β-catenin condensation facilitates clustering of the cadherin/catenin complex and formation of nascent cell-cell junctions

Domain: physics
Published: 2025-12-06
Editorial outcome: major_revision

Abstract
Cadherin-based junctions establish dynamically regulated adhesion between cells to coordinate tissue integrity and morphogenetic movements. Adhesion strength can be modulated by the organization of individual cadherin complexes into lateral clusters. Here, we identify a clustering mechanism of the cadherin complex established by its core component β-catenin. We show that the disordered termini of β-catenin drive the formation of condensates that incorporate other components of the cadherin complex in vitro. Using β-catenin mutants with hampered condensation, we demonstrate that β-catenin condensation nucleates the formation of submicron cadherin/catenin clusters that further develop into stable sites of adhesion. Furthermore, we show that β-catenin-dependent clustering ensures the efficient formation of de novo cell-cell adhesions. Our data thus indicate a role for β-catenin condensates in the supramolecular organization of the cadherin complex, and reveal that the function of β-catenin in the cadherin complex extends beyond connecting cadherin to α-catenin and the actin cytoskeleton.

Introduction
Cadherin-based adherens junctions are integral to epithelial cohesion, by establishing cell-cell adhesion through cadherin trans-interactions between neighboring cells and intracellular connections with the actin cytoskeleton1. During development and homeostasis, epithelial cohesion must be tightly coordinated with morphogenetic movements, which requires dynamic regulation of the adhesive properties of cadherin junctions2–4. This can be achieved by regulating the organization of cadherin complexes into lateral clusters on the cell surface, increasing the avidity of intrinsically weak cadherin trans-interactions5–8. Across different cell types and species various cadherin clusters have been identified, ranging from nano- to microscale supramolecular structures (reviewed by refs. 9,10). The formation of these clusters is mainly attributed to cis-interactions between ectodomains of E-cadherin and association with the actin cytoskeleton that, for instance, limit lateral diffusion11–18. Nonetheless, the molecular mechanisms regulating clustering, including the contribution of other cadherin-complex components, is incompletely understood10. Moreover, how clustering of the cadherin complex impacts adhesion dynamics in epithelial tissues remains to be answered.

Over the last years it has become clear that clustering of proteins at the membrane, including transmembrane receptor complexes, is facilitated by liquid-liquid phase separation19–21. This biophysical process concentrates proteins into biomolecular condensates through weak multivalent interactions, for instance between intrinsically disordered regions (IDRs) enriched for polar, charged, or aromatic amino acids22–24. Biomolecular condensates typically exhibit liquid-like properties, and by locally concentrating proteins, these dynamic structures can catalyze enzymatic reactions, sequester molecules, or serve as a structural organization platform25,26. The formation of condensates has recently been implicated in the assembly and regulation of cell adhesion complexes27. The tight junction (TJ) scaffolding zonula occludens (ZO) proteins form biomolecular condensates and thereby organize into membrane-attached compartments that locally enrich other components of this complex during formation of nascent TJs28,29. Multiple focal adhesion (FA) proteins exhibit condensation behavior, which contributes to integrin clustering, and the assembly and dynamics of FAs30–32. Whether formation of biomolecular condensates also contributes to the supramolecular organization of other adhesion complexes, including cadherin-based adhesions, remains to be determined.

β-Catenin is a central component of the cadherin complex1. It binds to E-cadherin immediately following synthesis of E-cadherin at the membrane of the endoplasmic reticulum, and establishes the link with α-catenin and consequently the actin cytoskeleton after their arrival at the plasma membrane33,34. The cellular function of β-catenin extends beyond cell-cell adhesion, as it also acts as transcriptional transactivator in the Wnt-signaling pathway35. The transcriptional role of β-catenin was recently shown to be supported by its capability to form biomolecular condensates, relying on weak intermolecular interactions through its structurally disordered N- and C-termini36–38. β-Catenin partitions into condensates at transcriptional enhancer elements, thereby promoting transcription of Wnt-target genes36. In this study, we investigated whether the condensation of β-catenin also impacts the formation and organization of cell-cell junctions. We find that β-catenin forms phase-separated droplets in vitro in the presence of E-cadherin and α-catenin and co-partitions these other cadherin complex components into condensates. Using β-catenin mutants with impaired condensate formation capacity, we demonstrate that β-catenin condensation promotes its clustering together with E-cadherin and α-catenin at the cell cortex and facilitates the formation of nascent cell-cell junctions. Our data thus indicate a role for β-catenin condensates in the supramolecular organization of the cadherin complex, and provide evidence for the importance of cadherin-complex clustering in establishing adhesion between two opposing cells.

Results
β-Catenin co-partitions with other components of the cadherin complex in vitro
The contribution of β-catenin condensate formation to its transcriptional function36 raises the question whether and how the condensation of β-catenin is linked to its function in cell-cell adhesion complexes (Fig. 1A, left). Incorporation in the cadherin complex through interactions with E-cadherin and α-catenin could impair β-catenin condensate formation, or alternatively, β-catenin may integrate these other components of the cadherin complex into condensates. To test these possibilities, we analyzed the formation of phase-separated droplets of purified β-catenin in vitro in the presence of E-cadherin and α-catenin, using recombinant β-catenin, the cytosolic tail of E-cadherin, and α-catenin, conjugated with fluorescent proteins (mEGFP-β-cateninWt, E-cadherincyto-mTagBFP2, and mCherry-α-catenin, respectively) (Fig. 1A, right).

We and others previously showed that upon induction of crowding by addition of 10% PEG-8000, mEGFP-β-catenin partitioned into spherical, micron-sized droplets in a concentration-dependent manner (refs. 36–38, Figs. 1B, C, and S1B). Under similar conditions, E-cadherincyto-mTagBFP2 did not form droplets (Figs. 1B, C and S1B). However, when combined at equimolar levels, similar to their stoichiometry at the plasma membrane in cells33,39, E-cadherincyto-mTagBFP2 incorporated into droplets of mEGFP-β-catenin (Fig. 1C, D). α-Catenin contains predicted IDRs (Fig. S1A), and formed droplets by itself at comparable concentrations as mEGFP-β-catenin (Figs. 1B, C and S1B). Furthermore, mCherry-α-catenin co-partitioned with β-catenin, both in the absence and presence of E-cadherin (Fig. 1C, D, and F). In contrast, unconjugated mTagBFP2 and mCherry controls were not able to form droplets nor partition into β-catenin droplets (Figs. 1C, D and S1C). Live-imaging revealed fusion of proximal droplets containing β-catenin, E-cadherincyto, and α-catenin, supporting their liquid-like properties (Fig. 1E and Movies S1 and 2). Altogether, these data demonstrate that β-catenin forms phase separated droplets in the presence of E-cadherin and α-catenin and these other cadherin complex components co-partition into these droplets.

