InteractiveFly: GeneBrief

Adherens junction protein p120: Biological Overview | References

BIOLOGICAL OVERVIEW

Correct patterning of cells within an epithelium is key to establishing their normal function. However, the precise mechanisms by which individual cells arrive at their final developmental niche remains poorly understood. An optimized system was developed for imaging the developing Drosophila retina, an ideal tissue for the study of cell positioning. Using this technique, the cellular dynamics of developing wild-type pupal retinas were characterized. Two mutants affecting eye patterning were analyzed and it was demonstrated that cells mutant for Notch or Roughest signaling were aberrantly dynamic in their cell movements. A role for the adherens junction regulator P120-Catenin in retinal patterning was establised through its regulation of normal adherens junction integrity. The results indicate a requirement for P120-Catenin in the developing retina, the first reported developmental function of this protein in the epithelia of lower metazoa. Based upon live visualization of the P120-Catenin mutant as well as genetic data, it is concluded that P120-Catenin is acting to stabilize E-cadherin and adherens junction integrity during eye development (Larson, 2008).

The precise spatial arrangement of the cells within tissues is essential for their function. In some tissues, spatial restriction of cell fate is sufficient to generate the final pattern. In other tissues, such as the developing mammalian brain, the vertebrate retina, and the intestinal epithelium, cells migrate from their original position to their final niche. These migrations can occur across significant distances but in most examples likely reflect more subtle cellular movements within the epithelium. Although some of the molecules that mediate these migrations are known, most tissues do not provide the needed accessibility to dissect individual cell movements in detail (Larson, 2008).

The Drosophila pupal retina is an ideal system in which to study cell positioning during development. The fully patterned retinal epithelium consists of a regular array of identical unit eyes. These 'ommatidia' are initially crudely arrayed within the larval eye field and are separated by a loose collection of 'interommatidial precursor cells' (IPCs). In the pupa, a precisely regulated combination of cell movements, death, and differentiation corrals these IPCs into their final positions, yielding a honeycomb pattern that re-organizes the ommatidia into a hexagonal array (Larson, 2008 and references therein).

These patterning steps are dependent on both signaling and adhesion. Cell-cell signaling regulates the number of cells within developing ommatidia as well as the specification of each cell type. For example, Notch pathway activity is required for differentiation of each of the 20 cell fates within the eye field. In addition, Notch activity is required within the IPCs for proper cell number as well as cell sorting. However, while the role of Notch in directing cell fate is well-established, less is understood of whether Notch also regulates cell morphogenesis (Larson, 2008).

The adhesion molecules Roughest and Hibris also play an essential role in patterning the retina as these two molecules are required to refine the IPC lattice to a hexagon. Single cell expression experiments with Roughest and Hibris indicate that both mediate the final positioning of cells within the hexagon through direct heterophilic adhesion and that both control adherens junction formation between IPCs. For example, as IPCs re-arrange into their final pattern they briefly reduce their adherens junctions; these junctions are then re-assembled as patterning is completed. Ectopic expression of Hibris in the developing hexagonal lattice resulted in the premature re-appearance of these junctions as well as mis-patterning. However, the mechanisms by which the adherens junctions are normally dynamically regulated are not known (Larson, 2008).

This study presents a method for visualizing development of the living pupal eye in situ. This method was use to extend previous observations on the cellular movements of the developing retina in wild-type and in two classical eye mutants that alter cellular positioning, one through cell signaling and a second through cell adhesion. Previous work has suggested that regulation of adherens junctions is important for patterning. To begin to address this issue, live visualization was used to demonstrate a role for P120-Catenin as a regulator of E-Cadherin as IPCs undergo the precise movements required to generate a hexagonal pattern within the eye field (Larson, 2008).

It has been proposed that adherens junctions play an important role in patterning the pupal eye. The Notch allele Notch^facet-glossy (N^fa-g), that specifically reduces Notch activity in the pupal eye and the truncation mutant rst^CT did not exhibit disruption of α-Catenin-GFP, suggesting that the adherens junctions were correctly regulated in Notch and rst mutants. Indeed, mutations specifically affecting the adherens junctions but not the integrity of the retinal epithelium have not been reported. P120-Catenin, encoded by p120ctn, is an armadillo repeat domain-containing protein that binds to the juxtamembrane domains of classical cadherins to regulate adherens junction stability and activity (reviewed in Anastasiadis, 2007 and Xiao, 2007). In mammals, it is essential for viability and modulates the levels and adhesive properties of cadherins (reviewed in McCrea, 2007). In Drosophila and C. elegans, deletion of the p120ctn locus enhanced mutations in cadherin but was non-essential for viability (Myster, 2003; Pacquelet, 2003; Pettitt, 2003). Recent work, however, has found that p120ctn is required to regulate neuron morphology (Li, 2005). In contrast, despite earlier reports to the contrary (Magie, 2002), recent studies ascribe no phenotype to p120ctn in Drosophila epithelia. This led to the suggestion that P120-Catenin solely plays a supporting role in cadherin-based adhesion (Fox, 2005; Myster, 2003; Pacquelet, 2003; Larson, 2008).

The surface phenotype of adult fly eyes homozygous for the null p120ctn allele p120ctn³⁰⁸ (Myster, 2003) was wild-type in appearance. However, examination of the pupal retina indicated that genotypically p120ctn mutant eyes have ectopic lattice cells and a partially penetrant mis-patterning of the 3° niche. Pupae bearing the p120ctn³⁰⁸ chromosome in trans to a deficiency covering the region (Df(2R)244) exhibited a phenotype similar to homozygous p120ctn³⁰⁸ retinas, consistent with previous reports (Myster, 2003) that p120ctn³⁰⁸ represents a null allele. The p120ctn³⁰⁸ mutation was generated by imprecise excision of a P-element found in the parent line KG01086 (Myster, 2003). Retinas bearing a single copy of p120ctn³⁰⁸ in trans to KG01086 exhibited a wild-type phenotype indicating that the p120ctn³⁰⁸ phenotype was a direct result of the excision event. Lastly, ubiquitous expression of a full-length p120ctn-GFP transgene in a p120ctn null background completely rescued the p120ctn null phenotype. Taken as a whole this data indicates that the eye phenotype is a direct result of a loss of p120ctn and represents the first reported developmental requirement of p120ctn in an epithelium of lower metazoa (Larson, 2008).

To better understand the role of p120ctn in patterning the fly eye, p120ctn null mutant development was imaged from 24 to 29 h a.p.f. (four retinas total). While the final p120ctn³⁰⁸ phenotype was fairly subtle, live imaging revealed surprisingly dramatic differences with wild-type development. In particular, live imaging of p120ctn³⁰⁸ pupal eyes showed consistent, transient separation of IPCs accompanied by a loss of α-Catenin-GFP fluorescence from the membranes at their contact face. This apparent breakdown of coherent junctions occurred almost exclusively between IPC:IPC and IPC:1° junctions and presumably accounted for their ability to achieve or maintain stable positions. To quantitate this difference, the dynamics of 3° emergence was followed throughout the stage of IPC patterning. A clear difference was observed in the ability of local IPCs to achieve and - in particular - to retain a position in the 3° niche. This instability and ectopic movement presumably accounts for the errors in 3° patterning observed in the mid-pupa. Other parameters such as cell movements were on the whole indistinguishable from wild-type. This result indicates that p120ctn³⁰⁸ IPCs are capable of forming adherens junctions but are unable to maintain them during dynamic cell movements (Larson, 2008).

No clear genetic interactions -- either as trans heterozygotes or as dominant modifier activity -- were observed between p120ctn and Egfr, wingless, roughest, Notch, shotgun, α-Catenin, or the small GTPases (reducing Rho1 or Cdc42). However, a closer genetic analysis of the relationship between p120ctn, shotgun, and Rho1 yielded surprising results. Based on both cell culture and in vivo data, mammalian P120-Catenin has been proposed to regulate both RhoA and E-cadherin (Anastasiadis, 2000; Davis, 2006; Grosheva, 2001; Noren, 2000; Perez-Moreno; 2006; Larson, 2008).

