Investigating the β-catenin nuclear import mechanism in yeast relies on the premise that a minimal, conserved β-catenin transport machinery exists in this organism. Therefore, we first tested whether β-catenin accumulates in the yeast nucleus. Specifically, we assessed the localization of a Xenopus β-catenin-GFP (xβ-catenin-GFP) in a wildtype yeast strain expressing an endogenously tagged nuclear envelope membrane protein, Heh2-mCherry, to help visualize the nuclear boundary. Indeed, xβ-catenin-GFP was enriched in the nucleus compared to GFP alone (Figure 1A). To quantify this steady state distribution, we measured the nuclear enrichment of xβ-catenin-GFP by relating the mean GFP fluorescence in the nucleus (N) and cytoplasm (C) (Figure 1A, plot at right). The xβ-catenin-GFP had a mean N:C ratio of ~1.6, which was significantly higher than GFP alone (1.1). As xβ-catenin-GFP is 119 kD, it would be unable to easily pass through the NPC diffusion barrier, suggesting that xβ-catenin-GFP can access a facilitated nuclear transport mechanism through the NPC (Popken et al., 2015; Timney et al., 2016).

In principle, three mechanisms of xβ-catenin nuclear import are possible: (1) xβ-catenin-GFP is imported by an NTR, (2) xβ-catenin-GFP piggybacks on an unknown binding partner that is itself imported by an NTR, or (3) xβ-catenin-GFP has an intrinsic ability to cross the NPC free of NTRs. To rule out the latter possibility, we tested whether xβ-catenin-GFP nuclear accumulation was dependent on a functional Ran gradient, which would specifically impact NTR-mediated transport (Schmidt and Görlich, 2016; Weis, 2003; Wente and Rout, 2010). We therefore assessed β-catenin-GFP localization in the mtr1-1 mutant strain, which is a temperature sensitive, loss of function allele in the gene encoding the yeast Ran-GEF (SRM1/MTR1/PRP20; Kadowaki et al., 1992). As Ran-GEF exchanges Guanosine-5’-diphosphate (GDP) for Guanosine-5’-triphosphate (GTP) on Ran in the nucleus, it is essential for the functioning of the nuclear transport system (Weis, 2003). At room temperature, xβ-catenin-GFP is localized in the nucleus with a N:C ratio similar to the wildtype strain (~1.6) (Figure 1B). In striking contrast, growth at 37°C, which is non-permissive for mtr1-1p function, resulted in the re-distribution of xβ-catenin-GFP such that it was evenly distributed between the nucleus and cytoplasm with N:C ratios identical to GFP alone (1.1) (Figure 1B). Importantly, xβ-catenin-GFP localization was not affected by the elevated temperature as wildtype cells showed N:C ratios of ~1.6 even at 37°C. Thus, nuclear accumulation of xβ-catenin-GFP is dependent on a functional Ran-GTP gradient raising the possibility that it requires a NTR-mediated pathway to accumulate in the nucleus.