We previously identified that condensate formation of β-catenin relies on weak intermolecular interactions through aromatic amino acids in its N- and C-terminal IDRs36–38, which do not overlap with the established high-affinity binding sites in β-catenin for E-cadherin (Fig. S1D)40–44. Mutation of these aromatic residues (from here on referred to as β-cateninIDRs*) diminished the partitioning of β-catenin, as well as co-partitioning of E-cadherin and α-catenin (Figs. 1F, G and S1B and E). The small fraction of β-cateninIDRs* droplets that did form contained all three proteins, indicating that the mutations impair the ability of β-catenin to form droplets but not its interactions with E-cadherin and α-catenin (Fig. 1G). These findings indicate that condensation of β-catenin drives the concentration of all three core cadherin complex components into droplets. In line with this, even though β-catenin and α-catenin both form droplets at similar concentrations, α-catenin did not co-partition with E-cadherincyto in the absence of β-catenin (Fig. 1C, D). Altogether, these data demonstrate that E-cadherin and α-catenin co-partition with β-catenin into phase-separated droplets formed by the IDR-dependent condensation of β-catenin.

IDR-dependent clustering of β-catenin at the cell cortex
Having established that β-catenin condensation co-partitions E-cadherin and α-catenin into droplets in vitro, we made use of β-cateninIDRs* with hampered condensation to investigate whether this behavior contributes to its organization at cell-cell contacts. Live-cell imaging of endogenously mEGFP-tagged β-catenin in the colorectal cancer cell line HCT116, mouse embryonic stem cells, and embryonic kidney cell line HEK293T displayed a distinct organization of β-catenin into small clusters (~250–600 nm in diameter) along the cell cortex (Figs. 2A and S2A, B and Movies S3, 5, and 6). Further characterization of HCT116 cells at varying cell densities revealed that mEGFP-β-catenin cluster formation was not limited to cell-cell contacts, but also occurred at free membranes lacking neighboring cell contacts (Fig. 2A and Movie S3). Moreover, we occasionally observed the coalescence of individual cortical mEGFP-β-catenin clusters (Fig. S2C and Movie S7).

We used the HCT116 cell line to test whether β-catenin condensation contributes to the formation of these clusters. For this, we generated β-catenin knock-out (β-catenin KO) lines of HCT116 and re-introduced either wildtype mScarlet-β-catenin (mSc-β-cateninWt) or mScarlet-β-catenin with mutated IDRs (mSc-β-cateninIDRs*; Figs. 2B and S2D). Both β-catenin variants were similarly able to localize to cell-cell contacts (Fig. S2E), in line with the notion that the IDRs are not required for β-catenin to bind E-cadherin (Fig. S1D)40–42. However, the presence of the distinct, small clusters at the cell cortex observed for β-cateninWt was strongly reduced in cells expressing β-cateninIDRs* (Fig. 2C–E). Moreover, exogenously expressed mSc-β-cateninIDRs* did not incorporate into clusters formed in cells expressing endogenous β-catenin conjugated to mEGFP, whereas the distribution of mSc-β-cateninWt closely overlapped with the clusters formed by endogenous mEGFP-β-catenin (Fig. 2F–H). Similarly, clusters of endogenous mEGFP-β-catenin at the free membrane selectively incorporated wildtype β-catenin and not β-cateninIDRs* (Fig. S2F). Thus, the IDRs of β-catenin are essential for the formation of β-catenin clusters at the cell cortex, and β-catenin with hampered condensation capacity fails to integrate into existing clusters of wildtype β-catenin.

To corroborate our findings indicating a role for β-catenin condensation in its clustering at the cell cortex and exclude potential confounding effects of the double-mutated IDRs, we designed additional β-catenin mutants with impaired condensate formation capacity. We previously showed that loss of a single β-catenin IDR severely affects the formation of droplets in vitro, implying both the N- and C-terminal IDR are essential for β-catenin condensate formation36. We mutated the aromatic amino acids in only the N- or C-terminal IDR of β-catenin (referred to as β-cateninnIDR* and β-catenincIDR*, respectively) and were sufficient to attenuate the formation of droplets (Fig. 3A, B). When these mutants were introduced into β-catenin KO HCT116 cells (Fig. S3A), we found that neither of the mutants efficiently formed clusters at the cell cortex (Fig. 3C, D), similarly to β-catenin with both IDRs mutated (Fig. 2D, E). These mutants independently link an inhibition of condensate formation in vitro to the absence of cluster formation in cells. Thereby, these mutants corroborate the conclusion that β-catenin condensation contributes to its clustering at the cell cortex.

IDR-dependent β-catenin clusters integrate other components of the cadherin/catenin complex to form stable adhesion complexes
Next, we aimed to investigate whether IDR-dependent clustering of β-catenin can drive the clustering of other components of the cadherin/catenin complex. To this end, we first co-expressed fluorescently tagged versions of E-cadherin and α-catenin in HCT116 mEGFP-β-catenin cells. E-cadherin-mCherry and mCherry-α-catenin colocalized within both the IDR-dependent mEGFP-β-catenin clusters at the plasma membrane (Fig. 4A–C). In contrast, mCherry non-specifically targeted to the plasma membrane by fusion to a C-terminal plasma membrane localization signal (CAAX-motif of K-Ras, see “Methods”), was not enriched at mEGFP-β-catenin clusters (Fig. 4A–C). Incorporation of E-cadherin into the IDR-dependent mEGFP-β-catenin clusters did not rely on its ability to form trans- or cis-interactions, as N-terminally truncated E-cadherin lacking these interaction regions was similarly integrated into mEGFP-β-catenin clusters (Fig. S4A). We further excluded that cortical β-catenin clusters were part of the β-catenin destruction complex, which mediates β-catenin degradation and was previously shown to assemble into supramolecular clusters at the cell cortex in other model systems45,46. However, the destruction complex component Axin1 was not detected in the β-catenin clusters at the cell cortex in HCT116 cells (Fig. S4B). Together, these findings demonstrate that the IDR-dependent mEGFP-β-catenin clusters represent clustered cadherin complexes. Next, we tested whether β-catenin condensate formation is essential for the clustering of the other cadherin complex components, or if these instead can independently form clusters. We therefore exogenously expressed mEGFP-α-catenin in β-catenin KO HCT116 cells rescued with either wildtype or the IDRs-mutated mSc-β-catenin, which showed that the α-catenin clusters were not formed in the presence of β-cateninIDRs* (Fig. 4D–F). Altogether, our data show that β-catenin condensation clusters other cadherin complex components and thereby contribute to their supramolecular organization at cell-cell contacts.

