Oncogene (2008) 27, 5808–5820 & 2008 Macmillan Publishers Limited All rights reserved 0950-9232/08 $32.00 www.nature.com/onc ORIGINAL ARTICLE Recruitment of adenomatous polyposis coli and b-catenin to axin-puncta

MC Faux, JL Coates, B Catimel, S Cody, AHA Clayton, MJ Layton and AW Burgess

Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, Parkville, Victoria, Australia

The adenomatous polyposis coli (APC)tumour suppressor coli (APC) form a complex that promotes the phos- is a multifunctional protein involved in the regulation of phorylation of b-catenin by glycogen synthase kinase-3-b Wnt signalling and cytoskeletal dynamics. Little is known (GSK3-b), which consequently targets b-catenin for about how APC controls these disparate functions. In this ubiquitination by bTrCP and destruction by the study, we have used APC- and axin-fluorescent fusion proteasome (Aberle et al., 1997; Orford et al., 1997; proteins to examine the interactions between these Kitagawa et al., 1999). Wnt stimulation and APC proteins and show that the functionally distinct popula- mutations disrupt the APC/axin complex and result in tions of APC are also spatially separate. Axin-RFP forms increased levels of b-catenin in the nucleus and cytoplasmic punctate structures, similar to endogenous cytoplasm (Munemitsu et al., 1995). b-Catenin is known axin puncta. Axin-RFP recruits b-catenin destruction to interact with members of the Tcf family of transcrip- complex proteins, including APC, b-catenin, glycogen tion factors to activate transcription of genes involved in synthase kinase-3-b (GSK3-b)and casein kinase-1- a proliferation and cellular transformation (Korinek (CK1-a). Recruitment into axin-RFP puncta sequesters et al., 1997). APC from clusters at cell extensions and this prevents its The function of APC in the regulation of the Wnt/ microtubule-associated functions. The interaction between b-catenin pathway has been studied in depth and the APC-GFP and axin-RFP within the cytoplasmic puncta is aberrant activation of b-catenin as a result of truncating direct and dramatically alters the dynamic properties of mutations in APC is recognized as a central event in APC-GFP. However, recruitment of APC to axin puncta colon cancer (Polakis, 1997, 2000; Nathke, 2004). is not absolutely required for b-catenin degradation. However, the spatial and temporal regulation of APC’s Instead, formation of axin puncta, mediated by the DIX interactions with Wnt signalling components and the domain, is required for b-catenin degradation. An cytoskeleton is less well understood. APC is localized at axinDDIX mutant did not form puncta, but still mediated the basal plasma membrane in association with micro- recruitment of destruction complex proteins and phos- tubules, to the peripheral ends of cell extensions (Nathke phorylation of b-catenin. We conclude that there are et al., 1996; Barth et al., 2002). This distribution of APC distinct pools of APC and that the formation of axin points to a major role for APC in cytoskeletal functions puncta, rather than the axin/APC complex, is essential associated with cell migration. APC binds to micro- for b-catenin destruction. tubules (Munemitsu et al., 1994; Su et al., 1995) and Oncogene (2008) 27, 5808–5820; doi:10.1038/onc.2008.205; regulates their growth and stability (Kita et al., 2006), published online 30 June 2008 and has been linked to the actin cytoskeleton via interactions with the Rac1/Cdc42 effector protein Keywords: APC; axin; b-catenin IQGAP1 (Watanabe et al., 2004), the Rac-specific exchange factor Asef (Kawasaki et al., 2000) and the Rho effector mDia (Wen et al., 2004). b-catenin has also recently been implicated in the clustering of APC at cellular protrusions (Sharma et al., 2006). However, it is Introduction not clear whether the ends of APC-loaded microtubules are the sites of the axin/b-catenin/GSK3-b/APC ‘de- Wnt signalling controls crucial stages in cell fate struction’ complex or whether this function takes place decisions, tissue development and maintenance and elsewhere in the cell. has been identified as a key signalling pathway in cancer The subcellular location of axin is less well defined (Logan and Nusse, 2004; Schneikert and Behrens, 2007). than APC. In Xenopus the physiological concentration When Wnt signalling is suppressed, the scaffold protein of axin is proposed to be exceedingly low (Lee et al., axin and the tumour suppressor adenomatous polyposis 2003), making detection at the subcellular level difficult. Endogenous axin has been detected in small, punctate Correspondence: Dr MC Faux, Epithelial Biology Laboratory, cytoplasmic structures that redistribute to large aggre- Ludwig Institute for Cancer Research, PO Box 2008, Royal gates close to the plasma membrane following treatment Melbourne Hospital, Parkville, Victoria 3050, Australia. with LiCl (which inhibits GSK3-b, mimicking Wnt E-mail: [email protected] Received 12 November 2007; revised 30 April 2008; accepted 30 May activation) (Levina et al., 2004; Wiechens et al., 2004). 2008; published online 30 June 2008 Ectopically expressed recombinant axin is localized in Recruitment of APC and b-catenin to axin puncta MC Faux et al 5809 cytoplasmic vesicles or puncta (Fagotto et al., 1999; destruction complex proteins and that formation of the Smalley et al., 1999; Schwarz-Romond et al., 2005). The axin puncta, rather than the axin/APC complex, is C-terminal DIX domain of axin is known to mediate critical for b-catenin degradation. self-association, which is thought to be important for the regulation of b-catenin (Hsu et al., 1999; Kishida et al., 1999; Sakanaka and Williams, 1999) and has recently been shown to associate into polymers that are Results required for puncta formation (Schwarz-Romond et al., 2007a, b). It has also been proposed that axin shuttles in To study the subcellular location and dynamic proper- and out of the nucleus (Wiechens et al., 2004) and that it ties of axin, we assessed the localization of both associates with microtubules and regulates their stability endogenous and recombinant axin fused to monomeric through dishevelled (Dvl) (Ciani et al., 2004). red fluorescent protein (RFP) (Figure 1a). Using a Although APC and axin have been shown to interact commercially available polyclonal axin antibody, we using immunoprecipitation (Hart et al., 1998), the observed endogenous axin in distinct puncta in the subcellular location of this interaction has not been well cytoplasm of Madin–Darby canine kidney (MDCK) defined. It is possible that only a small proportion of cells (Figures 1b and c). Both endogenous and APC associates with the axin complex. Recent biochem- recombinant axin puncta were evenly distributed ical data point to the existence of discrete populations of throughout the cytoplasm (Figures 1b–e), consistent APC where the APC/axin/b-catenin complex is separate with previous reports for endogenous axin in HEK293 from APC interactions with microtubules (Penman cells (Levina et al., 2004; Wiechens et al., 2004) and et al., 2005). These studies suggest that the spatial recombinant axin (Fagotto et al., 1999; Smalley et al., regulation of both APC and axin is a dynamic process 1999; Schwarz-Romond et al., 2005, 2007b). Axin-RFP but complex formation at the subcellular level has not was localized to cytoplasmic puncta when expressed been explored. In the present study we have used APC- in both MDCK (Figure 1e) and HEK293 T cells and axin- fluorescent fusion proteins to study axin (Supplementary Figures S1, S2). Similarly, epitope– regulation of APC subcellular localization and to tagged axin-HA (Figure 1d) formed cytoplasmic puncta, examine the dynamic interactions of these proteins in suggesting that the monomeric RFP tag did not affect its living mammalian cells. We examined whether other distribution. destruction complex proteins, b-catenin, GSK3-b and casein kinase-1-a (CK1a) were recruited to axin puncta Axin recruits APC to cytoplasmic puncta and whether axin puncta formation, mediated by the Endogenous APC concentrated in clusters at the ends of DIX domain of axin, is required for complex formation microtubules in subconfluent epithelial cells (Nathke and b-catenin degradation. Our data show that there are et al., 1996; Barth et al., 2002; Figure 2a). Transiently distinct subcellular pools of APC, that axin redistributes expressed APC-GFP was also concentrated in distinct APC from microtubule cell extensions to cytoplasmic clusters at the peripheral ends of microtubules in axin puncta, and that this prevents microtubule- MDCK cells (Figure 2b), mimicking the staining of associated functions of APC. We show that axin recruits endogenous APC in these cells (Figure 2a). In some

