BIOLOGICAL OVERVIEW

Because adenomatous polyposis coli (APC) was first identified in vertebrates, this review will initially focus on the vertebrate protein before dealing with specific information about the Drosophila homolog, termed APC-like, or simply, Apc. APC, a gene mutated in familial adenomatous polyposis and sporadic colorectal tumors, is a tumor suppressor and a component of the WNT pathway (see Drosophila Wingless) functioning as a negative regulator in signal transduction. The vertebrate APC protein binds to ß-catenin (the vertebrate homolog of Armadillo) and accelerates ß-catenin turnover. APC also binds to Zeste white-3 (the vertebrate homolog of Drosophila Shaggy) and is in fact a better substrate for Zw-3 than ß-catenin. A complex between these three proteins may keep ß-catenin levels low (Rubinfeld, 1996). Indeed, APC, Zeste white-3 and Axin are part of a multi-molecular complex termed "ß-catenin destruction complex." APC, E-cadherin, and TCF (the vertebrate homolog of Pangolin) can all bind to the middle domain of ß-catenin, the part that contains a stretch of 13-fold repeated sequences, but each partner binds to a specific subset of these repeats. Possibly, there is some competition for binding, and binding by one or the other partners is subject to some regulation (Nusse, 1997).

APC traces its history to tumors found in familial colon carcinomas; it is a tumor suppressor gene that is deleted in tumors or in germ line DNA. The findings on interactions between Arm and Pangolin in Drosophila have also led to a much better understanding of the mechanism of tumorigenesis as a result of APC loss. Korinek (1997) has shown that several colon carcinoma lines, derived from tumors with APC deletions, contain a complex between one of the human TCF homologs (hTCF-4) and ß-catenin. This complex activates expression of reporter constructs, indicating that the loss of APC function in these cells releases enough ß-catenin to team up with TCF to become a transcriptional activator. As predicted from the model in which APC is a gatekeeper of ß-catenin, transfection of full-length APC into those cells inhibits expression of the reporter gene constructs. Mutant forms of APC, when transfected, are incapable of blocking target gene expression. These experiments provide a plausible explanation of how loss of APC can activate gene expression and lead to cell transformation (although no target genes in oncogenesis are known). A surprise came when Morin (1997) found several colon carcinoma cell lines with normal levels of wild-type APC but nevertheless displaying strong expression of these reporter constructs). It appeared that ß-catenin in those cells had found another way to escape from APC control: by mutations in its own gene. These mutations are also present in several melanomas (Robbins, 1996 and Rubinfeld, 1997 ) and form a special class: they eliminate specific amino acids in the amino-terminal domain of the protein, in particular residues that become phosphorylated prior to the proteolytic down-regulation of ß-catenin. Hence, ß-catenin can turn into an oncogene in its own right. Together, these findings suggest a model in which APC acts to regulate ß-catenin negatively. Inactivation of APC, the model suggests, results in elevation of ß-catenin levels and subsequent cellular transformation. However, not all data are consistent with this model (Nusse, 1997).

Keeping in mind the hypothesized role for APC in vertebrates as the gatekeeper for ß-catenin, it is of interest to explore the role on APC in Drosophila, especially because of the in depth understanding of the role of Armadillo (the Drosophila homolog of ß-catenin) and Pangolin in Wingless signaling, described briefly, in the two paragraphs below.

The roles of Armadillo (Arm) in both Wingless signal transduction and cellular adhesion have been extensively studied, making the fly a particularly amenable model system for the analysis of the regulation of ß-catenin by Apc. The cytoplasmic localization of Arm is regulated by phosphorylation. Shaggy/Zeste-white 3, a serine threonine kinase, lies upstream of Arm, and positively regulates the phosphorylation of Arm. Tyrosine phosphorylation modifies the adherens junction, increases the level of cytoplasmic rather than junctional Arm, but probably does not result in disassembly of the junction. Wingless signal negatively regulates Arm phosphorylation (Peifer, 1994).

How do Wingless signals get to the nucleus? The pathway has now been worked out in Drosophila, based on studies with vertebrates. A maternally expressed Xenopus homolog of mammalian HMG box factors (XTcf-3) binds to ß-catenin (the vertebrate homolog of Armadillo). XTcf-3 (Drosophila homolog: Pangolin) is a transcription factor that mediates ß-catenin-induced axis formation in Xenopus embryos. It has been proposed that the ß-catenin-XTcf-3 complex is responsible for activation of targets genes in response to upstream Wnt signals, which allow cytoplasmic ß-catenin to interact with XTcf-3 (Molenaar, 1996). These observations suggest that Armadillo, plus the associated transcription factor, Pangolin, are the nuclear effectors of Wingless.

