Adenomatous polyposis coli tumor suppressor homolog 2



Reported here is the expression pattern during Drosophila development of a new adenomatous polyposis coli (APC) homolog called Apc2. Apc2 protein is expressed in all embryonic and larval cells examined. In the early blastoderm embryo, a striking concentration of Apc2 is seen in the cortical actin caps. Microtubules are closely associated with these caps. Since human APC has been reported to bind to microtubules, an investigation was carried out to see whether the cortical Apc2 co-localizes with tubulin. However, this is not the case, implying that the putative tubulin-binding property of human APC is not well conserved (Yu, 1999a).

Using a yeast two-hybrid screen for proteins that bind to Armadillo, the Drosophila beta-catenin homolog, a new Drosophila APC homolog, Apc2, has been identified. Apc2 also binds to Shaggy, the Drosophila GSK-3 homolog. Interference with Apc2 function produces embryonic phenotypes like those of shaggymutants. Interestingly, Apc2 is concentrated in apicolateral adhesive zones of epithelial cells, along with Armadillo and E-cadherin, which are both integral components of the adherens junctions in these zones. Various mutant conditions that cause dissociation of Apc2 from these zones also obliterate the segmental modulation of free Armadillo levels that is normally induced by Wingless signaling. It is proposed that the Armadillo-destabilizing protein complex, consisting of Apc2, Shaggy, and a third protein, Axin, is anchored in adhesive zones, and that Wingless signaling may inhibit the activity of this complex by causing dissociation of Apc2 from these zones (Yu, 1999b).

During embryonic epidermal morphogenesis, Wg activity results in cells adopting a smooth apical surface, suggesting a direct effect of the Wg pathway on cytoskeletal remodelling. Consistent with this interpretation, dAPC-1 and dAPC-2, two paralogous proteins that display overlapping roles in the down regulation of Wg signalling through shuttling between nucleus and cytoplasm, associate with the cytoskeleton in different cell contexts. In the epidermis, dAPC-2 (E-APC) localizes at the basis of actin bundles that support cell extensions. Since dAPC2 mutant embryos lack ventral denticles, dAPC2 has been suggested to be a direct effector of Wg on cytoskeleton dynamics. However, shavenbaby (svb) is shown to be the determinant of cytoskeletal reorganization that leads to F-actin bundling during epidermal morphogenesis. Furthermore, Svb expression is necessary and sufficient (regardless to Wg activity) to localize dAPC-2 protein at the base of the apical actin-rich extensions. This shows that dAPC-2 is not primarily directed to actin bundles through Wg signalling. It also strongly suggests that the loss of denticles in dAPC2 embryos results from overactivity of the Wg pathway, which represses svb transcription. This interpretation is further supported by the fact that, as in case of ectopic activation of Wg signalling, dAPC2 mutations do not prevent formation of dorsal trichomes, which is dependent upon svb activity. Thus, dAPC2 and svb display two distinct functional interactions during denticle morphogenesis: (1) cytoplasmic dAPC2 acts to inhibit the signalling activity of ß-catenin that represses svb transcription; (2) when svb is expressed in epidermal cells, svb activity triggers F-actin bundling and redirects a pool of dAPC2 protein to the base of microfilament bundles. These data demonstrate that, rather than acting directly on F-actin dynamics, the Wg pathway acts through a ß-catenin/TCF-dependent signalling pathway culminating, in the nucleus, in the regulation of svb transcription during epidermal morphogenesis. A growing accumulation of evidence also supports the theory that APC proteins have Wnt independent roles in cytoskeletal regulation during the Drosophila development, such as spindle anchoring in syncytial embryos, cell adhesion, and larval brain development. Further elucidation of a putative Wg-independent function of dAPC2 in F-actin dynamics during epidermal differentiation now awaits uncoupling of its signalling activity from its association with the cytoskeleton (Delon, 2003).

The actin-binding protein profilin is required for germline stem cell maintenance and germ cell enclosure by somatic cyst cells

Specialized microenvironments, or niches, provide signaling cues that regulate stem cell behavior. In the Drosophila testis, the JAK-STAT signaling pathway regulates germline stem cell (GSC) attachment to the apical hub and somatic cyst stem cell (CySC) identity. This study demonstrates that chickadee, the Drosophila gene that encodes profilin, is required cell autonomously to maintain GSCs, possibly facilitating localization or maintenance of E-cadherin to the GSC-hub cell interface. Germline specific overexpression of Adenomatous Polyposis Coli 2 (APC2) rescued GSC loss in chic hypomorphs, suggesting an additive role of APC2 and F-actin in maintaining the adherens junctions that anchor GSCs to the niche. In addition, loss of chic function in the soma resulted in failure of somatic cyst cells to maintain germ cell enclosure and overproliferation of transit-amplifying spermatogonia (Shields, 2014).

Chickadee, the only Drosophila profilin homolog, is required cell intrinsically for GSC maintenance in the testis. As profilin is a regulator of actin filament polymerization and filamentous actin (F-actin) plays a crucial role in the development and stabilization of cadherin-catenin-mediated cell-cell adhesion, profilin likely maintains attachment of Drosophila male GSCs to the hub through its effect on F-actin, which concentrates at the hub-GSC interface where localized adherens junctions anchor GSCs to hub cells. It is proposed that profilin-dependent stabilization of F-actin at the GSC cortex next to the hub may help localize E-cadherin and APC2 to the junctional region. E-cadherin and APC2 in turn may recruit β-catenin/Armadillo, stabilizing the adherens junctions that attach GSCs to the hub. Chickadee may thus facilitate maintenance of GSCs through a cascade of interactions leading to localization and/or retention of both E-cadherin and β-catenin at the hub-GSC interface (Shields, 2014).

E-cadherin plays a crucial role in maintaining hub-GSC attachment. GSC clones mutant for E-cadherin are not maintained. In addition, germline overexpression of E-cadherin delayed GSC loss in stat-depleted GSCs. The results indicate that profilin function is required in GSCs for proper localization of E-cadherin to the hub-GSC interface. Several studies have shown that the actin cytoskeleton plays a crucial role in assembly and stability of adherens junctions. A favored model in the field is that actin filaments indirectly anchor and reinforce E-cadherin-mediated cell junctions by forming an intracellular scaffold for E-cadherin molecules. Indeed, binding to F-actin stabilized E-cadherin and promoted its clustering. Furthermore, the actin cytoskeleton participates in proper localization of E-cadherin molecules to cell-cell contacts. In chic/profilin mutant GSCs, disruption of actin polymerization at the cell cortex leading to local F-actin disorganization may destabilize E-cadherin and reduce its ability to localize to the GSC-hub junction, form clusters and build adequate adherens junctions (Shields, 2014).