To further examine IDR-dependent β-catenin clusters, we applied electron microscopy (EM). We prepared 90–100 nm cryosections of mEGFP-β-catenin HCT116 cell monolayers flat-embedded to preserve the orientation of the cells47. Endogenous mEGFP-β-catenin expression was too low to discern clear sites of enrichment by immunogold labeling of GFP. To circumvent this, we imaged the same samples by correlative light-EM (CLEM) (Figs. 5A and S5A). Highly sensitive fluorescence microscopy revealed mEGFP signal was preserved and well visible, even in ultrathin sections. Hence, we used mEGFP fluorescence to guide us to β-catenin labeled regions by light microscopy and sequentially imaged the same regions by EM. By overlaying the two images we discerned that clusters of mEGFP-β-catenin identified by light microscopy represent cell-cell junctions, typically of a focal nature, with high protein densities at the adjacent cytosolic sites (Figs. 5A and S5A). The overall cell morphology and that of the identified cell-cell adhesions was similar in classical resin-embedded EM, validating our CLEM findings (Fig. S5B)48–50. These results indicate that the IDR-dependent β-catenin clusters at cell-cell contacts represent sites of intercellular adhesion. In line with this, mosaic cultures of mEGFP-β-catenin cells and mSc-β-catenin cells revealed that the IDR-dependent β-catenin clusters integrated β-catenin from both cells forming the cell-cell contact, and thus appear to be part of intercellularly connected adhesion complexes (Fig. 5B, C). Furthermore, disruption of the actin network by addition of Cytochalasin D altered the organization of the β-catenin clusters, with individual clusters coalescing into larger structures, which supports the idea that these clusters are engaged with the actin network (Fig. S5C and Movie S8).

The identification of cadherin/catenin clusters relying on β-catenin condensation provides a conundrum, as biomolecular condensates typically behave as dynamic, liquid-like structures that appear to be antithetical with the role of cadherin adhesions as force-bearing structures25,26. We therefore assessed the dynamics of β-catenin in the IDR-dependent clusters using fluorescence recovery after photobleaching (FRAP). This showed that mEGFP-β-catenin clusters at cell-cell contacts did not recover beyond the fluorescence levels of the surrounding membrane outside of the clusters, indicating that the observed IDR-dependent clusters at cell-cell contacts are immobile, stable structures (Figs. 5D and S5D and Movie S9). In contrast, photobleached clusters at the free membrane exhibited fluorescence recovery within the cluster, and showed recovery kinetics of τ1/2=17 s (Fig. 5E, F and Movie S10). These findings indicate the existence of dynamic IDR-dependent E-cadherin/β-catenin clusters at the cell cortex outside of cell-cell contacts. Altogether, our findings support a model in which IDR-dependent clustering drives the assembly of dynamic E-cadherin/β-catenin clusters at the membrane. These clusters can mature into stable structures upon the formation of cell-cell contacts, potentially involving additional anchoring interactions that render the complex immobile.

The formation of nascent cell-cell adhesions is promoted by IDR-dependent β-catenin clustering
Clustering of the cadherin complex increases the avidity of extracellular E-cadherin domains, which can enhance the probability of forming stable transmembrane interactions during the formation of de novo cell-cell adhesions6–8. Indeed, the formation of cadherin/catenin clusters has previously been observed during the formation of nascent cell-cell adhesions across different cell types, including epithelial Madin-Darby canine kidney (MDCK) cells51–54. To investigate the role of β-catenin IDR-dependent clustering during this process, we depleted endogenous β-catenin from MDCK cells and reintroduced either wildtype mSc-β-catenin or the different β-catenin-IDR mutants that are attenuated in their ability to form condensates (Figs. S6A, B and S6B).

By following sparsely seeded single cells over time during the establishment of de novo cell-cell contacts, we observed the emergence of cortical β-cateninWt in a clustered pattern within minutes after initial contact between protrusions of neighboring cells, similarly to what has been described previously for E-cadherin (Fig. 6A, B and Movies S11 and S15)55. In cells expressing either of the three β-catenin IDR mutants, the clustering of β-catenin in the initial phase of junction formation was significantly hampered. These mutant cells showed a strongly reduced presence or complete absence of cluster formation, with instead a more uniform appearance of β-catenin at the forming cell-cell contacts (Fig. 6A, B and Movies S12–14).

In wildtype cells, the emergence of β-catenin clusters is followed by lateral expansion of the new contact site, and the cell-cell contact reorganizes from dynamic protrusions into a linear junction in which β-catenin rearranges into a more uniform distribution (Figs. 6A–C and S6C and Movies S11 and S15). IDR-mutated cells were able to eventually establish cell-cell contacts after an extended period of time, indicated by the formation of a cohesive monolayer (Fig. S6C). However, mutant cells showed a significantly decreased efficiency of junction formation compared to wildtype cells. The rapid cortical accumulation of β-catenin to the novel contact site of two neighboring cells observed in wildtype cells was strongly delayed in all three mutant cell lines (Fig. 6C, D and Movies S12–14, S16). Furthermore, a significant fraction of cells expressing the β-catenin IDR mutants completely failed to form a linear cell-cell junction during the timespan of the experiment (3–4 h), with cells either maintaining dynamic protrusions without junction linearization or not extending the new contact and subsequently breaking their junction (Fig. 6B, E and Movies S17 and 18). Defects in junction formation in mutant cells predominantly occurred between cell-cell contacts devoid of clustering (Fig. 6B), substantiating that IDR-dependent clustering of the cadherin complex increases the probability of successful cell-cell contact formation. Overall, these data demonstrate that β-catenin condensation facilitates clustering of the cadherin/catenin cell-cell adhesion complex, and this clustering contributes to the formation of novel sites of cell-cell adhesion.

Methods
Antibodies
The following commercial antibodies were used at the indicated concentrations for Western blot (WB) and immunofluorescence (IF): rabbit anti-αE-catenin (Sigma-Aldrich; C2081; 1:500 IF); rabbit anti-β-catenin (Sigma; C2206; 1:2500 WB); mouse anti-β-catenin (BD Biosciences; 9018884; 1:1000 IF); mouse anti-α-tubulin (DM1A; Calbiochem; CP06; 1:5000 WB); rat anti-RFP (5F8; Chromotek; 1:2500 WB); and mouse anti-γ-catenin (Plakoglobin; Zymed; 13-8500 1:250 IF). For EM, biotin-anti-GFP (Rockland; 600-106-215; 1:300) and rabbit anti-biotin (Rockland; 100-4198; 1:10000) antibodies were used.

