Apc-like
The 20-amino acid repeat region in the central part of human APC down-regulates cytoplasmic beta-catenin levels in a colon carcinoma cell line (SW480), which lacks endogenous wild-type APC (Munemitsu, 1995). To test whether Drosophila Apc might have a similar function, an expression construct with a partial Apc cDNA containing the beta-catenin binding sites was generated and transiently transfected into the colon carcinoma cell line SW480. Forty-eight hours after transfection, the protein level and
cellular localization of beta-catenin were measured by Western blot analysis and immunostaining, respectively. Apc significantly reduces the concentration of beta-catenin protein, with an efficiency ~60% that of human APC. Immunostaining shows that intracellular cytoplasmic beta-catenin decreases significantly, after introduction of the Apc fragment, but beta-catenin localizing at the plasma membrane is not significantly altered. These results demonstrate that Apc can down-regulate intracellular beta-catenin levels similar to that of human APC, suggesting that Apc is also a functional homolog of the human APC (Hayashi, 1997).
The existence of homologous beta-catenin binding sites in Drosophila Apc raises a question whether Apc interacts with the Drosophila homolog of beta-catenin, the Armadillo protein. To test this possibility an in vitro binding assay was carried out using a bacterially expressed Apc fusion protein containing beta-catenin binding sites and Arm protein translated in vitro. Arm binds to the Apc fragment containing the beta-catenin binding sites, but not to a control composed of a beta galactosidase fusion protein, suggesting that binding between Arm and the Apc fragment is specific. Altogether these results indicate that the beta-catenin binding sites in Apc can substitute for human APC in the down-regulation of beta-catenin, and that the same region interacts directly with Arm (Hayashi, 1997).
Drosophila Armadillo and its vertebrate homolog beta-catenin
are key effectors of Wingless/Wnt signaling. In the current
model, Wingless/Wnt signal stabilizes Armadillo/beta-catenin,
that then accumulates in nuclei and binds TCF/LEF
family proteins, forming bipartite transcription factors
which activate transcription of Wingless/Wnt responsive
genes. This model was recently challenged. Overexpression
in Xenopus of membrane-tethered beta-catenin or its paralog
plakoglobin activates Wnt signaling, suggesting that
nuclear localization of Armadillo/beta-catenin is not essential
for signaling. Tethered plakoglobin or beta-catenin might
signal on their own or might act indirectly by elevating
levels of endogenous beta-catenin. These hypotheses
were tested in Drosophila by removing endogenous Armadillo. A series of mutant Armadillo proteins with
altered intracellular localizations were generated, and these were expressed in
wild-type and armadillo mutant backgrounds. Membrane-tethered Armadillo cannot signal on its
own; however it can function in adherens junctions. Mutant forms of Armadillo were generated carrying either
heterologous nuclear localization or nuclear export signals.
Although these signals alter the subcellular localization of
Arm when overexpressed in Xenopus, in Drosophila they
have little effect on localization and only subtle effects on
signaling. This supports a model in which Armadilloís
nuclear localization is key for signaling, but in which
Armadillo intracellular localization is controlled by the
availability and affinity of its binding partners (Cox, 1999).
Data in vivo suggest that among
Armís known partners, cadherins have the highest affinity, with
APC and dTCF (Pangolin) having lower and lowest affinities,
respectively. Thus, in embryos with reduced levels of Arm, the
remaining Arm is exclusively associated with cadherins, as
assayed by immunolocalization and by function. About 70% of cellular Arm is cadherin-associated. When cadherin binding sites are saturated, excess Arm
binds to APC/Axin, leading to its destruction and thus
preventing accumulation of free Arm. While APC levels, at
least in mammalian cells, are low, relative to the total
pool of beta catenin, Arm bound to APC is rapidly targeted for
destruction, thus opening the way for the binding of additional
Arm. Normally the destruction machinery can not only dispose
of all non-junctional Arm, but its resources will not even be fully employed, since
Arm synthesis can be increased several-fold without biological
consequences. However, when the destruction machinery is inactivated
either by Wg signal or mutation, Arm is synthesized
but not destroyed, and thus levels of Arm rise. APC can bind
Arm but in all probability, the APC is rapidly saturated, allowing accumulation of
sufficient Arm to allow dTCF to effectively compete for
binding. DE-cadherin, dAPC, dTCF and any other possible
unknown partners together account for virtually all the Arm in
a normal embryo; little if any free Arm is present.
