Adenomatous polyposis coli tumor suppressor homolog 2 (Apc2), a second Drosophila APC homolog, was identified nearly simultaneously in three laboratories (van Es, 1999; Yu, 1999a and McCartney, 1999). Whereas, the previously characterized APC-like regulates Arm signaling in the Drosophila larval photoreceptors (Ahmed, 1998), two features of APC-like were surprising, given the widespread expression and essential function of mouse APC. Embryonic expression of APC-like is largely confined to the central nervous system (Hayashi, 1997), and null mutations in APC-like are viable and fertile, with strong effects only in the larval photoreceptors (Ahmed, 1998). These observations suggested the existence of a second APC gene in flies, now identified as Apc2.

As well as having an intimate involvement in Wingless signaling, a function well documented for APC-like, APCs in general have additional cellular roles. When human APC (hAPC) is overexpressed in cultured cells, it decorates microtubules (MTs) and can bind and bundle MTs in vitro (Munemitsu, 1994; Smith, 1994). In cultured cells, APC localizes at the cell cortex in membrane puncta where bundles of MTs often terminate (Nathke, 1996). If one expresses a stabilized form of ßcatenin (Drosophila homolog: Armadillo) that cannot be phosphorylated by GSK (Drosophila homolog Shaggy), in cultured cells, mutant ßcatenin accumulates with APC in membrane puncta, and these cells display altered migratory behavior. These data have prompted the suggestion that APC may regulate cell migration via its interaction with MTs, and that this role is modulated by ßcatenin. APC may also influence cytoskeletal dynamics by binding to EB1 (Su, 1995), which associates with the MT cytoskeleton in mammalian cells (Berrueta, 1998; Morrison, 1998). Yeast EB1 homologs contribute to MT function and may form part of a cytokinesis checkpoint (Beinhauer, 1997; Schwartz, 1997; Muhua, 1998). In addition to connections with MTs, APC may associate with the actin cytoskeleton via ß- and alpha-catenin (McCartney, 1999).

Both actin and MT cytoskeletons are targets of the Wg/Wnt pathway. Signaling by Wnt family members or by their Frizzled (Fz) receptors (see Drosophila Frizzled) is required to orient certain cell divisions in both nematodes and flies. In C. elegans, Wnt signaling directs the orientation of mitotic spindles in specific early embryonic blastomeres and orients postembryonic asymmetric cell divisions (for review see Han, 1997), whereas in Drosophila, Fz is required for orientation of the mitotic spindles of bristle precursor cells (Gho, 1998). Fz also plays a key role in orienting the cytoskeleton during formation of hairs and bristles, polarized outgrowths of the cell membrane (for review see Shulman, 1998). Both the actin and MT cytoskeletons are required for hair positioning and growth (Wong, 1993; Turner, 1998).

Discovery of Drosophila Apc2, and investigation of its cytoskeletal connection (McCartney, 1999), raises the possibility that Apc2 acts as an effector molecule through which Wnt signaling influences the cytoskeleton. Because Apc2 associates with the actin cytoskeleton in contexts where no Wg signaling is thought to occur, such as in pre-blastoderm embryos, Apc2 may play more fundamental roles in cytoskeletal regulation. Such functions may now be revealed by further genetic analyses of APC-like and Apc2 (McCartney, 1999).

Anti-Apc2 antisera were used to characterize Apc2 expression and subcellular localization. During nuclear division cycles 10-13, which take place without cytokinesis in the peripheral cytoplasm of the embryo, Apc2 shows dynamic changes in subcellular localization, coincident with changes in actin. Sequential changes in MT organization as nuclei proceed through mitosis direct the reorganization of the cortical actin cytoskeleton. Before nuclei migrate to the periphery, actin is found at the cortex in a random reticulum. When nuclei reach the periphery, actin condensations appear in interphase and prophase above each nucleus, forming an actin bud that overlays a cytoplasmic bud. This separates the mitotic apparatus of one nucleus from that of its neighbor. As division proceeds to metaphase, actin redistributes from the crown of the bud to its lateral cortex, forming an oblong ring around each spindle. During anaphase, actin redistributes into discs above each newly formed nucleus. Centrosomes and their associated MTs direct the changes in actin distribution, although the mechanism responsible for this interaction is not known (McCartney, 1999).

