at the beginning of the third larval instar, but vein/intervein cell-shape differences are not observed before ~24 h APF. As such, the Rap1/Cno complex likely represents a pupal-specific target of Egfr signaling. This study has shown, therefore, that a single developmental signaling pathway can first determine a cell's fate, and later contribute towards its morphological differentiation. Critical to this process, therefore, are genetic and/or epigenetic factors that temporally regulate the output of Egfr/Ras signaling. In the future it will be important to identify such factors not only for the Egfr/Ras pathway, but other developmental signaling pathways as well (O'Keefe, 2012).

Finally, it is becoming increasingly clear that Rap1 affects cancer progression, often by promoting metastasis. In cancer cells, levels of Rap1 activity are typically high, which stimulates migration and metastasis by up-regulating integrin-based cell adhesion. Such is the case in pancreatic, prostate, and breast cancers. However, loss of Rap1 can also cause metastasis by down-regulating cadherin and disrupting the epithelial integrity of the tumor (e.g., ovarian and prostate cancer). Within this disease context, the Egfr/Ras and Rap1 signaling networks often interact. Most recently, Egfr activation of Rap1 has been shown to promote metastasis of human pancreatic carcinoma cells. The precise mechanisms by which Egfr/Ras signaling affects Rap1 activity (both during normal development and disease) must be deciphered, therefore, if these metastatic processes are to be understood and/or mitigated (O'Keefe, 2012).

EFFECTS OF MUTATION

Flies doubly mutant for cnomis1 and scabrous and those for cnomis1 and Notch always have rumpled, downward curving wings. The Notch/cnomis1 double mutant flies also exhibit a "giant socket" phenotype. These phenotypes are rarely observed in flies singly mutant for either cnomis1, scabrous or Notch. The wing vein gaps caused by another Notch allele producing an activated form of N protein, are dominantly suppressed by cnomis1. Heterozygosity for shaggy and myospheroid promotes formation of extra wing veins in cnomis1 homozygotes. The genetic interactions suggest that cno participates with members of the Notch pathway in regulating adhesive cell-cell interactions for the determination of cell fate. Since there appears to be a direct physical interaction between Notch receptor and Dishevelled, providing a link between Notch and wingless signaling, perhaps Canoe plays a role in modifying this interaction (Miyamoto, 1995).

The canoemisty1 (cnomis1) mutation was isolated by virtue of its severe rough eye phenotype from approximately 500 fly lines, each line harboring a single autosomal insertion of a P element (Bm delta w). Excision of the P element generated a lethal, null allele (cnomis10), together with many revertants with normal eye morphology. Ommatidia homozygous for cnomis10, produced in an otherwise wild-type eye by somatic recombination, typically contain a reduced number of outer photoreceptors. Some cnomis1 homozygous adults bear extra macrochaetes on the head, notum, humerus and/or scutellum. cnomis1 hemizygotes often show conspicuous wing phenotypes, such as a notched blade and the loss of a cross vein (Miyamoto, 1995).

Cone cells are lens-secreting cells in ommatidia, the unit eyes that compose the compound eye of Drosophila. Each ommatidium contains four cone cells derived from precursor cells of the R7 equivalence group which expresses the gene sevenless (sev). When a constitutively active form of Ras1 (Ras1V12) is expressed in the R7 equivalence group cells using the sev promoter (sev-Ras1V12), additional cone cells are formed in the ommatidium. Expression of Ras1N17, a dominant negative form of Ras1, results in the formation of 1-3 fewer cone cells than normal in the ommatidium. The effects of Ras1 variants on cone cell formation are modulated by changing the gene dosage at the canoe locus, which encodes a cytoplasmic protein with Ras-binding activity. An increase or decrease in gene dosage potentiates the sev-Ras1v12 action, leading to marked induction of cone cells. A decrease in cno+ activity also enhances the sev-Ras1N17 action, resulting in a further decrease in the number of cone cells contained in the ommatidium. In the absence of expression of sev-Ras1V12 or sev-Ras1N17, an overdose of wild-type cno (cno+) promotes cone cell formation, while a significant reduction in cno+ activity results in the formation of 1-3 fewer cone cells than normal in the ommatidium. It is proposed that there are two signaling pathways in cone cell development, one for its promotion and the other for its repression; Cno is thought to function as a negative regulator for both pathways. It is also postulated that Cno acts predominantly on a prevailing pathway in a given developmental context, thereby resulting in either an increase or a decrease in the number of cone cells per ommatidium. The extra cone cells resulting from the interplay of Ras1v12 and Cno are generated from a pool of undifferentiated cells, normally fated to either develop into pigment cells or undergo apoptosis (Matsuo, 1997).

Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).

Since ommatidial rotation is a cell motility process requiring cytoskeletal rearrangements, it was of interest to determine if effectors of Egfr other than the Raf/MAPK cascade play a role in this process. The Ras GTPase, the main transducer of Egfr signaling, can utilize distinct effectors in different contexts. In addition to nuclear signaling, mediated by the Raf/MAPK/Pnt cascade, Ras can affect cell growth and cytoskeletal rearrangements via its effectors Rgl/Ral, Phospho-inositol-3-Kinase (PI3K) and Canoe, whose human homolog (AF6) is known as the critical partner of ALL1 in a chimeric protein associated with myeloid leukemia (Gaengel, 2003).

A direct canoe (cno) requirement in ommatidial rotation was tested using LOF alleles. First, it was asked whether cno heterozygosity interacts with the Star^48-5/+ rotation phenotype. Strikingly, similar to the enhancement observed with Egfr or Ras, the cno^2/+ and cno^3/+ genotypes enhance the S^48-5/+ rotation phenotype. cno is required for cone cell and photoreceptor differentiation and thus clones of null and strong alleles cause a general disorganization of the eye and are difficult to analyze for rotation defects. However, the hypomorphic cno^mis1 allele is subviable in trans to the strong alleles cno² and cno³ with mildly rough eyes, allowing an analysis of ommatidial rotation. Eye sections of such transheterozygous cno flies (e.g. cno^mis1/cno²) reveal severe rotation defects. To test whether such defects are already observed at the time when rotation takes place, cno mutant third instar eye discs were analyzed. Strikingly, rotation defects, comparable in strength to the stronger aos alleles, are apparent in cno eye imaginal discs. The discs were counterstained with anti-Elav to ensure that the photoreceptor complement is normal in such cno mutant discs and the observed rotation abnormalities are primary defects, which was indeed confirmed. A similar analysis of Ral/Rgl is precluded by the lack of suitable alleles. In summary, these data indicate that cno plays a critical role in ommatidial rotation and acts as an effector of Egfr/Ras signaling in this context (Gaengel, 2003).

Since ommatidial rotation is a cell biological event, it is probable that among the main read-outs affected are cell-adhesion properties of the precluster cells and effects on cytoskeletal elements. This is further supported by observations that (1) Raf/MAPK-independent and thus transcription-independent Egfr/Ras signaling pathways are important, and (2) that canoe is required in this context. To address this further, two sets of experiments were performed. First, tests were performed for genetic interactions between the dosage-sensitive Star^–/+ rotation phenotype and selected factors required in cell adhesion and cytoskeletal regulation; and second, whether cell-adhesion components such as cadherins and integrins are normally localized in aos^rlt and cno^Mis1 mutant backgrounds was directly analyzed (Gaengel, 2003).