This model helps explain the differences in localization of
the Armadillo attached to a nuclear localization sequence (Arm-NLS) and Armadillo attached to a nuclear export signal (Arm-NES) in flies and frogs. In Xenopus,
added NLS or NES signals dramatically altered Armís
intracellular distribution as expected, while in Drosophila the
distribution of wild type Armadillo, Arm-NLS and Arm-NES are
indistinguishable. It is proposed that this reflects differences in
the level of expression. In flies, mutant Arm accumulates at
near wild-type levels, so its binding partners can accommodate
the additional protein. Arm bound to cadherin at the plasma
membrane is unavailable for nuclear import; likewise Arm in
a complex with dTCF is not available for export. Thus Arm-NLS
and Arm-NES localization is primarily determined by
their binding partners, resulting in a near normal localization.
In contrast, Arm-NLS and Arm-NES expression levels in
Xenopus likely exceed those of either endogenous beta-catenin or its
binding partners. Free Arm is thus accessible to the nuclear
import and export machinery, allowing alteration of its localization. Given this, is nuclear localization of Arm a regulated step in
Wg signaling in normal cells? The fact that a subset of cells
accumulate cytoplasmic but not nuclear Arm suggests that
nuclear import may be regulated. In the simplest situation,
addition of an NLS ought to promote Arm nuclear
accumulation and trigger signaling, while addition of an NES
should antagonize signaling. However, heterologous targeting
signals have only subtle effects on signaling. Arm-NES signals in the same fashion as does
Arm-WT, while only a subset of the Arm-NLS lines are
activated for signaling. In the case of Arm-NLS:
in cells in which the destruction machinery is on, no free Arm
is available for nuclear import or export. In cells with
intermediate levels of Wg signaling, the destruction machinery
may be slowed, allowing accumulation of cytoplasmic Arm in
complex with APC, but not to sufficient levels to saturate APC
and allow nuclear import. Only when signaling is fully
activated would sufficient free Arm accumulate for nuclear
import. Addition of an NLS would thus only alter the balance
in cells near the signaling threshold. Further, if nuclear Arm is
bound to dTCF, it may be inaccessible to the nuclear export
machinery. The mechanisms by which Arm/beta-catenin enters nuclei
remain unclear; dTCF-dependent and independent pathways
may exist. The
recent observation that beta Catenin may mediate its own nuclear
transport, independent of importins, further complicates the
issue. Additional levels of regulation may occur,
beyond the simple regulation of Arm/beta Catenin stability (Cox, 1999 and references).
Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the
amount of beta-catenin/Armadillo, which in turn activates gene transcription. The Drosophila
protein Axin is shown to interact with Armadillo and Drosophila APC. D-Axin was identified in a yeast two-hybrid screen for proteins that bind the Armadillo repeat domain of Arm. d-axin codes for a protein of 743 amino acids. A region near its N-terminus shows similarity to the regulator of G protein signaling (RGS domain), whereas its C-terminus contains a region homologous to a conserved sequence near the N-terminus of Dishevelled. Thus D-Axin has a domain structure very similar to that of proteins of the mammalian Axin family. Unlike mammalian Axin family members, which bind to GSK-3beta, D-Axin does not bind to the homologous protein Shaggy/Zeste white3. d-axin is expressed maternally and is ubiquitously expressed during development. Embryos devoid of maternal and zygotic d-axin have completely naked ventral cuticle, lacking all denticles (Hamada, 1999).