In cycle 10-13 embryos, Apc2 colocalizes with actin at all stages of mitosis. The Apc2/actin colocalization is most prominent in the microvillar projections at the surface of the bud in interphase and prophase. At metaphase and anaphase, Apc2 and actin condensations are observed at the lateral cortex of the bud; Apc2 staining is somewhat less intense here, relative to actin. Toward the base of the bud, condensations of actin and Apc2 are also found in the region of the centrosome and asters. These Apc2 condensations occur within 0.3-0.5 µm of the surface of the embryo, and thus are most prominent above the spindle apparatus; kinetochore MTs are not in uniform focus until ~1.25 µm from the surface of the embryo. The location of these Apc2/actin condensations above the plane of the spindle places them in a position to interact with the astral MTs as they reach toward the cortex. During later nuclear cycles when pseudocleavage furrows are present, more defined dots of actin and Apc2 staining are sometimes observed in the region of the centrosomes. In a wild-type stock infected with the bacterial endosymbiont Wolbachia (visible as small propidium iodide-positive bodies), an additional Apc2 localization is seen. Wolbachia associate with astral MTs in Drosophila and thereby disperse into newly formed cells. In infected embryos, Apc2 localizes with the actin cytoskeleton as in uninfected stocks, and also associates with bacteria at the asters. Another astral MT-associated protein, the kinesin-like protein KLP67A is known to associate with bacteria. EM studies have shown that the bacteria are encapsulated within a cytoplasmic vacuole attached to astral MTs via an electron-dense bridge, possibly composed of cellular MT-associated proteins. Apc2's localization to the aster region of noninfected embryos and its association with bacteria suggest that Apc2 may contribute to the binding of the vacuole to the asters (McCartney, 1999).

After cellularization, Apc2 is still enriched in the region of MTs. Increased levels of cytoplasmic Apc2 are observed in mitotic domains (groups of cells undergoing synchronous mitosis). Here, cytoplasmic condensations of Apc2 are observed in the region of the spindle in metaphase and anaphase, but are absent in prophase or telophase; serial sections reveal that these cytoplasmic condensations are most prominent within 2-4 µm of the cell apex. In mitotic domains of a Wolbachia-infected strain, punctate condensations of Apc2 are observed near the spindle poles, presumably astrally associated bacteria, consistent with Apc2 localization to bacteria associated with preblastoderm asters (McCartney, 1999).

Apc2 is also expressed in dividing cells of the larval brain. The optic lobes contain two proliferative regions, the inner and outer proliferative zones. Apc2 is highly expressed in dividing cells of the proliferative zones and in their immediate progeny, but not in differentiated neurons. In contrast, Arm is not enriched in the proliferative zones but is enriched in axons. In the ventral nerve cord, Arm is found in axons, whereas Apc2 is found in midline glial cells. In contrast, Drosophila APC-like localizes to axons, at least in embryos (Hayashi, 1997). However, in larval neuroblasts (neural stem cells) Apc2 and Arm share a striking asymmetric distribution. Neuroblasts divide asymmetrically to produce a large neuroblast and a smaller ganglion mother cell, which will divide symmetrically to produce two neurons. The asymmetric division requires specific orientation of the mitotic spindle. Inscuteable (Insc), localized in a crescent opposite the future daughter cell during prophase and metaphase, is required for both spindle orientation and localization of the neural determinants Prospero and Numb. In larval neuroblasts, both Apc2 and Arm colocalize to a cortical crescent next to the future daughter cell; this crescent also includes the neural determinant Prospero. In contrast to other asymmetric neuroblast components, the Apc2 and Arm crescents are present even at interphase. In some neuroblasts, cortical actin also accumulates in a crescent with Apc2, whereas in others this association is less apparent. To examine the relationship between Apc2 and the spindle, neuroblasts were triple labelled with antibodies against phosphohistone, ß-tubulin, and Apc2. One pole of the spindle apparatus colocalizes with the Apc2 crescent; Apc2 is enriched at this point relative to the rest of the crescent. At this stage of the cell cycle, low levels of Apc2 were also observed at the opposite cortex; this position often coincided with the other spindle pole. Whereas cortical Apc2 associates with spindle poles, neuroblasts do not have cytoplasmic condensations of Apc2 around the central spindle as are observed in epidermal cells. Apc2 is also asymmetrically localized in embryonic neuroblasts (McCartney, 1999).