The Egfr/Ras/Cno link is intriguing for several reasons. The cno gene was originally identified as a mutation affecting the dorsal closure process during embryogenesis. Cno shows a genetic and molecular link to Ras: it contains two Ras-interacting domains and binds both WT Ras and activated Ras-V12. In addition, Cno has been postulated to link cytoskeletal elements to cellular junctions via its ability to bind actin, its interaction with ZO-1/Pyd and its homology with kinesin and myosin-like domains. Thus Cno could directly mediate an Egfr/Ras signal to cytoskeletal and cell architecture elements through its association with adherens junctions and its kinesin and myosin-like domains. Interestingly, Zipper does not only show a similar interaction with Star, like Cno, but it is also required during embryonic dorsal closure, and thus a more general Cno-Zipper link might exist in cell motility contexts (Gaengel, 2003).

A second interesting feature of cno is that it has been genetically linked to sca and Notch signaling. First, the phenotype of the sca¹ allele is strongly enhanced by cno^–/+. Second, cno alleles also display Notch-like phenotypes in the wing and a GOF Notch allele, Notch^Abruptex, is suppressed by cno. Although the biochemical role of Sca remains obscure, it has been linked to Notch, possibly as a Notch ligand, in several contexts. Thus, since sca has recently been implicated in ommatidial rotation, the link between Cno and Sca/Notch is intriguing. Taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in this process, and relaying this to cytoskeletal elements. The Canoe link is also interesting from a disease point of view since its human homolog AF6 is the critical partner of ALL1 in a chimeric protein associated with myeloid leukemia. Thus, taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in ommatidial rotation, and relaying this to the regulation of cell adhesion and cytoskeletal elements in the context of a developmental patterning process or disease (Gaengel, 2003).

Thus Egfr/Ras signaling plays a general role in the regulation of ommatidial rotation. Canoe has been identified as an effector of Ras in this context. Although much is known about how ommatidial chirality and the associated R3/R4 cell-fate decision are regulated (Fz/PCP-Notch signaling), no clear link between the mechanistic aspects of ommatidial rotation and Fz/PCP signaling previously existed. This is the first link to be demonstrated between Egfr signaling and PCP genes, namely Fmi. A further connection between Egfr signaling and PCP establishment is provided by Zipper, which acts downstream of Fz/Dsh and Rok in wing PCP and modifies the Star rotation phenotype. The identification of the Egfr pathway and its regulation of Fmi/cadherin-mediated cell adhesion will serve as an important entry point to further such studies (Gaengel, 2003).

The cell adhesion molecules Echinoid and Friend of Echinoid coordinate cell adhesion and cell signaling to regulate the fidelity of ommatidial rotation in the Drosophila eye

Directed cellular movements are a universal feature of morphogenesis in multicellular organisms. Differential adhesion between the stationary and motile cells promotes these cellular movements to effect spatial patterning of cells. A prominent feature of Drosophila eye development is the 90° rotational movement of the multicellular ommatidial precursors within a matrix of stationary cells. This study shows that the cell adhesion molecules Echinoid (Ed) and Friend of Echinoid (Fred) act throughout ommatidial rotation to modulate the degree of ommatidial precursor movement. It is proposed that differential levels of Ed and Fred between stationary and rotating cells at the initiation of rotation create a permissive environment for cell movement, and that uniform levels in these two populations later contribute to stopping the movement. Based on genetic data, it is proposed that ed and fred impart a second, independent, `brake-like' contribution to this process via Egfr signaling. Ed and Fred are localized in largely distinct and dynamic patterns throughout rotation. However, ed and fred are required in only a subset of cells -- photoreceptors R1, R7 and R6 -- for normal rotation, cells that have only recently been linked to a role in planar cell polarity (PCP). This work also provides the first demonstration of a requirement for cone cells in the ommatidial rotation aspect of PCP (Fetting, 2009).

ed and fred also genetically interact with the PCP genes, but affect only the degree-of-rotation aspect of the PCP phenotype. Significantly, this study demonstrates that at least one PCP protein, Stbm, is required in R7 to control the degree of ommatidial rotation (Fetting, 2009).