During wing disc development, Wg signaling is induced along the dorsoventral compartment boundary in the wing imaginal disc. Arm accumulates in the cytoplasm, associates with its partner Pangolin, and activates expression of target genes such as Distal-less. Mutation of d-axin results in the
accumulation of cytoplasmic Armadillo and results in elevation of Distal-less.
Ectopic expression of d-axin inhibits Wingless signaling. Hence, D-Axin negatively regulates Wingless
signaling by down-regulating the level of Armadillo. It is speculated that the Axin family of proteins functions to establish a threshold to prevent premature signaling events caused by Wg/Wnt and to restrict areas that are capable of responding to Wg/Wnt. These results establish the importance of the Axin
family of proteins in Wnt/Wingless signaling in Drosophila (Hamada, 1999).
Asymmetric division is a fundamental mechanism for generating cellular diversity. In the central nervous system of Drosophila, neural progenitor cells called neuroblasts undergo asymmetric division along the apical-basal cellular axis. Neuroblasts originate from neuroepithelial cells, which are polarized along the apical-basal axis and divide symmetrically along the planar axis. The asymmetry of neuroblasts 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. This study shows that by disrupting adherens junctions the symmetric epithelial division can be converted into asymmetric division. It was further confirmed that the adenomatous polyposis coli (APC) tumour suppressor protein is recruited to adherens junctions, and demonstrated that 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. Previous studies identified 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).
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 were used expressing Pon and tau proteins fused with green fluorescent
protein (GFP). 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 orientated 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).
The uncoupling of spindle orientation with asymmetric protein localization
in epithelial cells might be due to either a lack of such a coupling mechanism
or the dominance of the coupling mechanism by yet another spindle-positioning
mechanism. One of the hallmarks of epithelial cells is the adherens junction,
which is composed of the cadherin-catenin complex and other associated
proteins, is connected to the cytoskeleton, and is thought to be important
in maintaining the planar organization of the epithelial monolayer. Therefore
the possible role of adherens junction in orientating epithelial division was tested.
The formation of adherens junction requires genes such as shotgun,
crumbs (crb) and stardust. RNA interference (RNAi) was used to disrupt Crb function and analysed the effect on epithelial division (Lu, 2001).
Double-stranded (ds) crb RNA was injected into transgenic embryos
expressing Pon-GFP and tau-GFP. In about 70%
of crb(RNAi) embryos, the organization of the ectodermal
epithelium was 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 revealed 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 indicated 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. Therefore the effect of overexpressing Crb-intra on epithelial division was examined. As observed in crb(RNAi) embryos, epithelial cells overexpressing Crb-intra showed 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. Nextthe function of Baz in epithelial division was investigated. 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 orientated in random directions. After cytokinesis,
two equally sized daughter cells were produced and Pon-GFP was equally
distributed between them (Lu, 2001).
Daughter cell size asymmetry in neuroblast division is largely unaffected in baz(RNAi) embryos. It was also observed that in crb(RNAi) epithelial cells Baz can still be localized into a crescent but the crescent is mispositioned
and that 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 looked 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 (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 homologue 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 the 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 orientate the mitotic spindle,
RNAi was performed on a closely related fly homologue of EB1 (dEB1
). In dEB1(RNAi) embryos, the epithelial divisions were also asymmetric,
producing two unequally sized daughter cells, with Pon–GFP segregated
to the smaller cell. It was observed that 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 homologues in the fly genome.
It has been noted that E-APC lacks the carboxy-terminal domain that is required
for interaction with EB1, and no direct interaction was observed between E-APC and EB1 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 orientate 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 orientate 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 insc mutant, suggests that Insc functions
by strengthening the apical-basal polarity instead of weakening the
planar polarity through changing the behaviour 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 orientating division
axis may have its precedents 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 highlights the importance of tumour suppressors in regulating not only cell growth but also polarity and asymmetric division (Lu, 2001).
