Adenomatous polyposis coli tumor suppressor homolog 2
The stem cell niche provides a supportive microenvironment to maintain adult stem cells in their undifferentiated state. Adhesion between adult stem cells and niche cells or the local basement membrane ensures retention of stem cells in the niche environment. Drosophila male germline stem cells (GSCs) attach to somatic hub cells, a component of their niche, through E-cadherin-mediated adherens junctions, and orient their centrosomes toward these localized junctional complexes to carry out asymmetric divisions. This study shows that the transmembrane receptor tyrosine phosphatase Leukocyte-antigen-related-like (Lar), which is best known for its function in axonal migration and synapse morphogenesis in the nervous system, helps maintain GSCs at the hub by promoting E-cadherin-based adhesion between hub cells and GSCs. Lar is expressed in GSCs and early spermatogonial cells and localizes to the hub-GSC interface. Loss of Lar function resulted in a reduced number of GSCs at the hub. Lar function was required cell-autonomously in germ cells for proper localization of Adenomatous polyposis coli 2 and E-cadherin at the hub-GSC interface and for the proper orientation of centrosomes in GSCs. Ultrastructural analysis revealed that in Lar mutants the adherens junctions between hub cells and GSCs lack the characteristic dense staining seen in wild-type controls. Thus, the Lar receptor tyrosine phosphatase appears to polarize and retain GSCs through maintenance of localized E-cadherin-based adherens junctions (Srinivasan, 2012).
This work identifies a role for the transmembrane receptor tyrosine phosphatase Lar, acting cell-autonomously to maintain attachment of Drosophila male GSCs to the hub. Lar function appears to promote the maintenance of robust adherens junctions between GSCs and hub cells and to localize and/or retain E-cadherin at the hub-GSC interface. Consistent with the recently demonstrated requirement for E-cadherin to polarize GSCs by localizing Apc2 at the hub-GSC interface and to establish centrosome orientation in GSCs (Inaba, 2010), Apc2 was often mislocalized around the GSC cortex and centrosomes were often misoriented in Lar mutant GSCs (Srinivasan, 2012).
Lar may function in parallel with other cell signaling pathways that are important for maintaining attachment of GSCs to the hub. Activation of the JAK-STAT pathway in Drosophila male GSCs maintains GSCs at the hub. However, Stat92E protein levels appeared normal in Lar mutant GSCs, suggesting that Lar function is not required for activation of the JAK-STAT pathway. The Rap1 GTPase/Rap1 guanine nucleotide exchange factor (Rap-GEF) signaling pathway also regulates hub-GSC adhesion. Like Lar mutants, Rap-GEF mutants have impaired adherens junctions at the hub- GSC interface resulting in GSC loss. However, Rap-GEF function is required in hub cells, whereas Lar functions in GSCs to promote hub-GSC adhesion. Interestingly, expression of E-cadherin-GFP in either hub cells or GSCs of Rap-GEF mutants resulted in wild-type numbers of GSCs and restored E-cadherin localization at the hub- GSC interface, whereas expression of Ecadherin-GFP in Lar mutant GSCs did not rescue the loss of GSCs, suggesting that the Rap-GEF and Lar signaling pathways might use different mechanisms to build and/or maintain adherens junctions between the hub cells and GSCs (Srinivasan, 2012).
The ability of some GSCs to persist next to the hub in Lar mutant testes might be due to partial redundancy between Lar and other tyrosine phosphatases such as the type IIA family receptor tyrosine phosphatase Ptp69D, which has overlapping functions with Drosophila Lar in the central nervous system and the visual cortex and shares common signaling mechanisms. Alternatively, weak hub-GSC adhesion in Lar mutant testes might enable CySCs to compete for attachment to the hub, displacing some, but not all, GSCs from the hub. CySCs normally have smaller regions of contact with the hub than do GSCs but can outcompete GSCs from the hub when provided with an advantage. For example, overexpression of components of the integrin-based adhesion system in CySCs resulted in displacement of GSCs from the hub by CySCs (Srinivasan, 2012).