Destabilization of E-cadherin may contribute to the mislocalization of APC2 seen in chic mutant GSCs, as E-cadherin recruits APC2 to cortical sites in GSCs. Raising possibilities of a more direct link, actin filaments have been shown to be required for association of APC2 with adherens junctions in the Drosophila embryo and ovary. Treatment of embryos with actin-depolymerizing drugs resulted in complete delocalization of APC2 from adhesive zones and diffuse APC2 staining throughout the cell. Moreover, in ovaries of chic1320/chic221 females, APC2 was substantially delocalized from the plasma membranes of nurse cells and their ring canals, and increased levels of cytoplasmic APC2 staining were observed. Similarly, this study found that APC2 was delocalized from the hub-GSC interface in larval testes of chic11/chic1320 hypomorphs (Shields, 2014).

In several studies, delocalization of APC2 from junctional membranes correlated with detachment of β-catenin/Armadillo from adherens junctions. APC2 co-localizes with Armadillo and E-cadherin at adherens junctions of Drosophila epithelial cells, nurse cells in Drosophila ovaries and at the hub-GSC interface in Drosophila testes. Disruption of APC2 function resulting in significant reduction of junctional APC2 was accompanied by delocalization of junctional Armadillo and increased levels of free cytoplasmic Armadillo in embryonic epithelial cells and ovaries. In a previous study, which used chic1320/chic221 strong loss-of-function mutants, the delocalizing effect on junctional Armadillo was variable, presumably due to incomplete penetrance of chic mutant effects. Although this study did not observe significant disruption in Armadillo staining along the hub-GSC interface of testes from chic hypomorphs, this may be due to incomplete penetrance. In addition, the Armadillo protein detected could be localized to the cortex of hub cells rather than GSCs (Shields, 2014).

The finding that germline specific overexpression of APC2 in chic11/chic1320 hypomorphs partially rescued GSC loss is consistent with a previously proposed model that actin filaments shuttle APC2 to adherens junctions and APC2 in turn recruits cytoplasmic Armadillo to junctional membranes, reinforcing the adherens junctions. It is possible that in chic11/chic1320 hypomorphs, residual actin filaments associated with adherens junctions between the hub and GSCs are sufficient to shuttle the increased amounts of cytoplasmic APC2 to adherens junctions. This APC2 may in turn recruit free cytoplasmic Armadillo to the hub-GSC interface, locally stabilizing the adherens junctions and anchoring GSCs to their niche. Notably, however, germline specific overexpression of APC2 in testes of strong loss-of-function chic1320/chic221 mutants failed to rescue GSC loss. Thus either, adequate levels of actin filament polymerization may be required for the proposed translocation of junctional proteins to the plasma membrane, or APC2 function/localization may not be the only or even the major cell-autonomous target of profilin function important for maintaining GSCs. Indeed, loss of APC2 function did not lead to GSC loss. It is suggested that the localized cortical F-actin underlying adherens junctions at the GSC-hub interface, best candidate for the most direct target of chic function, strongly stabilizes adherens junctions between GSCs and the hub, with high levels of cortical APC2 able to in part make up for weak chic function by also stabilizing adherens junctions (Shields, 2014).

Maintenance of hub-GSC attachment appears to be a key role of STAT in GSCs. The finding that STAT binds to a site near the upstream promoter of the chic gene raises the possibility that STAT might foster GSC attachment to the hub in part by ensuring high levels of transcription of profilin in GSCs. However, activation of STAT is clearly not the only regulatory influence on profilin expression as profilin is an essential gene expressed in many cell types, including those in which STAT is not active or detected. It is likely that transcription factors other than STAT turn on profilin expression in many cell types and that STAT acts along with other regulators to reinforce profilin expression in GSCs and CySCs. Conversely, overexpression of profilin was not sufficient to re-establish attachment of stat-depleted GSCs, suggesting that STAT probably regulates a number of genes to ensure that GSCs remain within the stem cell niche (Shields, 2014).

Loss of chic function in somatic cyst cells impaired the ability of cyst cells to build and/or maintain the cytoplasmic extensions through which they embrace and enclose spermatogonial cysts. Two somatic cyst cells normally surround each gonialblast and enclose its mitotic and meiotic progeny throughout Drosophila spermatogenesis. The cyst cells co-differentiate with the germ cells they enclose. Several lines of evidence support the model that either the ability of somatic cyst cells to enclose germ cells or their ability to send signals to adjacent germ cells is important to restrict proliferation and promote differentiation of germ cells. In either case, activation of EGFR in cyst cells is required for cyst cells to enclose germ cells and/or send the signals for germ cells to differentiate. The similarities in phenotype between loss of chic function and loss of EGFR activation in somatic cyst cells raise the possibility that chic/profilin may act downstream of activated EGFR to modulate the actin cytoskeleton for the remodeling of cyst cells to form or maintain the cytoplasmic extensions that enclose germ cells. Indeed, activated EGFR is known in other systems to tyrosine phosphorylate phospholipase C-γ1 (PLC-γ1), a soluble enzyme in quiescent cells like daughter cyst cells, activating it to catalyze hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which binds profilin protein with high affinity, which inhibits the interaction between profilin and actin. The hydrolysis of PIP2 by activated PLC-γ1 results in localized release of profilin and other actin-binding proteins, enabling them to interact with actin and participate in cytoskeletal rearrangement and membrane protrusion. Thus, based on biochemical analysis in other systems, a link between EGFR activation and profilin leading to local remodeling of the actin cytoskeleton is plausible in somatic cyst cells, although it remains to be directly tested (Shields, 2014).

Effects of Mutation or Deletion

Apc2 is removed by deficiencies Df(3R)crb89-4and Df(3R)crb87-4, but not by Df(3R) crb87-5. All three deficiencies remove crumbs and thus have a null crumbs phenotype; thus, the severe epidermal fragmentation made examination of cuticular pattern impossible. During a genetic screen for suppressors of wg, a temperature-sensitive mutation was isolated that maps to this genomic interval by complementation with the same deficiencies, and has a phenotype consistent with that of a negative regulator of Wg signaling. Thus, it was evaluated as a candidate Apc2 mutation, sequencing Apc2 from the mutant and comparing its sequence to that of Apc2 in the parental stock from which the mutant is derived, and in several other wild-type stocks. There is only a single difference between the parental chromosome and the mutant: deletion of three nucleotides, leading to deletion of serine 241. This serine residue falls within an alpha-helix in the third Arm repeat (by analogy to the Arm repeats of ß-catenin). The length of this alpha-helix is invariant among APC family members, and this residue is either serine or alanine (a conservative change) in all APCs. Thus, this allele is referred to as dAPC2DeltaS (McCartney, 1999).