Shortstop (Shot) is a Drosophila Plakin family member containing both Actin binding and microtubule binding domains. In Drosophila, it is required for a wide range of processes, including axon extension, dendrite formation, axonal terminal arborization at the neuromuscular junction, tendon cell development, and adhesion of wing epithelium. To address how Shot exerts its activity at the molecular level, the molecular interactions of Shot with candidate proteins was investigated in mature larval tendon cells. Shot colocalizes with the complex between EB1 and APC1 and with a compact microtubule array extending between the muscle-tendon junction and the cuticle. It is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. Shot forms a protein complex with EB1 via its C-terminal EF-hands and GAS2-containing domains. In tendon cells with reduced Shot activity, EB1/APC1 dissociate from the muscle-tendon junction, and the microtubule array elongates. The resulting tendon cell, although associated with the muscle and the cuticle ends, loses its stress resistance and elongates. These results suggest that Shot mediates tendon stress resistance by the organization of a compact microtubule network at the muscle-tendon junction. This is achieved by Shot association with the cytoplasmic faces of the basal hemiadherens junction and with the EB1/APC1 complex (Subramanian, 2003).
Tendon cells undergo maturation during larval stages. In third instar larvae, different tendon cells acquire distinct shapes according to their orientation and the type of muscles to which they are connected. Initially, the localization of Shot was characterized relative to MT and F-actin organization in mature tendon cells of flat, opened third instar larvae. Shot and Tubulin staining overlapped within the entire cell. A unique domain at the focal plane of the muscle-tendon junction exhibits a compact MT array, which overlapped Shot staining. This domain was not detected when the optical section was taken 0.5 microm more internal to the junction focal plane. An optical cross-section perpendicular to the muscle-tendon junction site shows that the MT-Shot array extends from the muscle-tendon junction to the cuticle. Moreover, the MT array is oriented in the same direction as the microfilaments of the muscle cells, as shown by EM analysis. Thus, Shot and MTs are colocalized within a unique subcellular domain in the tendon cell that connects the muscle-tendon junction and the cuticle (Subramanian, 2003).
These immunofluorescent localization studies suggest a distinct abundance of MTs and MFs at both sides of the muscle-tendon junction. While MFs are highly enriched at the muscle side, MTs are detected mainly at the tendon side. To address whether this organization reflects differences in the distribution of additional junction-associated proteins, the larval flat preps were stained for PSß-integrin, Paxillin, and P-tyrosine, and their relative distribution at the focal plane of the muscle-tendon junction was analyzed. In all preparations, Shot marks the outlines of the tendon cell. About half of the PSß-integrin staining overlaps Shot staining, whereas the other half is located at the muscle membrane. Paxillin is more abundant on the muscle side. Interestingly, staining for P-tyrosine, which marks the extent of tyrosine-phosphorylated proteins (including Paxillin, Src, and others) is restricted to the tendon side of the junction and is tightly associated with the plasma membrane. Thus, although PSß-integrin distribution appears to be equal at both sides of the muscle-tendon hemiadherens junction, the molecular composition on both sides of these junctions appears to be distinct, as demonstrated for EB1, APC1, Paxillin, and the extent of P-tyrosine reactivity. It is tempting to speculate that these unequal protein distributions might relate to the enrichment of MTs and Shot on the tendon cell side (Subramanian, 2003).
EB1 is an evolutionarily conserved protein that binds the plus ends of growing MTs. It was first identified as a binding partner for the adenomatous polyposis coli tumor suppressor, APC. The EB1/APC complex is involved in regulation of MT polymerization and MT association with distinct subcellular domains. For example, in yeast, the EB1 homolog (BIM1) has been shown to modulate MT dynamics and link MTs to the cortex. Drosophila EB1 (Rogers, 2002) is important for the proper assembly, dynamics, and positioning of the mitotic spindle. Its association with APC2 in apical cell-cell adherens junctions is suggested to be essential for parallel spindle orientation (Lu, 2002) and for neuroblast asymmetric cell division (Subramanian, 2003).