Having established that β-catenin import requires a functional Ran pathway, we next sought to map the sequence elements of β-catenin that confer nuclear localization. β-catenin can be divided into three domains: a central region rich in armadillo (ARM) repeats sandwiched between two unstructured domains (Figure 2A). We generated constructs where each of these domains was individually deleted and examined their localization in yeast. Removal of the C-terminus significantly reduced nuclear enrichment of xβ-catenin-(1-664)-GFP compared to constructs lacking either the N (xβ-catenin-[141-782]-GFP) or ARM (Δ141-664) domains, which accumulated in the nucleus at levels similar to the full-length protein (Figure 2A–C). These data suggested that the C-terminus contains sequence elements required for nuclear accumulation. Consistent with this idea, the C-terminus of β-catenin (xβ-catenin-[665-782]-GFP) was sufficient to confer nuclear accumulation of GFP to levels comparable to the full-length protein (mean N:C 1.6; Figure 2A, B and D). Of note, both the ARM repeats (xβ-catenin-[141-664]-GFP) and the N-terminus of β-catenin (xβ-catenin-[1-141]-GFP) could confer some nuclear enrichment of GFP but to a considerably lesser extent than the C-terminus (mean N:C of ~1.3; Figure 2A, B and D). Additionally, the ARM repeats had some affinity for the nuclear periphery (Figure 2; xβ-catenin-[141-664]-GFP, white arrows). Of future interest, even the weaker nuclear accumulation driven by the N-terminal and ARM repeat sequences is dependent on a functional Ran pathway as xβ-catenin-1–664 does not accumulate in the nucleus in the mtr1-1 strain at the non-permissive temperature (Figure 2—figure supplement 1). Thus, when taken together, there are several elements of xβ-catenin that, in isolation, can target to the nucleus in a Ran pathway dependent fashion, but the C-terminus contains a sequence that was both necessary and sufficient for nuclear accumulation at levels comparable to the full-length protein. Consistent with previous work (Koike et al., 2004; Mis et al., 2020), these data suggested that the C-terminus of xβ-catenin contains the dominant NLS in β-catenin, which we further mapped to amino acids 665–745 (Figure 2A, B and E).

We confirmed that nuclear accumulation of xβ-catenin-(665-745)-GFP was dependent on the Ran pathway using the mtr1-1 strain (Figure 2—figure supplement 2A). To ensure that this sequence did not confer binding to a yeast-specific factor, we also tested localization of xβ-catenin-(665-745)-GFP in HEK293T cells, a human embryonic kidney cell line. In line with the yeast results, xβ-catenin-(665-745)-GFP showed higher levels of nuclear accumulation compared to GFP alone (Figure 2—figure supplement 2B). Furthermore, to test if this NLS is required for β-catenin localization in human cells, we also examined human β-catenin that lacks aa 665–745 in HEK293T cells. Indeed, deletion of aa 665–745 (hβ-catenin-[∆665–745]-GFP) led to a significant reduction in nuclear to cytoplasmic ratio compared to the full-length hβ-catenin localization (Figure 2—figure supplement 2C).

Next, we investigated the function of the β-catenin NLS in the context of Wnt signaling using the secondary axis assay in Xenopus (McMahon and Moon, 1989; Smith and Harland, 1991; Sokol et al., 1991). Overexpression of Wnt effectors including β-catenin induces a secondary axis in Xenopus embryos. By injecting a moderate dose (200 pg) of xβ-catenin mRNA, secondary axes develop in roughly half of the embryos (Figure 2—figure supplement 3A-B) compared to none in the uninjected controls (UICs). If we delete the coding sequence for the NLS (xβ-catenin-[∆665–745]-GFP) then the number of embryos with secondary axes is significantly reduced (Figure 2—figure supplement 3A-B). To ensure that this loss of function is due to the inhibition of β-catenin nuclear import, we added the classical NLS of the SV40 large T-antigen (cNLS), which is imported by Kap-α/β1, to the N-terminus of this construct (cNLS-xβ-catenin-[∆665–745]-GFP). cNLS-xβ-catenin-(∆665–745)-GFP could induce secondary axes similarly to the full-length β-catenin (Figure 2—figure supplement 3B), consistent with the conclusion that xβ-catenin-(∆665–745)-GFP was still functional for Wnt signaling but lacked the nuclear localization element. Supporting this supposition, we imaged xβ-catenin-GFP in these Xenopus embryos and found that while xβ-catenin-GFP localizes to the cell membrane and nucleus, xβ-catenin-(Δ665–745)-GFP shows a reduction in nuclear accumulation, which is rescued by the addition of the cNLS (Figure 2—figure supplement 3C).