Interestingly, the phenotype observed with complete loss of p120ctn activity (using the null deletion allele p120ctn³⁰⁸) was further enhanced by removing a functional genomic copy of shotgun (shg^R69) or Rho1 (Rho1^72O). Removal of Rho1 resulted in additional ectopic cells and an increase in the frequency of patterning errors. In the case of the shg interaction, the hexagonal IPC pattern was disrupted with extra cells present in double layers around bristle cells. The severity of this interaction prevented its quantification. Neither shg^R69 nor Rho1^72O, both null alleles, gave a dominant phenotype on their own. The ability of mutations in shotgun or Rho1 to further enhance a null mutation in p120ctn indicates that both DE-Cadherin and Rho1 act, at least in part, through a pathway that is independent of P120-Catenin. However, a consistent difference was observed in E-cadherin localization. While full loss of p120ctn led to at most a slight decrease in Armadillo and E-cadherin, it was noted that E-cadherin protein was discontinuous at the membranes of p120ctn³⁰⁸ cells. This was best observed when comparing loss of P120-Catenin next to a rescue construct of P120-Catenin in neighboring clonal patches (Larson, 2008).

This study has further characterized the cell movements required to pattern the developing pupal retina. While many of these movements have been inferred from dissected tissue, it was observed that the developing retina was more dynamic than expected in both wild-type and mutant flies. For example, it has been speculated that the roughest phenotype was due to a loss of cell movement. However, rst^CT IPCs were observed to actively exchange contacts and neighbors despite the fact that this exchange did not productively pattern the retina. In an earlier study, scanning electron micrographs showed that rst^CT IPCs extend filopodia from their apical surface in a manner identical to wild-type. Combined with live visualization studies, this data indicates that rst^CT cells have an active cytoskeleton and can participate in cell rearrangement but cannot functionally recognize 1°s. Alternatively, rst^CT cells may fail to establish junctions that stabilize a final position; consistent with this latter possibility, the ability of ectopic Hibris to direct precocious adherens junctions has been reported (Bao, 2005). While SEM studies of N^fa-g remain to be conducted, the similarity of the movements of N^fa-g IPCs to rst^CT IPCs is striking. In fact, Notch is required for localization of Roughest protein perhaps accounting for their phenotypic similarity (Larson, 2008).

Cell adhesion plays a key role in patterning the developing pupal retina (Bao, 2005). In normal patterning, DE-cadherin staining between IPCs decreased during later stages of IPC re-arrangements, only to increase a few hours later as patterning was completed and cell contacts were finalized (Bao, 2005; Grzeschik, 2005). The loss of roughest resulted in uniform DE-cadherin staining during this time, suggesting that one method by which Roughest may affect retinal patterning is through modulation of E-cadherin levels. BMP family signaling was found to regulate retinal patterning, in part by positively regulating E-cadherin. Using live visualization, this study found that P120-Catenin positively regulated DE-cadherin-based junctions, further demonstrating a role for DE-cadherin regulation in fine cellular patterning within the eye (Larson, 2008).

The mechanism by which P120-Catenin regulates cadherin-based junctions in Drosophila remains unclear. In mammals, P120-Catenin has been shown to regulate cadherin by modulating its endocytosis (Hoshino, 2005; Miyashita, 2007; Xiao, 2005) and degradation (Davis, 2003; Ireton, 2002; Xiao, 2003). It is noted, however, that Drosophila cadherins lack the di-leucine motif that P120-Catenin masks to prevent endocytosis in mammals. Mammalian P120-Catenin also acts as an inhibitor of the small GTPase Rho by regulating RhoGAPs such as p190RhoGap (Wildenberg, 2006) or Rho itself (Anastasiadis, 2000; reviewed in Anastasiadis, 2007). During Drosophila embryogenesis, however, p120ctn failed to show functional interactions with mutations in Rho1 (Fox, 2005). In contrast, this study detected an interaction between p120ctn and Rho1 during eye development, but this interaction was also inconsistent with the mammalian data. If the p120ctn null phenotype was the result of a loss of Rho inhibition, then it would have been expected that removal of a functional genomic copy of Rho would suppress the p120ctn phenotype. Instead the results are consistent with a model in which P120-Catenin and Rho1 act in parallel pathways to regulate eye development (Larson, 2008).

p120 catenin is required for the stress response in Drosophila

p120ctn is a ubiquitously expressed core component of cadherin junctions and essential for vertebrate development. Surprisingly, Drosophila p120ctn (dp120ctn) is dispensable for adherens junctions and development, which has discouraged Drosophila researchers from further pursuing the biological role of dp120ctn. This study demonstrate that dp120ctn loss results in increased heat shock sensitivity and reduced animal lifespan, which are completely rescued by ectopic expression of a dp120ctn-GFP transgene. Transcriptomic analysis revealed multiple relish/NF-kappaB target genes differentially expressed upon loss of dp120ctn. Importantly, this aberrant gene expression was rescued by overexpression of dp120ctn-GFP or heterozygosity for relish. These results uncover a novel role for dp120ctn in the regulation of animal stress response and immune signalling. This may represent an ancient role of p120ctn and can influence further studies in Drosophila and mammals (Stefanatos, 2013).

Csk-deficient boundary cells are eliminated from normal Drosophila epithelia by exclusion, migration, and apoptosis: a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells

The construction and maintenance of normal epithelia relies on local signals that guide cells into their proper niches and remove unwanted cells. Failure to execute this process properly may result in aberrant development or diseases, including cancer and associated metastasis. This study shows that local environment influences the behavior of dCsk-deficient cells. Broad loss of dCsk leads to enlarged and mispatterned tissues due to overproliferation, a block in apoptosis, and decreased cadherin-mediated adhesion. Loss of dCsk in discrete patches leads to a different outcome: epithelial exclusion, invasive migration, and apoptotic death. These latter phenotypes required sharp differences in dCsk activity between neighbors; dE-cadherin, P120-catenin, Rho1, JNK, and MMP2 mediate this signal. Together, these data demonstrate how the cellular microenvironment plays a central role in determining the outcome of altered dCsk activity, and reveal a role for P120-catenin in a mechanism that protects epithelial integrity by removing abnormal cells (Vidal, 2006).

The mechanisms that regulate organ size and shape are not well understood, but recent studies have pointed to the importance of local interactions between neighboring cells. For example, in the process known as 'cell competition', cells with relatively higher proliferative rates actively eliminate their neighbors by programmed cell death. Conversely, apoptotic cells send proliferative signals to their neighbors to compensate for their loss. In this way, normal tissue size is achieved. The misregulation of such mechanisms may contribute to the development of cancer, since most solid tumors arise from intact epithelia and are resistant to size-control signals. Tumors are particularly dangerous when linked to metastasis, a process in which cells leave the primary tumor and invade distant tissues. These processes are best understood within the context of an intact epithelium, in which the full range of cell interactions is retained. Work in Drosophila has provided an important in situ view of the action of oncogenes within epithelia (Vidal, 2006).

Src family kinases (SFKs) are active in a broad range of cancer types, including tumors of the breast, colon, and hematopoietic systems. SFK activity typically increases as tumorigenesis progresses and is associated with metastatic behavior. The major inhibitor of SFK activity is C-terminal Src kinase (Csk) and its paralog Chk; these may act as tumor suppressors in, e.g., breast cancer, presumably through their ability to inhibit Src activity and perhaps other pathways. Drosophila Csk acts primarily or exclusively through Src pathway regulation, and the reduction of dCsk activity by itself led to increased organ size, organismal lethality, and increased cell proliferation due to a failure to exit the cell cycle. However, neither Csk loss nor Src activation has been clearly linked to early events in tumorigenesis, bringing into question the role of Csk/Src in proliferation in vivo. Instead, Src is currently thought to be a major player in the metastatic events that occur later in oncogenesis. How Csk or Src promotes the metastatic behavior of cells in situ remains largely unknown (Vidal, 2006).