Whereas homozygous mutant embryos accumulate normal levels of Apc2, mutant Apc2 migrates more rapidly on SDS-PAGE than wild-type protein. A portion of Apc2 in heterozygous mutants, which are wild-type in phenotype, also migrates abnormally, suggesting that this is an intrinsic property of mutant Apc2 rather than a consequence of the mutant phenotype. The subcellular localization of Apc2 in Apc2DeltaS mutants is dramatically altered at both the permissive (18°C) and restrictive (25°C) temperatures. At the restrictive temperature, Apc2 association with the cell cortex is essentially abolished, rendering the protein almost completely cytoplasmic. At the permissive temperature, some cortical Apc2 remains. In heterozygotes, Apc2 protein localization is intermediate between mutant and wild-type, as if mutant protein localizes incorrectly despite the presence of wild-type protein. The loss of phosphorylated Apc2 isoforms observed in these mutants may be a consequence of the loss of cortical association (McCartney, 1999).

The localization of dAPCDeltaS mutant protein has been examined at the restrictive temperature in other tissues. Although dAPCDeltaS is found in apical buds in the preblastoderm embryo, it no longer associates with actin structures as does the wild-type protein. Furthermore, Apc2DeltaS does not associate with the apical plasma membrane in the wing imaginal epithelia, marked by the presence of cortical actin. In the larval neuroblasts, Apc2DeltaS is largely cytoplasmic, although an association with the cortex is sometimes observed (McCartney, 1999).

Apc2 acts as a negative regulator of Wg signaling in the embryonic epidermis. The mutant Apc2DeltaS is viable and fertile at the permissive temperature (18°C). At the restrictive temperature (25°C), Apc2DeltaS homozygous mutants derived from heterozygous mothers are viable, indicating that maternal contribution of Apc2 is sufficient for embryonic development. Heterozygous embryos derived from homozygous mutant mothers are wild-type and survive to adulthood, suggesting that zygotic function is also sufficient. Mutant embryos derived from mutant mothers (referred to as Apc2DeltaS maternal/zygotic mutants) have severe abnormalities in their embryonic body plan. On the ventral surface, wild-type embryos show segmentally repeated denticle belts interspersed with naked cuticle. In Apc2DeltaS maternal/zygotic mutants, denticle belts are replaced with an almost uniform expanse of naked cuticle, as is observed when wg is ubiquitously expressed. The dorsal surface also has an array of pattern elements marking specific cell fates; cells receiving Wg signal secrete fine hairs. On the dorsal surface of Apc2DeltaS maternal/zygotic mutants, many more cells secrete fine hairs, which is what occurs when wg is ubiquitously expressed. Thus, maternal/zygotic loss of Apc2 function activates Wg signal transduction both dorsally and ventrally, suggesting that wild-type Apc2 helps negatively regulate this pathway (McCartney, 1999).

Perturbing Apc2 function at defined developmental time points supports this hypothesis. At the permissive temperature, Apc2 mutant embryos develop normally into adults and a homozygous mutant stock can be maintained. When homozygous mutant embryos were shifted up to the restrictive temperature at 4 h after egg laying (AEL), they secrete uniform naked cuticle, like animals at the restrictive temperature throughout development. Progressively later upshifts result in intermediate cuticle defects, with increasing numbers of denticles secreted, until by 10 h AEL the pattern is essentially wild-type. Conversely, shifts from the restrictive temperature down to the permissive temperature at 4 h AEL fully rescue the pattern, whereas progressively later downshifts result in more and more naked cuticle replacing the ventral denticle belts. Thus, Apc2 activity is required between 4-10 h AEL, the same time window during which wg acts. Somewhat surprisingly, Apc2 function may be dispensable for adult patterning; mutant embryos shifted up to the restrictive temperature after 10 h and cultured continuously at this temperature develop into apparently normal adults. This could be the result of partial activity of the Apc2DeltaS allele. However, it is suspected that Apc2DeltaS is at least a strong hypomorph, because placing this allele over a deficiency for the region both in the mother and the zygote, does not increase the severity of the embryonic mutant phenotype at restrictive temperature (McCartney, 1999).

Epistasis analysis was carried out to position Apc2 with respect to other components of the signal transduction pathway. wg; Apc2DeltaS double mutant embryos (with Apc2DeltaS mutant mothers) show a partial rescue of the wg phenotype, with restoration of the normal diversity of cuticular pattern elements and small expanses of naked cuticle, suggesting that Apc2 is downstream of wg. There are two possible explanations for the fact that the double mutant does not show the same phenotype as the Apc2 single mutant: either Apc2DeltaS is not null, or the negative regulatory machinery remains partially active in the absence of Apc2. If Apc2DeltaS is not null, it was reasoned that repeating the epistasis test with Apc2DeltaS in trans to a deficiency removing Apc2 (Df(3R)crb87-4) might further reduce Apc2 function, producing a double mutant phenotype more similar to that of Apc2DeltaS alone. However, when this was done, there was no change in the double mutant phenotype, suggesting that Apc2DeltaS may be genetically null for this function. Other components of the Wg signal transduction pathway act downstream of Apc2. Embryos maternally and zygotically mutant for both dishevelled (dsh) and Apc2 show a phenotype indistinguishable from the dsh single mutant, as do embryos maternally mutant for both dsh and Apc2 that are zygotically dsh/Y; Apc2DeltaS/Df(3R)crb87-4. Likewise, arm; Apc2 and Apc2; dTCF double mutants (derived from Apc2 homozygous mothers) are indistinguishable from arm or dTCF single mutants. Thus, dsh, arm, and dTCF all act genetically downstream of Apc2; this was expected for arm and dTCF, but was surprising for dsh (McCartney, 1999).

Loss of Apc2 also leads to ectopic activation of Wg-responsive genes. One target is wg itself. If the Wg pathway is constitutively activated by removing zw3 function or by expressing constitutively active Arm, an ectopic stripe of wg RNA is induced in each segment. A similar ectopic stripe of wg RNA is seen in Apc2DeltaS maternal/zygotic mutants. Similarly, the domain of expression of a second Wg target gene, engrailed (en), is expanded relative to wild-type, as is the case in zw3 mutants or in the presence of activated Arm. In addition, a novel phenotype has been observed. In Apc2DeltaS maternal/zygotic mutants, the levels of Wg protein are higher and Wg extends more cell diameters away from wg-expressing cells than in wild-type. These effects on Wg protein do not appear to be accounted for solely by ectopic activation of wg RNA, because they are detected beginning at stage 9 before induction of ectopic wg, and they are not observed in embryos expressing activated Arm. Thus, the efficiency of Wg protein transport appears to be enhanced in Apc2 mutants (McCartney, 1999).