Next, to define the NTR responsible for xβ-catenin-GFP nuclear import, we used the Anchor-Away approach (Haruki et al., 2008) to systematically inhibit 10 budding yeast NTRs, all of which have orthologs in human cells (Table 1). This strategy takes advantage of the rapamycin-induced dimerization of a FK506 binding protein (FKBP12) with the FKBP-rapamycin binding (FRB) domain (Figure 3A). In this system, NTR-FRB fusions are expressed in a strain harboring FKBP12 fused to a highly abundant plasma membrane protein (Pma1) (Figure 3A). The addition of rapamycin leads to the rapid (~15 min) trapping of the NTRs at the plasma membrane (Haruki et al., 2008). We systematically tested whether the addition of rapamycin (or the DMSO carrier alone) impacted the nuclear accumulation of xβ-catenin-(665-782)-GFP in each of the 10 NTR-FRB strains. Consistent with prior data (Fagotto et al., 1998), plasma membrane trapping of the Kapβ1 ortholog, Kap95-FRB, did not impact nuclear localization of xβ-catenin-(665-782)-GFP (Figure 3B). Indeed, trapping 9 of the 10 NTRs, including the IPO11 ortholog, Kap120, had no overt influence on xβ-catenin-(665-782)-GFP nuclear localization (Figure 3B and C, and Figure 3—figure supplement 1). In contrast, we observed a remarkable inhibition of nuclear accumulation, specifically when Kap104-FRB (ortholog of Kapβ2/Transportin-1[TNPO1]) was anchored away (Figure 3B and C). These data support a model in which Kap104 specifically mediates the nuclear import of xβ-catenin-(665-782)-GFP in the yeast system.

Having established that Kap104 mediates β-catenin nuclear transport in yeast, we compared the xβ-catenin-(665-782) protein sequence to established TNPO1 NLS (e.g. PY-NLS; Lee et al., 2006; Soniat and Chook, 2015; Soniat et al., 2013). By close inspection, the xβ-catenin amino acid sequence (665-703) does conform to the loose PY-NLS consensus with a hydrophobic methionine (M) in place of a tyrosine (Y; Figure 4A; Soniat and Chook, 2015); this NLS is conserved across vertebrate species (Figure 4A). We therefore mutated the PM motif in β-catenin by substituting the proline (P) and methionine (M) residues for tandem alanine (A) amino acids. We tested whether these changes impacted the ability of the xβ-catenin-(665-703) to import a GFP fusion to three maltose binding proteins. MBP(x3)-GFP is constitutively excluded from the nucleus due to its large molecular weight (149 kDa) (Figure 4B; Popken et al., 2015). Fusion of the xβ-catenin-(665-703) can confer nuclear localization of this large fusion protein. Furthermore, this localization is dependent on the PM motif as substitution of PM with AA abolishes nuclear localization (Figure 4B). These amino acids were also critical for human (h)β-catenin-(665-782)-GFP nuclear accumulation in human cell lines (HeLa) as the PM to AA substitution reduced the mean N:C ratios of this construct from 2.5 to 1.5 (Figure 4C). Thus, the PM sequence in the β-catenin PY-NLS is required for nuclear import in yeast and human cells.

To test that the β-catenin-NLS is directly recognized by TNPO1, we generated recombinant TNPO1 and Glutathione S-transferase (GST) fusions of human β-catenin (GST-hβ-catenin) and human β-catenin containing the PM-AA mutations (GST-hβ-catenin P687A, M688A). We immobilized these GST fusions (and GST alone) on GT Sepharose beads and tested binding to purified TNPO1. We observed specific binding of TNPO1 to the GST-hβ-catenin (Figure 5A), which was disrupted by the PM to AA mutations in the NLS. Furthermore, as NTR-NLS interactions are disrupted by the binding of Ran-GTP to the NTR in the nucleus, we tested the Ran-GTP sensitivity of the TNPO1:β-catenin complex. We generated recombinant RanQ69L, which cannot hydrolyze GTP and confirmed that RanQ69L-GTP specifically interacted with TPNO1 and not to β-catenin (Figure 5—figure supplement 1B). Next, we assessed the formation of the TNPO1:β-catenin complex in the presence or absence of RanQ69L-GTP. Adding RanQ69L-GTP specifically disrupted β-catenin binding to TPNO1, indicating that the TNPO1:β-catenin complex reflects the formation of a canonical NTR-NLS import complex (Figure 5B). When taken together, these data establish a model in which TNPO1 imports β-catenin through a direct interaction with its PY-NLS that is modulated by TPNO1-binding to Ran-GTP.