This study analyzed the phenotypes of dCsk in the context of developing epithelia. The outcome of a cell's loss of dCsk is linked to its cellular microenvironment. When dCsk activity is reduced broadly in the developing eye or wing, the result is overproliferation, inhibition of apoptosis, and decreased cell adhesion. Tissue integrity is retained, but dCsk cells become inappropriately mobile and fail to maintain their appropriate contacts. The outcome of these effects is an overgrown and mispatterned adult tissue. By contrast, loss of dCsk in discrete patches results in epithelial exclusion, invasive migration through the basal extracellular matrix, and eventual apoptotic death; these events occur exclusively at the boundary between dCsk and wild-type cells. Further emphasizing the unique nature of cells at this boundary, a specific requirement was found for a signal that includes Drosophila orthologs of E-cadherin, P120-catenin, RhoA, JNK, and the metalloprotease MMP2. Hence, this study explores the mechanisms by which the cellular microenvironment can direct different behaviors of cells, both in the regulation of apoptosis and epithelial integrity. It also uncovers a mechanism for the removal of abnormal cells from a normal epithelium (Vidal, 2006).

This study shows that reducing dCskactivity results in a blockade of apoptosis and downregulation of cellular adhesion. The work is consistent with the view that Csk is a tumor suppressor that acts at multiple steps. Mutations in the locus encoding the Csk paralog Chk have been described in breast tumors, and, in this study, it has been observed that human Chk can functionally replace dCsk. Therefore the experimental advantages of developing Drosophila imaginal epithelia were used to explore specific aspects of dCsk function that are relevant to the behavior of tumor cells (Vidal, 2006).

Visualization studies suggest that a reduction in dCsk activity leads to a failure of cells to stably retain associations with their neighbors, resulting in prolonged cell movement as cells slide across each other in a manner not observed in wild-type tissue. This may reflect a failure to establish stable junctions, excess cell motility, or both. Recent work has demonstrated a critical and dynamic role for the cadherin-based apical junctions in patterning the Drosophila retina. Misexpressing dE-cadherin prevents patterning defects in GMR>dCsk-IR retinas, suggesting that dCsk cells have reduced dE-cadherin function. Links between Csk, Src, cadherins, and junctional integrity have been reported in mammalian cell culture, and an association has been observed between Drosophila Src42A and dE-cadherin during embryonic development. The data are consistent with this view: misexpression of a kinase-dead form of Src42A leads to a disruption in the localization of the dE-cadherin-associated protein Armadillo; also, reduced Armadillo levels observed in dCsk retinas is rescued by dE-cadherin misexpression. Together, these data suggest that altering dCsk/Src activity affects cell movements by decreasing dE-cadherin adhesion (Vidal, 2006).

The mechanism by which dCsk alters dE-cadherin function is not clear, but it is relevant to note that Src activation can shift cadherin-based cell adhesion from a 'strong' to a 'weak' adhesive state in mammalian cultured cells. Phosphorylation of cadherins and catenins may mediate 'inside-out' signaling that can alter the adhesive strength of the homophilic bond between cells. Evidence for such a mechanism has been provided for integrin-mediated focal adhesions, and Src activity can alter focal adhesions. However, normal basal membrane architecture was observed in dCsk cells, as assessed both by anti-integrin staining and by transmission electron microscopy, indicating that at least the gross structure is not affected (Vidal, 2006).

The ability of dCsk to influence cell proliferation, apoptosis, and cell adhesion is consistent with its ability to direct tissue overgrowth: reducing dCsk activity throughout a tissue (or the entire organism) leads to significantly enlarged tissues. This ability demonstrates that dCsk can participate in the mechanisms that set tissue size. A small number of other proteins have been implicated in this process, including Salvador, Hippo, and Lats/Warts, which show phenotypes that are strikingly similar to dCsk. Furthermore, dCsk can directly phosphorylate Lats/Warts in vitro (Vidal, 2006).

However, reduction of dCsk activity shows some important differences. Mutations in salvador, hippo, or lats/warts lead to an increase in Diap1 levels, which, in turn, blocks apoptotic cell death. By contrast, reductions in dCsk does not significantly alter Diap1 protein levels. Furthermore, although both Hippo and dCsk are required to exit the cell cycle, the cell cycle profile from hippo mutant cells is normal, while dCsk cells contain a significant shift toward G2/M. Perhaps the most striking difference is the effects of these factors on discrete mutant patches. While broad loss of dCsk activity leads to expanded tissues, surprisingly discrete patches of dCsk tissue are eliminated by neighboring cells. Unlike salvador, hippo, or lats/warts, clonal patches of dCsk cells fail to survive to adulthood. The effects of dCsk reduction are more similar to those reported for the tumor suppressor gene scribble. The scribble locus encodes a component of the septate junction that regulates cell polarity and proliferation; mutant cells display neoplastic overgrowth in a homotypic environment, but are removed by JNK-dependent apoptosis in discrete clonal patches abutting wild-type tissue (Vidal, 2006).

This work provides evidence that neighboring wild-type tissue provides a locally nonautonomous signal that leads to the removal of dCsk mutant cells. For example, FRT-derived clones of dCsk cells were out-competed by neighbors with normal levels of dCsk: this was most easily seen by the clonally related 'twin spot' of wild-type tissue that was consistently larger than the few surviving dCsk clones. In contrast, FRT-mediated dCsk clones that encompassed the entire eye survived and overproliferated. In the developing wing, cells at the periphery of sd>dCsk-IR or ptc>dCsk-IR expression domains were preferentially removed by apoptosis. This death is dependent not on absolute dCsk activity, but on the juxtaposition of cells that are starkly different in their levels of dCsk. Small differences, for example across the ptc>dCsk-IR or omb>dCsk-IR graded expression domains, did not trigger cell death (Vidal, 2006).

This translocation and death of dCsk-IR cells at the patched/wild-type boundary requires at least two steps. At boundaries with wild-type tissue, dCsk cells initially lose their apical profile, shift downward, and eventually become basally excluded from the epithelium. Such excluded cells then migrate away from the boundaries in both directions and eventually die by apoptosis. These events are strikingly reminiscent of those described for tumor cells undergoing metastasis. Altered activity of both Csk and Src has been implicated in a broad variety of tumors. Typically, however, increased Src activity is associated with later events in tumorigenesis, particularly metastasis. Although the connections between high Src activity and metastases are not understood, they likely include Src's ability to break cell-cell junctions and increase cell motility. Another hallmark of metastatic behavior is the ability to degrade basal extracellular matrix: this study also demonstrate a functional requirement for MMP2 activity during the translocation of mutant cells out of the wing epithelium (Vidal, 2006).

While evidence supports the view that the activity of Csk -- and presumably Src and perhaps other effectors -- can regulate metastatic behavior, it alone is not sufficient. First, reducing dCsk activity by itself is not sufficient to allow migrating cells to survive; the data suggest that most or all eventually die. This is consistent with previous work highlighting the importance of a 'two-hit' model to allow for stable tumor overgrowth and metastasis. A second mutation that prevents apoptotic cell death would be minimally required. Second, all cells within a discrete dCsk patch are not equivalent: cells at the boundary of the clone that border cells of strongly differing dCsk levels are exclusively prone to release from the epithelium. This work predicts that cells at the borders of some human tumors are especially prone toward metastatic behavior. Metastasis is often the most serious aspect of a tumor, and approaches that address the metastatic behavior of cells may need to take into account the properties of cells at the periphery. Understanding whether and how these cells are unique may help to more effectively target therapeutic intervention (Vidal, 2006).

In addition to enabling a detailed examination of dCsk cells and their behavior within an epithelium, this model system permitted identification of signaling components that are necessary to execute the aberrant cell mobility and cell death. The results indicate important roles for dE-cadherin, dP120ctn, Rho1, dJnk, and MMP2 (Vidal, 2006).

JNK-dependent apoptosis is required for a broad palette of related mechanisms such as cell competition in developing tissues and the removal of scribble mutant cells. JNK signaling is also associated with the movement of cells within epithelia, including dorsal closure in Drosophila and in mammals. Interestingly, JNK activity is required for the synthesis of MMP2 by v-Src-transformed mammalian cells (Vidal, 2006).