This study demonstrates that ed and fred have partially overlapping functions during the two phases of ommatidial rotation. It is proposed that different levels of Ed and Fred in rotating and non-rotating cells modulate the adhesivity of these cells, a prerequisite for rotation to occur. In the second phase, Ed and Fred are required in R1, R6, R7 and the cone cells, where they are likely to regulate the Egf receptor to contribute to the slowing of rotation (Fetting, 2009).

There are two phases of rotation distinguishable by the rate at which the ommatidia rotate. The initial phase (rows 4-7) is fast, with ommatidia rotating 10-15° per row, whereas rotation slows to 5-10° per row in the slow phase (rows 7-15). The data demonstrate that Ed and Fred function during both phases and that they play unique roles in each phase (Fetting, 2009).

In the first phase, it is proposed that the tight regulation of Ed and Fred levels between rotating and stationary cells creates an environment that is permissive to rotation. Immediately before rotation starts, Ed begins to be endocytosed in the ommatidial precluster cells. Concurrently, Ed levels fall dramatically in these cells while remaining high in the stationary interommatidial cells (IOCs), setting up an imbalance in Ed levels between these two populations of cells. It is proposed that the resulting differential adhesion between these two cell populations enables the rotating cells to slide past their stationary neighbors in accordance with Steinberg's differential adhesion hypothesis (DAH) (Steinberg, 2007). The DAH suggests that cell populations maximize the strength of adhesive bonding between them and minimize the adhesive free energy, and use tension generated by adhesion between cells to drive events such as cell rearrangements during morphogenesis. Cells with equivalent levels of Ed (or Fred) adhere more tightly to one another and adhesion is reduced between cells with different levels of Ed (or Fred), thereby enabling the two groups to slide past one another. In support of this hypothesis, artificially equalizing levels of Ed or Fred significantly slows rotation (Fetting, 2009).

The data are consistent with Ed and Fred playing two key roles in the slow phase by both directly and indirectly (through Egfr signaling) affecting the physical component of the process. It is suggested that in both cases the outputs produce adhesive forces that slow/stop rotation. Ed and Fred are required in photoreceptors R1, R6 and R7 and the cone cells for normal ommatidial rotation. These cells do not become fully integrated into the ommatidial cluster until the second half of rotation. Furthermore, R1, R6 and R7 constitute the rotation interface until the cone cells are recruited, at which point the cone cells co-opt this position and role. Consequently, Ed and Fred are required in the right place (the subset of cells that lie at the rotation interface) and at the right time (the slower phase of rotation) to play a role in slowing rotation (Fetting, 2009).

It is proposed that Ed and Fred activity in R1, R6, R7 and the cone cells regulates Egfr signaling in these cells to slow/stop rotation as follows. Egfr signaling promotes rotation via the Ras/Cno and Ras/Mapk/Pnt effectors (Brown, 2003; Gaengel, 2003), so its output must be dampened to slow rotation. Ed binds and inhibits the Egf receptor, whereas Fred binds Ed and interferes with this inhibition. Therefore, cooperation between Ed and Fred precisely titrates Egfr activity in the cells in which Ed and Fred function. As R1, R6 and R7 are recruited into the ommatidial cluster, Ed levels are high in these cells, thereby decreasing Egfr signaling at their side of the rotation interface, thus impeding rotation. This inhibitory role switches to the cone cells when they are recruited, creating a new rotation interface (Fetting, 2009).

Rotation may be slowed through Egfr signaling activity via its effector Cno, the fly homolog of Afadin/AF-6, an actin-binding adherens junction (AJ) protein. Afadin and its binding partners, nectins and α-actinin, build and stabilize dynamic AJs that undergo remodeling (Ooshio, 2007). The majority of cno mutant ommatidia over-rotate, indicating that Cno inhibits ommatidial rotation. Since Egfr signaling promotes and Cno inhibits rotation, Egfr signaling is likely to suppress Cno activity during rotation thereby blocking stable junction formation. In this scenario, high levels of Egfr would be required during the early phase of rotation to prevent Cno from promoting stable junctions between rotating and non-rotating cells. Consistent with this hypothesis, levels of Ed, an Egfr inhibitor, are very low in ommatidial cells both when rotation commences and during the fast phase of rotation (Fetting, 2009).