To explore the function of TNPO1-mediated import of β-catenin in vertebrates, we applied two different Wnt signaling assays: (1) a TCF/LEF reporter and (2) Xenopus secondary axis development. First, once β-catenin enters the nucleus, it binds to the TCF/LEF complex to activate transcription of Wnt responsive genes (Molenaar et al., 1996; van de Wetering et al., 1997; van de Wetering et al., 1991). A well-established reporter assay (commonly known as TOPFLASH) places the TCF/LEF DNA-binding element upstream of a reporter such as GFP or luciferase (Molenaar et al., 1996; van de Wetering et al., 1991). In Xenopus tropicalis, the Tg(pbin7Lef-dGFP) line has seven tandem TCF/LEF DNA binding sites upstream of GFP and is an effective reporter of Wnt signaling (Borday et al., 2018; Denayer et al., 2008). We crossed heterozygous transgenic animals with a wildtype animal such that half of the resultant progeny had the transgene. In vertebrates, the tnpo1 gene is duplicated (tnpo1 and tnpo2), and both paralogs have nearly identical sequence and function (Figure 6—figure supplement 1; Dormann et al., 2010; Rebane et al., 2004; Twyffels et al., 2014). Therefore, we injected single guide RNAs (sgRNAs) targeting both tnpo1 and tnpo2 with Cas9 protein at the one cell stage and raised embryos to st10 before fixing them. Because GFP fluorescence is undetectable at these early stages, we used whole mount in situ hybridization (WMISH) to visualize GFP transcripts as an assay for Wnt reporter activation and used sibling embryos without the transgene as a WMISH negative control. When we depleted both tnpo1 and tnpo2 using F0 CRISPR, significantly more embryos had weak expression of the GFP transgene compared to UIC embryos (Figure 6A, see images of embryos stained for GFP transcripts as key to histogram). This result was specific as the second set of non-overlapping sgRNAs gave comparable results (Figure 6A, tnpo1/2 sgRNAs#2). Importantly, we detected deleterious gene modification at the appropriate targeted sites using Inference of CRISPR Edits (ICE) analysis (Figure 6—figure supplement 2).

We next tested the function of mouse Tnpo1/2 in a stable transgenic mouse fibroblast cell line in which luciferase is expressed under the control of TCF/LEF DNA-binding elements. To activate Wnt signaling, we transfected a full-length human β-catenin-GFP that increased luciferase signal 7.7-fold over transfection of GFP alone (Figure 6B). Then we measured luciferase activity in transgenic fibroblasts transfected with hβ-catenin-GFP in which we depleted transcripts of TNPO1 or TNPO2 (alone or simultaneously) using specific siRNAs. Compared to control siRNA, depletion of either TNPO1 or TNPO2 led to a 34% reduction in luciferase signal (Figure 6B). By targeting both transcripts, the luciferase signals were reduced by 64% (Figure 6B). By western blot, we observed the production of hβ-catenin-GFP and the specific reduction of TNPO1/2 (Figure 6—figure supplement 3). We next examined the dependence of β-catenin nuclear localization on TNPO1/2 in colorectal cancer cell lines, HCT-116 and DLD-1, which harbor an activating Ser45 β-catenin mutation and a deactivating frameshift mutation in APC, respectively. Similar to the TCF/LEF mouse fibroblast cell line, TNPO1/2 depletion by siRNA reduced β-catenin levels specifically in nuclear fractions compared to control siRNA condition (Figure 6—figure supplement 4). Taken together, TNPO1/2 is required for β-catenin nuclear localization across a wide range of species: Xenopus, mouse, and human cancer cell lines.