Plasmids
Plasmids for bacterial expression were generated by In-Fusion cloning of the indicated constructs into a pGEX-2T plasmid: mEGFP, mEGFP-β-cateninWt (NM_001904.4;36), mEGFP-β-cateninIDRs*36, mEGFP-β-cateninnIDR* and mEGFP-β-catenincIDR*, mTagBFP2, E-cadherincyto-mTagBFP2 (E-cadherin NM_009864.2, amino acids 736–783; ref. 73) mCherry and mCherry-α-catenin (NM_009818.1; ref. 73). Lentiviral plasmids were generated by In-Fusion cloning of the indicated constructs into a pLV-CMV plasmid: mScarlet-β-cateninWt (NM_001904.4, ref. 36), mScarlet-β-cateninIDRs*36, mScarlet-β-cateninnIDR*, mScarlet-β-catenincIDR*, E-cadherinΔecto-mScarlet (NM_001287125.2, ΔAA166-708), E-cadherin-mCherry (NM_001287125.2), mCherry-αE-catenin (NM_009818.1; ref. 74), mCherry-(KRAS)CAAX (amino acids KMSKDGKKKKKKSKTKCVIM; ref. 75) and mEGFP-α-catenin (NM_009818.176). pcDNA3.1-Axin1b-mRFP1 (NM_181050.3) was a kind gift from the Maurice lab (UMC Utrecht, The Netherlands).

Protein purifications
Recombinant proteins were expressed in the E. coli CK600K or BL21-DE3 Star strain and grown in Nutrient Broth n1 (Sigma-Aldrich). Following induction with 100 μM IPTG for 14–16 h at 25 °C, cells were harvested by centrifugation at 3470×g for 30 min at 4 °C, resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM EDTA, 5 mM β-mercaptoethanol, and 5% glycerol), lysed by sonication followed centrifugation at 51428×g for 30 min at 4 °C. Supernatants were loaded onto Glutathione Sepharose® 4B (Merck) beads in gravity columns (Bio-Rad). Columns were washed twice with 10 volumes of high salt washing buffer (50 mM Tris-HCl pH 7.5, 400 mM NaCl, 5 mM β-mercaptoethanol and 5% glycerol), once with 10 volumes of droplet formation buffer (50 mM Tris-HCl pH 7.5, 125 mM NaCl, 10% glycerol, 1 mM DTT) and once with TEV protease buffer (50 mM Tris–HCl pH 8.0, 0.5 mM EDTA and 1 mM DTT). TEV protease His-6 (Protean) cleavage was performed with 1.5 kU of the enzyme in 4 ml of TEV protease buffer overnight, and cleaved proteins were eluted with 2 volumes of TEV protease buffer. Proteins were further purified by gel filtration chromatography (Superdex 200 HiLoad 16/60 GE; Healthcare) in 20 mM Tris–HCl pH 8.0, 125 mM NaCl, and 1 mM DTT. For α-catenin, specifically, the monomeric pool was collected.

Droplet assays
Recombinant proteins were diluted at indicated concentrations in droplet formation buffer (50 mM Tris-HCl pH 7.5, 125 mM NaCl, 10% glycerol, 1 mM DTT). After addition of 10% PEG-8000, the protein solution was immediately loaded onto microscope slides as a single drop. Slides were imaged with spinning disk confocal microscope (Nikon Ti2) with Apo TIRF 60× Oil DIC N2 lens or spinning disk CSU ×1 Nikon Ti with Plan Apo VC 60× N.A. 1.40 oil. Images were post-processed to correct for channel misalignment.

Quantifications were performed according to a previously established pipeline77, the code for this analysis is available at the following Github link: https://github.com/krishna-shrinivas/2020_2_Henninger_Oksuz_Shrinivas_RNA_feedback/tree/master/Droplet_analysis. Droplets were segmented by intensity, size and circularity thresholds and their intensity was calculated. The mean intensity of each droplet (C-in) and of the bulk (C-out) were calculated for each channel. The partition ratio was computed as (C-in)/(C-out), as a proxy for average per droplet concentration of fluorescent protein inside the condensates over the concentration of fluorescent protein outside the condensates. For each image, the total intensity of each droplet was calculated and then the sum was computed (TC-in) as well as the total bulk intensity of the whole field (TC-out). The condensed fraction was computed as (TC-in)/(TC-out), as a measure of the total amount of fluorescent protein inside the condensates over the total amount of fluorescent protein in a given image. Quantification data was filtered to exclude droplets with partition ratios lower than 1.1 for mCherry and mEGFP channels and lower than 1.35 for mTagBFP2 to correct for mEGFP bleedthrough. Two types of analysis were performed, one in which all the droplets for all the channels were considered for the condensed fraction (Figs. 1C and 3B) and one in which the droplet mask of β-catenin was used as a scaffold (β-catenin co-condensed fraction; Fig. 1G).

Cell lines and culture
MDCK GII cells (gift from W. J. Nelson, Stanford University) were cultured at 37 °C and 5% CO2 in low-glucose DMEM (Sigma; D5523-10L) supplemented with 10% FBS (Sigma; F7524-500ML), 1× g/liter sodium bicarbonate, and Penicillin-Streptomycin (Lonza; LO DE17-602E). HCT116 (obtained from ATCC, CCL-247) and HEK293T (obtained from ATCC, CRL-3216) cells were cultured in high-glucose DMEM (Sigma; D6429-24X500ML) supplemented with 10% FBS and Penicillin-Streptomycin. V6.5 murine embryonic stem cells (gift from Rudolf Jaenisch, Whitehead Institute). were cultured at 37 °C and 5% CO2 on 0.2% gelatinized tissue culture plates in 2i+LIF media, composed of DMEM/F12 (Lonza; BE04-687F/U1), N2 supplement (GIBCO; 17502048), B27 supplement (GIBCO; 17504001), 0.5 mM L-glutamine (Lonza; LO BE17-605E), 0.5× non-essential amino acids (Lonza; LO BE13-114E), Penicillin-Streptomycin, 0.1 mM β-mercaptoethanol (Sigma; M6250), 1 uM PD0325901 (Selleckchem; S1036), 3 uM CHIR99021 (Selleckchem; S2924), and 1000 U/mL recombinant LIF (Millipore; ESG1107). Live-cell imaging experiments were performed under the same culture conditions. All cell lines were regularly tested for the absence of mycoplasma.

Cell lines in which β-catenin is endogenously tagged with 3× mEGFP have been previously described36. Knock-out lines for β-catenin in HCT116 and MDCK cells were generated by CRISPR-Cas9, by transfection of pSpCas9(BB)−2A-GFP (Addgene #48138) with gRNA sequence (5′-GTTCCCACTCATACAGGACTTGG-3′) targeting human and canine β-catenin, using Lipofectamine LTX or Lipofectamine 3000, respectively. Two days following transfection, GFP-positive cells were sorted by FACS in 96-well culture plates to obtain monoclonal lines. Clones were screened for successful knock-out of β-catenin by immunofluorescence, and validated by both western blotting and sequencing of the genomic locus.