With the cloning of Drosophila APC-like and the isolation of mutants, analysis of Drosophila Apc has begun in earnest. Drosophila Apc interacts in cell culture with vertebrate ßcatenin. Expression of the domain of Apc homologous to the region required for ß-catenin down-regulation results in down-regulation of intracellular ß-catenin in a mammalian cell line. This same region binds in vitro to the Armadillo protein. In spite of the demonstrated interaction of Drosophila Apc with Armadillo, no role for Apc could be demonstrated in Drosophila segmentation. APC mRNA and protein expression is very low, if detectable at all, during stages when Arm protein accumulates in a striped pattern in the epidermis of Drosophila embryos. Removing zygotic Apc expression does not alter Arm protein distribution, and the final cuticle pattern is not affected significantly (Hayashi, 1997). Of great interest is the finding of a second Drosophila Apc gene by the Berkeley Drosophila Genome Project (Ahmed, 1998). Perhaps this second Apc gene plays a role in segmentation.

Given the expectation of a role for Drosophila Apc in segmentation, the demonstration of a lack of Apc involvement in this process was truely a disappointment. Nevertheless, high levels of Apc expression have been detected in the central nervous system, suggesting a role for Apc in central nervous system development (Hayashi, 1997). A deficiency in the chromosomal region to which the Apc gene maps was used as the basis for genetic screens to isolate mutants in Apc. Potential mutants in Apc were identified by staining embryos from various mutant lines with a polyclonal antibody directed against the central portion of Apc. In wild-type embryos at early embryonic stages, Apc is detected in the pole cells, which ultimately give rise to the adult germline. This Apc expression is provided maternally and persists through the course of pole cell migration to the gonads. At later embryonic stages, strong zygotic Apc expression is found in both the CNS and peripheral nervous system, within the axon fiber tracts. Three mutants with a reduction in this zygotic expression were identified. Apc pole cell expression, provided by heterozygous mothers, persists in homozygous mutant embryos, thus demonstrating that Apc is provided maternally (Ahmed, 1998).

Phenotypic analysis of Drosophila Apc mutants has demonstrated a role for the gene in eye development. The normal compound eye structure consists of approximately 750 highly ordered eye units, termed ommatidia. Each ommatidium contains eight neuronal photoreceptor cells surrounded by retinal pigment cells. In contrast to the normal or wild-type animal, on eclosion the homozygous Apc mutant shows complete loss of all retinal neurons within all ommatidia, while the pigment cell lattice remains intact. Several days after eclosion, the pigment cells enlarge to the point of confluence (Ahmed, 1998).

Third instar larval eye imaginal discs (the precursors of the adult eye) from homozygous Apc mutants were analyzed prior to development of the photoreceptor phenotype, to determine whether photoreceptor cell loss is a consequence of defects in the initial formation of neurons or from defects in their subsequent differentiation. The mutant eye discs were examined for expression of Neurotactin, a neuronal-specific transmembrane protein that is used as a marker for photoreceptor cells. Both at the level of patterning of the ommatidial arrays and at the level of individual photoreceptor cells, the Neurotactin antigen staining is normal in the Apc mutant. As well, retinal axonal projections to the optic lobe are intact. This indicates that the initial photoreceptor cell formation proceeds normally in Apc mutants and that the defect seen in the mutant adult is the result of neuronal degeneration. Further studies have determined that the neuronal degeneration in the Apc mutant is a result of programmed cell death (apoptosis). Expression of p35, a baculoviral protein that interferes with apoptosis by inhibiting the function of caspase proteases, rescues the Apc mutant phenotype. Despite the dramatic rescue from death of Apc mutant retinal neurons by p35, there is a striking feature that distinguishes the rescued photoreceptors. In wild-type adult eyes, the retinal neurons extend the entire length of the ommatidia, tapering gradually in diameter from their apical to basal regions. In contrast, while the rescued Apc mutant photoreceptors display an intact morphology at the apical surface of the eye, at more basal levels, their diameters are dramatically shrunken, and they lose contact with their neighbors. This abnormal morphology is thought to reflect an arrest in differentiation and that this arrest accompanies the apoptotic death observed in the Apc mutant eye (Ahmed, 1998).

To test for Apc regulation of Arm activity genetically, Arm levels in the Apc mutant were lowered by replacing one wild-type copy of the arm gene with an null allele. When the wild-type arm gene dosage is reduced by one-half in the Apc mutant, many neurons survive. The overall efficiency of rescue achieved by halving the Arm dosage is similar to that obtained by ectopic p35 expression; however, one striking difference between the two is that the neurons rescued in the arm heterozygotes appear completely normal from apex to base. Thus, an inactivating arm mutation is a dominant suppressor of both the differentiation defect and the cell death of retinal neurons in the Apc mutant. These findings provide genetic evidence that Apc functions to regulate Arm negatively and suggest that in the absence of Apc, an increase in Arm activity results in both a differentiation defect and apoptotic cell death (Ahmed, 1998).