Having established the importance of TNPO1/2 for β-catenin nuclear localization in vertebrates, we next evaluated the specific impact of inhibiting β-catenin nuclear import by testing the function of the PY-AA mutant in the Xenopus secondary axis assay. We compared the number of secondary axes induced by the wildtype hβ-catenin mRNA to a PM to AA mutated version (P687A, M688A). We noted a significant reduction in the number of secondary axes induced by the PM-AA mutant β-catenin (Figure 6C). If we add the cNLS to the N-terminus of the PM-AA mutant then induction of secondary axes is rescued, suggesting that the PM-AA mutant fails to enter the nucleus to activate Wnt signaling (Figure 6C).

Having established that TNPO1 binds directly to a PY-NLS and imports β-catenin into the nucleus, we wondered whether direct perturbation of the β-catenin-TNPO1 interaction could, in principle, be a viable therapeutic strategy. We therefore took advantage of the prior design of a potent TNPO1 peptide inhibitor, M9M, that binds with high affinity to the TNPO1 NLS binding site (Cansizoglu et al., 2007). We tested whether this peptide could inhibit Wnt signaling in the mouse fibroblast TCF/LEF luciferase reporter cell line (Cansizoglu et al., 2007). Excitingly, transfection of the M9M peptide reduced luciferase activity in a dose-dependent manner, regardless of whether activation was induced with a Wnt ligand (Wnt3a) or by co-transfection with human β-catenin (Figure 7A, Figure 7—figure supplement 1A-B).

The M9M peptide is a chimera of the NLSs of heterogeneous nuclear ribonucleoprotein (hnRNP) M and A1 (Cansizoglu et al., 2007). To test the specificity, we leveraged the understanding of the key amino acids that confer binding to TNPO1 in each individual NLS to design a control M9M-A peptide. M9M-A contains seven amino acid substitutions that would reduce binding to TNPO1 (Figure 7—figure supplement 1C). The M9M-A peptide only reduced luciferase activity by 24% (compared to 45% by M9M at the similar dosage; Figure 7B).

As the M9M peptide inhibits the nuclear import of a multitude of TNPO1/2 cargos (Cansizoglu et al., 2007), we sought to ensure that the M9M-mediated inhibition of Wnt signaling was specifically due to the reduced nuclear import of β-catenin. We therefore transfected a human β-catenin with a cNLS, which would be imported by Kap-α/Kap-β1 and thus be insensitive to M9M inhibition. The cNLS-human β-catenin could drive the luciferase reporter to levels comparable to human β-catenin, but the M9M peptide only reduced this signal by ~15% (Figure 7B). Thus, these data support the conclusion that the M9M peptide’s impact on Wnt signaling is due, at least in part, to the inhibition of β-catenin nuclear transport via TNPO1/2. Together, Wnt signaling can be inhibited by blocking TNPO1-mediated import of β-catenin either by mutating the β-catenin NLS or competitive inhibition with the M9M peptide.

Further information and request for reagents may be directed to and will be fulfilled by the lead contact, Mustafa K. Khokha (Mustafa.khokha@yale.edu).

X. tropicalis and Xenopus laevis were maintained and cared for in accordance with the Yale University Institutional Animal Care and Use Committee protocols. In vitro fertilization was performed as per standard protocols (del Viso and Khokha, 2012; Sive et al., 2007).

All yeast strains used in this study are listed in Supplementary file 1. Yeast strains were grown at 30°C, unless indicated otherwise in yeast extract peptone dextrose (YPD) medium (1% bacto yeast extract, 2% bactopeptone, 2% glucose, and 0.05% adenine sulfate). Transformation of yeast was carried out using standard protocols (Amberg and Strathern, 2005).