Early in the second half of rotation, it is proposed that higher levels of Ed activity are necessary to repress Egfr signaling at the rotation interface, possibly increasing the amount of active Cno and consequently increasing the number of stable AJs between the moving and stationary cells. The more tightly the cells adhere to one another, the less permissive the environment is for movement, and the more difficult rotation becomes. Ed levels are high in the cells in which it would need to be high, i.e., R1, R6, R7 and the cone cells. Once rotation is complete, Ed and Fred are at high levels at the cell boundaries between the interommatidial and ommatidial cells, an indication that stable AJs now cement the fully rotated ommatidia in place (Fetting, 2009).

ed and fred interact genetically with the R3 and R4 genes, respectively, modifying only the degree-of-rotation aspect of the PCP phenotype. Genetic and molecular epistasis data suggest that ed and fred act in a pathway either downstream of, or parallel to, the PCP genes. First, localization of Ed and Fred does not require the PCP complex, nor do the PCP proteins require Ed and Fred for their localization. Second, mutations in ed and fred affect only one aspect of the PCP phenotype (Fetting, 2009).

Nectins and afadins have been implicated in numerous human diseases and developmental defects, including breast cancer, metastasis and cleft palate. Defective cell adhesion and cell signaling also underlie these problems. Given the interspecies conservation of AJ genes, similar mechanisms might control ommatidial rotation and contribute to these human diseases (Fetting, 2009).

The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled

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 pyd^MZ 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 pyd^MZ 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 pyd^MZ 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 pyd^MZ 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 pyd^MZ 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 pyd^MZ 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 pyd^MZ 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).

EVOLUTIONARY HOMOLOGS

Mammalian Ras proteins associate with multiple effectors, including Raf, Ral guanine nucleotide dissociation stimulator, phosphoinositide 3-kinase and AF-6. In the nematode Caenorhabditis elegans, LIN-45/Raf has been identified genetically as an effector of LET-60/Ras. To search for other effectors in C. elegans, a yeast two-hybrid screening was carried out for LET-60-associating proteins. The screening identified a novel protein, designated Ce-AF-6, which exhibits a strong structural homology with human AF-6, rat Afadin and Drosophila Canoe and possesses both a Ras-associating (RA) domain and a PSD-95/DlgA/ZO-1 (PDZ) domain. Ce-AF-6 associates with human Ha-Ras in a GTP-dependent manner, with an efficiency comparable to that of human Raf-1 Ras-binding domain. When the effects of mutations of the Ras effector region residues were examined for associations with various effectors, Ce-AF-6 was found to possess a distinct and the most rigorous requirement for the effector region residues. These results strongly suggest that Ce-AF-6 is a putative effector of Ras that possesses a distinct recognition mechanism for association with Ras (Watari, 1998).

Reciprocal chromosome translocations involving 11q23 are frequently associated with acute leukemias, with the t(4;11) translocation predominating among acute lymphoblastic leukemias, and the t(9;11), t(11;19) and t(6;11) translocations most common among acute myeloid leukemias. In each of these translocations the ALL-1 gene, located at 11q23 and constituting the human homolog of Drosophila trithorax, fuses to a specific gene on the partner chromosome to produce a chimeric protein. The partner gene from chromosome 6 (AF-6) is expressed in a variety of cell types and encodes a protein of 1612 amino acids. The protein contains short stretches rich in prolines, charged amino acids, serines, or glutamines. In addition, the AF-6 protein contains the GLGF motif shared with several proteins of vertebrates and invertebrates thought to be involved in signal transduction at special cell-cell junctions (Prasad, 1993).