Figure 1 Localization of axin to cytoplasmic puncta. (a) Schematic representation of domain structures of axin-, axinDRGS- and axinDDIX-RFP and APC-GFP fusion proteins. (b, c) Endogenous axin is localized to cytoplasmic puncta in Madin–Darby canine kidney (MDCK) cells. Cells were fixed and immunostained for endogenous axin with antibodies to axin-1. (c) High magnification of cytoplasmic puncta of endogenous axin. (d) MDCK cells transiently expressing axin-HA were fixed and immunostained with anti-HA antibodies. (e) MDCK cells expressing axin-RFP. Shown are single confocal sections.

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5810 cells, APC-GFP also decorated the microtubule network puncta affects microtubule-associated functions of APC. (see Figure 2f). In contrast, in cells expressing axin-RFP, The presence of APC on microtubule plus ends in cell endogenous APC was redistributed to cytoplasmic extensions has been reported to stabilize and promote puncta, coincident with axin (Figure 2c, arrowheads). the growth of microtubules (Kita et al., 2006). The loss In cells not expressing axin-RFP, APC was localized in of APC results in reduced cell protrusions and decreased clusters at cell extensions (Figure 2c, arrows). Expres- microtubule stability (Kroboth et al., 2007). We sion of the mutant axinDRGS-RFP protein (Figure 1a) examined microtubule assembly activity in untrans- that does not bind APC (Behrens et al., 1998; Hart et al., fected, axin-RFP and axinDRGS-RFP transfected 1998; Kishida et al., 1998) but forms similar puncta to MDCK cells. Cells were incubated on ice for 1 h to wild-type axin, had no effect on the localization of depolymerize microtubules and microtubule polymer- endogenous APC (Figure 2d, arrows), confirming that ization was then induced by incubation at 30 1C relocalization of APC depends upon binding to axin. followed by rapid fixation (Figures 2g–j). After 1 min Similarly, when APC-GFP was coexpressed with axin- incubation at 30 1C, microtubule asters formed in RFP, APC-GFP redistributed from the cell tips to the untransfected cells (Figure 2g) and at 5min, micro- punctate structures containing axin-RFP (Figure 2e). tubules had repolymerized (not shown). In contrast, in Again, coexpression of APC-GFP with the mutant axin-RFP expressing cells, microtubule assembly was axinDRGS-RFP did not lead to redistribution of APC reduced significantly and at 1 min, microtubule asters (Figure 2f). Similar results were obtained in HEK293 had not formed (Figure 2h). Microtubule assembly in T cells (Supplementary Figures S1 and S2). Thus, axin axinDRGS-RFP expressing cells was similar to control has the capacity to recruit APC to cytoplasmic puncta. cells (Figure 2i). We quantitated these differences by Although endogenous APC was detected in axin-RFP counting the number of cells with microtubule asters puncta, APC could not be detected in the endogenous (spindles; Figure 2j). Less than 25% of axin-RFP- axin puncta observed in MDCK cells (Figures 1b and c): expressing cells formed asters at 1 min compared to either endogenous axin is not at a sufficient concentra- more than 95% in untransfected cells and 75% in axin- tion (Lee et al., 2003) to allow recruitment of APC or DRGS expressing cells (Figure 2j). Expression of axin- APC is present at a level below the threshold of RFP therefore inhibited the microtubule assembly detection. Therefore, we examined the levels of axin activity of APC. This was not a direct effect of axin required to sequester APC away from cell extensions. overexpression, as APC binding and recruitment to axin The relative levels of expression of axin-RFP:APC-GFP puncta was required. Sequestering APC into axin puncta were measured based on total fluorescence intensity in therefore shifts the cellular population of APC away individual cells and were correlated with APC localiza- from its microtubule-associated location and function. tion in either the puncta or microtubules (Supplemen- tary Figures S2C and D). Scatter plots revealed two distinct data clusters corresponding to APC localized to Direct interaction between APC-GFP and axin-RFP the microtubule network only when axin expression was within puncta low, and APC localized to axin puncta as the level of To determine whether colocalization of APC and axin axin increases (Supplementary Figure S2D). These data within the axin puncta involves a direct interaction, we indicate a threshold level of axin expression is required measured fluorescence resonance energy transfer for APC to redistribute to puncta. (FRET) between APC-GFP and axin-RFP using We next investigated whether recruitment of APC fluorescence lifetime imaging microscopy (FLIM). A from clusters at the ends of microtubule tips to the axin decrease in APC-GFP lifetime in the presence of axin-

