Adenomatous polyposis coli tumor suppressor homolog 2
Human APC (hAPC) and Drosophila Apc-like (Hayashi, 1997) bind to ßcatenin (ßcat) and Armadillo (Arm), respectively. A test was performed to see whether Apc2 also interacts with Arm in vivo. Arm was immunoprecipitated (IPed) from embryonic extracts, and, in parallel, proteins were IPed with anti-myc, a control mAb. Apc2 specifically co-IPs with Arm from both early and older embryos, but does not co-IP with the control anti-myc antibody. Arm could not be detected in anti-Apc2 IPs. Because the antigen for the Apc2 antisera includes the Arm binding region, these sera might not recognize an Apc2-Arm complex. An Apc2 fragment containing the putative ßcat binding sites co-IPs with ßcat when expressed in the human colorectal cancer cell line SW480 (McCartney, 1999).
The hAPC-ßcat interaction is direct, and is mediated by the 15 and 20 amino acid repeats of hAPC and the Arm repeats of ßcat; the analogous region of Drosophila Apc-like binds Arm (Hayashi, 1997). To test whether Apc2 directly interacts with Arm, the yeast two-hybrid system was used to examin whether the 15 and 20 amino acid repeats of Apc2 interact with the full set of Arm repeats of Arm (R1-13), or with the centralmost Arm repeats (R3-8; the binding site for Drosophila E-cadherin and dTCF). For comparison, the 15 and 20 amino acid repeats of Drosophila Apc-like (Hayashi, 1997) were tested. The full 15 and 20 amino acid repeat regions of both Drosophila Apc-like and Apc2 strongly interact with the entire Arm repeat region and with R3-8. Thirty one and thirty four amino acid fragments carrying individual 15 or 20 amino acid repeats of dAPC and Apc2 (selected as good matches to the consensus) were also tested. Individual 15 amino acid repeats of either Drosophila Apc-like or Apc2 interact with both the entire Arm repeat region of Arm and with R3-8. An individual 20 amino acid repeat of Drosophila Apc-like also interacts with both Arm fragments. A single 20 amino acid repeat of Apc2 interacts strongly with Arm repeats 1-13; its interaction with R3-8 is much weaker (McCartney, 1999).
Since hAPC is phosphorylated, it was thought that various Drosophila Apc2 isoforms might be phosphorylation variants. To test this, Apc2 was immunoprecipitated (IPed) from embryos and the IPs were treated with protein phosphatase 2A (PP2A), a serine/threonine-specific phosphatase. PP2A treatment reduces the apparent molecular mass of Apc2; this effect is abolished if the PP2A inhibitor okadaic acid is included during incubation. Further, if embryonic cells are dissociated and incubated in tissue culture medium, the apparent molecular mass of Apc2 decreases; this effect is also abolished by okadaic acid, suggesting that it is mediated by endogenous phosphatases. Parallel alterations in Arm phosphorylation support this hypothesis. Taken together, these data suggest that the Apc2 isoforms reflect, at least in part, differential phosphorylation (McCartney, 1999).
Biochemical analyses suggest that Apc2 associates with the cell cortex. When 0 to 6 hour old embryos are fractionated into soluble (S100) and membrane-associated (P100) fractions, Apc2 partitions almost equally into these two fractions. In contrast, Arm is almost exclusively in the membrane fraction at this stage. The isoforms of Apc2 in the membrane fraction migrate more rapidly on SDS-PAGE than those in either the soluble fraction or the total cell lysate; because these isoforms are not detectable in total lysate, it is suspected that they may arise during fractionation by dephosphorylation. To examine whether Apc2 might associate with the membrane via a glycoprotein, Con A-Sepharose. Con A-Sepharose can be used to isolate membrane glycoproteins as well as proteins associated with them (e.g., Arm). A subset of Apc2 specifically binds to Con A in extracts from 0-6-h embryos. Thus, Apc2 may be anchored to the cortex via a transmembrane glycoprotein (McCartney. 1999).