In nondividing cells, Apc2 also associates with the cell cortex, and colocalizes with actin. In the embryo, Apc2 is most strongly expressed in the epidermis and other epithelial cells. In the epidermis, Apc2 is enriched at the cell cortex and is also found throughout the cytoplasm in a punctate distribution. At the cortex, Apc2 appears as numerous punctate condensations of protein that are most prevalent at the apical end of the lateral cell surface but are also found more basally. The most intense staining of Apc2 appears at points of contact between multiple epidermal cells. Apc2 condensations often colocalize with condensations of actin and phosphotyrosine, although actin and phosphotyrosine associate with the cortex more continuously. In fully polarized epithelial cells, like the embryonic hindgut or the larval imaginal discs, Apc2 is enriched in adherens junctions, where it colocalizes with Arm; Apc2 also accumulates on the apical plasma membrane. The intracellular distribution of Apc2, in contrast to that of Arm, is not modulated in a segmental fashion. A strikingly different localization of Apc2 occurs in the epidermis after stage 15. Apc2 becomes organized into very large apical structures in segmentally repeated subsets of ventral epidermal cells, just before the stage at which these cells initiate denticle formation. The Apc2 structures occur specifically in the anterior epidermal cells of each segment and colocalize with similar actin structures, which likely represent larval denticle precursors (McCartney, 1999).

Although Apc2 colocalizes with actin in many tissues, it does not colocalize with actin in all contexts. For example, during cellularization, actin is prominent at the cellularization front, whereas Apc2 is enriched at the apical cortex. In addition at the cortex of epidermal cells actin is present at the membrane in a continuous fashion, whereas Apc2 is restricted to regions of most intense actin staining. Finally, Apc2 is not found with actin in cytokinesis furrows. Thus, although Apc2 associates with the actin cytoskeleton, the context-dependent nature of this association suggests that this association is regulated (McCartney, 1999).

Thus Apc2 colocalizes with actin in many but not all cell types, suggesting a regulated interaction. The association between the actin cytoskeleton and Apc2 may occur via Arm and beta-catenin, although in some places where Apc2 and actin colocalize, there is little or no detectable Arm. The colocalization of Apc2 and actin is intriguing given the effects of Wnt/Fz signaling on planar polarity in Drosophila. In the wing, the best studied example, Frizzled signaling triggers asymmetric polymerization of actin, leading to development of an actin-based wing hair in the distal vertex of each hexagonal wing cell. The colocalization of actin and Apc2 during the onset of denticle formation is particularly striking in this context, because the process of denticle formation is very similar to that of wing hair formation. Similarities are found in the nature of the structure, its strict orientation in the plane of the tissue, and in its cell biological and genetic bases. This raises the possibility that Wg/Wnt signaling directly affects the actin cytoskeleton and thus tissue polarity, using Apc2 as an effector (McCartney, 1999 and references therein).

Although Apc2 does not contain the basic region thought to mediate MT association of hAPC, these data are consistent with the possibility that Apc2, like hAPC, may associate with MTs under certain circumstances. The data for a microtubule association of Apc2 are less robust than those suggesting association with actin. Whereas Apc2 does not prominently localize to most microtubule-based structures (nor does hAPC, unless overexpressed), Apc2 localizes to several places consistent with a role in anchoring microtubules. In preblastoderm embryos, when actin is essential for tethering the spindle to the membrane, Apc2 colocalizes with cortical actin and subcortical actin puncta. Subcortical Apc2 is concentrated just above the spindle, placing it in a position to interact with astral MTs as they reach toward the cortex. Both Apc2 and actin also localize to a dot-like structure, which may be the centrosome. In postblastoderm embryos, Apc2 is subtly enriched in the vicinity of the spindle (McCartney, 1999).