Figure 2 Recruitment of adenomatous polyposis coli (APC) to cytoplasmic axin puncta. (a) Endogenous APC and (b) APC-GFP localize to the ends of microtubules in clusters in Madin–Darby canine kidney (MDCK) cells. APC (anti-APC)/APC-GFP -green, b- tubulin -red, nuclei (4,6-diamidino-2-phenyl indole; DAPI) blue. Shown are single section confocal images. APC (left); composite image (middle), scale bar 20 mm; inset (right) at higher magnification of APC clusters, scale bar, 5 mm. (c) In the presence of axin-RFP endogenous APC redistributes to the axin puncta. (d) MDCK cells expressing axinDRGS-RFP. Cells were fixed and immunostained with antibodies to APC (green). Shown are single section confocal composite images, scale bar 20 mm. APC is detected at cell tips in untransfected and axinDRGS-RFP cells (c, d arrows). Distinct APC puncta are present in cells expressing axin-RFP (c, arrowheads), but no APC was associated with axinDRGS-RFP puncta (d). (e) APC-GFP is recruited to axin-RFP puncta in MDCK cells. (f) APC- GFP is not recruited to axinDRGS-RFP puncta. Shown are composite images of Z series projections of cells imaged by laser scanning confocal microscopy. Scale bar 20 mm. (g–j) Recruitment of APC to axin-RFP puncta prevents microtubule assembly following microtubule depolymerization. MDCK cells were incubated on ice for 1 h to depolymerize microtubules and then at 30 1C for 1 min, fixed and immunostained. (g) Microtubule spindle formation occurs in non-axin expressing cells. (h) In axin-RFP expressing cells, microtubule spindle assembly is decreased. (i) Microtubule spindle formation in axinDRGS-RFP expressing cells. (j) Untransfected, axin- and axinDRGS- RFP cells were scored for the presence or absence of microtubule spindles. Microtubule spindle assembly was reduced in axin-RFP but not in the axinDRGS-RFP cells. (k–n) Measurement of nano-scale interaction of APC-GFP and axin-RFP by frequency-domain FRET-FLIM. Shown are APC-GFP fluorescence images and fluorescence lifetime images for: (k) APC-GFP expressed alone; (l, m) APC-GFP coexpressed with (l) axin-RFP and (m) axinDRGS-RFP. APC-GFP fluorescence concentrated at cell tips (arrowheads); APC-GFP fluorescence in intracellular puncta with corresponding depressions in fluorescence lifetimes (arrows). Scale bar 20 mm. (n) Mean and s.e. of fluorescence lifetimes of APC-GFP in clusters at cellular extensions and in intracellular puncta in APC-GFP and axin-RFP co-expressing cells. Statistical analysis revealed that fluorescence lifetime in puncta was reduced compared to fluorescence lifetime in clusters at cellular extensions (***Po0.0001).

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5811 RFP indicates FRET, which implies that the two When axin-RFP was coexpressed with APC-GFP, the fluorophores are within 10 nm of each other (Figures redistribution of APC-GFP to intracellular axin puncta 2k–n). The fluorescence lifetime of the GFP fluorophore was accompanied by a reduction in APC-GFP fluores- in cells expressing APC-GFP alone was relatively cence lifetime (Figure 2l, arrows, average lifetime uniform, with lifetime values in the expected range of 1.61±0.03 ns, n ¼ 82; Figure 2n) indicating a direct 2–2.25ns for GFP (Figure 2k; average lifetime interaction. Coexpression of APC-GFP with the mutant 2.03±0.03 ns (n ¼ 11), Figure 2n). Fluorescence lifetime axinDRGS-RFP protein had no effect on the lifetime of was independent of the fluorescence intensity and the APC-GFP (Figures 2m and n). The interaction therefore the amount of APC-GFP in the clusters. between axin-RFP and APC-GFP is therefore specific,

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5812 0:00 6:25 12:25 0.3 14 2 APC m 0.2 12 µ 0.1 2 MSD 10 m

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Figure 3 Axin restricts the dynamic behaviour of adenomatous polyposis coli (APC). Time-lapse analysis of APC-GFP and axin- RFP. Still frames from (Supplementary material (movies 1A–C)) of APC-GFP and axin-RFP in living HEK293 T cells. Merged images of GFP and red fluorescent protein (RFP) signal are shown. Elapsed time is indicated in the top left of panel (a) (min:sec). (a) APC- GFP (Movie 1A). Dashed arrows indicate decoration of microtubule network; arrowheads indicate movement of GFP along microtubules. The APC-GFP-structures cluster in several specific regions (single asterisks); some clusters appear to grow and move along the microtubules before dispersing (double asterisks) or even disappearing (arrows). (b–d) APC-GFP and axin-RFP coexpression. (b) Movie 1B. GFP signal is now localized in cytoplasmic puncta coincident with axin, visualized as yellow in the merged image. Large (arrowheads) and smaller (arrows) axin puncta demonstrate oscillatory movement (Movie 1B). The asterisks denote puncta that appear to separate into red and green particles and then merge to yellow. (c) The cell on the left from Movie 1C (and at higher magnification in d), demonstrates two GFP puncta that overlap with RFP (visualized as yellow), denoted by open arrowhead symbol. This cell also displays APC-GFP decoration of the microtubule network (dashed arrows) and accumulation of GFP-particles (*) that appear to move along the microtubules before dispersing (**). The lower level of axin expression in this cell appears only to influence a relatively small proportion of APC-GFP; the APC-GFP not bound to axin moves along microtubules in a similar fashion to APC-GFP expressed alone. This tracking of APC-GFP is visualized in Movie 1C. (e) Single Particle Tracking (SPT) analysis of APC- GFP and axin-RFP. Plot of the mean-squared displacement (MSD) versus time for a highlighted particle (*) undergoing directed motion from (a) (green triangles) and a highlighted particle from (d) (open arrowhead) undergoing constrained motion (red diamonds and inset). (f) Diffusion and velocity coefficients from SPT analysis.