The asymmetry of neuroblast cell divisions might arise from neuroblast-specific expression of the proteins required for asymmetric division. Alternatively, both neuroblasts and neuroepithelial cells could be capable of dividing asymmetrically, but in neuroepithelial cells other polarity cues might prevent asymmetric division. By disrupting adherens junctions the symmetric epithelial division of epidermal cells can be changed into asymmetric division. The adenomatous polyposis coli (APC) tumor suppressor protein is recruited to adherens junctions, and both APC and microtubule-associated EB1 homologs are required for the symmetric epithelial division along the planar axis. These results indicate that neuroepithelial cells have all the necessary components to execute asymmetric division, but that this pathway is normally overridden by the planar polarity cue provided by adherens junctions (Lu, 2001).
Drosophila neuroblasts delaminate from a polarized epithelial layer in the ventral neuroectoderm and divide asymmetrically along the apical-basal axis to produce larger apical neuroblasts and smaller basal ganglion mother cells. Inscuteable (Insc) as a central protein in organizing neuroblast division. Insc provides positional information that couples mitotic spindle orientation with the basal localization of cell-fate determinants such as Numb and Prospero together with their respective adaptor proteins Partner of Numb (Pon) and Miranda (Lu, 2001 and references therein).
The apical localization of Insc involves both a Baz-dependent initiation step and a maintenance step that requires Baz and Partner of Inscuteable (Pins). The expression of Baz and Pins in both neuroblasts and neuroepithelial cells suggests that these cells share certain apical-basal polarity information. Consistent with this notion is the observation that, when Pon is expressed ectopically in epithelial cells it is localized to the basal cortex, as in neuroblasts. Unlike neuroblasts, however, epithelial cells divide symmetrically along the planar axis and segregate ectopic Pon equally between the two daughter cells. These observations raise further questions: do epithelial cells have the ability to couple spindle orientation with protein localization, and segregate proteins asymmetrically between two unequally sized daughter cells? If so, what prevents them from executing this asymmetric division (Lu, 2001)?
To characterize epithelial division by monitoring it in live embryos, transgenic embryos expressing Pon and tau proteins fused with green fluorescent protein (GFP) were used. During epithelial cell cycle, tau-GFP-labelled mitotic spindle is formed along the planar axis of the embryo, and Pon-GFP is initially uniformly associated with the cortex and then localized to a basal crescent. The mitotic spindle remains oriented along the planar axis throughout mitosis. After cytokinesis, the Pon-GFP crescent is bisected by the cleavage furrow and is equally distributed between two equally sized daughter cells. This in vivo analysis shows that the machinery for basal protein localization is intact in epithelial cells, but it is uncoupled from spindle orientation (Lu, 2001).
Double-stranded (ds) CRB RNA was injected into transgenic embryos expressing Pon-GFP and tau-GFP. In about 70% (n = 200) of crb(RNAi) embryos, the organization of the ectodermal epithelium is disrupted, with epithelial cells losing their columnar shape, adopting rounded morphology, and becoming separated from each other. Live imaging of epithelial divisions in these embryos reveals that nearly all the epithelial cells show a tight coupling between the positioning of Pon-GFP crescents and the orientation of the mitotic spindle. Pon-GFP crescents were found at basal and lateral positions and less frequently at apical positions on the cell cortex, and one of the spindle poles was positioned underneath the Pon-GFP crescent (Lu, 2001).
After cytokinesis, Pon-GFP was segregated to one of the two similarly sized daughter cells. Asymmetric segregation of Pon-GFP to one of two similarly sized daughter cells was also observed in crb zygotic mutant embryos. Immunostaining of crb(RNAi) embryos with antibodies against Asense, Prospero and Insc indicates that epithelial cells do not express these neuronal markers, suggesting that the ability of these cells to undergo asymmetric division is not a result of cell-fate change (Lu, 2001).