The asymmetric localization of Apc2 in dividing neuroblasts is also consistent with a possible role for Apc2 in linking the spindle to the cortex. During neuroblast mitosis, the spindle is specifically oriented. Insc, which localizes to a crescent opposite the future daughter cell from late interphase through metaphase, coordinates the neuroblast asymmetric cell division. Other proteins are likely to act in this process; e.g., Bazooka acts upstream of Insc (Kuchinke, 1998). In C. elegans and yeast, actin or actin-associated proteins localize asymmetrically in cells in which spindle orientations are specified, suggesting a role for actin in this process. The position of actin in a crescent next to the future daughter cell in a subset of the neuroblasts suggests that actin may affect spindle orientation in Drosophila neuroblasts as well. Apc2 may also play a role in this process. Within the crescent, Apc2 localization is strongest in the region of the spindle pole. During later stages of mitosis, although Apc2 remains enriched in a crescent next to the future daughter, Apc2 also localizes to the cortex on the opposite side of the cell, often in the region of the other spindle pole. In contrast to other asymmetrically localized components of the neuroblast, Apc2 localizes to a crescent during all stages of the cell cycle; in fact, the Apc2 crescent is most apparent during interphase and prophase. Apc2 and actin can also act as polarity markers for other proteins; actin is required for the asymmetric localization of Insc, Prospero, and Staufen. Nevertheless, the Apc2/MT connection remains speculative. In the future, tests will be made to see whether Apc2 associates with MTs, whether Apc2 can affect spindle orientation, and whether Apc2 and Arm function in the neuroblast asymmetric cell division (McCartney, 1999).

This issue is raised in light of the influence of Wnt signaling on mitotic spindle orientation in both C. elegans embryos (Thorpe, 1997) and in Drosophila sensory cells (Gho, 1998 ). In C. elegans, Wnt signaling controls spindle orientation independent of transcription (Schlesinger. 1999 ), suggesting that the Wnt pathway directly targets the cytoskeleton. Since Apc2 regulates Wg/Wnt signal transduction and appears to have connections to the cytoskeleton, it is a candidate for a direct effector of this process. RNA interference studies of C. elegans relatives of Arm (WRM-1) and APC (APR-1) do not reveal defects in spindle orientation (Schlesinger, 1999), suggesting the existence of a branch to the cytoskeleton upstream of APC and Arm. However, because RNA interference may not completely remove gene function, and because of the divergence between APR-1 and the APC family, the involvement of Arm and Apc2 in the pathway remains plausible. These data are also intriguing in light of studies of the hAPC binding protein EB1 (Su, 1995), which colocalizes with the spindle, centrosome, and asters (Berrueta, 1998 ; Morrison, 1998). Budding and fission yeast EB1 homologs are required for spindle assembly and stability (Beinhauer, 1997; Schwartz, 1997; Muhua, 1998). However, it is worth noting that Apc2 appears to lack the binding domain for EB1 identified in hAPC (McCartney, 1999).

cDNA clone length - 4044 bases

Bases in 3' UTR - 645

Amino Acids - 1067

Structural Domains

All APC family members share an NH2-terminal conserved domain, 6 Arm repeats, and a series of ßcat binding (15 and 20 amino acid repeats) and Axin binding (SAMP repeats) motifs. Drosophila Apc2 is shorter at its NH2 and COOH termini than other APCs. Apc2 lacks the COOH-terminal basic region (the putative MT binding site) found in hAPC and Drosophila Apc-like (Hayashi, 1997), as well as the hAPC region containing binding sites for Discs-large (DLG) and EB1. Substantial alternative splicing is unlikely, as there are only two small introns in coding sequences (63 and 197 nucleotides). Drosophila Apc2 is most similar to other APC family members in the Arm repeats, where it most closely resembles Apc-like; hAPC2 is more similar to hAPC (Apc2 is 81% identical to Drosophila Apc-like and 57% identical to hAPC). Thus, there is no correspondence between individual human and fly proteins, even though both phyla show neural-enriched isoforms (Drosophila Apc-like and hAPC2), suggesting independent gene duplications. All APCs have six Arm repeats; a putative seventh Arm repeat is much more divergent and is not identifiable in Drosophila Apc2. The NH2-terminal conserved region (61% identity to Drosophila Apc-like vs. 44% identity to hAPC) distantly resembles the Arm repeat consensus and may form one or two degenerate Arm repeats. APC family members also share similarity on the C-terminal side of the Arm repeats. hAPC has two sets of repeated ßcatenin binding sites, the 15 and the 20 amino acid repeats; hAPC2 lacks the 15 amino acid repeats). Apc2 shares two of the three 15 amino acid repeats of Apc-like. Drosophila Apc-like and Apc2 each have five 20 amino acid repeats, among which are interspersed SAMP repeats. Apc-like has four SAMP repeats, whereas Apc2 has two. Apc2 ends 40 amino acids after the last SAMP repeat (McCartney, 1999).

For information about APC homologs see Apc-like.

date revised: 12 February 2000

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

Please e-mail comments/corrections to brodyt@codon.nih.gov