HEK293T and HeLa cells were maintained and cultured with Dulbecco's Modified Eagle Medium (DMEM) medium + 10% fetal bovine serum + 1% penicillin and streptomycin in a T-75 flask. The engineered 3T3 mouse fibroblast cell line that stably expresses the TCF/LEF luciferase transgene was maintained with DMEM medium + Cell Growth Medium Concentrate (Enzo life sciences). Further experimental procedures for the 3T3 cell line can be found under TCF/LEF luciferase assay method section. Upon 70–80% cell confluency, cells were transfected with plasmids using jetPRIME (Polyplus-transfection) following the manufacturer’s instructions. Cells were fixed with 4% paraformaldehyde/PBS and further processed for immunofluorescence 24–48 hr post-transfection. Antibodies used in this study are listed in Key resources table. Cells were tested for mycoplasma using MycoAlert Detection Kit (Lonza).

Key resources and all plasmids used in this study are listed in Key resources table and Supplementary file 2, respectively. Xenopus β-catenin-GFP (Addgene #16839), Xenopus cNLS-β-catenin-GFP (Addgene #16838), GST-human β-catenin (Addgene #24193), and NLS-mCherry (Addgene #49313) plasmids were obtained from Addgene. GST-transportin 1 (TNPO1) and Gal-MBP(x3)-GFP plasmids were generous gifts from Dr. Yuh-min Chook at UTSW and Dr. Liesbeth M. Veenhoff at University of Groningen, respectively. Gibson Assembly (New England Biolabs) was used to generate GFP-tagged Xenopus β-catenin truncation constructs following the manufacturer’s instructions. Both the human and Xenopus β-catenin P687A, M688A variants were generated using Q5 site- directed mutagenesis (New England Biolabs) following the manufacturer’s instructions. Subsequently, the β-catenin constructs were sub-cloned into the pRS406 vector containing an ADH1 promotor for yeast studies or a pCS2+ vector for mammalian/Xenopus studies. mRNAs were generated using the SP6 mMessage machine kit (Thermo Fisher Scientific) and RNA clean & concentrator kit (Zymo Research) following the manufacturer’s instructions. We obtained siRNAs directed against mouse TNPO1 (s108857), mouse TNPO2 (s102754), and a control siRNA (4390843) from Thermo Fisher Scientific. To generate CRISPR sgRNAs, we used the EnGen sgRNA synthesis kit (NEB) following the manufacturer’s instructions with the following targeting sequences tnpo1 sgRNA#1 (5’-GGCATGGGGGCCACCTCTTG-3’), tnpo1 sgRNA#2 (5’- GGGTTACGTTTGTCCTCAAG-3’), tnpo2 sgRNA #1 (5’- GGGCGTTTAGCCGCGTTCTA-3’), and tnpo2 sgRNA #2 (5’- GGCGTCATGGATGAGTCCGA-3’) (designed using CRISPRscan Moreno-Mateos et al., 2015). CRISPR experiments in wildtype or transgenic X. tropicalis were performed as previously described (Bhattacharya et al., 2015). CRISPR gene editing efficiency was assessed using Synthego ICE (ice.synthego.com) as previously described (Sempou et al., 2018). The M9M peptide (GGSYNDFGNYNNQSSNFGPMKGGNFGGRFEPYANPTKR) and the M9M-A peptide (GGSYNDFGNYNNQSSNAAAAKGGNFGGAFEAAANPTKR) were synthesized by LifeTein.

Expression plasmids containing full length and fragments of the β-catenin-GFP coding sequence under the control of the ADH1 promoter were transformed into the W303, Heh2-mCherry::NAT (BWCPL1314) strain. A yeast colony that incorporated the plasmid sequence was cultured and mounted onto a coverslip for live imaging on a DeltaVision wide-field microscope (GE Healthcare) with a CoolSnapHQ² CCD camera. Yeast fluorescent images were deconvolved using the iterative algorithm sofWoRx. 6.5.1 (Applied Precision, GE Healthcare). β-catenin-GFP transfected HeLa or HEK293T cells were mounted on Pro-Long Gold coated coverslip for imaging on a Zeiss Axio Observer microscope. All fluorescent images were analyzed with Fiji software. For quantification, the oval selection tool was used to draw a circle in both the nuclear and cytosolic regions on the same image plane to measure florescence intensity.