To test this idea, the effects of elevated levels of Arm on photoreceptor differentiation were analyzed using the UAS/GAL4 system to overexpress Arm in retinal neurons. Neuronal-specific overexpression of Arm under control of the elav-GAL4 transactivator results in photoreceptor loss that is phenotypically similar to, but weaker than, that seen in Apc mutants, with some ommatidia losing all photoreceptor cells, and most others having a reduction in their number. This reveals that death of photoreceptors is sensitive to Arm dosage and suggests the requirement for titration of Apc activity. To examine further the effects of Arm levels on photoreceptor death, mutations in the amino terminus of Arm were used that reduce the rate at which it is degraded. Neuronal-specific overexpression of an amino-terminal deletion of Arm that results in its stabilization, directed by the elav-GAL4 transactivator, results in the loss of all neurons in all ommatidia, and only pigment cells remain. Overexpression of stabilized Arm under control of the sevenless-GAL4 transactivator, which directs strong expression in 3 of the 8 photoreceptor cells, and weak expression in 2 others, results in photoreceptor loss of only a fraction of cells per ommatidium. Together, these findings demonstrate that overexpression of Arm within retinal cells committed to a neuronal fate results in their death and suggest that the Apc mutant phenotype is mediated by elevation of Arm activity (Ahmed, 1998).

To characterize Arm's ability to activate cell death, and further examine the role of Apc in the Wingless pathway, effects brought about by other members of the Wingless signaling pathway on the apoptosis that is induced by Apc loss were also examined. One well-characterized negative regulator of Arm's signal transduction function is the serine/threonine kinase Zeste-white 3 (Zw3). Inactivation of Zw3 yields elevated levels of cytoplasmic Arm but has little effect on Arm's function in junctional complexes. Neuronal-specific overexpression of Zw3, directed by the elav-GAL4 transactivator, rescues many retinal neurons from apoptosis in the Apc mutant. Remarkably, the rescued cells are detected solely at the apical surface of the eye; more basal sections reveal no rescue. Thus, although the underlying differentiation defect persists, overexpression of Zw3 prevents retinal cell death in the Apc mutant, suggesting a role for cytoplasmic Arm in the activation of apoptosis (Ahmed, 1998).

The signaling mediated by Arm requires not only its cytoplasmic accumulation but also the activity of the DNA-binding protein Drosophila TCF known as Pangolin (Pan). Arm directly interacts with Pangolin through its central Armadillo repeats, a region that is critical in Arm's ability to induce cell death. Pangolin function in an Apc mutant background cannot be completely eliminated; however, flies heterozygous for two mutant alleles of pangolin, pan^ciD and pan¹³, were examined to determine if Pangolin is required in the activation of cell death that results from Apc loss. pan^ciD is mutant for two loci, ci and pan; it behaves as both a hypomorphic allele of ci and a null allele of pan. In flies heterozygous for pan^ciD, some retinal neurons are rescued from cell death in the Apc mutant. As was seen with ectopic Zw3 expression, the rescued cells are detected solely at the apical surface of the eye. The same rescue is found in Apc mutants that are heterozygous for the null allele pan¹³. These findings suggest that elevated Arm levels activate a cell death pathway via the Arm/Pangolin complex (Ahmed, 1998).

Apc mutations in Drosophila reveal a surprising phenotype that parallels the retinal defects induced by APC loss in humans. In both species, retinal lesions composed of degenerated photoreceptor neurons and enlarged pigment cells are formed during eye development, suggesting a particular sensitivity of these cell types to APC loss. There has been some debate as to whether the degeneration of retinal neurons found in congenital hypertrophy of the retinal pigment epithelium (CHRPE) is a secondary consequence of a functional defect in the pigment cells or vice versa. Both the cell death that results from overexpression of Arm specifically within neurons, as well as the curious expression pattern of Arm and Apc in retinal neurons, suggest that in the fly, retinal degeneration in the setting of APC loss is the result of a defect within the photoreceptor cells themselves (Ahmed, 1998 and references).

Is there a role for Armadillo and Pangolin in the induction of retinal cell death during the normal development of the Drosophila eye? Apoptosis occurs within every developing ommatidium to eliminate two to three extraneous cells, a requirement in the refining of the highly ordered compound eye structure. Disruption of this process results in the survival of additional pigment cells in each ommatidium, and a disordering of the retina. Despite the extensive apoptosis that occurs during retinal development, there is little cell death in photoreceptors, and there is no evidence supporting a role for Arm in normally occurring retinal cell death. Rather, the results of the current study suggest that the normal differentiation of these neurons requires tight regulation of Arm activity. In the absence of this regulation, a differentiation defect occurs. A model is favored in which Pangolin, via the Arm/Pangolin complex, acts as a sensor of unregulated elevation of Arm; in response to this elevation, a cell death pathway is triggered. This Pangolin sensing mechanism would provide an Arm-based molecular link between a gross aberration in neuronal differentiation and the concomitant activation of cell death (Ahmed, 1998).