In wild-type testes, Lar localizes to the hub-GSC interface, which is the region of cell cortex where localized adherens junctions anchor GSCs to their niche. Adherens junctions are formed by extended clustering of transmembrane cadherin proteins that form homotypic interactions with cadherins on opposing cell membranes. The highly conserved cytoplasmic tail of E-cadherin acts as an anchor for β-catenin and p120-catenin and indirectly for α- catenin through its interaction with β-catenin. Lar also localizes to adherens junctions in epithelial cells and in neuronal synapses that are enriched in cadherin-catenin complexes. Lar physically associates with the cadherin-catenin complex in cultured cells and with N-cadherin in Drosophila embryos (Srinivasan, 2012).
Adherens junctions are associated with underlying arrays of cortical F-actin, organized by the high local concentration of β-catenin dimers. F-actin filaments, in turn, regulate the stability and strength of adherens junctions. Biochemical and genetic analyses of Lar indicate a role in regulating the actin cytoskeleton. Loss of Lar function in Drosophila oocytes results in defects in follicle formation, egg elongation and anterior-posterior polarity that are correlated with defects in actin filament organization. Lar might help to maintain hub- GSC adhesion by interacting with and modulating the function of regulators of F-actin. Drosophila Lar and its homologs physically and genetically interact with Ena, a member of the Ena/VASP family of actin regulators. Drosophila Ena and its mammalian homologs localize to adherens junctions and have been implicated in the formation and strengthening of adherens junctions in several cell types. However, although Ena localized to the hub-GSC interface, where adherens junctions are present, its function was not absolutely required for GSC maintenance, suggesting that other F-actin regulators in addition to Ena function to maintain hub-GSC adhesion (Srinivasan, 2012).
Lar protein localized to the hub-GSC interface might instead, or in addition, regulate the tyrosine phosphorylation state of components of the adherens junctions to maintain strong adhesion between hub cells and GSCs. Regulation of the tyrosine phosphorylation of components of adherens junctions plays an important role in modulating the adhesive state of cells. Tyrosine phosphorylation of E-cadherin in epithelial cells induces loss of cell-cell contacts and the endocytosis of E-cadherin. A possible role of Lar is to maintain adherens junctions by dephosphorylating E-cadherin. Alternatively, Lar might target E-cadherin to the membrane to build adherens junctions, as has been shown for the mammalian homolog of Lar in cultured hippocampal neurons, where it promotes the accumulation of cadherin-catenin complexes at the synapse to enhance cell adhesion. Alternatively, or in addition, Lar might regulate tyrosine phosphorylation of the catenins associated with E-cadherin at the hub-GSC interface. Tyrosine phosphorylation of β-catenin leads to loss of cadherin-β-catenin interaction and to internalization of Ecadherin, reducing the strength of adherens junctions. Mammalian Lar has been shown to dephosphorylate β-catenin in vitro, suggesting that in vivo Lar might promote cell adhesion by regulating the phosphorylation of β-catenin (Srinivasan, 2012).
In addition to GSCs, Lar protein was also detected in two-, four- and eight-cell transit-amplifying spermatogonial cysts, which have the ability to dedifferentiate and reoccupy the hub to replace lost GSCs. Under conditions that promote dedifferentiation, spermatogonial cells send out dynamic, actin-rich, thin protrusions, suggesting acquisition of motility. An intriguing possibility is that Lar might facilitate the ability of dedifferentiating spermatogonial cells to reorganize their actin cytoskeleton to recognize and build adherens junctions with the hub, similar to the role of Lar in the nervous system, where it promotes axonal migration, possibly by facilitating reorganization of the actin cytoskeleton. One of the ligands of Lar identified in the nervous system, the heparan sulfate proteoglycan Dally-like (Dlp), is expressed by hub cells and helps maintain GSCs in their undifferentiated state. At Drosophila neuromuscular junctions, Dlp interacts with and inhibits the phosphatase activity of Lar to regulate active zone morphology and function at synapses. Similarly, in dedifferentiating spermatogonial cells, interaction of Lar in GSCs with Dlp on hub cells might inhibit cell motility and promote the formation of adherens junctions between hub cells and the dedifferentiating germ cells (Srinivasan, 2012).