Stable lines of HCT116 and MDCK cells expressing indicated constructs were generated using lentiviral transductions followed by selection with Puromycin (Bio Connect; SC-108071B) or Blasticidin (BioConnect; ant-bl-1). In HCT116 and MDCK β-catenin knockout lines in which mSc-β-cateninWt or mSc-β-cateninIDRs* were re-expressed, cells were sorted by FACS to obtain monoclonal lines. Plasmids E-cadherinΔecto-mScarlet and Axin1-mRFP1 were transiently transfected in HCT116 cells using Lipofectamine LTX and imaged two days later.

Live-cell microscopy and analyses
For live-cell imaging, cells were seeded on glass-bottom dishes (Lab-Tek II), precoated with Rat Tail Collagen I (Corning). Cells were imaged on a Nikon Spinning Disc confocal microscope using a 60× objective (NA=1.49). Imaging was performed at 37 °C and 5% CO2 in temperature- and CO2-controlled incubators, using NIS-Elements software. To study the effect of actomyosin disruption, Cytochalasin D was added during image acquisition at a concentration of 2 µg/ml.

For the detection of β-catenin clusters at cell-cell contacts, lines (4 pixels wide) were manually drawn over cell-cell contacts with a clear linear morphology using ImageJ Fiji software78. Only cells with comparable levels of exogenous protein expression were included in the analyses. Line profiles were normalized to the 20th–40th lowest intensity values. Individual clusters were detected using BAR plugin Find Peaks79, with a minimal peak amplitude of 50% (for endogenously tagged mEGFP-β-catenin) or 25% (for exogenously expressed mEGFP-β-catenin). For the quantification of number of peaks per µm, detected peaks were manually verified, blindly. The fraction of overlapping peaks was determined by counting the number of mEGFP peaks per cell-cell contact that had an mSc or mCherry peak detected within 3 pixels distance. The size of mEGFP-β-catenin clusters was determined by fitting a Gaussian function to line profiles of individual clusters and calculating the full-width at half-maximum (FWHM=2 2ln(2)σ) from the extrapolated standard deviation (σ).

For the observation of in vitro droplets fusion events, samples were prepared as explained in the droplet assays method and images were acquired every 3.16 s.

Cell-cell junction formation
To analyze de novo junction formation, sub-confluent MDCK cells were trypsinized and sparsely seeded as single cells on collagen-coated glass-bottom dishes. After attachment and spreading of cells (~2 h), cells were imaged at a time interval of 2–3 min for 3–4 h, allowing single cells to migrate towards other cells and form new contacts. De novo contacts were manually scored for the duration from the first contact between cells until the start of cortical β-catenin signal appeared, for the presence of clusters during junction formation, and for the successful establishment of a linear junction (i.e., extension of the contact with no junction breakage or failure to reduce dynamic protrusions).

Immunostainings
For immunostainings, confluent cell monolayers grown on collagen-coated glass-bottom dishes were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich); permeabilized with 0.2% Triton X-100 (Sigma-Aldrich); blocked in buffer containing 1% BSA (Sigma-Aldrich), 1% goat serum (Life Technologies), and 1% donkey serum (Jackson Immunoresearch); and incubated with the indicated primary and Alexa-conjugated secondary antibodies (Life Technologies), together with DAPI (Sigma-Aldrich) where indicated. Cells were analyzed on a Zeiss LSM880 scanning confocal microscope using a 63× objective (NA=1.15), using Zen image acquisition software.

Fluorescence recovery after photobleaching (FRAP)
FRAP experiments were performed on a Zeiss LSM880 scanning confocal microscope using a 63× objective (NA=1.15), using Zen image acquisition software, or on a Nikon Spinning Disc confocal microscope with a FRAP module (OMS) using a 60× objective (NA=1.42), using NIS-Elements software. Imaging was performed at 37 °C and 5% CO2 in temperature- and CO2-controlled incubators. Individual clusters were bleached using 100% laser power and imaged over time (5–9.5 min) with a 10 s interval in a z-stack (5×0.5 µm interval). Fluorescent intensities were measured using ImageJ78 by manually drawing ROIs over individual clusters and a concomitant distal region at the junction within maximum projections for each time frame. Background-subtracted florescent intensity values were normalized to the pre-bleaching and immediate post-bleach mean intensities. To calculate the mobile fraction (Fmob) and the half-life recovery time (τ1/2), the average of 15 replicates was fitted to a single exponential curve in Prism 8 software (GraphPad):
Y(t)=Y(0)+ Fmob*Y(0) * (1-e^(-t/τ1/2))

Correlative light electron microscopy (CLEM)
CLEM was performed as described in detail in refs. 80,81. HCT116 cells expressing endogenously tagged 3× mEGFP-β-catenin were seeded on carbon- and collagen-coated coverslips and fixed in 2% PFA and 0.2% glutaraldehyde (GA). Cells were washed with PBS, incubated in PBS/0.15% glycine for 10 min, washed with PBS/0.1% BSA, and incubated with 12% gelatin (Merck; G1890) at 37 °C for 30 min. The 12% gelatin was then solidified at 4 °C for 30 min, and the coverslips were incubated with 2.3 M sucrose for 48 h at 4 °C. After incubation, the gelatin, including embedded cells, had spontaneously detached from the coverslip. The gelatin-embedded cell layer was then cut into blocks of ~1 mm3 and mounted on pins with cell bottoms facing up and snap-frozen and stored in liquid nitrogen.

Samples were cryosectioned into ribbons of 90–100 nm ultrathin sections and deposited on grids (Cell Microscopy Core, UMC Utrecht). The grids were incubated with PBS/0.15% glycine for 30 min at 37 °C, rinsed with PBS and incubated with 10 µg/mL DAPI in 0.1% BSA-c (Aurion; 900.099) and 0.5% fish skin gelatin (Sigma-Aldrich; G7765) in PBS (BSA-c/FSG) for 20 min. Grids were then washed 5× in PBS and 2× in 50% glycerol in 0.1 M PHEM (EMS; 11162) before being submerged in 50% glycerol in 0.1 M PHEM and sandwiched between a coverslip and glass slide. The sections were imaged for mEGFP signal and DAPI in this configuration on a Leica Thunder widefield microscope with 100× objective (NA=1.47), Photometrics Prime 95B sCMOS camera and LAS X software. After image collection of the whole ribbon of sections, the grids were retrieved, washed 3× in PBS and incubated with BSA-c/FSG) for 10 min. Next, we labeled grids with biotin-anti-GFP antibody (Rockland; 600-106-215; 1:300) in BSA-c/FSG for 1 h. Grids were then washed 5× in PBS/0.1% BSA and incubated with bridging rabbit anti-biotin antibody (Rockland; 100-4198; 1:10000) in BSA-c/FSG for 20 min, followed by 5 more PBS/0.1% BSA washes and incubation with Protein-A conjugated to 10 nm gold particles (Cell Microscopy Core, UMC Utrecht; PAG10; 1:50) in BSA-c/FSG for 20 min. Grids were then washed 5× with PBS, incubated with 1% GA in 0.1 M phosphate buffer, 2× PBS, 8× milliQ, and contrasted using uranyl acetate pH 7 (5 min) and uranyl acetate:methylcellulose pH 4 (10 min, 4 °C). Excess uranyl was blotted away and grids were dried using the “loop-out” method81. The dried grids were imaged in a FEI Tecnai T12 transmission electron microscope (TEM) with Veleta VEL-FEI-TEC12-TEM camera and SerialEM software. Regions selected by the fluorescence images were correlated and imaged at 50,000× magnification in TEM. The obtained image tile sets were correlated based on DAPI signal and nuclear outlines using ImageJ Fiji software78.