Apc2 mutant embryos still respond to Wg signaling, because segmental stripes of stabilized Arm remain. In Apc2DeltaS maternal/zygotic mutants, levels of cytoplasmic Arm in all cells are elevated, but cells receiving Wg signal continue to accumulate more Arm than their neighbors. In contrast, zw3 loss of function results in uniform accumulation of cytoplasmic Arm in all cells, eliminating the Arm stripes. Immunoblot analysis of Arm protein from Apc2DeltaS maternal/zygotic mutants reveals an accumulation of hypophosphorylated Arm. This effect is not as dramatic as that seen in a zw3 mutant, but is similar to that seen upon ubiquitous expression of Wg using the e22c-GAL4 driver. Thus, the effect of Apc2DeltaS on Arm levels is intermediate between that of wild-type and that of zw3 loss of function, suggesting that negative regulation of Arm is reduced but not completely abolished in Apc2DeltaS (McCartney, 1999).

Because Apc2DeltaS activates Wg signaling, it was asked whether the change in its localization would simply be a consequence of pathway activation. When Wg signaling is activated by ubiquitous Wg expression (via the e22c-GAL4 driver) or by removing zw3 function, the localization of Apc2 is essentially unchanged, suggesting that pathway activation is not sufficient to eliminate cortical Apc2 . There is also no apparent change in Apc2 protein levels or isoforms in zw3 mutants relative to wild-type; this is somewhat surprising, since it is known that mammalian GSK phosphorylates mammalian APC, and suggests that Drosophila Apc2 can be phosphorylated by another kinase (McCartney, 1999).

These epistasis tests between Apc2 and other components of the Wg pathway generally conform to earlier models of APC function, but also suggest further complexity. As expected, Apc2 acts downstream of wg and upstream of arm and dTCF. However, the suppression of wg by dAPC2DeltaS is incomplete. This may be because dAPC2DeltaS is not null, because dAPC2 is not completely essential for Arm downregulation, or because of redundancy. In contrast to zw3, dAPC2DeltaS is genetically upstream of dsh. However, the relative positioning of dAPC2 and Dsh will not be definitive until a protein-null allele of Apc2 is available. Most current models place Dsh upstream of the destruction machinery, but the recent discovery that Dsh (along with Axin, APC, and Zw3/GSK) is a component of the destruction complex reveals that these proteins may function as a network rather than in a linear series, making the results of epistasis tests more difficult to interpret. For example, the epistasis relationships might be explained if Apc2 regulates assembly of Dsh into the destruction complex. In the absence of Apc2, Dsh might constitutively turn off the destruction complex, activating signaling; thus, loss of Apc2 would have no effect if Dsh is also absent (McCartney, 1999).

naked cuticle (nkd) is an embryonic lethal recessive zygotic mutation that produces multiple segmentation defects, the most prominent of which is the replacement of denticles by excess naked cuticle. This phenotype is also seen in embryos exposed to excess Wg, as well as in embryos lacking both maternal and zygotic contributions from any of three genes that antagonize Wg: zeste-white3/glycogen synthase kinase 3beta (zw3/gsk3beta), D-axin and D-Apc2. In nkd embryos, hh and en transcripts initiate normally but accumulate in broad stripes, including cells further from the source of Wg, which suggests that these cells are hypersensitive to Wg. Next, a stripe of new wg transcription appears just posterior to the expanded Hh/En stripe. This extra wg stripe requires both wg and hh activity and is required for the excess naked cuticle seen in nkd mutants. The death of cells producing Hh/En contributes to the marked shortening of nkd mutant cuticles (Zeng, 2000).

Inactivation of the Adenomatous Polyposis Coli tumor suppressor triggers the development of most colorectal carcinomas. APC is required for targeted degradation of ß-catenin, the central transcriptional activator in the Wnt/Wingless (Wg) signal transduction pathway; however, the precise biochemical functions of APC remain uncertain. The two Drosophila homologs of APC (Apc1 and Apc2) appear to have predominantly different tissue distributions, different subcellular localizations and mutually exclusive phenotypes upon inactivation. Unexpectedly, despite these differences, simultaneous reduction in both Drosophila Apc proteins results in the global nuclear accumulation of ß-catenin and the constitutive activation of Wg transduction throughout development. This redundancy extends even to functions previously thought to be specific to the individual Apc homologs. Together, these results reveal that the combined activity of Apc1 and Apc2 allows a tight regulation of transcriptional activation by ß-catenin and suggest that APC proteins are required for the regulation of Wnt transduction in all cells (Ahmed, 2002).

The in vivo analyses of loss-of-function mutations in the two Drosophila homologs of Apc have been crucial in providing conclusive evidence that transcriptional transactivation by ß-catenin can in fact be negatively regulated by APC. However, previous studies using loss-of-function mutations in either of the two Drosophila Apc genes have failed to establish an absolute requirement for Apc in regulating Wg signaling throughout development, since many Wg transduction events proceed normally, particularly during post-embryonic stages. These findings raised questions as to whether Apc is required to prevent the constitutive activation of Wg transduction in only a subset of cells, and whether Apc function could be compensated for by other mechanisms elsewhere. Simultaneously reducing the activities of both Drosophila Apc proteins is reported in this study. An absolute requirement is found for Apc proteins in preventing the constitutive activation of Wg signaling in many epithelial cells throughout development. In those limited situations for which the inactivation of one of the two Drosophila Apc proteins does lead to hyperactivation of transcriptional activation by Arm, the other Apc protein can functionally substitute if provided in sufficient quantity. This result argues against a specific function for either Apc protein in regulating Wg transduction (Ahmed, 2002).

Apc1 is highly (though not exclusively) expressed in neurons, while Apc2 is highly (though not exclusively) expressed in most epithelial cells, leading to the proposal that the two Apc proteins function in a tissue-specific manner. The data presented here argue against a tissue-specific division in Apc expression or function. The dramatic and global constitutive activation of Wg transduction that is revealed only by simultaneous reduction in both Drosophila Apc proteins demonstrates that both Apc proteins are found and function in many tissues that are not restricted by cell type or developmental stage (Ahmed, 2002).

These results reveal that the combined activity of Apc1 and Apc2 within the same cell enables these two proteins to tightly regulate Arm levels. Thus, specific phenotypes that are found upon inactivation of either Apc1 or Apc2 singly (leading to cell death in pupal retinal neurons and cell fate transformation in the embryonic epidermis, respectively) denote the relatively rare situations in which the activity of one of the two Apc proteins is not sufficient to compensate for reduction in the other. The data reveal that even in the embryonic epidermis, Apc1 and Apc2 function to prevent the ectopic activation of Wg transduction. When Apc2 activity is reduced, ectopic Wg transduction is very sensitive to the dose of Apc1, since cutting the wild-type dose of Apc1 in half either maternally or zygotically has dramatic effects. In this tissue, Apc1 has a subsidiary role though, and the normal levels of Apc1 are not sufficient to compensate for Apc2 loss. These data, coupled with the rescue of Apc2 reduction by Apc1 overexpression, suggest that the absolute levels of Apc1 and Apc2 are important in enabling the two Apc proteins to compensate for each other (Ahmed, 2002).