X. laevis embryos were injected with a mixture of either 200 pg of Xenopus or human β-catenin-GFP mRNA and cNLS-mCherry mRNA in one of four cells (targeting the ventral side). Embryos were assessed for a secondary axis via stereomicroscopy at stage 17–19. For the β-catenin localization experiments by fluorescence, stage 10 embryos were fixed in 4% paraformaldehyde/PBS at 4°C overnight on a nutator. Embryos were washed in 1× PBS + 0.1% TritonX-100, and the dorsal blastopore lips were sectioned with a razor blade and mounted on Pro-Long Gold (Invitrogen) coated coverslip before imaging on a Zeiss 710 confocal microscope.

To employ the Anchor-Away approach, we used a yeast strain that harbors a FKBP12 fusion of the endogenous plasma membrane H⁺-ATPase (PMA1 gene), a Heh2-mCherry fusion to mark the nuclear envelope and a mutated TOR1 gene (HHY110: HEH2-mCherry::KAN, PMA1-2xFKBP12, fpr1::NAT tor1-1). In this strain, individual, endogenous NTRs are tagged with the FRB domain at the C-terminus by homologous recombination of a PCR product that contains an FRB sequence, a 3× HA epitope, and a selective marker, HIS3, flanked by a 60 bp homology arm of endogenous NTR coding sequence (Figure 3—figure supplement 2 and Supplementary file 3 for FRB cloning primers; Haruki et al., 2008; Longtine et al., 1998). Integration of an FRB sequence is confirmed by colony PCR using a gene-specific forward and a plasmid specific reverse primer and rapamycin-induced cell death for essential NTRs (Figure 3—figure supplement 2 and Supplementary file 3 for colony PCR primers). Subsequently, an expression plasmid containing the coding sequence for xβ-catenin (665-782)-GFP under the control of the ADH1 promoter was transformed into the Anchor-Away line. These lines were treated with 1 mg/ml of rapamycin (5–15 min of incubation) or vehicle alone (DMSO) before imaging.

A 3T3 mouse fibroblast cell line that has a stable integration of the luciferase reporter gene under the Wnt responsive TCF/LEF promoters was used for this assay (Enzo life sciences). Cells were maintained with DMEM medium + Cell Growth Medium Concentrate (Enzo life sciences). Prior to the transfection, cells were seeded on a 24 well plate in media containing DMEM + Cell Assay Medium Concentrate (Enzo life sciences). Subsequently, cells were transfected with siRNA (25 pmol) first, then GFP or human β-catenin-GFP DNA (0.5 µg) for 48 hr and 24 hr, respectively, using JetPRIME per the manufacturer’s instructions. Luciferase was quantified using the Luciferase Assay System (Promega) and the Promega Glomax luminometer according to the manufacturer’s instructions. For the M9M peptide experiment, cells were transfected with an M9M or M9M-A peptide dose ranging from 0.635 µg to 0.5 µg using ProteoJuice Protein transfection following the manufacturer’s instructions for 20 hr, and Wnt signaling was activated either by Wnt3a ligand (50 ng/ml) or human β-catenin-GFP DNA (0.5 µg) for 16–24 hr.

Heterozygous X. tropicalis Tg(pbin7Lef-dGFP) were crossed with wildtype X. tropicalis. Fertilized embryos were injected with sgRNAs targeting tnpo1 and tnpo2 and Cas9 protein at one-cell stage and collected at stage 10 for in situ hybridization as previously described (Khokha et al., 2002). Digoxigenin-labeled anti-sense GFP probe was used to detect GFP transcript expression. Progeny that did not carry the transgene was used as a negative control.