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).
Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells. This often involves interactions between spindle and astral microtubules and cortical actin. In yeast and C. elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious. This study reports that the tumour suppressor homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos. Armadillo, alpha-catenin and APC2 all localize to sites of cortical spindle attachment. APC2-Armadillo complexes often localize with interphase microtubules. Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2-Armadillo complexes. Together, these data suggest that APC2, Armadillo and alpha-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase (McCartney, 2001).
Stem cell self-renewal can be specified by local signals from the surrounding microenvironment, or niche. However, the relation between the niche and the mechanisms that ensure the correct balance between stem cell self-renewal and differentiation is poorly understood. This study shows that dividing Drosophila male germline stem cells use intracellular mechanisms involving centrosome function and cortically localized Adenomatous Polyposis Coli tumor suppressor protein to orient mitotic spindles perpendicular to the niche, ensuring a reliably asymmetric outcome in which one daughter cell remains in the niche and self-renews stem cell identity, whereas the other, displaced away, initiates differentiation (Yamashita, 2003).
Adult stem cells maintain populations of highly differentiated but short-lived cells such as skin, intestinal epithelium, or sperm through a critical balance between alternate fates: Daughter cells either maintain stem cell identity or initiate differentiation. In Drosophila testes, germline stem cells (GSCs) normally divide asymmetrically, giving rise to one stem cell and one gonialblast, which initiates differentiation starting with the spermatogonial transient amplifying divisions. The hub, a cluster of somatic cells at the testis apical tip, functions as a stem cell niche: Apical hub cells express the signaling ligand Unpaired (Upd), which activates the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway within GSCs to maintain stem cell identity (Yamashita, 2003).
Analysis of dividing male GSCs by expression of green fluorescent protein (GFP)-α-tubulin in early germ cells revealed that in 100% of the dividing stem cells observed, the mitotic spindle was oriented perpendicular to the hub-GSC interface throughout mitosis, with one spindle pole positioned within the crescent where the GSC contacted the hub. Stem cell division was rare, averaging one dividing stem cell observed per 5 to 10 testes (~2% of total stem cells) in 0- to 2-day-old adults. Spindles were not oriented toward the hub in gonialblasts (Yamashita, 2003).
Drosophila male GSCs maintained a fixed orientation toward the hub throughout the cell cycle, unlike Drosophila embryonic neuroblasts or the Caenorhabditis elegans P1 cell, in which spindle orientation is established during mitosis by a programmed rotation of the spindle. The single centrosome in early interphase GSCs was consistently located adjacent to the hub. After centrosome duplication, one centrosome remained adjacent to the hub, whereas the other migrated to the opposite side of the nucleus. The mechanisms responsible for Drosophila GSC spindle orientation may differ between sexes. In female GSCs, the spectrosome, a spherical intracellular membranous structure, remains localized next to the apical cap cells, where it may help anchor the spindle pole during mitosis. In interphase male GSCs, in contrast, the spectrosome was often located to the side, whereas at least one centrosome held the stereotyped position adjacent to the hub (Yamashita, 2003).
To investigate centrosome function in orientation of male GSCs, males were analyzed that were null mutant for the integral centrosome component centrosomin (cnn), which is required for normal astral microtubule function. In cnn mutant males, mitotic spindles were not oriented toward the hub in ~30% of the dividing GSCs examined. In an additional 10% to 20%, spindles were properly oriented, but the proximal spindle pole was no longer closely associated with the cell cortex at the hub-GSC interface and the entire spindle was displaced away from the hub. The frequency of spindle orientation defects was highest in metaphase. Loss of function of cnn also partially randomized the interphase centrosome positioning in male GSCs. In more than 35% of the cnn mutant GSCs with duplicated centrosomes that were scored, neither centrosome was positioned next to the hub (Yamashita, 2003).