Electron microscopy of resin-embedded cells
HCT116 3× mEGFP-β-catenin cells (the same cell line as used in the CLEM experiments) were seeded in 3 cm dishes 24 h prior to fixation. Fixative consisted of 2.5% GA and 2% PFA in 0.1 M PHEM buffer and was added 1:1 to cell culture medium for 5 min at RT. Culture medium and fixative were then replaced with fixative alone for 2 h at RT. Samples were then stored in 0.5 PFA in 0.1 M PHEM at 4 °C. For contrasting, 1% OsO4 was used for 1 h. After a dehydration series in ethanol, samples were further processed in EPON in increasing concentrations and finally embedded in 100% EPON. EPON was polymerized for 5 d at 60 °C.

The resin-embedded samples were prepared for thin sectioning by removal from the culture dish, cutting out a rectangle of ∼0.5 by 1 mm and mounting, bottom side up, on an EPON stub. We proceeded by cutting 60–70 nm sections on a Leica Ultracut S (Leica Microsystems) using a DiATOME Ultra Diamond Knife 45°. Sections were placed on Formvar- and carbon-coated copper grids and post stained in a Leica EM AC20 (Leica Microsystems) using uranyl and lead citrate. Samples were imaged on a Tecnai T20 (FEI Tecnai) TEM using Radius software.

PONDR disorder score prediction
Disorder scores were calculated with the PONDR web tool: http://www.pondr.com.