Mammalian ß-catenin is proteolytically processed in apoptotic cells, in a manner that appears caspase dependent. As the cleavage of ß-catenin renders it unable to function in adherens junctions, and the disruption of such junctions is an early event in programmed cell death, this processed form has been proposed to be an effector of apoptosis (Brancolini, 1997). In contrast, the data for Drosophila suggest that at least in retinal neurons, ß-catenin could function not only as an effector but also as an activator of the cell death machinery. The processing of ß-catenin in apoptotic cells generates a deletion of the amino terminus that is predicted to result in its stabilization (Brancolini, 1997). If caspase-mediated proteolytic processing of Arm occurs in apoptotic photoreceptor cells, it might generate a positive feedback loop, whereby elevated levels of Arm would activate an apoptotic pathway; this induction of apoptosis would result in the cleavage and stabilization of Arm, which in turn would reinforce the activation of the apoptotic pathway. APC itself is specifically proteolytically processed to generate a 90 kDa amino-terminal fragment in colon carcinoma cells undergoing apoptotic death (Browne, 1994). As such, cleavage would likely eliminate APC's ability to regulate ß-catenin negatively, this would further reinforce a positive feedback loop, increasing ß-catenin levels in apoptotic cells. In this regard, the markedly increased levels of apoptosis found in the colonic polyps, but not in the surrounding mucosa, from humans with germline mutations in APC are intriguing (Sträter, 1995). Together, these findings from varied experimental systems implicate ß-catenin and APC as important regulators not only of cell differentiation but also of programmed cell death (Ahmed, 1998).

Adenomatous polyposis coli is present near the minimal level required for accurate graded responses to the Wingless morphogen

The mechanisms by which the Wingless (Wg) morphogen modulates the activity of the transcriptional activator Armadillo (Arm) to elicit precise, concentration-dependent cellular responses remain uncertain. Arm is targeted for proteolysis by the Axin/Adenomatous polyposis coli (Apc1 and Apc2)/Zeste-white 3 destruction complex, and Wg-dependent inactivation of destruction complex activity is crucial to trigger Arm signaling. In the prevailing model for Wg transduction, only Axin levels limit destruction complex activity, whereas Apc is present in vast excess. To test this model, Apc activity was reduced to different degrees, and the effects were analyzed on three concentration-dependent responses to Arm signaling that specify distinct retinal photoreceptor fates. It was found that both Apc1 and Apc2 negatively regulate Arm activity in photoreceptors, but that the relative contribution of Apc1 is much greater than that of Apc2. Unexpectedly, a less than twofold reduction in total Apc activity, achieved by loss of Apc2, decreases the effective threshold at which Wg elicits a cellular response, thereby resulting in ectopic responses that are spatially restricted to regions with low Wg concentration. It is concluded that Apc activity is not present in vast excess, but instead is near the minimal level required for accurate graded responses to the Wg morphogen (Benchabane, 2008).

Previous genetic studies have provided conclusive evidence that the two Drosophila Apc proteins are crucial negative regulators of Arm signaling. Simultaneous inactivation of both Apc proteins results in ectopic Arm signaling in nearly all, if not all, cells, indicating that Apc is required to prevent Arm signaling in the absence of Wg stimulation. In contrast with the prevailing model for Wg transduction, which proposes that Apc is present in vast excess, the work presented in this study reveals that a less than twofold reduction in Apc activity can shift the threshold for the response to Wg. It is concluded that by negatively regulating Arm, Apc prevents ectopic Arm activity not only where Wg is absent, but also within the range of the Wg gradient (Benchabane, 2008).

Translation of a gradient of Wg morphogen activity to quantitatively distinct levels of Arm signaling is required to induce concentration-dependent cellular responses, although the mechanisms by which this occurs remain uncertain. The current results reveal that in regions of low Wg concentration, reducing total Apc activity by less than twofold results in aberrant cell fate specification. A morphogen model predicts that the low Wg concentration present in this region of the gradient is below the threshold necessary to trigger a detectable cellular response. This is the only region within the Wg gradient where a relatively small reduction in total Apc activity elicits an ectopic cellular response, and this response is characteristic of intermediate-level Arm signaling. Thus, these results reveal that Apc activity is in excess in regions where Wg is absent, but is not in vast excess within the range of the Wg gradient. Together, these data indicate that Apc activity is present near the minimal level required to prevent ectopic Arm signaling and thereby ensure accurate graded responses (Benchabane, 2008).

In Xenopus egg extracts, the levels of Axin are several magnitudes lower than the levels of other proteins in the destruction complex, suggesting that only Axin is a limiting component in Arm proteolysis, whereas Apc is present in vast excess. How can these biochemical data be reconciled with the current in vivo data, which indicate that Apc is not present in excess within the range of the Wg gradient? One possibility is that the levels of Apc in Xenopus eggs are much greater than those present in Drosophila photoreceptors. Alternatively, total Apc levels could be present in excess regardless of cell type or organism, but the relevant pool contributing to destruction complex activity, distinguished by either post-translational modification and/or intracellular localization, might be present near threshold levels. A correlation between the degree of reduction in the activity of the fly and mammalian Apc proteins with the level of β-catenin/Arm signaling has been demonstrated in several other developmental contexts and in tumorigenesis. Thus data from diverse experimental models indicate that the level of Apc contributes to the level of β-catenin/Arm signaling (Benchabane, 2008).