The dynamic rearrangement of cell-cell junctions (such as tight junctions and adherens junctions) is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions contain associated proteins such as occludin and its associated ZO-1 and ZO-2 and adherens junctions contain associated adhesion proteins such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. The ALL-1 fusion partner from chromosome 6 (AF-6) has been identified as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes, such as cell-cell contact sites. The roles of Ras and AF-6 were investigated in the regulation of cell-cell contacts and it was found that AF-6 accumulates at the cell-cell contact sites of polarized MDCKII epithelial cells and has a distribution similar to that of ZO-1 but somewhat different from that of catenins. Immunoelectron microscopy reveales a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacts with ZO-1 in vitro. ZO-1 interacts with the Ras-binding domain of AF-6, and this interaction is inhibited by activated Ras. AF-6 accumulates with ZO-1 at the cell-cell contact sites in cells lacking tight junctions, such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 fibroblasts results in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells, and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras (Yamamoto, 1997).

In infantile leukemias and therapy-related leukemias, the MLL gene is frequently found to be disrupted and fused to various translocation partner genes, such as AF4/FEL, LTG9/AF9 and LTG19/ENL as a result of 11q23 translocations. The N-terminal portion common to various chimeric MLL products, as well as to MLL-LTG9 and MLL-LTG19, localizes in the nuclei, and therefore might play an important role in leukemogenesis. In the present study, MLL-AF6 chimeric products found in the t(6;11)(q27;q23) translocation were analysed, since AF6, a Ras-binding protein, exhibits a different subcellular localization from that of LTG9/AF9 and LTG19/ENL. Immunofluorescence staining data and cell fractionation analyses demonstrate that MLL-AF6 chimeric products localize in the nuclei despite the fact that AF6 itself localizes in the cytoplasm, confirming the importance of the nuclear localization of chimeric MLL products. The region in the N-terminal portion of MLL responsible for this nuclear localization is a region containing AT-hook motifs (Joh, 1997).

The Drosophila fat facets and canoe genes regulate non-neural cell fate decisions during ommatidium formation. The FAM (Fat facets in mouse) de-ubiquitinating enzyme regulates the function of AF-6 (mammalian Canoe homolog) in the MDCK epithelial cell line. The expression of the FAM and AF-6 proteins overlaps extensively in the mouse eye from embryogenesis to maturity, especially in the non-neural epithelia including the retinal pigment epithelium, subcapsular epithelium of the lens and corneal epithelium. Expression is not limited to the epithelia however, because FAM and AF-6 also co-localize during lens fiber development as well as in sub-populations of the neural retina (Kanai-Azuma, 2000).

Par-3 is a cell-polarity protein that regulates the formation of tight junctions (TJs) in epithelial cells, where claudin is a major cell-cell adhesion molecule (CAM). TJs are formed at the apical side of adherens junctions (AJs), where E-cadherin and nectin are major CAMs. Nectin first forms cell-cell adhesions, and then recruits cadherin to nectin-based cell-cell adhesion sites to form AJs and subsequently recruits claudin to the apical side of AJs to form TJs. The cytoplasmic tail of nectin binds afadin and Par-3. Afadin regulates the formation of AJs and TJs cooperatively with nectin. This paper deals with the role of Par-3 in the formation of these junctions by using Par-3-knockdown MDCK cells. Par-3 is necessary for the formation of AJs and TJs but was not necessary for nectin-based cell-cell adhesion. Par-3 promotes the association of afadin with nectin, whereas afadin is not necessary for the association of Par-3 with nectin. However, the association of afadin with nectin alone is not sufficient for the formation of AJs or TJs, and Par-3 and afadin cooperatively regulates it. This paper describes these novel roles of Par-3 in the formation of junctional complexes (Ooshio, 2007).