JNK activity can be triggered by several upstream signaling factors, including the small GTPases of the Rho family, and genetic data provide a link between dCsk, dJnk, and Rho1. Rho family proteins are key regulators of cell shape and motility. They also promote the cytoskeletal rearrangements required for epithelial-to-mesenchymal transitions (EMTs), and it is noted that dCsk boundary cells show a number of features that are reminiscent of EMTs. In Drosophila, Rho1 was found to induce an 'invasive' phenotype in wing disc cells, but, in this study, it was demonstrated that, similar to dCsk boundary cells, ptc>Rho1 misexpressing cells also undergo apoptotic death. Most importantly, halving the genetic dose of Rho1 strongly suppresses discrete loss of dCsk, but does not appreciably affect broad loss. Thus, Rho1 activity is linked to dCsk, and activation of Rho1 is sufficient to phenocopy both the apoptotic and migratory phenotypes of dCsk cells located near wild-type tissue (Vidal, 2006).

Previous work in mammalian cell culture has provided direct links between Src and P120-catenin, between cadherins and P120-catenin, and between RhoA and P120-catenin; the latter two interactions have been reported in Drosophila tissue culture systems as well. This study further supports links between these factors in dCsk boundary cells. Interestingly, although normal levels of both dP120ctn and Rho1 were required for the efficient removal of dCsk boundary cells, they were not required for the phenotypes resulting from broad loss of dCsk. The requirement for p120ctn specifically in boundary cells may explain why, although it is the only ortholog present in Drosophila, dP120ctn (Drosophila p120-catenin) is not required for organism viability (Vidal, 2006).

Both Src and P120-catenins are known to directly interact with cadherins, and, in fact, a role was demonstrated for dE-cadherin/Shotgun in the removal of dCsk cells. A model is postulated in which the loss of dCsk results in the remodeling of the zonula adherens, presumably by the phosphorylation of catenins and dE-cadherin itself by Src. Src activation is known to switch cadherin from a strong adhesive state to a weak one, providing one potential explanation for why dCsk retinal cells displayed reduced cell adhesion in situ. One critical question regarding cadherins is whether they have signaling roles that are independent of their adhesive properties. Perhaps relevant to this point, it was surprising to find that reducing dE-cadherin function leads to a suppression of the effects of dCsk-IR at the boundary. A simple dCsk-IR-mediated reduction in dE-cadherin adhesion would be enhanced by further reducing dE-cadherin activity, suggesting that dE-cadherin may provide an active signal that promotes boundary cells' release from the epithelium. If such a signal does exist, neighboring wild-type cells must trigger it, either through their own endogenous dE-cadherin or through a separate, local signal. Why are multiple (3-4) rows affected? The results are consistent with the creation of a successive new boundary as the previous row of cells descends, although other longer-range signals cannot be ruled out (Vidal, 2006).

It is noted that reducing dCsk activity by itself is not sufficient to direct stable tumor overgrowth, supporting the importance of a 'two-hit' model in Drosophila. Loss of the junction protein Scribble showed similar phenotypes to dCsk, including apoptosis, but was found to confer survival and metastatic-like behavior to cells in the presence of an activated Ras isoform. Interestingly, coexpression of dE-cadherin prevents this metastatic behavior (Vidal, 2006).

Finally, how can dP120ctn and Rho1 promote release of dCsk near wild-type boundaries but not act similarly with other dCsk cells? One source of information is the cadherins themselves: the boundary creates an interface of cadherins that have been exposed to different levels of Csk and, presumably, Src activity. This unusual interface may generate the needed dE-cadherin signal. Importantly, recent work has noted a change in the subcellular localization of P120-catenin and E-cadherin specifically at the border of human tumor tissues. At the time that ptc>dCsk-IR boundary cells lose their apical profiles, this study found that dP120ctn is relocalized to the cytoplasm. These results again emphasize the possibility that cells at tumor boundaries pose a special risk of undergoing epithelial-to-mesenchymal-like transitions and metastatic behavior. Metastasis is often the most serious complication of progressing tumors. Targeting therapies to this aspect of cancer may benefit from considering boundary cells and their potentially distinctive properties (Vidal, 2006).

Abelson, enabled, and p120 catenin exert distinct effects on dendritic morphogenesis in Drosophila

Neurons exhibit diverse dendritic branching patterns that are important for their function. However, the signaling pathways that control the formation of different dendritic structures remain largely unknown. To address this issue in vivo, the peripheral nervous system (PNS) of Drosophila was used as a model system. Through both loss-of-function and gain-of-function analyses in vivo, it has been shown that the nonreceptor tyrosine kinase Abelson (Abl), an important regulator of cytoskeleton dynamics, inhibits dendritic branching of dendritic arborization (DA) sensory neurons in Drosophila. Enabled (Ena), a substrate for Abl, promotes the formation of both dendritic branches and actin-rich spine-like protrusions of DA neurons, an effect opposite that of Abl. In contrast, p120 catenin (p120 ctn) primarily enhances the development of spine-like protrusions. These results suggest that Ena is a key regulator of dendritic branching and that different regulators of the actin cytoskeleton exert distinct effects on dendritic morphogenesis (Li, 2005).

Previous studies implicated Ena in the actin-dependent process of axon guidance. Ena, one of the founding members of Ena/VASP family proteins, is present at the leading edge of lamellipodia and at the tips of filopodia and directly binds to the actin monomer-binding protein profilin (Krause, 2003). In cultured hippocampal neurons, Ena/VASP activity is required for the normal formation of filopodia on growth cones and neurite shafts. Using both loss-of-function and gain-of-function approaches, it has been demonstrated that Ena regulates the dendritic branching of different subtypes of DA sensory neurons in Drosophila. Dendritic filopodia may be involved in dendritic growth and branching. As shown by time-lapse analysis, numerous filopodia-like thin processes at the tips of lateral dendrites of DA neurons undergo rapid extension and retraction during Drosophila embryogenesis. Some are stabilized and eventually become lateral branches. These immunostaining studies showed that Ena was localized in dendrites of DA sensory neurons and that the number of dendritic filopodia is significantly reduced in ena mutant embryos. Therefore, it is highly likely that Ena plays analogous roles in dendrites to control the formation of filopodia, precursors of stable dendritic branches (Li, 2005).

Abl, a nonreceptor tyrosine kinase, plays a key role in several developmental processes including axon guidance and epithelial morphogenesis and has a cell-autonomous function in limiting dendritic branching, opposite that of Ena. Loss of Abl activity results in an increased number of dendritic branches and spine-like protrusions, while overexpression of Abl inhibits the formation of dendritic branches and spine-like protrusions. A mutant Abl construct that abolishes its kinase activity has no effect on dendritic development, suggesting that phosphorylation of its substrates is required for its activity in this process. Thus, the extent of dendritic branching appears to be controlled, in part, by the balance between the activities of Ena and Abl (Li, 2005).

Several lines of evidence indicate that Abl also interacts with cadherin complexes. Abl mutations in Drosophila significantly enhance the axonal defects found in armadillo (Arm, the ß-catenin homologue) mutants. Drosophila E-cadherin interacts with Abl in controlling epithelial morphogenesis. Furthermore, Abl forms a complex with the axon guidance receptor Robo and N-cadherin, and the kinase activity of Abl is essential for phosphorylation of ß-catenin. Abl also interacts with and phosphorylates ß-catenin, a member of the p120ctn subfamily in mammals. Several components in the N-cadherin complex regulate various aspects of neuronal morphogenesis (Li, 2005 and references therein).

The potential interaction between Abl and p120ctn prompted an examination of the role of p120ctn in dendritic branching. Drosophila p120ctn mutant embryos do not show defects in adherens junctions, and p120ctn is not required for DE-cadherin function in vivo. However, studies in mammalian systems indicate that p120ctn plays a key role in maintaining normal levels of cadherin. In this study, morphological and molecular differences between dendritic branches and spine-like protrusions on some DA neurons are described. Although those spine-like protrusions do not make synaptic connections with other neurons, they do share some similarities with mammalian spines. Most notably, these processes are more or less perpendicular to dendritic shafts and are highly enriched in actin. Evidence is provided that p120ctn has a function in neural development. It was found that loss of p120ctn activity reduced the number of spine-like protrusions on dendrites of some DA sensory neurons, while overexpression of p120ctn promotes the formation of these fine dendritic structures. Since overexpression of ß-catenin, another cadherin-associated protein, increases spine density in cultured mammalian neurons, it remains possible that p120ctn regulates actin cytoskeleton dynamics through modulating the cadherin complex. Interestingly, ena and p120ctn interacted genetically, revealing a supporting role for p120ctn in modulating dendritic branching of a subset of DA neurons, which was not obvious when p120ctn was mutated alone. Taken together, these genetic analyses suggest that different regulators of the actin cytoskeleton exert their specific effects on dendritic morphogenesis through interactive molecular pathways (Li, 2005).