Overexpression of the membrane-bound cytoplasmic tail of Crb (Crb-intra) causes similar disorganization of the epithelium as seen in crb mutants. The effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra show coupling of the mitotic spindle with the Pon-GFP crescent and asymmetric segregation of Pon-GFP to one of the daughter cells. Thus, when the formation of the adherens junction is disrupted, epithelial cells switch from a symmetric to an asymmetric division pattern (Lu, 2001).
In addition to its function in localizing Insc and regulating division axis in the neuroblasts, Baz is also required for the formation of adherens junction and the maintenance of epithelial polarity. The function of Baz in epithelial division was examined. The baz(RNAi) embryos showed overall disruption of epithelium organization similar to that observed in crb(RNAi) embryos. Unlike in crb(RNAi) embryos, however, epithelial cells in baz(RNAi) embryos divide in a symmetric fashion, with Pon-GFP distributed uniformly around the cell cortex throughout mitosis and the mitotic spindle orients in random directions. After cytokinesis, two equally sized daughter cells are produced and Pon-GFP is equally distributed between them (Lu, 2001).
Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. In crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned and Pon-GFP is always localized to the opposite side of the Baz crescent. This suggests that, although mispositioned, Baz is still functional in directing Pon-GFP localization in crb(RNAi) embryos. To test whether the coupling of Pon-GFP localization with spindle orientation observed in crb(RNAi) embryos is Baz dependent, double RNAi was performed by co-injecting a mixture of baz and crb dsRNAs. Epithelial divisions in the co-injected embryos appeared similar to baz single-injected embryos, with Pon-GFP segregated equally between two equally sized daughter cells. It is therefore concluded that epithelial cells depend on Baz to couple spindle orientation with protein localization when the adherens junction is disrupted (Lu, 2001).
To investigate the molecular mechanism underlying the planar positioning of spindles by the adherens junction, the function of proteins associated with the adherens junction was examined. A ubiquitously expressed, epithelial-cell-enriched APC (Apc2, termed here E-APC) is localized to the adherens junction, and, in shotgun and crb mutants, this adherens junction localization of E-APC is disrupted. The human APC protein interacts with a microtubule-associated EB1 protein, and the yeast homolog of EB1 (Bim1), together with the cortical marker Kar9, has been implicated in a search-and-capture mechanism of spindle positioning. Therefore, the function of E-APC in epithelial cell division was tested (Lu, 2001).
In about 60% of E-APC(RNAi) embryos, the positioning of Pon-GFP crescent and orientation of mitotic spindle became tightly coupled during epithelial division. At cytokinesis, epithelial cells divided asymmetrically to produce two unequally sized daughter cells, and Pon-GFP was always segregated to the smaller daughter cell. The asymmetric segregation of Pon-GFP and the ability to undergo unequal cytokinesis all depend on Baz, because in baz and E-APC double RNAi embryos, Pon-GFP is equally segregated to two similarly sized daughter cells. Therefore, in the absence of E-APC, epithelial cells divide asymmetrically in a Baz-dependent fashion. This suggests that adherens-junction-associated E-APC promotes spindle positioning along the planar axis and prevents the coupling of spindle positioning with asymmetric basal protein localization (Lu, 2001).
To test whether E-APC functions with EB1 to orient the mitotic spindle, RNAi was performed on a closely related fly homolog of EB1 (dEB1). In dEB1(RNAi) embryos, the epithelial divisions are also asymmetric, producing two unequally sized daughter cells, with Pon-GFP segregated to the smaller cell. The penetrance of dEB1(RNAi) phenotype (20%) is lower than that of E-APC(RNAi). Since there is strong maternal contribution of dEB1, the low penetrance might be due to a perdurance of maternal dEB1 protein. Alternatively, it might be due to functional compensation by two other distantly related EB1 homologs in the fly genome. It has been noted that E-APC lacks the carboxy-terminal domain, which is required for interaction with EB1, and no direct interaction between E-APC and EB1 could be detected in in vitro binding assays. It therefore remains to be determined whether the two are functionally linked together in vivo through some cofactor(s), or whether E-APC functions mainly to maintain adherens junction integrity and EB1 interacts with other unidentified molecules to orient spindles (Lu, 2001).