Biochemical data suggest that the association of the C-terminal EF-GAS2 domain with EB1 is MT independent. A direct physical interaction between the C-terminal EF-GAS2 domain and alpha-tubulin had been suggested by a yeast two-hybrid screen and by the ability to precipitate purified Tubulin by the GAR and GSR domains (both included within the C terminus of the mammalian ACF7). These domains lack the EF-hands motif. EB1 is detected in association with the entire Shot C-terminal domain containing EF-hands. At this stage, no additional information is available regarding the site responsible for EB1 association (Subramanian, 2003).
The data suggest that Shot association with the basal muscle-tendon junction is EB1 independent (since it is detected even in the absence of EB1/APC1); hence, it is suggested that EB1 and APC1 become associated with the muscle-tendon basal hemiadherens junction in postmitotic tendon cells following their association with Shot. The assembly of the MT-rich domain may be induced by either their direct association with Shot or with EB1/APC1, or with both. Interestingly, EB1, APC1, and Shot are not observed at the cell-cell adherens junctions formed between the tendon cell and its neighboring ectodermal cells (Subramanian, 2003).
There is no direct evidence for the polarity of MTs within the compact Shot/MT-rich domain, since no differential EB1 localization was detected in this domain. Other studies suggest that MTs in the entire epidermis are arranged at a polar orientation in which their plus ends face the basal pole and their minus ends face the cuticle. Similarly, in the pupal wing, MTs have been shown to be arranged with their plus ends facing the basal hemiadherens junctions. Thus, it is likely that in the tendon cells (which are part of the epidermal layer), MTs are similarly arranged, i.e., with their plus ends facing the basal hemiadherens junction (Subramanian, 2003).
The experiments show that reduced Shot activity leads to a significant tendon cell elongation, occurring presumably following muscle contractions. What is the mechanism allowing the MTs to elongate in the mutant tendon cell? An interesting possibility is that the MTs are connected to the muscle-tendon junction through their plus ends via their association with EB1 and Shot, and that this arrangement arrests further MT polymerization and maintains the MTs in a polarized arrangement. Following dissociation of the Shot/EB1/APC1 complex and the reduction of Shot activity, MTs undergo further polymerization and extension, leading to the significant elongation of the tendon cell. The newly formed MTs are not well connected to the cell cortex, thus leading to cell breakdown upon further muscle contraction. Support for the involvement of Shot in mediating MT-polarized organization emerges from recent analysis of Shot function in mushroom body neurons of the Drosophila adult brain. Distinct Nod-ßgal reactivity suggests that MT polarity within the axons is distinct from that of dendrites in wild-type mushroom body neurons. In neurons mutant for shot, the polarity of MTs in the axons is reversed and resembles that of dendrites (Subramanian, 2003).
Shot may perform a similar function in the organization of a compact and polarized array of MTs in the adult wing epithelium, as well as within the ligament cells of embryonic sensory chordotonal organs (Subramanian, 2003).
Studies with the different Shot domains show that the Actin binding domain, but not the Plakin domain, is capable of driving specific localization of both domains to the F-actin layer at the muscle-tendon junction. The thin Actin layer at the muscle-tendon junction may therefore be essential for the recruitment of Shot into the cytoplasmic faces of the hemiadherens domains. The C terminus containing the EF-hands and GAS2 domains is also capable of localizing at the muscle-tendon junction domain. This localization may be attributed to its association with endogenous EB1, as well as with MTs that are already arranged in the larval tendon cells, or with endogenous Shot. The Plakin domain on its own did not show specific subcellular localization, suggesting that it does not bind to proteins that are highly localized in the tendon cell. Alternatively, proteins that may form a complex with this domain may be engaged in existing protein complexes and therefore are not accessible to the exogenous Plakin protein. None of the Shot structural domains show a dominant-negative effect when overexpressed in tendon cells or in wing imaginal discs; this finding suggests that the proteins to which they bind are not present in limited amounts (Subramanian, 2003).