Rho1 regulates Drosophila adherens junctions independently of p120ctn: Genetic interactions with shotgun

During animal development, adherens junctions (AJs) maintain epithelial cell adhesion and coordinate changes in cell shape by linking the actin cytoskeletons of adjacent cells. Identifying AJ regulators and their mechanisms of action are key to understanding the cellular basis of morphogenesis. Previous studies (Magie, 2002) linked both p120catenin and the small GTPase Rho to AJ regulation and revealed that p120 may negatively regulate Rho. This study examined the roles of these candidate AJ regulators during Drosophila development. It was found that although p120 is not essential for development, it contributes to morphogenesis efficiency, clarifying its role as a redundant AJ regulator. Rho has a dynamic localization pattern throughout ovarian and embryonic development. It preferentially accumulates basally or basolaterally in several tissues, but does not preferentially accumulate in AJs. Further, Rho1 localization is not obviously altered by loss of p120 or by reduction of core AJ proteins. Genetic and cell biological tests suggest that p120 is not a major dose-sensitive regulator of Rho1. However, Rho1 itself appears to be a regulator of AJs. Loss of Rho1 results in ectopic accumulation of cytoplasmic DE-cadherin, but ectopic cadherin does not accumulate with its partner Armadillo. These data suggest Rho1 regulates AJs during morphogenesis, but this regulation is p120 independent (Fox, 2005).

Analysis of Rho1 and Rho1 shg mutants is consistent with the hypothesis that Rho1 regulates AJs, but suggests that their interactions are complex. A weak shg allele was enhanced, but stronger alleles were suppressed. There are several possible explanations for these contrasting results. Weak alleles (e.g. shg^G119) make protein with reduced but residual function. If Rho1 negatively regulates cadherin endocytosis, more mutant DE-Cad protein might be endocytosed in Rho1's absence, further reducing functional DE-Cad and enhancing the phenotype. However, null or very strong shg alleles accumulate no functional DE-Cad at AJs, rendering regulation of cadherin endocytosis a moot point. The slight suppression by Rho1 of strong shg alleles may result from a reduction of morphogenetic movements, reducing cuticle disruption. Alternatively, some mutant DE-Cad proteins may be capable of coupling to Rho1 while others are not. Rho1 can bind alpha-catenin (Magie, 2002), and active Rho1 may be recruited to AJs by that interaction. shg^G119 has a wild-type cytoplasmic domain and could presumably couple to Rho1; reducing Rho1 might further impair its function. By contrast, the shg² mutation may impair Arm and/or alpha-catenin binding and thus Rho1 recruitment; if so this mutant protein would not be further impaired by Rho1 removal. Finally, the complex genetic interactions might reflect different requirements for Rho1 during neuroblast delamination and head involution, which are affected by strong or weak reduction in DE-Cad function, respectively. Future studies of Rho regulation of and by AJs will help distinguish between these possibilities (Fox, 2005).

Binding site for p120/δ-catenin is not required for Drosophila E-cadherin function in vivo

Homophilic cell adhesion mediated by classical cadherins is important for many developmental processes. Proteins that interact with the cytoplasmic domain of cadherin, in particular the catenins, are thought to regulate the strength and possibly the dynamics of adhesion. beta-catenin links cadherin to the actin cytoskeleton via alpha-catenin. The role of p120/δ-catenin proteins in regulating cadherin function is less clear. Both beta-catenin and p120/δ-catenin are conserved in Drosophila. This study addresses the importance of cadherin-catenin interactions in vivo, using mutant variants of Drosophila epithelial cadherin (DE-cadherin) that are selectively defective in p120ctn (DE-cadherin-AAA) or beta-catenin-armadillo (DE-cadherin-Delta beta) interactions. This study analyzed the ability of these proteins to substitute for endogenous DE-cadherin activity in multiple cadherin-dependent processes during Drosophila development and oogenesis; epithelial integrity, follicle cell sorting, oocyte positioning, as well as the dynamic adhesion required for border cell migration. As expected, DE-cadherin-Delta beta did not substitute for DE-cadherin in these processes, although it retained some residual activity. Surprisingly, DE-cadherin-AAA was able to substitute for the wild-type protein in all contexts with no detectable perturbations. Thus, interaction with p120/δ-catenin does not appear to be required for DE-cadherin function in vivo (Pacquelet, 2003. Full text of article).

To specifically disrupt the interaction between DE-cadherin and p120ctn, a conserved triplet of glycine in the juxtamembrane region of DE-cadherin was mutated to alanine (DE-cadherin-AAA). The corresponding mutation in human E-cadherin disrupts the interaction with p120 without affecting the interaction with ß-catenin. Also, a version of DE-cadherin that should be unable to interact with ß-catenin was made by deleting the COOH-terminal ß-catenin interaction domain (DE-cadherin-δß). The mutant DE-cadherin molecules were tested by transfection in Schneider cells as well as by overexpression with the UAS-Gal4 system in the follicular epithelium of egg chambers (Pacquelet, 2003).

Wild-type (DE-cadherin-wt) as well as mutant cadherin proteins were enriched at the cell cortex, presumably the plasma membrane, in Schneider cells as well as in follicular cells. As a measure of the ability of the different forms of DE-cadherin to bind ß-catenin and p120ctn, their ability to recruit p120ctn and ß-catenin to the cortex was examined. Schneider cells were cotransfected with tagged versions of p120ctn and ß-catenin. Without coexpressed cadherin, HA-p120ctn and ß-catenin were cytoplasmic; ß-catenin was present only at a low level in the cytoplasm due to its instability. Coexpression of wild-type DE-cadherin resulted in recruitment of HA-p120ctn and in recruitment and/or stabilization of ß-catenin at the cortex. In follicular cells, overexpressed HA-p120ctn and endogenous ß-catenin were examined. A small amount of HA-p120ctn and endogenous ß-catenin was present at the cell cortex, presumably due to association with endogenous DE-cadherin. Overexpression of DE-cadherin-wt resulted in efficient recruitment of HA-p120ctn and of more ß-catenin. In both Schneider cells and follicular cells, DE-cadherin-AAA recruited ß-catenin, but not HA-p120ctn, whereas DE-cadherin-δß recruited HA-p120ctn, but not ß-catenin. Immunoprecipitation from Schneider cell extracts confirmed an interaction between HA-p120ctn and DE-cadherin-wt, but not DE-cadherin-AAA. Thus, DE-cadherin-AAA and DE-cadherin-δß appear to be good tools to selectively affect DE-cadherin interaction with p120ctn or with ß-catenin (Pacquelet, 2003).

Cell aggregation is commonly used to measure cadherin-dependent adhesion and importance of cytoplasmic interactions in mammalian cells. As a first functional analysis of the DE-cadherin mutants, the ability was compared of DE-cadherin-wt, DE-cadherin-AAA, and DE-cadherin-δß to induce aggregation of Schneider cells. A form of DE-cadherin lacking the full cytoplasmic tail (DE-cadherin-δcyt) was also tested. All four constructs mediated DE-cadherin-dependent aggregation, and the degree of aggregation was indistinguishable. Thus, DE-cadherin-mediated aggregation of Schneider cells is independent of its cytoplasmic tail. This does not appear to be due to lack of p120ctn; Schneider cells express some p120ctn as detected by Northern blot. Also, co-overexpression of HA-p120ctn or ß-catenin had no effect (Pacquelet, 2003).