How is a gradient of Wg concentration translated into quantitatively distinct levels of Arm activity? Upon Wg stimulation, inactivation of the Axin/Zw3/Apc destruction complex is the primary event that triggers Arm signaling. Inactivation of Axin is important for downstream signal transduction in response to Wg stimulation, and is likely to be mediated by the translocation of Axin to the plasma membrane, and/or the degradation of Axin. Thus the local Axin concentration is likely to have a significant role in determining whether the destruction complex is assembled, and consequently is important in regulating Arm stability. The current findings provide in vivo evidence that the level of destruction complex activity is crucial for accurate patterning in response to Wg, and is dependent not only on Axin, but also on the maintenance of Apc activity above a minimal level. It is concluded that within the range of the Wg gradient, both Axin and Apc are present near threshold levels, and that, together, they achieve the precise levels of destruction complex activity required for accurate graded responses (Benchabane, 2008).

Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites

In many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, this study used this system to understand how cells can maintain dynamic arrays of polarized microtubules. Growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time, and growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, this study shows that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. It is propose that Kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie, 2010).

Most cells in multicellular organisms contain polarized noncentrosomal microtubule arrays. In interphase mammalian cultured cells, microtubules are nucleated at the centrosomal microtubule organizing center (MTOC), and plus ends grow towards the cell periphery. However, in many differentiated cells, minus ends are not focused at a centrosomal MTOC. In epithelial cells, a major population of microtubules has minus ends focused at the apical side and plus ends at the basal side. In muscle cells, minus ends spread out around the nuclear envelope, and neurons have perhaps the simplest and most strikingly polarized noncentrosomal microtubule arrays. The mechanisms that organize these noncentrosomal microtubule arrays are poorly understood (Mattie, 2010).

Neurons have two types of processes that extend from the cell body: axons and dendrites. Dendrites primarily receive signals from other neurons or the environment, and axons send signals to other neurons or output cells. One basic difference between axons and dendrites is the arrangement of microtubules. In axons microtubules are arranged into an overlapping array of uniform polarity plus-end-out microtubules. In dendrites of cultured mammalian neurons microtubules have mixed orientation near the cell body. In dendrites of Drosophila neurons 90%-95% of microtubules have minus ends distal to the cell body. As the dendritic array in Drosophila is very simple, and extremely different from a centrosomal array, This study used it as a model system to identify mechanisms that organize polarized noncentrosomal microtubules (Mattie, 2010).

It is not known how uniform dendrite microtubule polarity is established or maintained. Models for generating the plus-end-out axonal microtubule array focus on sliding of microtubules by motor proteins. In mammalian neurons, microtubules are thought to be nucleated in the cell body at the centrosome, then released from the centrosome and transported down the axon in the correct orientation by motors including dynein. Models to account for mixed orientation microtubules in dendrites of cultured neurons have also been proposed. In this case, the kinesin MKLP1 (Kif23) has been proposed to transport minus-end-out microtubules into dendrites along plus-end-out microtubules. The current study identified a new mechanism that is required for uniform microtubule polarity in dendrites (see Interactions between kinesin-2 and +TIPs, and localization of Apc2-GFP to dendrite branch points). As it uses conserved, generally expressed proteins, it could play a role in maintaining microtubule polarity in many other cell types (Mattie, 2010).

Using Drosophila dendrites as a model system, this study demonstrates that growing microtubule plus ends almost always turn towards the cell body at branch points, and that they track stable microtubules through branches. Kinesin-2, EB1 and APC are all required to maintain microtubule polarity and are linked in an interaction network. Based on these results, a model is proposed for directed growth of microtubules in dendrites. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Apc can interact with the Kap3 subunit of kinesin-2, and so could increase concentration of the motor near the branch. EB1-GFP does not concentrate at branches, so it is proposed that a growing microtubule plus end coated with EB1 is transiently linked to kinesin-2 as it passes through the branch, through the interaction between Apc and the EB1 tail. SxIP motifs in Apc and Klp68D could also contribute to this interaction. As both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail, the motor domain would be free to walk along a nearby stable microtubule towards the plus end and cell body (Mattie, 2010).

Even a very brief application of force pulling the growing microtubule towards the cell body should be sufficient to steer growth towards the cell body. Once the tip of the microtubule turns, growth would be constrained by the dendrite walls. The association of the growing plus end with stable microtubule would probably only need to be maintained over a distance of a micron. This model is consistent with the observations that kinesin-2 has shorter run lengths than kinesin-1, and that individual EB1 interactions with the microtubule plus end persist less than a second (Mattie, 2010).

Observations of plus end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved, because plus ends turning sharply are frequently seen. Stable microtubules do not accommodate such sharp turns, so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Instead they likely represent a switch from a freely growing plus end to one that is following a track (Mattie, 2010).

Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. This mechanism is also likely necessary to establish uniform microtubule polarity in branched dendrites, but probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity and a similar mechanism could play a role in dendrites. For example, a kinesin anchored to the cell cortex by its C-terminus could shuttle minus-end-out microtubule seeds into dendrites. Alternately, polarized microtubule nucleation sites could be localized within dendrites. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie, 2010).

Because kinesin-2, APC, EB1 and polarized microtubules are found in many cell types, directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity. This is a newly identified function for both the +TIP proteins and kinesin-2. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells, and sliding of microtubule tips along the side of microtubules was also seen in this system, but there was no association with a particular direction of movement or overall microtubule polarity (Mattie, 2010).