as shown by coprecipitation and deletion mapping (Mimori-Kiyosue et al., 2000) and it also accumulated at studies (Behrens et al., 1998; Hart et al., 1998), and specific structures, possibly associated with microtubules occurs within cytoplasmic puncta rather than at the ends (Figure 3a; arrowheads; Supplementary material of microtubules. (movie 1A)). In contrast, when APC-GFP was coexpressed with axin-RFP and relocalized to puncta, there was no longer Dynamic behaviour ofAPC-GFP is influenced by any detectable tracking, or coordinated or directed axin-RFP movement of APC-GFP (Figures 3b–d; Supplementary GFP-tagged APC has previously been shown to move material (movies 1B and C)). Instead, both APC-GFP along the microtubule network towards their plus ends and axin-RFP were restricted to the puncta and appear in the periphery of cell extensions (Mimori-Kiyosue to oscillate on the spot. Single particle tracking (SPT) et al., 2000). We therefore examined whether binding to analysis from time-lapse images revealed rapid diffusion axin altered the dynamics of APC movement within the of free APC-GFP, whereas APC-GFP particles in the cell (Figure 3; Supplementary material (movies 1A–C)). axin-RFP puncta moved threefold more slowly APC-GFP was primarily localized at the microtubule (Figure 3f). Our analysis includes both combined tips with some decoration of the microtubule network diffusion and directed motion. Particle trajectories were when present at higher levels (Figure 3a, dashed arrow; determined from time-lapse images, represented as Supplementary material (movie 1A); see also Supple- mean-squared displacement (MSD) as a function of mentary Figure S2B). The movement of human time (Figure 3e). The shape of the MSD versus time plot APC-GFP was consistent with that of Xenopus APC provides information on the speed and type of motion.

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5813 A linear plot indicates random Brownian diffusion an correspondingly restricted. In cells expressing low levels upward curvature indicates directed motion and a of axin-RFP, only a small proportion of total APC-GFP downward curvature indicates constrained motion. was colocalized with axin-RFP, however, all of this SPT analysis demonstrated that there was a directional proportion had restricted movement (Figures 3c and d, component to the movement in 11 out of 17 APC-GFP- open arrowheads). In contrast, the majority of APC- containing spots analysed, as evidenced by the upward GFP in these cells was not bound to axin and the curvature in the MSD versus time plot (Figure 3e, green dynamics of this proportion was equivalent to that of triangles). In contrast, when APC-GFP was colocalized APC-GFP in cells not transfected with axin-RFP with axin-RFP, the levelling of the MSD versus time (Supplementary material (movie 1C); Figures 3c and d, plot indicates constrained motion (Figure 3e, red dashed arrow). Thus, axin influences the dynamics of diamonds, and inset). Both larger (Figure 3b, arrow- axin-bound APC but has no effect on the proportion heads) and smaller puncta (Figure 3b, arrow) displayed that is not bound. This suggests that two populations of similar oscillatory behaviour. Thus, the location and APC, one microtubule associated and the other axin- movement of APC-GFP bound to axin-RFP, was puncta associated, can coexist in the cell. restricted compared to the rapid, directed movement of free APC-GFP. The dynamic behaviour of APC-GFP coexpressed Recruitment ofdestruction complex proteins into axin with axin-RFP was analysed in cells that expressed puncta different levels of axin-RFP (Figures 3c and d; To determine whether axin puncta represent the Supplementary material (movie 1C)). Regardless of b-catenin destruction complex (Schneikert and Behrens, axin-RFP expression levels, APC-GFP dynamics were 2007), we examined the ability of axin-RFP to recruit controlled by whether or not APC-GFP was bound to endogenous b-catenin, GSK3-b and CK1-a to axin axin-RFP. In cells expressing high levels of axin-RFP puncta (Figure 4). Initially, we confirmed that transient (Figure 3c, arrowhead), the majority of APC-GFP was expression of recombinant axin resulted in downregula- colocalized with axin-RFP and its movement was tion of b-catenin in SW480 cells, a colon cancer cell line

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Figure 4 Recruitment of b-catenin, glycogen synthase kinase-3-b (GSK3-b) and casein kinase-1-a (CK1-a) in cells expressing axin- RFP. (a) SW480 and (b) Madin–Darby canine kidney (MDCK) cells expressing axin-RFP were treated with the proteasome inhibitor MG132, and immunostained with antibodies to b-catenin for SW480 cells (a) or phospho-b-catenin (P-b-cat) for MDCK cells (b). Shown are confocal images of b-catenin or P-b-cat, axin-RFP and composite images (b-catenin or P-b-cat—green, axin-RFP—red, nuclei—blue in (a)) for control and treated cells. Arrows indicate axin-RFP expressing cells; arrowheads indicate b-catenin or P-b-cat coincident with axin-RFP. (c) GSK3-b and (d) CK1-a are recruited to axin-RFP puncta in MDCK cells. Shown are confocal images of MDCK cells expressing axin-RFP and immunostained with antibodies to GSK3-b (c) or CK1-a (d). Composite images show GSK3-b or CK1-a—green, axin-RFP—red. Scale bars, 20 mm. (e) Fluorescence intensity of GSK3b from individual MDCK cells with and without axin-RFP expression was measured. Values shown are mean and s.e.m. of GSK3b fluorescence intensity from at least 50 cells.