The number of germ cells associated with the hub was increased 20% to 30% in cnn mutant males, from an average of 8.94 GSCs per hub in the wild type to 11.89 GSCs per hub in cnnHK21/cnnHK21 and 10.69 GSCs per hub in cnnHK21/cnnmfs3. Hub size was not significantly different in cnn compared with wild-type males. In cnn testes with many stem cells, GSCs appeared crowded around the hub and often seemed attached to the hub by only a small region of cell cortex. Finite available physical space around the hub may limit the increase in stem cell number in cnn mutant males (Yamashita, 2003).
As suggested by the increased stem cell number, there were several cases in both live and fixed samples from cnn males in which a stem cell that had recently divided with a mitotic spindle parallel to the hub-GSC interface produced two daughter cells that retained contact with the hub, a finding that was not observed in the wild type. GSCs were also observed dividing with a misoriented/detached spindle that lost attachment to the hub, probably explaining the mild increase in stem cell number relative to the frequency of misoriented spindles (Yamashita, 2003).
The normal close attachment of one spindle pole to a region of the GSC next to the hub and the effects of cnn mutants on centrosome and spindle orientation suggest that a specialized region of the GSC cell cortex touching the hub might provide a polarity cue toward which astral microtubules from the centrosome and spindle pole orient. High levels of DE-cadherin (fly epithelial cadherin) and Armadillo (Arm; fly ß-catenin) colocalized at the hub-GSC interface, as well as at the interface between adjacent hub cells, marked by high levels of Fas III. High levels of DE-cadherin and Arm were not detected around the rest of the GSC surface. Forced expression of DE-cadherin-GFP specifically in early germ cells confirmed that DE-cadherin in GSCs colocalized to the hub-GSC interface (Yamashita, 2003).
DE-cadherin and Armadillo at the hub-GSC interface may provide an anchoring platform for localized concentration of Apc2, one of two Drosophila homologs of the mammalian tumor suppressor gene Adenomatous Polyposis Coli (APC), which in turn may anchor astral microtubules to orient centrosomes and the spindle. Immunofluorescence analysis revealed Apc2 protein localized to the hub-GSC interface. In apc2 mutant males, GSCs were observed with mispositioned centrosomes, misoriented spindles, or detached spindles. Both the average number of stem cells and hub diameter increased in apc2 mutant males compared with that of the wild type. Unlike in cnn mutants, GSCs did not appear crowded around the hub in apc2 males, perhaps as a result of the enlarged hub (Yamashita, 2003).
The second Drosophila APC homolog, apc1, may also contribute to normal orientation of the interphase centrosome and mitotic spindle. Apc1 protein localized to centrosomes in GSCs and spermatogonia during late G2/prophase, after centrosomes were fully separated but before nuclear envelope breakdown. Apc1 was not detected at centrosomes from prometaphase to telophase. Spindle orientation and centrosome position were perturbed in GSCs from apc1 males, and the number of stem cells per testis and the diameter of the hub both slightly increased in apc1 mutant testes compared with those of the wild type (Yamashita, 2003).
It is proposed that the reliably asymmetric outcome of male GSC divisions is controlled by the concerted action of (1) extrinsic factor(s) from the niche that specify stem cell identity, and (2) intrinsic cellular machinery acting at the centrosome and a specialized region of the GSC cortex located at the hub-GSC interface to orient the cell division plane with respect to the signaling microenvironment. Astral microtubules emanating from the centrosome may be captured by a localized protein complex including Apc2 at the GSC cortex where it interfaces with the hub, similar to the way in which cortical Apc2 may orient mitotic spindles in the syncytial embryo or epithelial cells (Yamashita, 2003).