3T3 mouse fibroblast cells were lysed in radioimmunoprecipitation assay (RIPA) buffer to harvest protein samples. Nuclear and cytoplasmic proteins were extracted from colorectal cancer cell lines (HCT-116 and DLD-1) following the manufacturer’s instructions (NE-PER Thermo Fisher). Protein levels are normalized, and immunoblots were carried out in bolt 4–12% Bis-Tris plus gels following standard protocols. Antibodies used in this study are listed in Key resources table.

pGEX-6P1, pGEX-human β-catenin, pGEX-human transportin 1, or pET28a-His6-RanQ69L were transformed into the BL21 Escherichia coli strain and cultured in Lysogeny broth (LB) with antibiotics to mid-log phase (OD₆₀₀ 0.6–0.8). To induce expression of the recombinant proteins (GST alone, GST-hβ-catenin, GST-hTNPO1, and His6-hRanQ69L), isopropyl β-d-1-thiogalacto pyranoside was added at a final concentration of 1 mM for 3 hr. All cultures were harvested in 50 ml batches and stored at –80°C until further use. Glutathione Sepharose (GT) beads (Millipore Sigma) were washed and equilibrated in lysis buffer 1 (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM MgCl₂, 10% glycerol, 0.05% NP-40, 1 mM DTT, and protease inhibitor cocktail mix [Millipore Sigma]). Bacterial pellets containing GST tagged proteins were resuspended with the ice cold lysis buffer 1, sonicated, and spun down at 4°C at 30,000× g for 20 min. The supernatant was collected into a new 50 ml conical tube and incubated with 200 µl of GT bead slurry for 1 hr at 4°C. Subsequently, the GST-GT bead slurry was collected and washed with lysis buffer 1 (excluding the protease cocktail mix). The GST tag was removed from hTNPO1 using proTEV Plus Protease (Promega), and the protease enzyme was further removed from hTNPO1 protein by Ni-NTA Magnetic Beads (NEB) as per the manufacturer’s instructions. To pulldown His6-RanQ69L, bacterial pellets were resuspended with ice cold lysis buffer 2 (50mM Tris pH 7.4, 500 mM NaCl, 2 mM MgCl2, 20 mM Imidazole, 10% glycerol, 0.05% NP-40, and protease inhibitor cocktail mix), sonicated, and spun down at 4°C at 30,000× g for 20 min. The supernatant was collected into a new 50 ml conical tube and incubated with 200 µl of equilibrated Ni NTA bead slurry for 1 hr at 4°C. The His-Ni bead slurry was collected and washed with lysis buffer 2. His6-RanQ69L was eluted from Ni beads by adding the elution buffer (50 mM pH Tris 7.4, 500 mM NaCl, 2 mM MgCl2, 500 mM Imidazole, 10% glycerol, 0.05% NP-40, and protease inhibitor cocktail mix) and rotating at 4°C for 1 hr. The supernatant containing His-RanQ69L was collected by centrifugation at 500× g for 3 min and dialyzed in the buffer (50 mM pH Tris 7.4, 150 mM NaCl, 2 mM MgCl2, 10% Glycerol, and 1 mM PMSF) at 4°C overnight. Subsequently, RanQ69L was incubated in GTP buffer (400 uM GTP in 5 mM EDTA) 4°C for 1 hr. hNTPO1 protein was incubated with GT beads preloaded with GST fusion protein (GST alone or GST-hβ-catenin) for 1 hr at 4°C. Then, RanQ69L protein (dialysis buffer + GTP or RanQ69L) was added to the mix for 1 hr at 4°C. The beads were washed with the lysis buffer and eluted with SDS-PAGE sample buffer. Protein samples were separated by SDS-PAGE and detected with Coomassie (BioRad).

Statistical significance was defined as p<0.05 (*), 0.002 (**), 0.0002 (***), and 0.0001 (****). The double axis assay and in situ data were analyzed by Fisher’s exact tests. Otherwise, unpaired two-tailed Student’s t-tests or a one-way ANOVA test were used to determine significance of mean ratio of nuclear to cytosolic fluorescence intensity in GraphPad Prism 8.4.3.