It was not possible to determine whether the converse situation is also true -- whether reducing levels of endogenous Apc2 would exacerbate defects resulting from mutations in Apc1, because a hypomorphic allele of Apc1 to use as a sensitized background for genetic interaction tests is unavailable. Retinal neuronal apoptosis is exquisitely sensitive to total Apc2 activity, since increasing the dose of Apc2 by only one copy is sufficient to prevent apoptosis in the Apc1 mutant. Together, these data suggest that the absolute levels, or total 'dose' of intracellular Apc1 and Apc2 is important in preventing the hyperactivation of Arm. Whether the dose sensitivity that is revealed in these situations reflects differences not only in total levels, but also in the relative binding affinities of the two Apc proteins for Arm, Axin or Zw3 remains to be investigated (Ahmed, 2002).

The functional redundancy in the Apc proteins suggests that the C-terminal half of Apc1 might not be required for targeted degradation of Arm, since this region of the protein is completely lacking in Apc2. However, this region of Apc1 might be important in previously proposed roles for APC that might be independent from ß-catenin degradation. These include the alteration of cell migration through regulation of the actin cytoskeleton, the planar positioning of mitotic spindles with respect to the polarized epithelial cell membrane, and in kinetochore-microtubule attachment. While the data demonstrate that both Drosophila Apc proteins function in the regulation of Wg transduction, further analysis employing the Apc1;Apc2 double mutant will be required to address their possible redundancy in functions that are independent of ß-catenin degradation (Ahmed, 2002).

Is there an absolute requirement for APC in the targeting of ß-catenin to a degradation pathway? In cell culture experiments, overexpressed Axin is able to downregulate ß-catenin levels even in cells that lack wild-type APC. Furthermore, even after deletion of its RGS domain, which is required for the interaction of Axin with APC, overexpressed Axin is still able to induce the degradation of ß-catenin. These data have led to the hypothesis that APC may facilitate, but not be absolutely necessary for, the Axin-mediated degradation of ß-catenin. If APC were to merely facilitate Axin mediated degradation of ß-catenin, it would be expected that phenotypes found upon reduction in APC would not be as severe as those found upon inactivation of Axin, since residual Axin-mediated degradation of ß-catenin would persist in the absence of APC. Instead, it is found that inactivation of APC results in phenotypes that completely mimic inactivation of Axin, with respect to both their scope and their severity. These data argue against a secondary role for APC in the degradation of ß-catenin, and provide in vivo evidence for an absolute requirement for APC in preventing the constitutive activation of Wg transduction in virtually all epithelial cells (Ahmed, 2002).

Human APC has been found to shuttle between the nucleus and cytoplasm. Nuclear export of human APC is dependent on both nuclear export sequences within APC and on the CRM1 export receptor. Treatment of cells in culture with the CRM1-specific export inhibitor leptomycin B results in the nuclear accumulation of APC, as well as the nuclear accumulation of ß-catenin. These findings have led to the proposal that APC is required for the nuclear export of ß-catenin. However this hypothesis must be reconciled with studies employing oocytes and semipermeabilized cultured cells to investigate ß-catenin export, which reveal that ß-catenin can be exported from the nucleus in a manner that is independent of the CRM1 pathway and independent of APC (Ahmed, 2002).

In epithelial cells that lack both wild-type Drosophila Apc1 and Apc2, Arm accumulates within the nucleus. Nuclear accumulation of Arm is found only in the Apc1Q8;Apc2d40 maternal/zygotic double mutant, and occurs during gastrulation. The nuclear localization of Arm, and the temporal pattern of the nuclear accumulation of Arm in the absence of wild-type Apc1 and Apc2, is similar to that seen upon inactivation of Axin, and in contrast to that seen upon inactivation of Zw3, in which the increased levels of Arm appear uniformly dispersed between nucleus and cytoplasm. These data are therefore completely consistent with the model that there is a second role for APC in the nuclear export of ß-catenin, in addition to the role APC serves in the targeting of ß-catenin to degradation (Ahmed, 2002).

However, an alternate model for APC function in Arm localization incorporates three observations: (1) a similar temporal pattern of nuclear Arm accumulation is seen in Axin mutants and in Apc1;Apc2 double mutants; (2) the interaction of Axin with ß-catenin is critically dependent on APC, and (3) ß-catenin is freely diffusible from nucleus to cytosol. In this model, an Axin/APC complex would serve as a cytoplasmic anchor for ß-catenin and would dictate, in part, the steady-state subcellular localization of ß-catenin. Axin would serve as the primary cytoplasmic anchor for ß-catenin, but its physical interaction with ß-catenin would be greatly enhanced by APC. The elimination of either Axin or APC, or their functional inactivation in the presence of Wg transduction, would not only increase the total levels of ß-catenin, but would also shift the steady state localization of ß-catenin to the nucleus. While further experiments will be necessary to distinguish between roles for APC in the nuclear export and/or cytoplasmic anchoring of ß-catenin, these data suggest that together, APC and Axin exercise two levels of control of ß-catenin activity: APC and Axin not only initiate the destruction of ß-catenin, but also modulate the ability of ß-catenin to accumulate in the nucleus where it can serve as a transcriptional activator (Ahmed, 2002).

The results of this study reveal an absolute requirement for APC in the targeting of ß-catenin for destruction and may have implications for the function of the human APC proteins in the regulation of Wnt transduction. In mouse and humans, as in Drosophila, there are two known APC homologs: APC and APC2/APCL. The mammalian APC homologs are expressed at high levels in the nervous system, with lower levels in many other tissues analyzed. Although human APC is widely expressed, germline mutations in APC result in a relatively narrow spectrum of disease. This includes the development of adenoma in the gastric and small and large bowel epithelia, as well as osteomas, desmoid fibromatosis, and lesions in retinal neurons and pigment epithelium. While hyperactivating mutations in ß-catenin are also associated with colonic carcinoma and desmoid fibromatosis, these hyperactivating mutations have been found in several carcinomas that are not detected in people with germline mutations in APC. Several scenarios could account for this discrepancy in sites of disease induced by APC loss versus ß-catenin hyperactivation. Perhaps human APC has a key role in controlling the degradation of ß-catenin in only a subset of epithelial tissues. Alternatively, in a manner directly analogous to that found for the two Drosophila Apc proteins, inactivation of one human APC homolog might be compensated for by the activity of the other in most tissues. Homozygous inactivation of human APC would induce disease states in only those tissues in which APC, rather than APC2, is the predominantly expressed gene, and would be dependent on the absolute levels of the two APC proteins in any given cell (Ahmed, 2002).