Interestingly, in larvae tendon cells, both the Actin-Plakin domain of Shot and the Shot C-terminal EF-GAS2 domain exhibit similar distribution. When transfected into Schneider cells, each domain shows a distinct subcellular localization. The Actin-Plakin-GFP was detected at the leading edge and in most cases did not overlap EB1, while the EF-GAS2 domain overlapped EB1 and decorated MTs. Similar studies with Drosophila Shot and ACF-7, the mammalian Shot ortholog, show that the Actin binding domain and the EF-GAS2 domain are associated with Actin MFs and with MTs, respectively, in transfected cells. However, the M1 domain in ACF-7 (similar to the Plakin domain) has been shown to be associated with MTs in the transfected cells, while the Shot-Plakin domain does not exhibit significant association with MTs. These differences may reflect differential distribution of yet uncharacterized ACF-7 binding proteins within the mammalian cells (Subramanian, 2003).
What could be the connection between Shot activity and reduced F-actin content? Recent studies suggest that MT disassembly activates Rho by the release of GEFs that are specifically associated with and inhibited by MTs. In tendon cells, no unique association was detected of GEF (Pebble) or Rho with the MT-rich domain. Therefore, the relevance of these factors is not clear. Recently, it was shown that in Drosophila embryonic tracheal cells, activated RhoA mimicks the Shot loss-of-function phenotype; this finding suggests a similar inverse correlation between Actin polymerization by RhoA and the loss of shot function. Thus, the activity of Shot in organizing MTs to special subcellular sites via its association with EB1/APC1, and the inhibition of F-actin in these sites, may be relevant to other tissues in which Shot plays an essential role (Subramanian, 2003 and references therein).
The MT network is essential for a wide array of cellular functions. Shot, a multidomain Plakin family member, is essential for arranging a compact network of MTs in tendon cells. This is achieved by the association of Shot with the cytoplasmic faces of the muscle-tendon junction and presumably by the subsequent recruitment of the EB1/APC1 complex to these sites. In tendon cells, this unique MT organization is essential to resist muscle contraction (Subramanian, 2003).
Wnt signaling causes changes in gene transcription that are pivotal for normal and malignant development. A key effector of the canonical Wnt pathway is ß-catenin, or Drosophila Armadillo. In the absence of Wnt ligand, ß-catenin is phosphorylated by the Axin complex, which earmarks it for rapid degradation by the ubiquitin system. Axin acts as a scaffold in this complex, to assemble ß-catenin substrate and kinases (casein kinase I [CKI] and glycogen synthase kinase 3ß [GSK3]). The Adenomatous polyposis coli (APC) tumor suppressor also binds to the Axin complex, thereby promoting the degradation of ß-catenin. In Wnt signaling, this complex is inhibited; as a consequence, ß-catenin accumulates and binds to TCF proteins to stimulate the transcription of Wnt target genes. Wnt-induced inhibition of the Axin complex depends on Dishevelled (Dsh), a cytoplasmic protein that can bind to Axin, but the mechanism of this inhibition is not understood. This study shows that Wingless signaling causes a striking relocation of Drosophila Axin from the cytoplasm to the plasma membrane. This relocation depends on Dsh. It may permit the subsequent inactivation of the Axin complex by Wingless signaling (Cliffe, 2003).
The subcellular distribution of Axin-GFP was studied at late embryonic stages, i.e., in epidermal cells that are no longer stimulated by Wingless ('-Wg cells'). In these -Wg cells, conspicuous green dots are seen throughout the cytoplasm. Similar dots have been observed in vertebrate cells expressing tagged Axin; these dots are associated with vesicles. Interestingly, most of the Axin-GFP dots coincide with dots of E-APC staining. E-APC is the main APC protein expressed in the Drosophila embryonic epidermis; many of the E-APC dots accumulate in apicolateral regions along the plasma membrane. This can be seen in young embryos that have just begun to express Axin-GFP, but, in older embryos in which Axin-GFP has accumulated to high levels, E-APC is largely delocalized from the plasma membrane and is recruited into the cytoplasmic Axin-GFP dots, presumably by direct binding. It is likely that these dots represent the Axin destruction complex. Thus, in -Wg cells, this complex appears to be located predominantly in the cytoplasm, where it actively promotes the degradation of Armadillo (Cliffe, 2003).