These results indicate that two sets of polarity cues exist for spindle positioning in epithelial cells: a planar polarity cue mediated by the adherens junction and an apical-basal polarity cue regulated by Baz. The division pattern of wild-type epithelial cells suggests that the planar polarity cue is normally dominant over the apical-basal polarity cue. Epithelial cells within the procephalic neurogenic region (PNR) that express endogenous Insc or epithelial cells outside of the PNR that express ectopic Insc are known to orient their mitotic spindle along the apical-basal axis during division. This suggests that the dominance of planar polarity over apical-basal polarity can be overcome by the expression of Insc. The normal appearance of the adherens junction in epithelial cells in the PNR, together with the observation that these cells divide along the planar axis and maintain their normal monolayer organization in an insc mutant, suggests that Insc functions by strengthening the apical-basal polarity instead of weakening the planar polarity through changing the behavior of the adherens junction (Lu, 2001).
When neuroblasts delaminate from the epithelium layer, they undergo morphological changes from columnar to round shape, lose their contacts with the surrounding cells and thus the adherens junction structures. This situation may be reminiscent of epithelial cells in adherens-junction mutants in which the planar polarity cue is lost. In both cases, the Baz-mediated polarity pathway takes over. That one polarity cue can dominate over another cue in orienting axis division may have its precedent in other organisms. Budding yeast can divide in either an axial or a bipolar pattern. Mutations in genes such as AXL1, BUD3, BUD4 and BUD10/AXL2 result in loss of polarity cue for axial bud formation and the cells divide in a bipolar fashion. This suggests that axial and bipolar cues coexist and that the axial cue is normally dominant over the bipolar cue. During mammalian cortical neurogenesis, neural progenitors switch from early symmetric divisions to later asymmetric divisions. It will be interesting to determine whether similar mechanisms and molecules are used to control this division symmetry switch in mammals. These results on E-APC highlight the importance of tumor suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).
The adenomatous polyposis coli (APC) protein is an important tumor suppressor in the colon. It promotes the destabilization of free cytoplasmic ß-catenin (the vertebrate homolog of the Drosophila protein Armadillo), a critical effector of the Wnt signaling pathway. The ß-catenin protein is also a component of adherens junctions, linking these to the actin cytoskeleton. The fruit fly has two APC genes: one encodes the ubiquitous E-APC (also known as dAPC2) and the other is mainly expressed in neuronal cells. In Drosophila epithelial cells, the ubiquitous form of APC, E-APC, is associated with adherens junctions. This association appears to be necessary for E-APC to function in destabilizing Armadillo. Using actin-depolymerizing drugs, it has been established that an intact actin cytoskeleton is required for the association of E-APC with adherens junctions in the Drosophila embryo. From an analysis of profilin mutants in which the actin cytoskeleton is disrupted, it was found that E-APC also requires actin filaments to associate with adhesive cell membranes in the ovary. Notably, conditions that delocalize E-APC from membranes, including a mutation in E-APC itself, cause partial detachment of Armadillo from adhesive membranes. It is concluded that actin filaments are continuously required for E-APC to be associated with junctional membranes. These filaments may serve as tracks for E-APC to reach the adherens junctions. The failure of E-APC to do so appears to affect the integrity of junctional complexes (Townsley, 2000).