Testing hypotheses for the functions of APC family proteins using null and truncation alleles in Drosophila

Adenomatous polyposis coli (APC) is mutated in colon cancers. During normal development, APC proteins are essential negative regulators of Wnt signaling and have cytoskeletal functions. Many functions have been proposed for APC proteins, but these have often rested on dominant-negative or partial loss-of-function approaches. Thus, despite intense interest in APC, significant questions remain about its full range of cellular functions and about how mutations in the gene affect these. Six new alleles of Drosophila APC2 were isolated. Two resemble the truncation alleles found in human tumors and one is a protein null. Ovaries and embryos null for both APC2 and APC1 were generated, and the consequences of total loss of APC function was assessed, allowing several previous hypotheses to be tested. Surprisingly, although complete loss of APC1 and APC2 resulted in strong activation of Wingless signaling, it did not substantially alter cell viability, cadherin-based adhesion, spindle morphology, orientation or selection of division plane, as predicted from previous studies. The hypothesis that truncated APC proteins found in tumors are dominant negative was tested. Two mutant proteins have dominant effects on cytoskeletal regulation, affecting Wnt-independent nuclear retention in syncytial embryos. However, they do not have dominant-negative effects on Wnt signaling (McCartney, 2006).

Despite substantial interest in Wnt signaling and its regulation in development and disease, important questions remain about the nature of the null phenotype and thus the full range of processes in which APC family proteins play a crucial role. Experiments in vitro, in cultured cells and in Drosophila suggested novel roles for APCs in cadherin-based adhesion, spindle structure and chromosome segregation. Although some of these effects are subtle, APC family function was not completely eliminated, suggesting that APCs may play essential roles in one or more of these processes. Alternatively, because these phenotypes were assessed in cells expressing truncated or otherwise mutant proteins, or expressing transfected APC fragments, it is possible that these effects result from dominant interference with binding partners of APC that work in a process in which APC proteins themselves are not essential (McCartney, 2006).

To distinguish between these possibilities, null mutations removing the function of both APCs must be characterized. In mammals, all work has been done in single mutants and most was done with cells or animals expressing one truncated APC allele. Recently, Cre-lox technology was used to generate mouse APC alleles that may be null; these delete exon 14, and are predicted to truncate APC before the Arm repeats. Although the phenotype of homozygous animals has not been reported, Cre induction was used to create homozygous mutant clones of colon cells. This triggers polyp formation, with mutant cells assuming stem cell properties consistent with Wnt activation. Other phenotypes were not assessed, however, and tests to confirm that this allele is protein null were not reported, so splicing variations might produce residual mutant protein (McCartney, 2006).

Ovaries and embryos for APC2, or double null for both APC2 and APC1, were examined null for essential roles in cadherin-based adhesion. No phenotypes were observed consistent with substantial disruption of cadherin-catenin function, which disrupts both oogenesis and embryonic epithelial integrity. In ovaries, loss of APC2 and APC1 had no apparent effect on adhesion, and in embryos no significant alterations were observe in DE-cadherin or alpha-catenin localization at adherens junctions. Thus APC family proteins do not play an essential role in cell adhesion. However, subtle modulatory effects cannot be rule doult (McCartney, 2006).

Proposed roles for APC proteins in spindle assembly and orientation were tested. Embryos null for both APC proteins had no defects in spindle structure in syncytial embryos, apart from those in regions of spindle detachment or defective metaphase furrows, and no defects in spindle orientation or cell division symmetry in the ectoderm during gastrulation. Thus APC family proteins are not essential for spindle function in these tissues. A subtle but significant lengthening of syncytial spindles was seeen during cycle 13. Subtle defects were not assessed in chromosome segregation, which might lead to a slow accumulation of aneuploid cells in tumors - this will require other assays. How can the earlier data suggesting that APC family proteins have roles in adhesion and cytoskeletal regulation be reconciled when full loss-of-function experiments indicate that they do not? One possibility is that truncated fragments of APC may have dominant effects on processes in which APC does not play an essential role - the data on the phenotype of APC2DeltaS in spindle tethering, which is discussed in more detail below, provide an example of this (McCartney, 2006).

Unlike most other tumor suppressors, APC homozygous null colon tumors are either rare or non-existent. Instead, one allele encodes a protein truncated in the 'mutation-cluster region' (MCR), lacking the SAMP repeats and all sequences located between that region and the C-terminal end, suggesting strong selection for this event during tumor development. Several models propose that the truncated APC proteins found in tumors are dominant negative. One suggests that this affects Wnt signaling, with truncated APC proteins promoting stem cell proliferation. Most models suggest that truncated proteins affect cytoskeletal functions. Different studies come to different conclusions, however. For example, some suggest that truncated APC interferes with microtubule-kinetochore attachments, leading to genomic instability, but others suggest these effects are subtle. A dominant-negative role of truncated APC is not essential for disease, as some FAP patients inherit germline-null APC mutations. Their adenomas carry truncating mutations in the other allele; in this case, there was no wild-type APC to be affected by a dominant-negative truncation. Furthermore, the putative dominant-negative effect is not sufficient for oncogenesis - mice engineered to express truncated APC in a wild-type background do not develop polyps or tumors (McCartney, 2006 and references therein).

Genetic data provide new insight into this question. Little evidence for dominant-negative effects on Wg signaling. Heterozygotes are viable and adults are wild type in phenotype, and wild-type paternal APC2 effectively rescues eight of the nine mutants, suggesting that mutant proteins cannot be strongly dominant negative. The exception is APC2c9, where there appears to be some interference with paternal rescue. Likewise, the data suggest that truncated APC2 does not substantially affect spindle structure (whether APC1 truncations behave dominantly was not assessed) (McCartney, 2006).

Compelling evidence was found for dominant-negative effects on nuclear retention in syncytial embryos. APC2DeltaS and APC2d40 heterozygotes exhibit elevated levels of nuclear loss, and the frequency of abnormal embryos is higher in APC2DeltaS homozygotes than in APC2 null mutants. Thus, the cytoskeletal functions of APCs may be more sensitive to dominant-negative effects of truncated proteins, and this may affect chromosome segregation and contribute to tumor progression (McCartney, 2006).

These data also illuminate the mechanisms of dominant-negative activity. Loss of APC1 did not enhance the nuclear-loss phenotype of APC2 null embryos, suggesting that APC1 does not play a significant role in this process. This suggests that the dominant-negative effect is not on maternally contributed APC1. As nuclear retention is not completely disrupted in double null mutants, alternative mechanisms of nuclear retention partially compensate for the lack of APC1 and APC2. Because APC2DeltaS has a nuclear retention defect more severe than that of embryos M/Z null for both APCs, this mutant protein may not only block residual APC2 function, but may also interfere with a parallel, APC2-independent means of nuclear retention (McCartney, 2006).

The data also provide insights into the domains of APC2 required for nuclear retention, suggesting roles for the Arm repeats and the C terminus. Because APC2DeltaS mutants exhibit much more nuclear loss than do APC2N175K and APC2c9, Arm repeat 5 may have a special importance, perhaps by interfering with binding of a particular partner; APC2DeltaS may also more profoundly affect the overall structure of the Arm repeats (McCartney, 2006).