To analyze the biological activity of the mutant DE-cadherin proteins during development, the following strategy was used to replace endogenous DE-cadherin with mutant forms: Transgenic flies were generated that express DE-cadherin-wt, DE-cadherin-AAA, or DE-cadherin-δß ubiquitously, under the control of the tubulin-α1 promoter. Endogenous DE-cadherin was removed from specific cells by generating homozygous shg mutant clones or zygotic expression was removed by analyzing shg homozygous mutant embryos. Both shg alleles used in this study are strong alleles, one (shg^R69) is a molecular null allele. Next, whether the transgene-encoded DE-cadherin could substitute for endogenous DE-cadherin, that is, rescue the shg mutant phenotype was determined. Membrane localization and expression levels were checked by immunostaining of egg chambers containing patches of shg mutant cells. These cells expressed only the transgene-encoded DE-cadherin, allowing comparison with endogenous levels. In follicle cells, DE-cadherin-wt transgenes and one of the DE-cadherin-AAA transgenes (#6) were expressed at somewhat higher levels than endogenous DE-cadherin. Another DE-cadherin-AAA transgene and DE-cadherin-δß transgenes gave similar levels of expression, slightly lower than endogenous DE-cadherin. In nurse cells, DE-cadherin-δß transgenes gave expression levels similar to endogenous DE-cadherin, whereas DE-cadherin-wt and both DE-cadherin-AAA transgenes were expressed at much higher levels (Pacquelet, 2003).

It was of particular interest to determine in how DE-cadherin might be regulated during border cell migration, which occurs during stage 9 of oogenesis. Border cells are a group of 6 to 10 somatic follicle cells that delaminate from the anterior tip of the egg chamber, invade the germ line cluster, and migrate to the oocyte. Border cells migrate in between the nurse cells and apparently use these cells as substratum. Absence of DE-cadherin from border cells or from nurse cells results in total lack of invasive migration, suggesting that border cells adhere to the nurse cell substratum through homophilic DE-cadherin interaction. Adhesion to a substratum must in some way be dynamic for cells to move productively across it. Relative to the well-characterized function of cadherin in epithelial cell adhesion, DE-cadherin function in border cell migration might therefore require additional regulatory mechanisms. In addition, border cells normally express higher levels of DE-cadherin than other follicle cells, and migration is quite sensitive to reduction of DE-cadherin level; even weak shg alleles cause detectable delays in migration. For these reasons, it seemed likely that border cell migration would be sensitive to perturbations of cadherin-catenin interactions. To test this, the activity of mutant DE-cadherin proteins was analyzed in border cells as well as in the substratum, the nurse cells. In both border cells and nurse cells, expression of DE-cadherin-wt as well as DE-cadherin-AAA-#6 completely rescued the migration. Thus, DE-cadherin-p120ctn interaction does not appear to be required for the migration. The less active DE-cadherin-AAA-#7 gave full rescue in nurse cells, but incomplete rescue in border cells. In border cells, DE-cadherin-AAA-#6 was expressed at levels similar to endogenous DE-cadherin, whereas DE-cadherin-AAA-#7 was expressed at lower levels. Given that border cell migration is sensitive to cadherin expression level, this can explain the incomplete rescue by cadherin-AAA-#7. DE-cadherin-δß expression in border cells did not rescue the shg phenotype at all, suggesting that DE-cadherin must be anchored to the actin cytoskeleton via ß-catenin to function in the migrating cells. DE-cadherin-δß could partially compensate for endogenous DE-cadherin in nurse cells. Strong linkage to the cytoskeleton via ß-catenin may be less critical for cadherin to function as substratum than in the actively migrating cell itself (Pacquelet, 2003).

Next, whether the DE-cadherin-p120ctn interaction was required for other types of adhesion was addressed. Early in oogenesis, DE-cadherin-mediated adhesion is necessary to correctly position the oocyte at the posterior pole of the egg chamber. If either the germ line or the somatic cells are mutant for shg, the oocyte is often mispositioned. Both DE-cadherin-wt and DE-cadherin-AAA-#6 rescued the shg defects in both germ line and follicular cell clones. Thus, adhesion between the oocyte and the follicular cells does not require the DE-cadherin-p120ctn interaction. DE-cadherin-AAA-#7 failed to rescue the lack of endogenous DE-cadherin in follicular cells, suggesting that levels of expression are important. DE-cadherin-δß was completely unable to rescue the mispositioning phenotype (Pacquelet, 2003).

Within the follicular epithelium, shg mutant cells sort away from wild-type cells generating a smooth boundary at the interface. This is thought to reflect preferential adhesion between DE-cadherin expressing cells. If DE-cadherin-p120ctn interaction alters adhesion, cells expressing only DE-cadherin-AAA might sort away from cells also expressing wild-type DE-cadherin. However, this was not observed. In contrast, cells expressing only DE-cadherin-δß did sort from cells also expressing wild-type DE-cadherin. Thus, the lack of interaction between DE-cadherin and ß-catenin resulted in a decrease of adhesion strong enough to obtain sorting, whereas the lack of interaction between DE-cadherin and p120ctn did not. At early to mid-oogenesis, shg mutant follicle cells maintained correct cell shape within the epithelium. This may reflect functional overlap between DE-cadherin and N-cadherin at these stages, as arm mutant clones have stronger defects. Late in oogenesis, shg mutant follicle cells showed cell shape defects in the epithelium. This defect was also rescued by DE-cadherin-AAA, but not DE-cadherin-δß (Pacquelet, 2003).

Embryos were examined in order to analyze additional types of DE-cadherin mediated adhesion in other tissues. DE-cadherin is maternally supplied, but zygotic expression is required for embryonic development, for example, in the embryonic epidermis. Homozygous shg¹ mutant embryos lack the head and ventral epidermis, which results in the lack of head and ventral cuticle. Both DE-cadherin-wt and DE-cadherin-AAA transgenes fully rescued this phenotype. Rescue of the shg zygotic lethality was tested. Despite the use of a heterologous promoter to express DE-cadherin, 10%-25% of shg¹/shg¹ embryos carrying a DE-cadherin-wt or DE-cadherin-AAA-#6 transgene hatched, and the resulting larvae looked normal. A few percent of shg^R69/shg^R69 mutants were even rescued to adulthood. The rescued females allowed testing of the ability of DE-cadherin-AAA to substitute for maternally provided DE-cadherin. Females expressing only DE-cadherin-AAA-#6 gave rise to viable progeny also carrying only DE-cadherin-AAA. Thus, DE-cadherin-AAA rescued both maternal and zygotic DE-cadherin functions. p120ctn protein is present in the embryo and is expressed in the ovary, in a pattern identical to that of DE-cadherin. However, the results indicate that the DE-cadherin-p120ctn interaction is not essential for any critical process during development (Pacquelet, 2003).

In an attempt to independently compromise p120ctn function, double-stranded RNA-mediated interference (RNAi) was used. Two p120ctn-RNAi transgenes (RNAi-700 and RNAi-2200) were able to severely reduce the levels of co-overexpressed HA-p120ctn, suggesting that levels of endogenous p120ctn would also be strongly reduced. Expression of p120-RNAi in somatic cells did not cause any detectable phenotype in the ovary. This is consistent with p120ctn not being important for DE-cadherin function during oogenesis. However, the lack of phenotype may also reflect incomplete removal of p120ctn. Maternal and zygotic expression of p120ctn-RNAi transgenes did not affect embryonic viability. Severe defects have been observed on injection of p120ctn double-stranded RNA in embryos (Magie, 2002). Resolving the discrepancy between these results awaits analysis of a p120ctn mutant (Pacquelet, 2003).

The general inability of DE-cadherin-δß to replace endogenous DE-cadherin probably reflects that ß-catenin is an important, often essential, interaction partner for cadherin. This supports the view that linkage to the actin cytoskeleton through ß-catenin and α-catenin is critical for cadherin function. It cannot be rule out that other proteins bind to the deleted COOH terminus of DE-cadherin, but the requirement for ß-catenin interaction is supported by the cellular defects in arm (ß-catenin) mutants (Pacquelet, 2003).

In contrast to DE-cadherin-δß, DE-cadherin-AAA was able to replace wild-type DE-cadherin in all assays. To investigate different modes of cadherin function, different types of DE-cadherin function were tested; stable interactions within an epithelium and between cell layers, as well as dynamic adhesion required for migration. Given that the AAA mutation disrupts or strongly attenuates the interaction with p120ctn, the results indicate that interaction with p120ctn is not required for DE-cadherin function. It remains possible that the interaction has a subtle modulatory effect on DE-cadherin function or a role in a nonessential process that was not tested directly. In chick embryos, experiments based on N-cadherin overexpression suggested a regulatory role of the juxtamembrane domain, but not the p120 binding site. Thus, a limited role of the p120ctn-cadherin interaction may not be restricted to Drosophila E-cadherin (Pacquelet, 2003).

Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component

Cadherin-catenin complexes, localized to adherens junctions, are essential for cell-cell adhesion. One means of regulating adhesion is through the juxtamembrane domain of the cadherin cytoplasmic tail. This region is the binding site for p120, leading to the hypothesis that p120 is a key regulator of cell adhesion. p120 has also been suggested to regulate the GTPase Rho and to regulate transcription via its binding partner Kaiso. To test these hypothesized functions, Drosophila, which has only a single p120 family member, was studied. p120 localizes to adherens junctions and binds the juxtamembrane region of DE-cadherin (DE-cad). Null alleles of p120 were generated, and it was found that mutants are viable and fertile and have no substantial changes in junction structure or function. However, p120 mutations strongly enhance mutations in the genes encoding DE-cadherin or Armadillo, the beta-catenin homologue. Finally, the localization of p120 during embryogenesis was studied. p120 localizes to adherens junctions, but its localization there is less universal than that of core adherens junction proteins. Together, these data suggest that p120 is an important positive modulator of adhesion but that it is not an essential core component of adherens junctions (Myster, 2003).

The cadherin-catenin complex plays an essential role in cell-cell adhesion. The classic cadherins, ß-cat/Arm and α-cat are core components of AJs, which are required for adhesion, assembly, and maintenance of epithelia. It is not clear whether these are the only core proteins of AJs or whether other proteins are essential for junctional assembly or function. Other junctional proteins are also likely to regulate adhesion, allowing cells to behave dynamically during development (Myster, 2003).

A growing body of evidence supports the idea that the JM domain of classic cadherins is a target of mechanisms that modulate adhesion. One model suggests that p120 family members act as critical regulators of adhesion by binding to the JM region. Other experiments suggested possible cadherin-independent roles of p120, as a Rho regulator and as a transcriptional modulator. To date, these hypothesized functions of p120 are largely based on indirect arguments. The postulated roles in adhesion regulation rest largely on the effects of mutating the JM region. The effects on Rho regulation rest largely on effects of p120 overexpression, and the interaction with Kaiso remains without a known biological function. Furthermore, most studies of p120 were performed in cultured cells, and thus little is known about its expression or localization during the complex events of embryogenesis. Models of p120 function were tested by characterizing the expression and function of the single Drosophila p120 (Myster, 2003).

It was hypothesized that p120 would be an essential gene, reflecting its proposed roles as a core AJ component and/or a critical regulator of adhesion, Rho, and transcription. However, Drosophila p120 is not essential: null mutants are viable and fertile under laboratory conditions. Thus, fly p120 is not an essential core component of cell-cell AJs, as are classic cadherins and the other catenins (Myster, 2003).

p120 has also been suggested to be a Rho regulator and thus a regulator of the actin cytoskeleton. Disruption of a key Rho regulator would be predicted to have severe consequences for cytoskeletal regulation and thus morphogenesis: alterations in Drosophila Rho function disrupt many developmental events. The data suggest that p120 is not an essential Rho regulator, since mutants are viable and no drastic defects are seen in the actin cytoskeleton in its absence. However, subtle defects are seen in the progress of cells during dorsal closure that may reflect subtle underlying defects in actin organization. The initial studies suggesting that p120 regulates Rho were done in cells expressing elevated levels of p120 and might not reflect a normal physiological function. However, it is more likely that p120 is one of several Rho regulators and that loss of p120 alone does not result in Rho misregulation. This is supported by the work of Magie (2002) who found that both p120 and α-cat bind to Rho. If these two proteins are redundant Rho regulators, loss of one may be compensated for by the other. In one respect, the data conflict with those of Magie who used double-stranded RNA interference (RNAi) to remove p120. Magie reported severe morphogenetic defects that were not seen in the current study. RNAi removal of p120 might fail to trigger a compensatory mechanism that does come into play in the mutant. However, Pacquelet (2003) reports that p120 RNAi has no effect. Attempted to perform p120 RNAi and did not reveal morphogenetic defects in p120 RNAi-injected embryos that were not also seen in controls (Myster, 2003).

Although animals homozygous mutant for p120 are viable and fertile, its conservation through 600 million yr of evolutionary time suggests it plays a role sufficient for natural selection to act on it. The data suggest that one function of p120 is as an important positive modulator of adhesion. In its absence, cells and animals are much more sensitive to reductions in cadherin or catenin function. These data are consistent with data generated in earlier experiments in cultured mammalian cells, though not necessarily consistent with models derived from these data that suggested an essential role for p120 in cell adhesion. Mutational alteration of the JM domain could often be compensated for by overexpressing cadherins or by treating the cells with reagents altering tyrosine phosphorylation. These data are consistent with the idea that the JM domain, and by extension p120, play modulatory roles rather than essential ones (Myster, 2003).

Another paper casts further light on this issue. Ireton (2002) examined a mammalian cell line that expresses only mutant forms of p120 and expresses these at low levels. Ireton found that this cell line exhibited impaired cell-cell adhesion and that this was rescued by restoration of wild-type p120. Further, Ireton could also restore adhesion by overexpressing either wild-type cadherin or a cadherin that could not bind p120. These data are quite consistent, suggesting that p120 positively promotes adhesion but that this deficit can be overcome by elevating cadherin levels (Myster, 2003).

The mechanism by which p120 modulates adhesion remains less clear. No clear or consistent differences were seen in the levels or localization of other AJ proteins in animals lacking p120 (though the data were consistent with the possibility that junctional localization of these proteins might be mildly reduced). The lack of a developmental phenotype further suggests that any difference in adhesion is likely to be subtle. The defects seen in the dorsal closure front may reflect such subtle defects. In a cultured cell system, Ireton (2002) observed that loss of p120 reduced the stability of E-cadherin. Although no differences were observed in steady-state levels of DE-cad, cadherin stability may not be a limiting factor in wild-type Drosophila. Ireton (2002) also observed reduced junctional accumulation of both ß-cat and α-cat. Although no striking effects were observed, the genetic interaction observed with arm is also consistent with reduced assembly of AJ complexes. Given the data and those of Ireton (2002), p120 could act in a wide variety of ways. For example, it might modulate assembly of cadherin-catenin complexes or their lateral clustering, it might alter cadherin stability (e.g., by competing for binding with presenilins), it might alter trafficking of cadherin-catenin complexes to or from the membrane, or it might modulate actin assembly at junctions via Rho or other mechanisms. Further work will be needed in flies and cultured mammalian cells to address these issues (Myster, 2003).

The data also provide the first comprehensive look at the localization of a p120 family member during embryogenesis. Overall, Drosophila p120 localization is largely consistent with what is known about its mammalian homologues. In most tissues, p120 localizes both to AJs and the cytoplasm, largely paralleling DE-cad and Arm. In addition to looking for junctional localization, localization of p120 to other cellular structures was examined. No accumulation of p120 in nuclei was noted, with the exception of the accumulation of p120-GFP in nuclei of syncytial embryos. However, tissue survey was not exhaustive, and this does not rule out a shuttling role in which steady-state levels of nuclear p120 are low. p120-GFP localizes to centrosomes in early embryos. The meaning of this remains to be determined (Myster, 2003).

Perhaps the most striking observation was that accumulation of p120 in AJs and other junctional structures was not as uniform as that of the core AJ proteins. These data are consistent with the suggestion that p120 is not an essential AJ component as are Arm, DE-cad, and α-cat. Instead, p120 appears to be targeted to a subset of AJs and junctional structures, suggesting that cells differ in their requirements for p120. Consistent with this, p120 mRNA accumulates at elevated levels in some cells undergoing morphogenetic movements. Thus, p120 may confer upon junctions in which it accumulates properties that facilitate certain morphogenetic events, though the genetic analysis suggests it does so partially redundantly with other regulators. A challenge in the future will be to determine the mechanisms by which p120 regulates adhesion and the identity of other regulators with which it may be redundant, and use this information to understand why some cells accumulate high levels of junctional p120 while others do not (Myster, 2003).