Statistics and reproducibility
All statistical analyses as indicated in the Figure legends, were performed using Prism 8 software (GraphPad) or R (version 4.5.1). Data were tested for normality, with two-sided tests employed unless otherwise specified, and corrections for multiple comparisons were made when indicated. No statistical method was used to predetermine sample size. Sample sizes are indicated in the legend of each experiment and were selected to provide adequate statistical power. For the selection of cell-cell contacts for analyses, only cells showing a proper expression of the transfected constructs were included. For the comparison between wildtype and mutant proteins, we analyze cell-cell contacts showing comparable expression levels. Data were either analyzed using automated image analysis, or manually analyzed and under blinded conditions when possible.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors summarized and discussed a novel clustering mechanism of the cadherin complex established by its core component β-catenin.On this basis, they further investigated the co-partition of β-catenin and other cadherin complex components, the IDR-dependent β-catenin clusters, and the importance of cadherin-complex clustering in establishing adhesion between two opposing cells. This work provides new insight and opinion into the function of β-catenin, which plays an essential role in the supramolecular organization of the cadherin complex. The manuscript is well-organized and clearly stated. The following issues need to be resolved before the article is published:\n1. The authors should determine the material properties (viscosity) of the droplets in vitro and ideally compare them to nascent adhesions in cells, e.g. by FRAP or observing fusion events of droplets.\n2. Page 7 Line 161. “similarly to β-catenin with both IDRs mutated (Figs. 3G and H)”, there are no graph G and graph H in Figure 3. According to the content of the statement, it should be corrected as “similarly to β-catenin with both IDRs mutated (Figs. 2D and E)”.\n3. Page 7 Line 172. “fusion to a CAAX-motif...”, there is a lack of explanatoin of what the CAAX-motif is.\n4. In Fig. 4G, to avoid misunderstood, it is suggested to replace “mEGFP-β-catenin + DNA” with “mEGFP-β-catenin + DAPI”.\n5. Page 9 Lines 204-206. The authors mention “We postulate that liquid-like assemblies of β-catenin guide initial cadherin complex clustering, and is followed by maturation of these clusters into a more stable state”. The data are not enough to support this hypothesis, which should be demonstrated. Whether the dynamics of β-catenin differ with time or component composition, and incomplete fluorescence recovery after photobleaching may be attributed to plasma membrane regionalization rather than maturation of β-catenin clusters.", "reviewer_2": "The authors propose an interesting model in which beta catenin assembles into phase separated liquid droplets to promote the clustering of cadherin adhesion receptors. The authors describe solution studies with different proteins and with beta catenin mutants, and they then correlate the solution behavior with observed cadherin clustering at sites of adhesion between cells. The solution studies are noteworthy and there are some interesting findings.\nThe study makes some very strong claims about the role of beta catenin that are not convincingly supported by the data. The conclusion that liquid droplets drive cadherin clustering is also undermined somewhat by the FRAP data indicating that the puncta at cell adhesions are not in fact liquid like. The paper is also missing some key controls, and additional key studies are needed to rule out other proposed mechanisms. These points are described in more detail below.\nIf beta catenin undergoes liquid-liquid phase separation when associated with cadherin at the membrane, the process should be controlled entirely by the local protein concentration on the cytoplasmic face of the membrane. Phase separation is driven by concentrations, solubility, and the surface tension of the droplet. The physics should be independent of whether the cells are adhering or not. I did not see any evidence that these droplets form at free cell membranes. They seem to be restricted to adhesions, suggesting that that other mechanisms are more important.\nA related experiment would be to test whether proposed condensates form in cells, if the adhesion complexes are decoupled from the cytoskeleton. Does force or reduced cadherin mobility have any effect? If clustering is due to beta catenin phase separation driven by the local concentration, then force and mobility shouldn’t have much effect. Along these lines, mutations that disrupt adhesion should not affect droplet formation.\nIs the stoichiometry of components that form droplets in solution the same as at the membrane in the cell? Conversely, if the stoichiometric amounts of the components at the membrane are mixed in solution, do the proteins still form droplets in solution? This is needed to confirm that the same physics in solution is also operating in the cell.\nDid they examine the effect of additives that disrupt phase segregated protein droplets, such as hexanediol?\nWhat is the effect of extracellular domain cadherin mutants that disrupt lateral interactions? If their hypothesis that beta catenin plays a key role is correct, the intracellular phase segregation should be independent of weak interactions between the extracellular regions.\nThe proposed model is difficult to reconcile with the role of p120 catenin in clustering (Yap et al. 1998 J Cell Bio and Vu et al 2021 Curr Biol). If they eliminate p120 catenin, beta catenin should still bind, form droplets and cluster cadherins. This should be tested.", "reviewer_3": "Remarks to the Author:\nThis paper by Monster et al. addresses whether protein condensation of β-catenin is a prerequisite for clustering of proteins of the E-cadherin complex and subsequent formation of cell-cell junctions that mediate adhesion between cells in culture. Specifically, the authors use droplet formation assays and show that mutations in the intrinsically disordered N- and/or C-terminals of β-catenin inhibit the formation of phase separated droplets, that also can contain E-cadherin and α-catenin. By live-cell confocal microscopy they show that the same mutations that inhibit phase separation also inhibit the accumulation of cadherin/catenin complexes that form clusters along the cell-cell contact regions of the membrane in HCT116 cells. In addition, CLEM and FRAP analyses are used to determine the cluster´s ultrastructure and stability, respectively. Lastly, live-cell confocal microscopy of sparsely seeded MDCK cells is used to visualize the dynamics of cluster organization during formation of cell-cell contact.\nIn general, I find this paper interesting, and most findings are both clear and well described. I have, however, suggestions and comments that might improve the manuscript.\nMajor points\n1. Since β-catenin is part of adherens junctions, it is appropriate to analyze if there is an association between this type of cell-cell junction and individual mEGFP-β-catenin clusters. The authors address this by a CLEM analysis (Fig. 4G). Unfortunately, the fluorescence associated with membrane region shown is weak. It is similar to the weak intensity in regions between clusters (Fig. 4B, E) or alternatively, the weak fluorescence intensity before onset of β-catenin enrichment (Fig. 5C). Moreover, the EM resolution is low and the cell membranes with relative high electron density are separated with cell membranes that appear not to be intact. Together this makes it hard to agree with a conclusion that fluorescence intensity and electron density show an accumulation of β-catenin to adherens junctions. I would recommend that the authors show CLEM images of another region with intense mEGFP-β-catenin fluorescence. A suggestion is the intensely fluorescent cell-cell contact region located ~10 µm to the left of the region boxed in yellow. The analyses should also include a statement about independent biological replicates and if possible, a reference to a paper showing electron micrographs of cell-cell junctions between HCT116 cells.\n2. The authors combine CLEM with electron microscopy detection of mEGFP-β-catenin by protein-A-conjugated gold particles. The result of the gold particle detection does not show an association between β-catenin clusters or putative adherens junctions. This is due to the choice of analyzed region (commented above, pnt. 1) as well as an insufficient number of gold particles detected. As little as 5 cold particles are detected and only 3 of these appear to be located intracellularly and at proximity to the cell membrane. Accordingly, no quantification of immunogold staining is given that supports an increased accumulation β-catenin to the clusters or adherens junctions. This experiment should also be described in the result section and not only in M&M and figure legend.\nMinor comments\n3. I interpret the term “ectopic expression” to mean that a gene product is present at an abnormal location. Not that a gene is transcribed from a genetically modified and abnormally located genomic locus. However, I notice that the same terminology is used by Zamudio et al., who generated the endogenously tagged mEGFP-β-catenin (ref. 36). I therefore leave it to the editor to decide if terms “ectopic” and “ectopically”, which are mentioned 22 times in the manuscript, are used correctly.\n4. I don´t understand why the authors make a distinction between stably integrated “ectopically” expressed transgenes and the endogenously tagged mEGFP-β-catenin gene. The chimeric proteins generated by both types of transgenes are most likely not regulated or expressed in a manner that mimics their corresponding endogenous proteins during the dynamic events that establish cell-cell junctions. This issue is not addressed in the manuscript but is important for the discussion about which biomolecular interactions that are the driving force of cadherin cluster formation under physiological conditions (e.g. page 13, line 312)\n5. The experiments shown in Fig. S4A lack a positive control, i.e. a figure showing that transiently expressed Axin1 is associated with clusters of β-catenin destruction complexes at the cell cortex. Perhaps the Axin1 expressed is sequestered into the cytosolic biomolecular condensates shown.