Mechanisms that orient the mitotic spindle by attachment of astral microtubules to specific cortical sites may be evolutionally conserved. In budding yeast, spindle orientation is controlled by capture and tracking of cytoplasmic microtubules to the bud tip, dependent on Kar9, which has weak sequence similarity to APC proteins. Kar9 has been localized to the spindle pole body and the cell cortex of the bud tip, reminiscent of the localization of Drosophila Apc1 at centrosomes and Apc2 at the cell cortex (Yamashita, 2003).
Polarization of Drosophila male GSCs toward the hub could result simply from the geometry of cell-cell adhesion. GSCs appear to be anchored to the hub in part through localized adherens junctions. Homotypic interactions between DE-cadherin on the surface of hub cells and male GSCs could concentrate and stabilize a patch of DE-cadherin. The resulting localized DE-cadherin cytoplasmic domains could then provide localized binding sites for ß-catenin and Apc2 at the GSC cortex. Although binding of E-cadherin and APC to ß-catenin is thought to be mutually exclusive, APC could be anchored at the cortical patch through the actin cytoskeleton, which in turn could interact with ß-catenin/α-catenin (Yamashita, 2003).
Orientation of stem cells toward the niche appears to play a critical role in the mechanism that ensures a reliably asymmetric out-come of Drosophila male GSC divisions, consistently placing one daughter within the reach of short-range signals from the hub and positioning the other away from the niche. Oriented stem cell division may be a general feature of other stem cell systems, helping maintain the correct balance between stem cell self-renewal and initiation of differentiation throughout adult life (Yamashita, 2003).
The rearrangement of cytoskeletal elements is essential for many cellular processes. The tumor suppressor Adenomatous polyposis coli (APC) affects the function of microtubules and actin, but the mechanisms by which it does so are not well understood. This study reports that Drosophila syncytial embryos null for Apc2 display defects in the formation and extension of pseudocleavage furrows, which are cortical actin structures important for mitotic fidelity in early embryos. Furthermore, the formin Diaphanous (DIA) functions with APC2 in this process. Colocalization of APC2 and DIA peaks during furrow extension, and localization of APC2 to furrows is DIA-dependent. Furthermore, APC2 binds DIA directly through a region of APC2 not previously shown to interact with DIA-related formins. Consistent with these results, reduction of dia enhances actin defects in Apc2 mutant embryos. Thus, an APC2-DIA complex appears crucial for actin furrow extension in the syncytial embryo. Interestingly, EB1, a microtubule +TIP and reported partner of vertebrate APC and DIA1, may not function with APC2 and DIA in furrow extension. Finally, whereas DIA-related formins are activated by Rho family GTPases, these data suggest that the APC2-DIA complex might be independent of RHOGEF2 and RHO1. Furthermore, although microtubules play a role in furrow extension, this analysis suggests that APC2 and DIA function in a novel complex that affects actin directly, rather than through an effect on microtubules (Webb, 2009).
APC family proteins have many well-documented effects on the microtubule cytoskeleton, whereas APC functions with actin are much less well understood. The Drosophila syncytial embryo is an excellent in vivo system in which to study the role of APC2 and its partners in organizing the cytoskeleton. Drosophila APC2 localizes to actin in syncytial embryos, and a roles has been suggested role for APC2 in tethering cortical microtubules to actin (McCartney, 2001; Webb, 2009 and references therein).
This study report the cytoskeletal consequences of eliminating all APC2 in the syncytial embryo. Apc2-null mutants exhibit incomplete actin rings and a failure of actin furrow extension. Apc2δS mutants exhibit more nuclear loss than do null mutants, but have weaker actin defects, suggesting that the APC2δS protein might interfere with a tethering process for which APC2 is not essential. The presence of actin furrow defects in embryos that are mutant for multiple alleles of Apc2, including a null, strongly suggests that APC2 functions in the normal organization of actin furrows (Webb, 2009).