Next, Axin-GFP was expressed in embryos without APC function, i.e., in embryos that express a mutant E-APC protein (N175K) and lack the second APC protein (dAPC) that acts redundantly with E-APC. These APC double mutants show very few Axin-GFP dots, and the green fluorescence appears mostly diffuse or grainy. Indeed, the staining of the mutant E-APC N175K protein itself appears grainy and is much less dotty than the staining of wild-type E-APC. The few remaining dots colocalize with Axin-GFP dots. Thus, E-APC is required for the formation of the Axin-GFP dots, indicating that the N175K mutant cannot promote Axin complex formation (Cliffe, 2003).
The N175K mutant bears a missense mutation in a surface residue of its Armadillo repeat domain, and its loss of function is due to its inability to associate with the plasma membrane. This results in naked cuticles, the hallmark of ubiquitous Wingless activation. Intriguingly, the N175K mutant is a fully stable protein that retains its Axin binding site. It binds to Axin as efficiently as wild-type E-APC in vitro. Thus, the inability of the N175K mutant protein to associate with the plasma membrane appears to be the sole reason for its failure to promote Axin complex assembly (Cliffe, 2003).
Expression of Axin-GFP in the APC double mutant embryos restores their mutant phenotype partially toward normal. Thus, Axin-GFP is less active in these mutants; this finding confirms that Axin function depends on APC. This dependence is strong but not absolute, and it is likely to reflect the role of APC in promoting Axin complex assembly. Moreover, overexpression of Axin-GFP compensates to some extent for the loss of APC. This parallels the results in APC mutant cancer cells in which overexpressed Axin proteins can bypass the function of APC; this finding suggested that APC has a regulatory role with regard to Axin. This regulatory role could be to target Axin to a specific subcellular location: one would expect APC-mediated targeting to be less critical at elevated levels of Axin expression (Cliffe, 2003).
Axin-GFP expression was examined next in the epidermis of 3- to 6-hr-old embryos; at this stage, stripes of +Wg cells alternate with stripes of -Wg cells. As in older embryos, conspicuous dots of Axin-GFP are scattered throughout the cytoplasm of -Wg cells. Strikingly, in +Wg cells, these dots are associated almost exclusively with apicolateral regions of the plasma membrane. This is observed neither in the epidermis of older embryos that lack Wingless expression nor in wingless mutants. Conversely, coexpression of Wingless with Axin-GFP causes a relocation of virtually all Axin-GFP dots to the plasma membrane and also restores the membrane-associated staining of E-APC in older embryos. Thus, Wingless signaling is both necessary and sufficient for relocation of the Axin-GFP dots to the plasma membrane. Notably, a FRET signal between Axin and LRP-5 has been observed in Wnt-stimulated mammalian cells; this result suggested a Wnt-induced recruitment of Axin to the plasma membrane. This result is the first direct demonstration that Wnt signaling triggers a relocation of Axin to the plasma membrane (Cliffe, 2003).
Axin-GFP levels were examined by Western blot analysis to confirm that Axin-GFP is expressed at moderate levels as an intact full-length fusion protein. Coexpression with Wingless does not change these levels of Axin-GFP, although this analysis can only detect a maximal reduction to 50%. The exposure of these embryos to ubiquitous Wingless was 0-8 hr, so the inability to detect a decrease in Axin-GFP levels in response to Wingless is not inconsistent with the previously determined half-life of tagged mammalian Axin of 4 hr under Wnt signaling conditions. Under these experimental conditions, the main effect of Wingless signaling is clearly a relocation of Axin to the plasma membrane rather than a destabilization of Axin (Cliffe, 2003).