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5814 containing only truncated APC (Figure 4a), as has been recruited to the destruction complex, APC is not reported previously (Behrens et al., 1998; Hart et al., absolutely required for the formation of a functional 1998). This downregulation was due to proteasome- destruction complex. mediated degradation, as inhibition of the proteasome by MG132 resulted in accumulation of b-catenin in axin-containing puncta (Figure 4a, arrowheads). In Formation ofaxin puncta is necessary for b-catenin MDCK cells, the proportion of b-catenin targeted for degradation but not phosphorylation degradation was masked by the large population of The DIX domains of both Dvl2 and axin have recently membrane-associated b-catenin, therefore we used been shown to mediate both self-polymerization and phospho-specific b-catenin antibodies that detect pS45/ puncta formation (Schwarz-Romond et al., 2007a, b). pT41/pS37/pS33-b-catenin (phospho-b-catenin) to vi- Therefore, we investigated whether axin polymerization sualize the b-catenin pool that is targeted for degrada- and puncta formation were required for assembly of the tion (Figure 4b). Again, inhibition of the proteasome destruction complex, and whether they influence recruit- allowed phospho-b-catenin to accumulate in axin- ment and b-catenin degradation. Previous studies have containing puncta (Figure 4b, arrowheads), implying shown that the DIX domain is important for b-catenin that axin puncta represent the destruction complex. regulation (Kishida et al., 1999; Sakanaka and Williams, Likewise, inhibition of GSK3-b in SW480 cells resulted 1999), however, it has also been reported that deletion of in increased b-catenin coincident with axin puncta the DIX domain does not affect the ability of axin to (Supplementary Figure S3A), further implicating the alter b-catenin localization (Nakamura et al., 1998). We axin puncta as the site of the destruction complex. tested the ability of axinDDIX-RFP to recruit b-catenin Endogenous GSK3-b and CK1-a were also recruited to in cells treated with proteasome inhibitors (Figure 6). As axin-RFP-containing puncta in MDCK cells (Figures 4c we had observed previously, expression of axin-RFP and d). Similarly, GSK3-b was recruited to axin-RFP resulted in destruction of b-catenin (Figure 6a, left) that puncta in SW480 cells (Supplementary Figure S3B). was prevented by inhibition of the proteasome, resulting Unlike b-catenin, levels of GSK3-b were not regulated in detection of b-catenin in axin puncta (Figure 6a, by the destruction complex as quantitation of total right). We found that axinDDIX did not form puncta GSK3-b fluorescence intensity shows no difference but instead was distributed diffusely in the cytoplasm between untransfected and axin-RFP-transfected cells (Figure 6b), consistent with previous reports that the (Figure 4e). Collectively, these results show that the DIX domain is required for efficient puncta formation endogenous destruction complex proteins, GSK3-b, (Schwarz-Romond et al., 2007b). Importantly, expres- CK1-a and APC, are all recruited to axin puncta. sion of axinDDIX-RFP did not result in reduced b- Recruitment of b-catenin was only observed if its catenin, but instead b-catenin was predominantly degradation was inhibited by switching off the protea- cytosolic with a distribution that mirrored that of some, implying that puncta participate in a functional axinDDIX (Figure 6b). Thus, axinDDIX appears to destruction complex. sequester b-catenin but this does not lead to its degradation. Inhibition of the proteasome did not have a marked effect on b-catenin distribution or levels Recruitment of b-catenin into axin puncta implying that the axin puncta formation is required for does not require full-length APC efficient degradation of b-catenin. To determine whether The degradation of b-catenin following transient ex- axin puncta formation is required for b-catenin phos- pression of axin in SW480 cells (Behrens et al., 1998; phorylation, we examined whether phospho-b-catenin Hart et al., 1998; Figure 4a) is somewhat surprising was sequestered in the cytoplasm following expression given that SW480 cells contain truncated APC protein of axinDDIX. Indeed, we found increased cytoplasmic that is not able to bind axin. This implies that expression phospho-b-catenin in axinDDIX cells compared to of axin allows a functional destruction complex to be untransfected surrounding cells, in both SW480 and formed in the absence of intact APC, but the relative MDCK cells (Figures 6c and d). Quantitation of importance of axin and APC in forming the destruction fluorescence intensity confirms that the levels of complex and the mechanism by which expression of axin cytoplasmic phospho-b-catenin are increased in axinD leads to b-catenin degradation has not been elucidated. DIX-expressing cells (Figure 6e). AxinDDIX therefore We explored the requirement for APC further in cells recruited b-catenin, GSK3-b and CK1 (as implied by other than SW480. The axinDRGS mutant, which phosphorylation of axinDDIX-associated b-catenin). cannot bind APC, was still able to downregulate AxinDDIX is also capable of recruiting APC. APC b-catenin in SW480 cells (Supplementary Figure S4) translocates from clusters at the ends of microtubules in and to recruit phospho-b-catenin and GSK3-b in a proportion of axinDDIX-expressing MDCK cells. MDCK cells (Figure 5). As with wild-type axin-RFP, This recruitment is not as efficient as recruitment by phospho-b-catenin was detected in axinDRGS puncta axin puncta (Supplementary Figure S5). These (Figure 5a) and levels of phospho-b-catenin were results imply that axinDDIX, which still contains increased in these axinDRGS-containing puncta in the binding sites for b-catenin, APC, GSK3-b and CK1-a, presence of proteasome inhibitors (Figure 5b). Similarly, still facilitates b-catenin phosphorylation by GSK3-b, GSK3-b was also recruited to axinDRGS-containing but proteasomal degradation is blocked in the absence puncta (Figure 5c). We conclude that, despite being of puncta.