Junctional adhesion molecule-A (JAM-A) is a transmembrane tight junction protein that has been shown to regulate barrier function and cell migration through incompletely understood mechanisms. JAM-A regulates cell migration by dimerization of the membrane-distal immunoglobulin-like loop and a C-terminal postsynaptic density 95/disc-large/zona occludens (PDZ) binding motif. Disruption of dimerization results in decreased epithelial cell migration secondary to diminished levels of beta1 integrin and active Rap1. This study reports that JAM-A is physically and functionally associated with the PDZ domain-containing molecules Afadin and PDZ-guanine nucleotide exchange factor (GEF) 2, but not zonula occludens (ZO)-1, in epithelial cells, and these interactions mediate outside-in signaling events. Both Afadin and PDZ-GEF2 colocalize and coimmunoprecipitate with JAM-A. Furthermore, association of PDZ-GEF2 with Afadin is dependent on the expression of JAM-A. Loss of JAM-A, Afadin, or PDZ-GEF2, but not ZO-1 or PDZ-GEF1, similarly decreases cellular levels of activated Rap1, beta1 integrin protein, and epithelial cell migration. The functional effects observed are secondary to decreased levels of Rap1A because knockdown of Rap1A, but not Rap1B, results in decreased beta1 integrin levels and reduced cell migration. These findings suggest that JAM-A dimerization facilitates formation of a complex with Afadin and PDZ-GEF2 that activates Rap1A, which regulates beta1 integrin levels and cell migration (Severson, 2009).

Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth

Afadin/AF6, an F-actin-binding protein, is ubiquitously expressed in epithelia and has a key role during development, through its regulatory role in cell-cell junction organization. Afadin loss of expression in 15% of breast carcinoma is associated with adverse prognosis and increased risk of metastatic relapse. To determine the role of afadin in breast cancer, the functional consequences were studied of afadin protein extinction using in vitro and in vivo models. Three different breast cancer cell lines representative of the major molecular subtypes were stably repressed for afadin expression [knockdown of afadin (afadin KD)] using RNA interference. Collective and individual migrations as well as Matrigel invasion were markedly increased in afadin KD cells. Heregulin-beta1 (HRG-beta1)-induced migration and invasion were increased by twofold in afadin KD cells. Conversely, ectopic expression of afadin in the afadin-negative T47D cell line inhibited spontaneous and HRG-beta1-induced migrations. RAS/MAPK and SRC kinase pathways were activated in afadin KD cells. Activation levels positively correlated with migration and invasion strength. Use of MEK1/2 (U0126) and SRC kinases (SU6656) inhibitors reduced afadin-dependent migration and invasion. Afadin extinction in the SK-BR-3 cell line markedly accelerated tumor growth development in mouse mammary gland and lung metastasis formation. These results may explain why the loss of afadin expression in tumors correlates with high tumor size and poor metastasis-free survival in patients (Fournier, 2011).

REFERENCES

Search PubMed for articles about Drosophila canoe

Asha, H., et al. (1999) The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J. 18: 605-615. 9927420

Boettner, B., et al. (2003). The AF-6 homolog Canoe acts as a Rap1 effector during dorsal closure of the Drosophila embryo. Genetics 165: 159-169. 14504224

Boettner, B. and Van Aelst, L. (2007). The Rap GTPase activator Drosophila PDZ-GEF regulates cell shape in epithelial migration and morphogenesis. Mol Cell Biol 27: 7966-7980. PubMed ID: 17846121

Bonello, T. T., Perez-Vale, K. Z., Sumigray, K. D. and Peifer, M. (2018). Rap1 acts via multiple mechanisms to position Canoe and adherens junctions and mediate apical-basal polarity establishment. Development 145(2). PubMed ID: 29361565

Brown, K. E. and Freeman, M. (2003). Egfr signalling defines a protective function for ommatidial orientation in the Drosophila eye. Development 130: 5401-5412. PubMed Citation: 14507785

Choi, W., et al. (2011). The single Drosophila ZO-1 protein Polychaetoid regulates embryonic morphogenesis in coordination with Canoe/afadin and Enabled. Mol. Biol. Cell 22(12): 2010-30. PubMed Citation: 21508316

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