The experiments provide an in vivo test of the function of proteins truncated in the MCR. APC2d40 and APC2g41 strongly reduce the ability to regulate Wg signaling, but are not as strong as the null APC2g10, or APC2f90, truncating APC2 at the end of the Arm repeats. A similar severely truncated allele of mammalian APC led to higher levels of Wnt reporter activity in cultured cells than did a truncation in the MCR. The data support the 'just right' hypothesis (Albuquerque, 2002), which posits selection in tumors for mutations in which Wnt signaling is elevated, but not too much. In this model, proteins truncated in the MCR retain some ability to regulate ß-catenin, resulting in levels of Wnt signaling that are above the threshold for polyp formation but not 'too high', which might be cell lethal. This study is the first direct test of this hypothesis using null alleles (McCartney, 2006).

The nine alleles also allow the roles of different domains in Wg regulation to be assessed. The role of the Arm repeats of APC has been unclear. In APC mutant tumor cells, transfection of constructs encoding the 15- and 20-amino acid repeats and the SAMP repeats alone restores ß-catenin turnover. However, because these cells retain the Arm repeat-containing truncated protein found in the tumor, these two APC fragments might exhibit intra-allelic complementation. The data suggest that the Arm repeats are crucial for full function of APC2 in the destruction complex. Since missense mutations are not null for Wg regulation, APC proteins may retain residual function in Wg signaling without the Arm repeats. Alternatively, the mutations that were isolated may not fully disrupt the repeats. Each Arm repeat comprises three alpha-helices. Together, the Arm repeats form a superhelical structure with a large groove where partner proteins bind. Structure-based sequence alignments indicate that four out of the five missense mutations (APC2e90, APC2N175K APC2c9 and APC2b5) are in helix 3 of Arm repeats 3, 6 and 7. These mutations affect residues predicted to be in the protein core rather than those predicted to form the binding surface. They may thus destabilize individual Arm repeats, or the Arm repeat domain, without totally eliminating its function. This is consistent with the temperature sensitivity of four out of the five Arm repeat mutations (McCartney, 2006).

APC2 and APC1 function redundantly in Wg signaling throughout Drosophila development, despite differences in domain structure and subcellular localization. This redundancy suggests that the shared domains - the Arm repeats, the 15- and 20-amino acid repeats, the SAMP repeats and the conserved sequences A and B - are sufficient for Wg regulation. It is hypothesized that the Arm repeats are the docking site for a binding partner important for destruction-complex function. Using the temperature-sensitive allele APC2DeltaS, it was found that the phenotype and the membrane association of the mutant protein vary in parallel. Two of the weakest new alleles also exhibit residual membrane association, thus the Arm repeats may bind a partner mediating cortical localization of the destruction complex. However, this does not explain how APC1 and APC2 can have different predominant localizations and yet be redundant. Perhaps low-level cortical accumulation of APC1, especially in the absence of APC2, is sufficient for function. Future tests of this model and identification of the relevant binding partner are needed (McCartney, 2006).

APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation

To ensure the accurate transmission of genetic material, chromosome segregation must occur with extremely high fidelity. Segregation errors lead to chromosomal instability (CIN), with deleterious consequences. Mutations in the tumor suppressor adenomatous polyposis coli (APC) initiate most colon cancers and have also been suggested to promote disease progression through increased CIN, but the mechanistic role of APC in preventing CIN remains controversial. Using fly embryos as a model, this study investigated the role of APC proteins in CIN. The findings suggest that APC2 loss leads to increased rates of chromosome segregation error. This occurs through a cascade of events beginning with incomplete centrosome separation leading to failure to inhibit formation of ectopic cleavage furrows, which result in mitotic defects and DNA damage. Several hypotheses related to the mechanism of action of APC2 were tested, revealing that APC2 functions at the embryonic cortex with several protein partners, including Axin, to promote mitotic fidelity. These in vivo data demonstrate that APC2 protects genome stability by modulating mitotic fidelity through regulation of the cytoskeleton (Poulton, 2013).

This study has used Drosophila embryos to explore how APC proteins regulate mitotic fidelity in vivo. The findings corroborate studies in mammalian cells suggesting that APC promotes genomic stability through its cytoskeletal functions. Mammalian APC may also promote genomic stability by regulating Wnt signaling, but the current data indicate that the role of APC2 in preventing CIN in syncytial embryos does not involve this. Furthermore, although APC2 mutants have increased mitotic defects, most mitoses proceed without error. Thus, APC2 is not a central part of the mitotic apparatus in vivo, which contrasts with studies suggesting that APC proteins are key mitotic regulators. Instead, the data suggest that APC2 ensures high-fidelity mitosis. Although this might reflect functional differences between human and fly APCs, it may also suggest that cultured cells represent a sensitized situation with an elevated mitotic error rate, in which removal of fidelity regulators such as APC has a greater impact; consistent with this, cultured cells are prone to karyotypic anomalies. It is important to note that the precise series of events triggered by APC2 loss in syncytial fly embryos are likely to be specific to that system, with APC playing diverse cytoskeletal roles at different times and places, both within the same animal and between species (Poulton, 2013).

The small but significant increase in mitotic failure in APC2 mutants prompted an analysis backwards from nuclear removal, with the idea that the primary defect might be significantly more frequent, but might not always trigger nuclear removal. Mechanistically, the data are consistent with a model wherein APC2 promotes high-fidelity chromosome segregation through a primary role in facilitating centrosome separation. In APC2 mutants, centrosome separation defects are highly elevated, with 12% of nuclei having separation reduced more than two standard deviations from the WT mean. Although this did not have apparent effects on spindle structure, it had an unexpected consequence. APC2 mutants had a dramatic increase in ectopic cleavage furrows, the likelihood of which was highly correlated with reduced centrosome separation. The possible causal connection between incomplete centrosome separation and ectopic furrowing was further supported by an analysis of EB1 knockdown, as EB1 acts independently of APC2 in syncytial embryo. Although it is contended that the similar effects of Eb1-RNAi and APC2 loss on centrosome separation and ectopic furrowing support the role of these phenotypes in downstream chromosome mis-segregation and nuclear fallout, it should be noted that EB1 appears to play a direct role in chromosome segregation. Thus, it is possible that this shared defect in chromosome segregation is responsible for nuclear fallout in both mutants, irrespective of their similarities in centrosome separation defects and ectopic furrows (Poulton, 2013).