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Figure 5 Axin recruitment of b-catenin and glycogen synthase kinase-3-b (GSK3-b) does not require adenomatous polyposis coli (APC). (a) Control or (b) MG132 treated Madin–Darby canine kidney (MDCK) cells expressing axinDRGS-RFP were immunostained with antibodies to phospho-b-catenin (P-b-cat). (c) Cells expressing axinDRGS-RFP were immunostained with antibodies to GSK3-b. Shown are confocal images of P-b-cat or GSK3-b, axinDRGS-RFP and composite images (P-b-cat or GSK3-b—green, axin-RFP— red). Arrowheads indicate phospho-b-catenin or GSK3-b coincident with axinDRGS-RFP. Scale bars: 20 mm.

Discussion in the recruitment of APC, b-catenin and GSK3-b and that axin puncta are crucial for b-catenin degradation. In this paper, we suggest a new paradigm for the The ability of axin to form puncta has been reported assembly of the b-catenin destruction complex. By previously (Fagotto et al., 1999; Smalley et al., 1999; expression of axin-RFP, we were able to detect the Schwarz-Romond et al., 2005), and recent studies show recruitment of proteins away from other subcellular that puncta formation is a dynamic process mediated by locations and formation of the b-catenin destruction protein oligomerization or polymerization (Schwarz- complex in association with axin puncta. Our results Romond et al., 2007a, b). Our data provide evidence for demonstrate that axin recruits APC into cytoplasmic the existence of endogenous axin puncta, consistent with puncta, where its movement becomes restricted as a the detection of small punctate structures or spots with result of axin binding, and its ability to regulate axin antibodies in 293 cells (Levina et al., 2004; microtubule assembly is decreased significantly. How- Wiechens et al., 2004). The DIX domain of axin has ever, in cells overexpressing axin, recruitment and been shown to mediate self-assembly in vitro and degradation of b-catenin in axin puncta does not contribute to the signalling function of axin (Kishida absolutely require APC. The recruitment of b-catenin, et al., 1999; Sakanaka and Williams, 1999), but is not GSK3-b and CK1-a to axin puncta implies that axin required for recruitment into Dvl2 puncta (Schwarz- puncta form the site of the classical b-catenin destruc- Romond et al., 2007b). Instead, our data now show that tion complex. Axin puncta, mediated by the DIX the DIX domain of axin is essential for axin-puncta domain, are not required for recruitment of destruction formation and b-catenin degradation. Polymerization of complex proteins or b-catenin phosphorylation, how- Dvl2 has been suggested to provide a high local ever, b-catenin degradation does not occur unless concentration of Dvl2, thus promoting recruitment of puncta are formed. We believe that axin is important its binding partners (Schwarz-Romond et al., 2007b). In

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5816 contrast, axin recruited other components of the essential for recruitment of its binding partners. b- destruction complex in the absence of puncta formation, Catenin is also correctly phosphorylated when bound to suggesting that dynamic polymerization into monomeric axinDDIX. Monomeric axinDDIX can assemblies with high local axin concentration is not therefore still act as a scaffold for complex assembly

SW480 cells Control +MG132 β-catenin β-catenin Axin-RFP β-catenin β-catenin Axin-RFP

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β-catenin β-catenin Axin∆DIX-RFP β-catenin β-catenin Axin∆ DIX-RFP

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P-β-cat P-β-cat Axin∆DIX-RFP P-β-cat P-β-cat Axin ∆DIX-RFP

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MDCK cells P-β-cat P-β-cat Axin∆ DIX-RFP P-β-cat P-β-cat Axin ∆DIX-RFP

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SW480 cells MDCK cells

500 200 400 150 300 100 -cat cytoplasmic -cat cytoplasmic 200 β β P- P- fluorescence intensity fluorescence intensity 100 50

0 0 Cell number 21 77 21 72 19 90 18 72 Axin∆DIX Untransfected Axin∆DIX Untransfected Axin∆DIX Untransfected Axin∆DIX Untransfected Control +MG132 Control +MG132