It was asked whether relocation of Axin-GFP to the plasma membrane might be sufficient for its inactivation. If so, overexpressed Wingless should block the excessive activity of Axin-GFP. This is only partly true: some restoration of naked cuticle (predominantly along the midline) is seen in embryos coexpressing Wingless and Axin-GFP compared to embryos expressing Axin-GFP alone. Thus, a component upstream of Axin but downstream of Wingless may be limiting in the inactivation of Axin. The relocation of Axin to the plasma membrane may be a necessary first step toward its inactivation (Cliffe, 2003).
To identify further components of the Wingless pathway that are required for this relocation, Axin-GFP was examined in various mutants. In sgg mutants, there are no significant changes in the subcellular distribution of the Axin-GFP dots, and their relocation to the plasma membrane in +Wg cells appears normal. Likewise, the few residual GFP-Axin in +Wg cells of APC double mutants are associated with the plasma membrane. Thus, neither GSK3 nor APC are required for relocation of Axin-GFP to the plasma membrane. Interestingly however, none of the Axin-GFP dots are associated with the plasma membrane in dsh mutants; Wingless is still expressed in these mutants at this stage). This is the case even if Wingless is coexpressed with Axin-GFP in these mutants. Thus, Dsh is the most downstream-acting component of the Wnt pathway that is required for the relocation of Axin-GFP to the plasma membrane (Cliffe, 2003).
Membrane bound forms of activated Armadillo ('Arm*', i.e., forms lacking their N termini) show significantly more signaling activity than Arm* without a membrane-targeting domain; this finding led to the suggestion that Armadillo exerts its signaling function in the cytoplasm rather than in the nucleus. However, overexpression of membrane-targeted Arm* causes a dramatic relocation of Axin-GFP, and of E-APC, to the plasma membrane throughout the embryonic epidermis, presumably by direct binding. This mimics the Wingless-induced membrane relocation of Axin-GFP, except that the membrane-targeted Arm* relocates Axin-GFP and E-APC to the entire lateral membrane where it itself is localized. No such relocation is seen under conditions of ubiquitous high levels of untargeted Arm*. The striking relocation of Axin-GFP to the plasma membrane by the membrane-targeted Arm* may cause its inactivation even in cells that are only weakly stimulated by Wingless; thus, this finding provides an alternative explanation for the increased activity of membrane bound Armadillo (Cliffe, 2003).
This work provides evidence that the assembly of Axin complex in the cytoplasm depends on a membrane-targeting function of E-APC. This function may also affect targeting to internal membranes, or vesicles, suggesting that the Axin complex may be associated with vesicles. In support of this, overexpressed Axin is associated with vesicles in Xenopus embryos. Furthermore, Dsh (which is required for the Wingless-induced membrane relocation of Axin) is also associated with vesicles, and to some extent with the plasma membrane, in vertebrate and Drosophila cells. Indeed, Axin and Dsh colocalize after overexpression in vertebrate cells. Notably, the DIX domain of the mammalian Dsh protein Dvl-2 contains a phospholipid binding motif that is conserved in the DIX domain of Axin, and targeting of Dvl-2 to vesicles by this motif is essential for its function in controlling the degradation of β-catenin (Cliffe, 2003).
Therefore, a possible model is that the Axin complex and Dsh are associated with the same vesicles, which may be recycling endocytic vesicles. Dsh may target these vesicles constitutively to the plasma membrane, where the Axin complex can interact potentially with Wnt receptors. This complex may be retained at the plasma membrane as a result of a Wnt-induced interaction between Axin and LRP/Arrow, and this retention may allow its subsequent inactivation. It is noted that LRPs are thought to recycle to the plasma membrane through endocytic vesicles, like their rapidly recycling LDL receptor relative. Recycling vesicles may thus provide a platform for APC-mediated assembly of the Axin complex and may convey this complex to the plasma membrane for inactivation by Wnt receptors (Cliffe, 2003).
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