As kinesin-2 has previously been shown to be enriched in tips of growing axons in cultured mammalian neurons, as has APC, it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. In fact, this type of microtubule tracking behavior has been observed in axonal growth cones under low actin conditions. Thus directed growth of microtubules could also be important during axon outgrowth. Indeed, the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be used to align microtubules in many circumstances (Mattie, 2010).

Wnt pathway activation by ADP-ribosylation

Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multi-protein 'destruction complex', assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. This study used an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. For nearly all Wingless-driven developmental processes, a three- to four-fold increase in Axin was found to be insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, both the tumor suppressor Adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) were found to have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and separable domains were defined in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation (Yang, 2016).

The Wnt/β-catenin signal transduction pathway directs fundamental processes during metazoan development and tissue homeostasis, whereas deregulation of Wnt signalling underlies numerous congenital disorders and carcinomas. Two multimeric protein complexes with opposing functions -- the cytoplasmic destruction complex and the plasma membrane-associated signalosome -- control the stability of the transcriptional co-factor β-catenin to coordinate the state of Wnt pathway activation. In the absence of Wnt stimulation, β-catenin is targeted for proteasomal degradation by the destruction complex, which includes the two tumour suppressors: Axin and Adenomatous polyposis coli (APC), and two kinases: casein kinase α (CK1α) and glycogen synthase kinase 3 (GSK3). Engagement of Wnt with its transmembrane receptors, Frizzled and low-density lipoprotein receptor-related protein 5/6 (herein LRP6), induces rapid LRP6 phosphorylation, recruitment of Axin to phospho-LRP6, and assembly of the signalosome, which includes two other Axin-associated components, GSK3 and Dishevelled (Dvl). Signalosome assembly results in the inhibition of β-catenin proteolysis; consequently stabilized β-catenin promotes the transcriptional regulation of Wnt pathway target genes (Yang, 2016).

As a key component in both the destruction complex and the signalosome, Axin is tightly regulated. Under basal conditions, Axin is maintained at very low levels, and serves as the concentration-limiting scaffold for assembly of the destruction complex. Following Wnt exposure, the rapid association of phospho-Axin with phospho-LRP6 triggers Axin dephosphorylation, inducing a conformational change that inhibits Axin's interaction with both the destruction and signalosome complexes. Axin is subsequently degraded; however, Axin proteolysis occurs several hours after Wnt exposure, and thus does not regulate Axin's essential role during the initial activation of the Wnt pathway (Yang, 2016).

The mechanisms that rapidly reprogram Axin from inhibitory to stimulatory roles following Wnt exposure remain uncertain. In current models, Wnt stimulation induces Axin's dissociation from the destruction complex, thereby promoting its interaction with the signalosome. As Wnt stimulation induces Axin dephosphorylation, decreased phosphorylation was postulated to facilitate the dissociation of Axin from the destruction complex; however, recent work revealed that the interaction of Axin with LRP6 precedes Axin dephosphorylation, and that dephosphorylation serves to inhibit, rather than enhance this interaction (Kim, 2013) Furthermore, some findings have challenged prevailing models, providing evidence that Axin's interaction with the destruction complex is not diminished upon Wnt stimulation. Thus, whereas the rapid switch in Axin function following Wnt stimulation is essential for the activation of signalling, the underlying mechanisms remain uncertain (Yang, 2016).

During investigation of this critical process, an unanticipated role was discovered for the ADP-ribose polymerase Tankyrase (Tnks) in the reprogramming of Axin activity following Wnt exposure. As Tnks-mediated ADP-ribosylation is known to target Axin for proteolysis, small molecule Tnks inhibitors have become lead candidates for development in the therapeutic targeting of Wnt-driven cancers. This study identified a novel mechanism through which Tnks regulates Axin: by promoting Axin's central role in rapid Wnt pathway activation. Wnt stimulation was found to modulate Axin levels biphasically in both Drosophila and human cells. Unexpectedly, Axin is rapidly stabilized following Wnt stimulation, before its ultimate proteolysis hours later. In an evolutionarily conserved process, the ADP-ribosylated pool of Axin is preferentially increased immediately following Wnt exposure. ADP-ribosylation enhances Axin's association with phospho-LRP6, providing a mechanistic basis for the rapid switch in Axin function following Wnt stimulation. These results thus indicate that Tnks inhibition not only increases basal Axin levels, but also impedes the Wnt-dependent interaction between Axin and LRP6, suggesting a basis for the potency of Tnks inhibitors in Wnt-driven cancers. Thus, Tnks not only targets Axin for proteolysis independently of Wnt stimulation, but also promotes Axin's central role in Wnt pathway activation, which may be relevant to the context-dependent activation of Wnt signalling and the treatment of Wnt-driven cancers with Tnks inhibitors (Yang, 2016).