A novel role has been demonstrated for an APC2-DIA complex in the organization of the actin cytoskeleton. Formins such as DIA are best known for their ability to nucleate unbranched actin filaments and accelerate filament elongation. Drosophila DIA functions in actin-based furrow assembly during cellularization and conventional cytokinesis. dia mutant syncytial embryos have defects in the initiation and elongation of actin furrows, consistent with DIA subcellular localization and known roles for formins. This study shows that APC2 and DIA colocalize together and with actin specifically at times when furrows are elongating. The fact that APC2 and DIA bind directly in vitro, but their colocalization is cell cycle-dependent, suggests that the interaction is regulated in vivo (Webb, 2009).
The simplest model for the function of an APC2-DIA complex in actin furrow formation is that DIA-dependent nucleation and elongation of unbranched actin filaments is essential for furrow extension, and that APC2 promotes DIA activity. The fact that the dia-null phenotype is more severe than that of Apc2, coupled with the enhancement of the Apc2-null phenotype by a reduction of dia, support the model that APC2 is not essential for, but might enhance, DIA activity. The dependence of APC2 on DIA for localization indicates that DIA may directly affect the regulation of its own activity (Webb, 2009).
One mechanism regulating the activity of formins has been extensively studied. DIA-related formins (DRFs) are autoinhibited through the binding of the N-terminal DIA inhibitory domain (DID) to the C-terminal DIA autoregulatory domain (DAD). Binding of the GTPase-binding domain (GBD) by RHO-GTP relieves the autoinhibition and activates DRFs, which function as dimers. Although RHO1 and RHOGEF2 have been reported to act upstream of DIA during Drosophila cellularization and embryonic morphogenesis, other reports suggest that they are in a parallel pathway during these times. The distinct actin defects in Rho1 and RhoGEF2 mutants as compared with Apc2 and dia mutants suggest that RHO1 is not the GTPase that directly activates DIA during furrow formation in the syncytial embryo. However, because Rho1-null embryos cannot be generated genetically owing to requirements during oogenesis, it is possible that there is a role for RHO1 in activating DIA independent of RHOGEF2. In addition, although the activity of other GEFs and GTPases cannot be ruled out, these observations, along with those of formins in other systems, suggest the existence of alternative mechanisms for DIA-related formin activation (Webb, 2009).
This study shows that APC2 and DIA can bind directly to each other. Thus, APC2 could directly affect the function of DIA, perhaps by stabilizing the open conformation required for optimal DIA activity. Alternatively, APC2 could enhance the activity of DIA by binding to and recruiting other DIA-activating factors to the complex. Once DIA is activated it might dissociate from this complex, resulting in two pools of DIA. This notion is supported by the observation that DIA, but not APC2, is enriched at the furrow tip. It is proposed that in the absence of APC2, the efficiency of DIA activation is reduced, resulting in a decrease in the amount of unbranched actin filaments and a consequent production of shallow furrows. Consistent with this model for APC2 function, APC proteins are thought to play a scaffolding role in the Wnt regulatory 'destruction complex'. Furthermore, vertebrate APC binds ASEF and IQGAP, activators of RAC and RAC/CDC42, respectively, through its N-terminal Armadillo repeats. Thus, APC2 may promote the association of DIA with GEFs and GTPases, or with other proteins that promote the open conformation or otherwise enhance DIA activity (Webb, 2009).
EB1 regulates microtubule function in many organisms including Drosophila, in which its disruption affects spindle positioning and dynamics in the early embryo. Mouse EB1 can bind both DIA1 and APC1 and the binary interactions identified have suggested a ternary complex. This study tested the possibility that EB1 plays a role in an APC2-DIA complex. Although Eb1-null syncytial embryos exhibit weak defects in furrow extension, the lack of a genetic interaction with Apc2, and the lack of direct binding to APC2, argue that EB1 function in the syncytial embryo is independent of the APC2-DIA complex (Webb, 2009).