Ectopic furrows form where cleavage furrows would form in standard mitotic divisions. Indeed, formation of normal cytokinetic furrows is prevented in syncytial divisions by preventing Rho activation in the spindle midzone; although centralspindlin proteins localize there, RhoGEF2 does not, and artificial elevation of Rho activity triggers ectopic furrows. The data suggest how ectopic furrows are normally prevented in early embryos. They are consistent with the hypothesis that proper centrosome separation leads to furrow formation around the dividing 'cell', rather than over the metaphase plate where it would form in normal cytokinesis. When centrosome separation is reduced, the ectopic RhoGEF2 recruitment observed at the spindle midzone might be sufficient to trigger ectopic furrowing. The model suggests that failure to fully separate centrosomes and subsequent formation of ectopic furrows then leads to mitotic defects. Nuclei and associated mitotic spindles were found to be able to be physically displaced by ingressing ectopic furrows, perhaps disrupting chromosome segregation. This provides strong selective pressure for the existing mechanism preventing ectopic furrowing (Poulton, 2013).

How then does this trigger nuclear removal? Nuclear removal can be initiated by DNA damage, monitored by the DNA damage sensor CHK2. Strikingly, APC2 mutants had more chromosome segregation errors than wild type, many of which were followed by nuclear removal. Furthermore, nuclei undergoing fallout accumulated the DNA damage marker γH2Av. These data are consistent with the hypothesis that in APC2 mutants, chromosome mis-segregation generates DNA damage, thus triggering CHK2-mediated nuclear removal. Indeed, nuclear removal was blocked in chk2;APC2 double mutants. Recent studies in mammalian cells indicate that chromosome mis-segregation can cause DNA damage; intriguingly, one suggests cytokinetic furrow ingression on lagging chromosomes damages DNA. Given the significant increase in lagging chromosomes and the ectopic cleavage furrows over spindles in APC2 mutants, it is tempting to speculate that a similar mechanism for generating DNA damage might be involved (Poulton, 2013).

Of course, APC2 loss also might lead to chromosome segregation errors by other mechanisms. For example, centrosome separation defects are sufficient to generate merotelic microtubule attachments, thus leading to chromosome mis-segregation. The rapidity of syncytial divisions may make early embryos particularly sensitive to this type of error. Furthermore, in syncytial embryos the shared cytoplasm might make it difficult to alter timing of the metaphase-anaphase transition locally at an affected nucleus to allow repair of microtubule attachment errors. Together, these data suggest that although APC2 loss may lead to chromosome mis-segregation and nuclear fallout by multiple means, most if not all of these pathways begin with defects in centrosome separation. It is thought that these findings on the role of APC2 in mitotic fidelity in syncytial embryos may extend to other cell types, as fly neural stem cells lacking APC proteins have a significantly longer mitotic cycle, suggesting mitotic defects. It will be interesting to see whether human APC promotes mitotic fidelity, at least in part, by regulating centrosome positioning; consistent with this, human APC facilitates centrosome movement in migrating neurons by stabilizing microtubule interactions with cortical actin Furthermore, in human colon cancer cells, merlin (neurofibromin 2), ezrin and APC2 govern centrosome and spindle positioning by regulating astral microtubule attachment to cortical actin, suggesting a conserved role for APC in positioning centrosomes and spindles. These findings may also help in understanding better the role of APC mutations in cancer. In most colon cancers, one APC allele bears a premature stop codon in the mid-region, truncating the protein. A fly mutant (APC2d40) that mimics these truncations has increased nuclear fallout, like APC2 null embryos. APC2d40 mutants were also found to have reduced centrosome separation. This suggests that at least some aspects of the model describing the mechanistic role of APC2 in promoting mitotic fidelity may apply to colon tumors with truncated APC (Poulton, 2013).

The data also test key aspects of models describing where and how APC2 regulates the cytoskeleton. These analyses suggest that APC2 acts at the embryonic cortex, where it binds βcat. These findings, together with the role of APC2 in centrosome separation, led to revision of a previous model. It is proposed that APC2 facilitates stable interaction of astral microtubules with cortical actin to promote centrosome separation. This model is consistent with numerous studies indicating that cortical microtubule attachment helps mediate centrosome separation by generating pulling forces on astral microtubules through cortical dynein. It is now important to explore the effects of APC2 on microtubule dynamics directly (Poulton, 2013).

These analyses of APC2 domain-deleted mutants revealed two additional mechanistic insights. First, the Arm repeats of APC2 are necessary for syncytial cytoskeletal function, suggesting that they bind an important partner. Based on the model that APC2 facilitates microtubule-cortex interactions, one attractive candidate is KAP3. Second, the SAMP repeats, which allow APC2 to bind the destruction complex scaffold Axin, are also required for the cytoskeletal role of APC2. Furthermore, Axin mutants, like APC2 mutants, had reduced centrosome separation, and increased ectopic furrows and nuclear fallout. These data suggest that in syncytial embryos, APC2 acts as part of a multiprotein complex sharing many components with the Wnt-regulatory destruction complex. However, comparing APC2 mutants rescuing mitotic fidelity (this study) or Wnt signaling strongly suggests that in syncytial embryos this complex has a distinct role from its Wnt-regulatory function. It will be important to determine the role of Axin in this complex, as this study found it is not required to localize APC2 to the cortex, unlike the destruction complex component GSK3 (Poulton, 2013).

These in vivo studies indicate that APC helps to ensure mitotic fidelity via cytoskeletal regulation, but show that, at least in syncytial fly embryos, this contribution is relatively subtle. Furthermore, the data suggest that embryos possess mechanisms to help compensate for defects caused by APC2 loss: although centrosomal separation defects affect 12% of nuclei, only 7% have ectopic furrows and only 2% experience chromosome segregation defects or nuclear fallout. It will be important to identify mechanisms buffering the effects of APC2 loss and thus reducing mitotic defects (Poulton, 2013).

Although the precise role that APC2 plays in syncytial embryos and the cascade of consequences of APC2 loss are likely to be confined to that system, it is hypothesized that APC proteins play analogous roles in other tissues, subtly regulating the cytoskeleton to promote mitotic fidelity. This is consistent with observations in cultured mammalian cells, which suggest that APC loss exerts diverse effects on the cytoskeleton, reducing mitotic fidelity. Such roles for APC proteins in mitotic fidelity and the possible mechanisms compensating for APC loss may have interesting implications for cancer progression. In colon polyps and early adenomas, prior to accumulation of additional mutations present in carcinomas, cells prone to massive mitotic defects would probably be eliminated by surveillance processes, such as DNA damage checkpoints, whereas cells with subtle perturbations of mitotic fidelity like those caused by APC2 loss might persist. Occasional mitotic errors may then help induce genomic instability spurring tumor progression. Furthermore, APC mutation might sensitize cells to checkpoint loss, as cytoskeletal defects caused by APC loss may be less effectively buffered, thereby elevating CIN. This provides a clear, testable model for how APC mutation contributes to tumor initiation and progression (Poulton, 2013).


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Adenomatous polyposis coli tumor suppressor homolog 2: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 April 2014

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