The discovery of a link between Drosophila E-APC and the actin cytoskeleton contrasts with the work in vertebrate cells that uncovered a link between APC and microtubules. This may be explained as follows: (1) there may be genuine differences between APC proteins in their ability to utilize cytoskeletal elements. Notably, the carboxy-terminal third of human APC, which spans the microtubule-binding domain (but which, however, does not mediate tracking), is conserved in other vertebrate APCs, and is also found in the neuronal Drosophila APC, but is absent in E-APC. It is not known whether the neuronal Drosophila APC binds to or colocalizes with microtubules. (2) Evidence for the ability of vertebrate APC to utilize the actin cytoskeleton for its subcellular localization may have been missed so far. This could be because, in the vertebrate studies, cytochalasin D was used and its actin-depolymerizing effect is much weaker than that of latrunculin A. Indeed, there is a significant effect of latrunculin A on the subcellular distribution of human APC in transfected mammalian cells. Also, there may be a subtle effect of cytochalasin D on the subcellular distribution of APC in mammalian cells. (3) Perhaps most important, the cells in which the various APC proteins have been studied are substantially different from one another. The vertebrate work was carried out in migrating tissue culture cells whereas the Drosophila work has focused on stationary cells that adhere tightly to one another within tissues; these are cells that do not exhibit any obvious migratory behavior. Although human and mouse APC are associated with cell membranes in the intestinal epithelium, the requirement for this association is not known. Using a polarized tissue-culture cell model, it has been discovered that human APC associates in an actin-dependent way with the apical cell membrane compartment. Perhaps the mechanism mediating the fast transport of APC to, and the transient association with, distal sites in migrating cells is fundamentally different from the mechanism mediating its stable association with junctional membrane compartments in tissue. Microtubules may be more suitable for the former; actin filaments for the latter (Townsley, 2000).
In the embryo, the ability of E-APC to associate with junctional compartments appears to be critical for the destabilization of Armadillo, perhaps because the Armadillo-destabilizing Axin complex is localized in these apical compartments. The failure of E-APC to reach the Axin complex would explain the observed embryonic phenotypes that mimic stabilization of Armadillo; according to the shuttling model, this would result in a failure of E-APC to deliver Armadillo to this complex, and consequently in a failure of Armadillo to be earmarked by this complex for degradation. Ultimately, stabilized Armadillo would translocate into the nucleus and alter the transcription of TCF target genes (Townsley, 2000).
This work provides evidence that the failure of E-APC to associate with membranes may not only elicit an indirect nuclear response, but may also directly affect the junctional integrity of these membranes. The delocalization of junctional E-APC correlates with detachment of junctional Armadillo in three different situations: in chic mutant ovaries, in LMB-treated embryos and, most importantly, in E-APC mutant ovaries and embryos. Furthermore, a mild effect on junctional Armadillo has been observed in embryos in which E-APC is depleted by RNA interference. These observations indicate that the failure of E-APC to associate with junctional compartments may affect the junctional integrity. Ultimately, this would also affect the associated actin filaments, an expectation that is borne out by the observations in the E-APC mutants. In any case, the loss of Armadillo and actin filaments from cellular junctions appears to be a consequence of the failure of E-APC to associate with, or to reach, these junctions. This is consistent with the shuttling model, which ascribes a function to APC in shuttling Armadillo from the cytoplasmic to the junctional compartment, for incorporation into cadherin junctions. Note that this putative effect of the delocalized mutant E-APC on the junctional integrity might weaken the junctional anchorage of the Axin complex. This would thus aggravate further its own junctional delocalization, and the cytoplasmic Armadillo would accumulate to yet higher levels (Townsley, 2000).
The mild mutant phenotypes in E-APC mutant ovaries could indeed be due to failure of adhesion between germ cells. Adhesion mediated by E-cadherin and Armadillo is critical for normal shaping and positioning of the nurse cells and of the oocyte during oogenesis. Furthermore, oogenesis involves massive growth of the germ cells, and it is thus reasonable to assume that the adhesive junctional zones in the germ-cell membranes undergo considerable remodelling during oogenesis. The association of E-APC with these junctional membranes may therefore reflect a function of E-APC in the process of junctional growth and/or remodelling. Strong loss-of-function mutations of E-APC are required to establish whether this is the case (Townsley, 2000).
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