Taken together, the lack of post-translationally modified, stabilized microtubules in the syncytial embryo, the lack of discernable microtubule defects in the Apc2 mutant, and the lack of APC2 interactions with EB1, strongly suggest that the Drosophila APC2-DIA complex has a distinct cytoskeletal function from that of the mouse DIA1-APC-EB1 complex that stabilizes microtubules in cultured cells. In addition, the colocalization of APC2 and DIA specifically when furrows are extending during prophase and metaphase, rather than during the critical period of microtubule function in anaphase, supports the model that the APC2-DIA complex primarily affects actin. It is intriguing that interactions between APC proteins and formins have been conserved, and that such complexes can affect both the actin and microtubule networks (Webb, 2009).
Unlike conventional cytokinesis that uses actomyosin-based contraction to drive membrane invagination, Myosin II function is dispensable for pseudocleavage furrow formation. Many proteins are known to affect the dynamic organization of syncytial actin. Centrosomin, a core centrosome component, Sponge, a putative unconventional RacGEF, and Scrambled, a novel protein, all have roles in normal cap formation. Cap formation and expansion require the Arp2/3 complex and its activator SCAR. Well-known actin regulators such as RHO1, RHOGEF2 and Abelson (Abl tyrosine kinase) may not affect furrow extension directly, but rather regulate the overall balance of actin activity. The Myosin VI protein Jaguar appears to play a specific role in furrow extension, where it might stabilize actin filaments, as it does in the Drosophila testis (Webb, 2009).
The best-understood mechanism for pseudocleavage furrow extension is the NUF (Arfophilin)-RAB11-DAH (Dystrophin) pathway, which utilizes microtubule-based transport from the recycling endosome to move membrane and actin to the site of furrow extension. Once actin has been delivered to this site, APC2-DIA might promote the nucleation and elongation of unbranched filaments necessary for furrow extension. Interestingly, it has been shown that in the absence of dia, and in Apc2 mutant embryos reduced for dia, some actin remains cap-like. This intriguing observation suggests that there is a relationship between the dissolution of caps and the formation of furrows, and that these distinct pools of branched and unbranched actin might be in a balance. The APC2-DIA complex has emerged as a key factor affecting actin organization in the early embryo, and further study will reveal how the many regulatory pathways converge to influence dynamic changes in actin organization (Webb, 2009).
The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor Adenomatous Polyposis Coli (APC) is an essential negative regulator of Wnt signaling inactivated in over 80% of colorectal cancers. APC participates in a multi-protein 'destruction complex' that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high affinity β-catenin binding-sites (20 amino acid repeats; 20Rs) on APC. This study tested these models by generating a Drosophila APC2 mutant lacking all β-catenin binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. The results are inconsistent with current models, as it was found that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, an APC2 mutant was generated lacking all β-catenin binding-sites (including the 15Rs), and a direct β-catenin/APC interaction was found to be also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, these findings support a model whereby β-catenin binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes a separation of β-catenin destruction and Wg/Wnt signaling outputs was observed, and it is suggested that cytoplasmic retention of β-catenin likely accounts for this difference (Yamulla, 2004).
The tumor suppressor Adenomatous polyposis coli (APC) is an essential negative regulator of Wnt signaling through its activity in the destruction complex with Axin, GSK3beta and CK1 that targets β-catenin/Armadillo (β-cat/Arm) for proteosomal degradation. The destruction complex forms macromolecular particles termed the destructosome. While APC functions in the complex through its ability to bind both β-cat and Axin, it is hypothesized that APC proteins play an additional role in destructosome assembly through self-association. This study shows that a novel N-terminal coil, the APC Self-Association Domain (ASAD), found in vertebrate and invertebrate APCs, directly mediates self-association of Drosophila APC2 and plays an essential role in the assembly and stability of the destructosome that regulates β-cat degradation in Drosophila and human cells. Consistent with this, removal of the ASAD from the Drosophila embryo results in β-cat/Arm accumulation and aberrant Wnt pathway activation. These results suggest that APC proteins are required not only for the activity of the destructosome, but also for the assembly and stability of this macromolecular machine (Kunttas-Tatli, 2014).
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