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5817 and b-catenin phosphorylation, but not b-catenin alter the dynamic behaviour of axin, possibly degradation. Puncta formation may be required for changing the affinity for axin and some of its ligands the recruitment, positioning and/or activation of the (Schwarz-Romond et al., 2007b). The constrained Skp1/Cul 1/F box ubiquitination machinery (Hart et al., motion of APC within axin puncta possibly reflects the 1999; Latres et al., 1999; Winston et al., 1999). size of the polymerized axin complex and/or an altered Experiments are currently underway to determine conformational state as a result of interactions that whether puncta formation is required for b-TrCP differ from the population of APC interacting with recruitment, b-catenin ubiquitination or proteasomal microtubules. degradation. Intriguingly, recruitment of APC to axin appears We propose that axin promotes the formation of an dispensable for b-catenin degradation (this study; Hart APC/axin complex at a precise subcellular location that et al., 1998; Kishida et al., 1999). However, APC has is distinct from the concentration and function of APC been shown to influence levels of b-catenin (Munemitsu at microtubule tips. This concept is supported by the et al., 1995). It may be that endogenous levels of axin are identification of at least two functionally different APC- not sufficient to regulate the levels of b-catenin in the containing protein complexes (Penman et al., 2005). absence of wild-type APC but increased axin can Here, we provide evidence for two distinct subcellular independently promote b-catenin degradation. Indeed, populations of APC with different functions. At overexpression of Dvl proteins results in Wnt-indepen- microtubule extensions, APC regulates microtubule dent signalling (Kishida et al., 1999; Smalley et al., 1999; assembly dynamics whereas in axin puncta, the APC/ Park et al., 2005) that is dependent on the Dvl DIX axin complex potentiates the destruction of b-catenin. domain (Schwarz-Romond et al., 2007b). In Xenopus, Our demonstration that axin redirects APC away from the local concentration of Dvl is important for microtubule ends, preventing microtubule assembly, canonical signalling (Park et al., 2005). Similarly, forced suggests that the functions of APC in the regulation of oligomerization of LRP6 induces Wnt signalling and microtubule dynamics and b-catenin stability are bypasses the requirement for Dvl (Cong et al., 2004; spatially separate. Bilic et al., 2007). Axin can direct the location and movement of APC, The role of APC in the regulation of b-catenin is well but APC does not appear to influence axin distribution. documented: truncating mutations in APC result in In Drosophila, APC is also recruited into cytoplasmic elevated b-catenin levels (Schneikert and Behrens, 2007). puncta following overexpression of axin (Cliffe et al., However, we have shown previously that stable expres- 2003). It appears that the amount of axin in the cell sion of APC in SW480 cells results in reduced b-catenin/ alters the balance between APC bound to axin and APC Tcf transcriptional signalling as a result of b-catenin associated with microtubules. Although the reported translocation to the cell periphery and not significant physiological concentration of axin is low (Lee et al., changes in the total amount of b-catenin (Faux et al., 2003), even low concentrations of axin (including 2004). We propose that axin, rather than APC, is the endogenous axin) form puncta, implying a high local crucial player in b-catenin degradation and APC may concentration. It is likely that the populations of contribute by regulating the cytoplasmic b-catenin pool APC are governed by whether or not axin is bound, available to the destruction complex or by influencing rather than by axin concentration. Importantly, the rate of b-catenin degradation. APC is mutated in the our data show that binding to axin restricted the majority of colon cancers and is the foremost tumour diffusion and velocity of APC-GFP and prevented suppressor in the colon. Given the functions for APC in directed motion (Mimori-Kiyosue et al., 2000; cell migration, cell adhesion and chromosomal stability Dayanandan et al., 2003; Langford et al., 2006). The (Fodde, 2003), the tumour suppressor activity of APC altered dynamics of APC bound to axin supports and its role in Wnt signalling is likely to be more our findings that the axin/APC complex is distinct from complex than simply regulating the nuclear levels of b- the rapidly moving APC population at microtubules. catenin (Polakis, 2000; Clevers, 2006). The challenge Indeed, we found that axin expression altered now is to determine how the activities of APC at the microtubule function and this was dependent on APC cytoskeleton are associated with Wnt signalling and binding. Recently, Dvl2 has been reported to tumour suppression.

Figure 6 Axin puncta formation is required for b-catenin degradation but not b-catenin phosphorylation. (a) Axin-RFP expression in SW480 cells resulted in b-catenin degradation in control cells (left panels) whereas b-catenin was recruited to axin puncta in MG132 treated cells (right panels). (b) AxinDDIX-RFP did not form puncta and resulted in sequestration of b-catenin in the cytoplasm, but not degradation, in both control and MG132-treated SW480 cells. The DIX domain is required for both puncta formation and b-catenin degradation. AxinDDIX expression in (c) SW480 and (d) Madin–Darby canine kidney (MDCK) cells resulted in sequestration of P-b-cat in both control (left panels) and MG132-treated cells (right). Shown are confocal images of b-catenin (a, b)or P-b-cat (c, d) and composite images (b-catenin, P-b-cat—green, axin-RFP, axinDDIX-RFP—red, nuclei (46-diamidino-2-phenyl indole; DAPI)—blue). Arrows indicate axinDDIX expressing cells where cytoplasmic P-b-cat is increased, dashed arrows denote untransfected cells. We found little evidence for axinDDIX-RFP puncta formation in either SW480 or MDCK cells. Scale bar, 20 mm. (e) Fluorescence intensity of cytoplasmic P-b-cat from individual SW480 (left graph) and MDCK (right) cells with and without axinDDIX was measured. Values shown are mean±s.e.m. of cytoplasmic P-b-cat fluorescence intensity. Note that proteasome inhibition had little effect on the cytoplasmic sequestration of b-catenin.