Rho1 interacts with p120^ctn and α-catenin, and regulates cadherin-based adherens junction components in Drosophila

Rho GTPases are important regulators of cellular behavior through their effects on processes such as cytoskeletal organization. Interactions have been studied between Drosophila Rho1 and the adherens junction components α-catenin and p120^ctn. While Rho1 protein is present throughout the cell, it accumulates apically, particularly at sites of cadherin-based adherens junctions. Cadherin and catenin localization is disrupted in Rho1 mutants, implicating Rho1 in their regulation. p120^ctn has recently been suggested to inhibit Rho activity through an unknown mechanism. This study found that Rho1 accumulates in response to lowered p120^ctn activity. Significantly, Rho1 was found to bind directly to α-catenin and p120^ctn in vitro, and these interactions map to distinct surface-exposed regions of the protein not previously assigned functions. In addition, both α-catenin and p120^ctn co-immunoprecipitate with Rho1-containing complexes from embryo lysates. These observations suggest that α-catenin and p120^ctn are key players in a mechanism of recruiting Rho1 to its sites of action (Magie, 2002).

The results indicate that in addition to Rho1's ubiquitous cytoplasmic expression, it accumulates at adherens junctions and is involved in regulating the proper localization of AJ components. Further, direct physical interactions occur between Rho1 and the catenins, p120^ctn and α-catenin. Isoprenylation at the C-terminal CAAX motif is involved in regulating the subcellular localization of Rho, however, binding to the catenins may represent another mechanism of recruiting Rho1 to its sites of action (Magie, 2002).

Rho1 activity is required to properly localize DE-cadherin during development, consistent with data from mammalian cell culture experiments implicating Rho and Rac in cadherin assembly and maintenance. The defects observed in cadherin localization are most prevalent in and around the leading edge (LE) cells undergoing dorsal closure. Previously Rho1 had been implicated in dorsal closure via its regulation of the LE actin cytoskeleton in cells flanking the segment borders. However, the disruption observed in cadherin distribution suggests that regulation of cell-cell adhesion may play a role in the dorsal closure phenotype observed in these embryos. Thus Rho1's effects on cadherin localization could be the result of a direct role in DE-cadherin clustering, or an indirect effect on the cortical actin cytoskeleton. The process of AJ formation in keratinocytes has been shown to require actin polymerization and the interdigitation of filopodia from neighboring cells. A similar interdigitation of filopodia is seen during dorsal closure in Drosophila and is likely involved in forming adhesive contacts between the two epithelial fronts. Since Rho and Cdc42 have been shown to act antagonistically in the formation of cellular processes in neurons, it is possible that disrupting the balance of Rho1 and Cdc42 function in LE cells results in inappropriate regulation of filopodial extensions. This could partially explain the disruption of DE-cadherin localization observed in Rho1 mutants. Alternatively, Rho1's primary role could be in directly regulating the adhesion of cells near the LE, with Rac and Cdc42 acting as the major organizers of the acto-myosin network (Magie, 2002).

In addition to the accumulation of Rho1 protein at sites of cadherin localization, a direct physical interaction was observed between Rho1 and both p120^ctn and α-catenin. The catenin family of proteins is important in regulating cadherin-based adhesion and linking cadherins to the actin cytoskeleton. ß-catenin binds to the catenin-binding domain of the cadherin molecule as well as to α-catenin. α-catenin, in turn, acts as a link to the actin cytoskeleton, either by directly binding actin filaments or through association with other actin-binding proteins. α-catenin also has been shown to bind spectrin, a major component of the membrane skeleton underlying the plasma membrane involved in stabilizing it and determining cell shape. Human colon carcinoma Clone A cells that contain mutant α-catenin have defects in spectrin assembly. Consistent with this, a breakdown of the α-spectrin cytoskeleton was oberved in embryos injected with α-catenin dsRNA, especially in morphogenetically active cells early in gastrulation. α-catenin protein is enriched at adherens junctions, but is not as strictly localized to them as is DE-cadherin. Binding of α-catenin to Rho1 may be a general mechanism through which Rho1 is recruited to the plasma membrane (Magie, 2002).

p120^ctn regulates the adhesive properties of cadherin complexes through its binding to the juxtamembrane domain of the cadherin molecule, although the precise mechanisms underlying this function are not known. p120^ctn also acts in the cytoplasm where it has been proposed to negatively regulate Rho activation in a manner similar to the GDI proteins, which prevent Rho from exchanging GDP for GTP, although it shares no sequence homology with them. The binding of p120^ctn to cadherins and its effects on Rho function have been shown to be mutually exclusive, such that once p120^ctn binds a cadherin molecule, it is no longer capable of inhibiting Rho activity or function. Rho would then be accessible to activating regulatory proteins such as GEFs, and could carry out its downstream functions. The physical interaction observed between Rho1 and p120^ctn suggests that this negative regulation of Rho1 is due to direct binding of p120^ctn to GDP-Rho1. Interestingly, this is the same face of the Rho protein that has been shown to bind to classical GDIs, consistent with the idea that despite the lack of sequence homology, p120^ctn may be acting in a similar way. Overexpression of p120^ctn in mammalian cells leads to an inhibition of Rho activity. Overexpression of p120^ctn in the system used in this study enhances the Rho1 mutant phenotype, as would be expected for a negative regulator. Embryos homozygous for a deficiency uncovering the p120^ctn locus show an accumulation of Rho1 protein at the leading edge and exhibit a severe dorsal open phenotype. A similar accumulation of Rho1 protein is observed in embryos injected with p120^ctn dsRNA. A positive feedback mechanism may be functioning whereby the relief of p120^ctn-mediated regulation in those cells results in the upregulation of Rho1 protein or an increase in Rho1 stability. It has recently been shown that overexpression of a RhoGDI in the hearts of mouse embryos results in the upregulation of RhoA expression, suggesting the existence of a negative feedback mechanism in the regulation of RhoA levels, although there are no other instances in which a positive feedback mechanism has been linked to Rho expression. Excess Rho activity disrupts cellular migration; cells at the leading edge in embryos that lack p120^ctn function remain cuboidal, rather than elongating as they would during normal dorsal closure, suggesting that Rho1 may be involved in regulating these cell shape changes. Alternatively, p120^ctn has been suggested to activate Rac and Cdc42 in the cytoplasm through an interaction with the GEF Vav2, and this could account for some of its effects on cell morphology. The observation that Rho1 can bind both p120^ctn and α-catenin and that their binding sites are not overlapping suggests that either could be involved in recruiting Rho1 to AJs or the plasma membrane in general. The data indicating that overexpression of α-catenin enhances the Rho1 mutant phenotype to a greater degree than p120^ctn suggests an important role for α-catenin in Rho1 function, perhaps as a factor generally involved in localizing Rho1 to its sites of action, while p120^ctn plays a more specific role at AJs (Magie, 2002).

The data suggest a model in which p120^ctn or α-catenin or both are involved in recruiting Rho1 to sites of cadherin localization, where it can then be activated and carry out its functions, including proper AJ formation. If Rho1 is not recruited properly, as in the case of a Rho1 mutant, this results in mislocalization of AJ components. The binding of p120^ctn to Rho1, either in the cytoplasm or while Rho1 is tethered at AJs through its interaction with α-catenin, inhibits the exchange of GDP for GTP and keeps Rho1 in an inactive state. The binding of p120^ctn to the juxtamembrane domain may release Rho1, allowing it to be activated by GEFs. GTP-Rho1 could then bind its downstream effectors and either directly regulate DE-cadherin assembly or maintenance, or indirectly affect AJ formation through its effects on the actin cytoskeleton. Rho1 localization at AJs could then be mediated either through continued association with α-catenin or through isoprenylation and insertion into the plasma membrane. Mutational analysis aimed at distinguishing between these models will provide further insight into this important feature of Rho1 function during morphogenesis (Magie, 2002).

Search PubMed for articles about Drosophila Adherens junction protein p120

Anastasiadis, P. Z., et al. (2000). Inhibition of RhoA by p120 catenin. Nat. Cell Biol. 2: 637-644. PubMed ID: 10980705

Anastasiadis, P. Z. (2007). p120-ctn: A nexus for contextual signaling via Rho GTPases. Biochim. Biophys. Acta 1773: 34-46. PubMed ID: 17028013

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date revised: 15 April 2014

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