Wnt exposure induces biphasic regulation in the level of Axin, and a large increase in the level of ADP-ribosylated Axin immediately after stimulation. ADP-ribosylation enhances the interaction of Axin with phospho-LRP6, and promotes the activation of Wnt signalling. These findings lead to three major revisions of the current model for the role of Tnks in the activation of the Wnt pathway. First, Tnks serves bifunctional roles under basal conditions and after stimulation, revealing a remarkable economy and coordination of pathway components. Second, the results provide a mechanistic basis for the rapid reprogramming of Axin function in response to Wnt stimulation, and thereby reveal an unanticipated role for Tnks in this process. These findings suggest that Wnt exposure either rapidly increases the ADP-ribosylation of Axin or inhibits the targeting of ADP-ribosylated Axin for proteasomal degradation, through mechanisms yet to be elucidated. Finally, pharmacologic inactivation of Tnks was shown to diminish the interaction of Axin with LRP6, revealing a previously unknown mechanism through which small molecule Tnks inhibitors disrupt Wnt signalling, distinct from their known role in stabilizing the destruction complex inhibitors (Yang, 2016).

In the absence of Wnt stimulation, the concentration-limiting levels of Axin regulate its scaffold function in the destruction complex. As components of the destruction complex participate in other signalling pathways, the low levels of Axin were proposed to maintain modularity of the Wnt pathway. The new findings indicate that Axin levels are not only regulated in the absence of Wnt, but also regulated biphasically following Wnt stimulation. This sequential modulation of Axin divides activation of the pathway into an early, fast phase and a delayed long-term phase. During embryogenesis, the earliest expression of Wg triggers the rapid appearance of Axin in segmental stripes, which is a novel hallmark for the initial activation of the pathway. The findings reveal that Wnt exposure induces a rapid increase in the total level of Axin, and importantly, a preferential increase in the level of the ADP-ribosylated Axin. The early Axin stripes are absent in Tnks null mutant embryos and are also absent when the Tnks binding domain in Axin is deleted. Therefore, it is proposed that Axin ADP-ribosylation contributes to Axin stabilization and to the rapid response to Wg stimulation (Yang, 2016).

It is postulated that the initial increase in levels of ADP-ribosylated Axin jump-starts the response to Wnt stimulation by enhancing the Axin-LRP6 interaction, whereas the subsequent decrease in Axin levels prolongs the duration of signalling by reducing destruction complex assembly. Thus, Wnt stimulation induces rapid increases in the levels of not only cytoplasmic β-catenin, but also ADP-ribosylated Axin. Previous work that coupled mathematical modelling with experimental analysis revealed that several Wnt signalling systems were responsive to the relative change in β-catenin levels, rather than their absolute value. This dependence was proposed to impart robustness and resistance to noise and cellular variation. The current data raise the possibility that a similar principle applies to changes in Axin levels on the Axin-LRP6 interaction, as the marked increase in ADP-ribosylated Axin levels following Wnt stimulation is evolutionarily conserved. Thus, the relative change in levels of ADP-ribosylated Axin may promote signalling following Wnt exposure by facilitating the fold change in β-catenin levels (Yang, 2016).

The current findings have relevance for the context-specific in vivo roles of Tnks in Wnt signalling suggested in previous studies. Tnks inhibition disrupts Wnt signalling in a number of cultured cell lines, but in vivo studies in several model organisms suggested that the requirement for Tnks in promoting Wnt signalling is restricted to specific cell types or developmental stages. In mice, functional redundancy exists between the two Tnks homologues, such that Tnks single mutants are viable and fertile, whereas double mutants display embryonic lethality without overt Wnt-related phenotypes. However, a missense mutation in the TBD of Axin2 that is predicted to disrupt ADP-ribosylation resulted in either activating or inhibiting effects on Wnt signalling that were dependent on developmental stage. Tnks inhibitors resulted in the same paradoxical effects, suggesting complex roles in mouse embryonic development. Analogously, treatment of fish with Tnks inhibitors resulted in no observed defects in Wnt-mediated processes during development; however, the regeneration of injured fins in adults, a process that requires Wnt signalling, was disrupted (Yang, 2016).

Similarly, the finding that Drosophila Tnks null mutants are viable (Wang, 2016a; Wang, 2016b; Feng, 2014) was unexpected, as Tnks is highly evolutionarily conserved, and no other Tnks homologues exist in fly genomes. Nonetheless, the current studies reveal that a less than twofold increase in Axin levels uncovers the importance of Tnks in promoting Wg signalling during embryogenesis. Therefore, it is postulated that Tnks loss can be compensated during development unless Axin levels are increased, but that the inhibition of Wg signalling resulting from Tnks inactivation cannot be attributed solely to increased Axin levels. Furthermore, Drosophila Tnks is essential for Wg target gene activation in the adult intestine, and exclusively within regions of the gradient where Wg is present at relatively low concentration. Thus, the context-specific roles of Tnks observed in different model organisms may reflect the mechanisms described in this study, which reveal that the Wnt-induced association of Axin with LRP6 occurs even in the absence of Axin ADP-ribosylation, but is markedly enhanced in its presence. It is postulated that by enhancing this interaction, Tnks-dependent ADP-ribosylation of Axin serves to amplify the initial response to Wnt stimulation, and thus is essential in a subset of in vivo contexts (Yang, 2016).