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5818 Materials and methods antibodies separately and together with identical results, hence staining with combined antibodies are shown. Where indi- Antibodies and plasmids cated, nuclei were visualized using 46-diamidino-2-phenyl The following antibodies were used: anti-APC (APC2, indole (Molecular Probes). produced in our laboratory (Catimel et al., 2006)), anti-APC (H290; Santa Cruz, Santa Cruz, CA, USA), anti-CK1a Confocal fluorescence microscopy and image analysis (Santa Cruz), anti-axin-1 (Zymed, San Francisco, CA, USA), Direct fluorescence (GFP and RFP) and immunofluorescence anti-phospho-b-catenin (Cell Signaling, Danvers, MA, USA, staining were detected in successive focal planes using Bio-Rad 9561, pS33, pS37, pT41 and 9565, pT41, pS45), anti-b-catenin MRC-1024 dual system (Hemel Hempstead, UK) and Nikon (19920/610153; BD Transduction Laboratories, San Jose, CA, C1 confocal microscopes. Double- and triple-label images were USA), anti-GSK3b (G22320; BD Transduction Laboratories), detected using standard filter sets and laser lines. Cells were anti-actin (clone AC-40; Sigma, St Louis, MS, USA), anti-b- imaged with Nikon Plan Apo  60 (NA1.4) or Nikon TIRF tubulin (clone 2.1; Sigma) and anti-HA (262K#2362; Cell  100 (NA1.45) oil immersion lenses. Fluorescence intensity Signaling). To generate C-terminally tagged APC-GFP, measurements were performed using ImageJ (NIH, Bethesda, enhanced GFP was subcloned as a BamH1/Not1 fragment USA). Following background subtraction, individual cells into pEF-mycAPC (Faux et al., 2004) after removing the were selected and mean fluorescence intensity measured and STOP. Expression of GFP at the C terminus did not multiplied by selected area to obtain total fluorescence. For compromise the localization of APC nor, as shown previously quantitative cytoplasmic fluorescence, the fluorescence inten- (Sharma et al., 2006), its colocalization with binding proteins sity of four randomly selected areas of identical size taken b-catenin, EB1, KAP3a and DLG-1, suggesting that the from the cytoplasm of individual cells were measured and C-terminal GFP does not interfere with APC interactions at its mean fluorescence intensity determined using ImageJ software. C terminus. For expression of axin-mRFP, the cDNA encoding monomeric RFP (mRFP1) (Campbell et al., 2002), gift from Dr Mark Prescot (Monash University, Australia), was subcloned Fluorescence lifetime imaging micrsoscopy analysis into pDsRed1-N1 (Clontech, Mountain View, CA, USA), FLIM experiments were carried out as described (Clayton replacing DsRed1 (referred to as pDsRed-mRFP1). The cDNAs et al., 2005; Hanley and Clayton, 2005). The phase and encoding full-length mouse axin1 isoform1, 125–956 aa (Zeng modulation were determined from a series of images taken at et al., 1997), was amplified from pCS2-6myc mouse axin (gift of 12 phase settings using software provided by the manufacturer Dr Frank Costantini, Columbia University Medical Centre, NY, and converted to phase lifetimes with correction for photo- USA) and an N-terminally extended mouse axin1 isoform1 bleaching as described (van Munster and Gadella, 2004). (12–956), was amplified from pCS2_MT-mouse axin (gift of Dr Fluorescein dianion (lifetime 4.1 ns) was used as a reference Daniel Capelluto, Denver, USA) and both were subcloned into (Magde et al., 1999). Two approaches were used to analyse the pDsRed-mRFP1. Both forms of axin-RFP formed puncta FLIM data. In the first approach the decrease in APC-GFP and were capable of recruiting APC-GFP. AxinDRGS-RFP lifetime in a FLIM image was used to infer the existence of (aa 190–353 deleted) and axinDDIX-RFP (aa 875–956 deleted) FRET between APC-GFP and axin-mRFP in axin puncta but deletion constructs were created in two parts and subcloned into not elsewhere in the cell or not in control cells. In the second pDsRed-mRFP1 (Figure 1). approach APC-GFP at cellular protrusions were distinguished from cytoplasmic axin-puncta-associated APC-GFP and the Cell culture, transfections and treatments mean phase lifetime in those two regions were determined for a MDCK and HEK293 T cells were grown in Dulbecco’s number of cells. The cellular population average and standard modified Eagle’s medium supplemented with 10% fetal calf deviations were used in an unpaired t-test to determine serum (FCS) and 1% penicillin/streptomycin. SW480 cells whether the lifetimes in the different regions were statistically (Leibovitz et al., 1976) were grown in RPMI supplemented different with a 95% confidence interval. with 0.00108% 10–2 thioglycerol, 0.1 U/ml insulin, 50 mg/ml hydrocortisone, 10% FCS and 1% penicillin/streptomycin. Time-lapse imaging and analysis Cells were plated on 1.5mm optical glass coverslips in 35mm APC-GFP and N-terminally extended axin(12–956)-RFP dishes. HEK293 T cells were plated on coverslips coated with transfected HEK293 T cells were imaged 24 h post transfection fibronectin (1 mg/ml). MDCK cells were transfected using in indicator-free DME containing 10% FCS in an humidified Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA); SW480 atmosphere containing CO2 saturation at 37 1C using a and HEK293 T cells were transfected using FuGENE 6 BioRad MRC-1024 dual system confocal microscope (see (Roche Molecular Biochemicals, Mannheim, Germany) with above) equipped with a Nikon Plan Apo  60 water 0.1–2 mg plasmid DNA per dish. Microtubule assembly immersion with coverslip correction collar (NA1.2). Time- experiments were performed as described (Nakamura et al., lapse images were collected sequentially over 60–90 cycles 2001). Transfected MDCK cells were incubated on ice for every 12–15s and saved as uncompressed AVI files. Single 60 min, and then at 30 1C for 0.5, 1, 2 and 5 min and cells were particles from time-lapse images were filtered and tracked fixed rapidly and immunostained as described (Faux et al., using the ImageJ plugin ‘Spot Tracker’ (Sage et al., 2005). The 2004). For inhibition of the proteasome, cells were treated with analysis of the trajectories follows essentially the same 15 mM MG132 (Sigma) or dimethyl sulphoxide control, 6 h at approach as the Cherry laboratory (Wilson et al., 1996). For 37 1C. Antibody dilutions were as follows: 1:200 for axin, each track through i images, the MSD is calculated for APC, GSK3-b, CK1-a; 1:400 for b-catenin; anti- different time intervals ndt, n ¼ 1,2,y, n being the number of pS33,pS37,pT41- and pT41,pS35-phospho-b-catenin antibo- images between which the displacement is computed, and dt is dies were diluted 1:200 and combined to assess phosphorylated the time interval between images. These calculations were b-catenin (P-b-cat), 1:500 for fluorescently-labelled Alexa- made up to noi/2 to prevent insufficient averaging from Fluor-488, -405or -532secondary antibodies (Molecular overlapping segments of the track. The MSD values were Probes Invitrogen, Carlsbad, CA, USA). In the case of plotted as a function of time (ndt) and the particle mobility phospho-b-catenin antibodies, all experiments were performed assigned to simple diffusion (diffusion coefficient D, Equation with anti-pS33,pS37,pT41- and pT41,pS35-phospho-b-catenin b), diffusion þ directed motion (velocity v, Equation b) (Saxton

Oncogene Recruitment of APC and b-catenin to axin puncta MC Faux et al 5819 and Jacobson, 1997) motion or an approximation to con- Acknowledgements strained diffusion (v is an imaginary number in Equation b). We thank Dr M Prescott, Dr F Costantini and Dr D Capelluto MSD ¼ 4D ðndtÞðaÞ for cDNA constructs. MCF, MJL and AWB are supported by NH&MRC program grant 234703; AHAC is an NH&MRC MSD ¼ 4D ðndtÞþv2 ðndtÞ2 ðbÞ RD Wright Research Fellow.

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