APC mutations disrupt β-catenin destruction complex condensates organized by Axin phase separation

The Wnt/β-catenin pathway is critical to maintaining cell fate decisions. Recent study showed that liquid–liquid-phase separation (LLPS) of Axin organized the β-catenin destruction complex condensates in a normal cellular state. Mutations inactivating the APC gene are found in approximately 80% of all human colorectal cancer (CRC). However, the molecular mechanism of the formation of β-catenin destruction complex condensates organized by Axin phase separation and how APC mutations impact the condensates are still unclear. Here, we report that the β-catenin destruction complex, which is constructed by Axin, was assembled condensates via a phase separation process in CRC cells. The key role of wild-type APC is to stabilize destruction complex condensates. Surprisingly, truncated APC did not affect the formation of condensates, and GSK 3β and CK1α were unsuccessfully recruited, preventing β-catenin phosphorylation and resulting in accumulation in the cytoplasm of CRCs. Besides, we propose that the phase separation ability of Axin participates in the nucleus translocation of β-catenin and be incorporated and concentrated into transcriptional condensates, affecting the transcriptional activity of Wnt signaling pathway. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-023-05068-0.

penicillin and 100 mg/mL streptomycin; the other cells were grown in RPMI 1640 (Gibco, 11875176) with 10% FBS, 100 units/mL penicillin and 100 mg/mL streptomycin.All cells were cultured at 37°C in a humidified atmosphere of 5% CO2.Transient transfections were performed with Lipofectamine 2000 (Invitrogen, 11668019) following the manufacturer's guidelines.When double transfection was needed, equal amounts of both plasmids were simultaneously transfected.

CRISPR/Cas9 knock-in in cells
For introduction of pScarlet at the N-terminus of Axin CRISPR/Cas9 lentivirus and Axin donor lentivirus were purchased from Obio Technology (Shanghai, China).Axin guide RNAs (gRNAs) were designed using the online CRISPR design tool (http:// CRISPR.mit.edu/).The Axin gRNA sequences was GTGAGCGACGAGTTTGACTGTGG.The plasmids were 3FLAG pLenti-U6-spgRNA v2.0-CMV-Puro-P2A-3Flag-spCas9 and pAdenoMCMV-MCS.The experiment was performed according to the lentivirus manual.Cells were selected and plated as single cells in a 96-well plate using a BD Fortessa system.After several weeks of incubation, wells containing single-cell colonies were used to expand the culture, and DNA was extracted for genotyping using the Animal Tissue DNA Isolation Kit (Fore Gene).For determination of the copy number of correct gene edits, the genomic sequence of the genes of interest was sequenced by Ruibiotech.
To obtain single clones of Axin、CTNNB1 KO cells, cells selected with puromycin for 3 days and isolated by colony formation assay or single cell culture.The single clones were validated by immunoblotting analysis and DNA sequencing.

TOP/FOP-Flash reporter assay
The TOP/FOP-Flash was co-transfected into cells along with wild-type Axin or NLS-mutated Axin in Axin KO SW480 cells.After transfection for 48 h, the cells were harvested for analysis with the Dual-Luciferase Reporter Assay System (Promega, USA) according to the manufacturer's instructions.The luciferase activity was measured using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer, USA).The luciferase intensity was normalized to the Renilla luciferase activity to standardize for transfection efficiency.
For Co-IP, SW480 cells were cultured in a 100 mm culture dish.Axin-Flag, APC R876* -GFP and APC R1450* -GFP were transfected respectively.1× lysis buffer supplemented with PMSF.After centrifuging at 12,000×g for 15 min at 4 °C, per 300 μL of the supernatant was added with 20 μL Protein A/G PLUS-Agarose (Santa Cruz) and incubated with the Anti-Flag antibody (Dia-an) or anti-GFP antibody (ImmunoWay) overnight at 4 °C.The next day, the immunoprecipitants were washed five times at 2000×r for 2 min at 4 °C and boiled with 1× Loading Buffer.At last, Western blotting assay was conducted according to the protocol above.Here, IgG (Proteintech) was used as a negative control.
Nuclei were washed once in buffer A and then lysed.The resulting two fractions were resuspended in the Laemmli buffer, boiled for 15min and resolved by SDS-PAGE for western analysis.

Immunofluorescence (IF) and confocal microscopy
Cells were seeded in a 24-well plate (Corning) with a coverslip in each well.The following day, the cells were fixed for 30 minutes with 4% PFA and treated with 0.5% Triton X-100 for 20 minutes at room temperature.After normal goat serum (ZSGB-Bio) blocking for 30 minutes at 37°C, the cells were incubated with primary antibody for 16 h at 4°C.After PBS washes, the corresponding fluorochrome-labeled secondary antibody was applied for 30 minutes at room temperature in the dark, and the samples were washed and then mounted with DAPI (Sigma, D9542).Following washes in PBS, coverslips were mounted onto slides with Vectashield (Solarbio) and sealed using transparent nail polish.Images were acquired on a confocal laser-scanning microscope (Zeiss LSM880, 63× oil objective, NA 1.4, 1 Airy Unit).
For multiplexed IF, it was carried out by using the PerkinElmer-Opal-Kit (Akoya Biosciences) according to the manufacturer's instructions.Briefly, these FFPE tissues were dewaxed with xylene, then rehydrated through a graded ethanol series, and fixed with neutral-buffered formalin prior to antigen retrieval that was performed with AR6 Buffer using high-pressure incubation.This step was followed by cooling, blocking, and serial staining with primary antibodies, HRPconjugated polymers, and opal fluorophores; cycles were repeated until all markers were stained.
Finally, the nuclei were counterstained with DAPI.Images were acquired on a confocal laserscanning microscope (Zeiss LSM880).

Video 1 legend
Axin overexpression in SW480 cells induces cytoplasmic liquid condensates, related to Supporting Figure 3F.

Figure S3 (
Figure S3 (A-B) The CRISPR/Cas9 system was applied to generate CTNNB1 KO cell from HEK293T (A) cells and Axin KO cell lines from SW480 cells (B).The efficiency of CTNNB1 KO and Axin KO were examined by Western-blotting.CTNNB1 KO-1 and Axin KO-1 are the two KO clones used in this study.(C) Representative images during OptoDroplet activation in β-catenin KO HEK293T cells expressing Cry2-mcherry-β-catenin and Cry2-mcherry.Time intervals between illuminating blue light and image

Figure S4 (
Figure S4(A-B) Predictions and annotation for intrinsically disordered APC protein by the MobiDB database (A) and IUPred web (B).The result indicates that APC is also a complex multidomain scaffolding protein

Figure S5 (
Figure S5 (A)The graphical view shows the APC protein domains and the positions and species of specific mutations.The length of the line connecting the mutation annotations to the protein domains indicates

Figure S7 (
Figure S7 (A-B) The NLS (A) and NES (B) amino acid sequence of β-catenin were predicted by PSORT II.(C) Schematic diagram of NLS-mutated or NES-mutated β-catenin derivatives tagged EYFP.Ectogenic NLS, predicated NLS and NES are indicated by the green box, red box and blue box, respectively.NLS, nuclear localization signals; NES, nuclear export signals; WT, wild type.(D) Representative images of NLS wild-type but NES-mutated (up) or NLS-mutated (down) β-catenin derivatives in SW480 and HCT116.