The recent discovery that Tnks enhances signalling in Wnt-driven cancers has raised the possibility that Tnks inhibitors will offer a promising new therapeutic option. Indeed, preclinical studies have supported this possibility. Tnks inhibitors were thought previously to disrupt Wnt signalling solely by increasing the basal levels of Axin, and thus by increasing destruction complex activity. However, the current findings indicate that the degree to which the basal level of Axin increases following Tnks inactivation is not sufficient to disrupt Wnt signalling in some in vivo contexts. Instead, the results reveal that Tnks inhibition simultaneously disrupts signalling at two critical and functionally distinct steps: by promoting activity of the destruction complex and by diminishing an important step in signalosome assembly: the Wnt-induced interaction between LRP6 and Axin. On the basis of these findings, it is proposed that the efficacy of Tnks inhibitors results from their combined action at both of these steps, providing a rationale for their use in the treatment of a broad range of Wnt-driven cancers. Therefore, these results suggest that in contrast with the current focus on tumours in which attenuation of the destruction complex aberrantly activates Wnt signalling (such as those lacking APC), the preclinical testing of Tnks inhibitors could be expanded to include cancers that are dependent on pathway activation by Wnt stimulation. These include the colorectal, gastric, ovarian and pancreatic cancers that harbour inactivating mutations in RNF43, a negative Wnt feedback regulator that promotes degradation of the Wnt co-receptors Frizzled and LRP6 (Yang, 2016).

PROTEIN STRUCTURE

Amino Acids - 2416

Structural Domains

Similar to mammalian APCs, Drosophila Apc contains several potential glycosylation sites and phosphorylation sites and lacks a signal sequence or transmembrane domain. The deduced Apc protein has 27% identity and 46% similarity over all to human APC. Furthermore, there is striking homology in domains previously identified in m-APC: like m-APCs, the amino-terminal third of Apc contains seven armadillo repeats with approximately 60% identity to those observed in the human protein. The repeats lie outside the region recognized by the human antibody used to screen the expression library; their presence provides further support that the gene described here is an APC homolog. In addition, Apc has at least 6 of the 10 beta-catenin binding sites previously identified in human APC, with ~50% identity at the amino acid level. Apc contains both types of beta-catenin binding sites observed in the human protein, one 15-amino acid repeat with the PXXYS motif thought to bind beta-catenin, and 5 of the 20-amino acid repeats with the PXXFS motif thought to provide beta-catenin down-regulating activity. Such repeats are not found in other beta-catenin binding proteins, such as E-cadherin or alpha-catenin, and are thought to be hallmarks of m-APCs. There are two additional regions in Apc that contain a PXXXS motif. One of these regions shows 40-50% identity to various 20-amino acid repeats, and the other region shows 40-50% identity to various beta-catenin binding sites and 20-amino acid repeats in the human protein, though these repeats do not align with any particular mammalian repeat. One of the regions with the PXXFS motif in Apc could be a 15-amino acid repeat, based on its alignment to the second 15-amino acid repeat of human APC. Because their exact function is not known, one of the PXXFS motifs and two PXXXS motif sites could raise the number of beta-catenin binding sites (15-amino acid repeat) from one to three, or the number of 20-amino acid repeats from five to seven. A potential GSK (Shaggy/zeste white 3) phosphorylation site has been observed in five out of seven 20-amino acid repeats. These results also suggest that overall homology between Apc and m-APCs could be underestimated due to lack of alignment in some conserved domains.

The carboxy terminus of Drosophila Apc and human APC do not show significant homology -- 21% amino acid identity in the carboxyl-terminal 72 amino acids containing the Discs large (Dlg) binding site. At carboxyl-terminal end, Apc lacks the S/T(X)V motif, a sequence required for the binding to the Discs large homology repeat domain. However, the carboxy terminal 270 amino acids of Apc show a charged basic character as observed in m-APCs between amino acids 2200 and 2400. This basic region has been implicated in the binding of m-APCs to microtubules. The basic domain of Apc has weak homology to the microtubule binding domain of microtubule-associated protein 4. This finding suggests that the carboxy terminus of Drosophila Apc may function like that of human APC in binding to microtubules. In addition to the domains mentioned above, a well conserved region of amino acids not previously identified as a separate entity is located slightly amino terminal to the armadillo repeats between amino acids 308 and 417. This conserved domain has 50% amino acid identity between Drosophila Apc and m-APCs, indicating an interesting region yet to be studied functionally. A very limited homology of Apc to m-APCs was observed in the oligomerization domain, as well as the Discs large binding site. This finding does not exclude the possibility that Apc may dimerize or interact using a different site. It remains possible that alternative splicing might produce as-yet-undetected Apc variants with high homology to m-APCs in this region. Alternative splicing forms of the cDNA in 5' end of human APC have been reported. Altogether, this similarity of domain structure of Drosophila Apc to m-APCs, including armadillo repeats, beta-catenin binding sites, and the basic domain suggests that Apc may have significant functional similarity to m-APCs (Hayashi, 1997).

date revised: 15 July 98

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