\n6. Page 7, line 161. ………… β-catenin with both IDRs mutated (Figs. 3G and H). Should be (Figs. 2G and H).\n7. Page 7, line 162-164. This sentence needs to be edited.\n8. Fig. 2H, The unit on the Y-axis should be “…. per cell-cell contact” and not “.... per junction”."}, "author_response": "We thank all Reviewers for their positive evaluation of our work and their constructive criticisms, which helped us to improve the manuscript. We have performed additional experiments and analyses, and revised the manuscript to address their criticisms, as detailed below in our point-to-point reply to their specific comments. We hope the Reviewers share our enthusiasm for our revised manuscript and find it suitable for publication in Nature Communications.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this manuscript, the authors summarized and discussed a novel clustering mechanism of the\ncadherin complex established by its core component β-catenin. On this basis, they further\ninvestigated the co-partition of β-catenin and other cadherin complex components, the IDR-\ndependent β-catenin clusters, and the importance of cadherin-complex clustering in establishing\nadhesion between two opposing cells. This work provides new insight and opinion into the\nfunction of β-catenin, which plays an essential role in the supramolecular organization of the\ncadherin complex. The manuscript is well-organized and clearly stated.\nThe following issues need to be resolved before the article is published:\n1. The authors should determine the material properties (viscosity) of the droplets in vitro and\nideally compare them to nascent adhesions in cells, e.g. by FRAP or observing fusion events of\ndroplets.\nTo address this point of the reviewer, we performed live-imaging of droplets containing β-\ncatenin, E-cadherin, and α-catenin formed in vitro. This showed that these droplets underwent\nfusion events (Fig. 1E). Similarly, we found evidence in cells that IDR-dependent clusters of β-\ncatenin at the cortex fused upon contact between clusters (Fig. S2C).\nThese new data have been included in the revised manuscript. Additionally, we acknowledge\nthe limitations of comparing β-catenin-formed droplets in vitro with cellular clusters, as these\nconditions are not directly comparable (for example, cellular clusters likely contain additional\ncomponents absent in vitro).\n2. Page 7 Line 161. “similarly to β-catenin with both IDRs mutated (Figs. 3G and H)”, there are\nno graph G and graph H in Figure 3. According to the content of the statement, it should be\ncorrected as “similarly to β-catenin with both IDRs mutated (Figs. 2D and E)”.\nWe thank the Reviewer for pointing out this mistake and have now corrected it.\n3. Page 7 Line 172. “fusion to a CAAX-motif...”, there is a lack of explanatoin of what the CAAX-\nmotif is.\nWe acknowledge that the description of the CAAX motif required further clarification. A CAAX\nmotif is a sequence consisting of a cysteine (C), two aliphatic amino acids (AA), and a terminal\namino acid (X). It is found at the C-terminus of various proteins and facilitates their targeting to\nmembranes. Specifically, we used the CAAX motif of K-Ras, which includes both this CAAX motif\nand an N-terminal polylysine sequence that directs K-Ras to the plasma membrane. The fusion\nof this motif to other proteins, such as the fluorescent protein mCherry in our case, is a commonly\nused strategy for plasma membrane targeting (e.g., https://doi.org/10.1074/jbc.M007194200).\nTo enhance clarity, we have revised the text and now introduce this as “mCherry non-specifically\ntargeted to the plasma membrane by fusion to a C-terminal plasma membrane localization signal\n(CAAX-motif of K-Ras, see Methods)”. Additionally, in the Methods section, we provide further\ndetails and the exact sequence used.\n4. In Fig. 4G, to avoid misunderstood, it is suggested to replace “mEGFP-β-catenin + DNA” with\n“mEGFP-β-catenin + DAPI”.\nWe have adapted this in all Figures of the manuscript.\n5. Page 9 Lines 204-206. The authors mention “We postulate that liquid-like assemblies of β-\ncatenin guide initial cadherin complex clustering, and is followed by maturation of these clusters\ninto a more stable state”. The data are not enough to support this hypothesis, which should be\ndemonstrated. Whether the dynamics of β-catenin differ with time or component composition,\nand incomplete fluorescence recovery after photobleaching may be attributed to plasma\nmembrane regionalization rather than maturation of β-catenin clusters.\nWe have extended our FRAP-based analysis of the IDR-dependent clusters at the cell cortex in\nHCT116 cells. In our original submission, we characterized the recovery kinetics after\nphotobleaching specifically of clusters at cell-cell contacts. Based on Reviewer 2’s comments,\nwe now demonstrate that IDR-dependent clusters also form at the free cell membrane (Figs. 2A\nand S2E). This prompted us to re-examine our initial FRAP data and extend our analysis by\ncomparing clusters at cell-cell contacts with those at the free membrane.\nThis analysis revealed that photobleached GFP-β-catenin clusters at cell-cell contacts did not\nshow recovery above the fluorescence levels at the membrane outside of the clusters (Fig. 5D\nand S5B; and as was also seen in the Figure included in the original submission). In contrast,\nphotobleached clusters at the free membrane show prominent fluorescent recovery within the\ncluster, with fast recovery kinetics (t1/2 = 17s) (Fig. 5E and F). These data imply that IDR-\ndependent clusters at the free membrane show more diffusivity of β-catenin, whereas this is\nreduced in clusters at cell-cell contacts.\nThese newly added FRAP data with comparison of the molecular dynamics of IDR-dependent\nclusters at the free membrane and cell-cell contacts does align with the model that initial IDR-\ndependent clustering drives the early assembly of E-cadherin/β-catenin clusters at the\nmembrane, which subsequently mature into a stable state after forming cell-cell contacts.\nNonetheless, the reviewer’s argument remains relevant, as other factors could influence\ndifferences in photorecovery kinetics. We therefore fully agree with this reviewer that we should\nbe cautious in interpreting our findings. Therefore, we have toned down this statement and now\ndescribe that “These findings indicate the existence of dynamic IDR-dependent E-cadherin/β-\ncatenin clusters at the cell cortex outside of cell-cell contacts. Altogether, our findings support a\nmodel in which IDR-dependent clustering drives the assembly of dynamic E-cadherin/β-catenin\nclusters at the membrane, which can mature into a stable state upon forming cell-cell contacts.”\nReviewer #2 (Remarks to the Author):\nThe authors propose an interesting model in which beta catenin assembles into phase\nseparated liquid droplets to promote the clustering of cadherin adhesion receptors. The authors\ndescribe solution studies with different proteins and with beta catenin mutants, and they then\ncorrelate the solution behavior with observed cadherin clustering at sites of adhesion between\ncells. The solution studies are noteworthy and there are some interesting findings.\n(*) The study makes some very strong claims about the role of beta catenin that are not\nconvincingly supported by the data. The conclusion that liquid droplets drive cadherin clustering\nis also undermined somewhat by the FRAP data indicating that the puncta at cell adhesions are\nnot in fact liquid like.\nIn our manuscript, we describe:\ni) that the IDRs of β-catenin drive the formation of phase-separated droplets in vitro and are\nessential for integrating E-cadherin and α-catenin into these droplets.\nii) that the IDRs of β-catenin drive the formation of clusters at the cell cortex and are essential\nfor integrating E-cadherin and α-catenin into these clusters.\nThe most parsimonious interpretation of these findings, in our view, is that β-catenin’s ability to\nform IDR-dependent condensates contributes to the formation of β-catenin/E-cadherin clusters\nin cells. However, we acknowledge that we do not have direct evidence for the liquid-like\nbehavior of cortical β-catenin clusters in cells. To prevent overinterpretation, we have revised\nthe manuscript to further emphasize that our conclusions are based on a combination of in vitro\nand cellular experiments.\nAdditionally, in response to other comments from this Reviewer, we have expanded our FRAP-\nbased analysis of IDR-dependent clusters at the cell cortex in HCT116 cells. In our original\nsubmission, we characterized the recovery kinetics after photobleaching specifically in clusters\nat cell-cell contacts. Following comment 1 of this Reviewer, we now demonstrate that IDR-\ndependent clusters also form at the free cell membrane (Figs. 2A and S2E). This prompted us to\nre-examine our initial FRAP data and extend our analysis by comparing clusters at cell-cell\ncontacts with those at the free membrane. This analysis revealed that photobleached GFP-β-\ncatenin clusters at cell-cell contacts did not recover beyond the fluorescence levels of the\nsurrounding membrane outside of the clusters (Fig. 5D and S5B; and as was also seen in the\nFigure included in the original submission). In contrast, photobleached clusters at the free\nmembrane exhibited prominent fluorescence recovery within the cluster, with fast recovery\nkinetics (t1/2 = 17s) (Fig. 5E and F). These results suggest that IDR-dependent clusters at the\nfree membrane are more dynamic, while those at cell-cell contacts show reduced diffusivity.\nWhile we refrain from making claims about liquid-like behavior of these cellular clusters, our\nnewly added FRAP data support the model in which IDR-dependent clustering drives the\nassembly of dynamic E-cadherin/β-catenin clusters at the membrane, which can mature into a\nstable state upon forming cell-cell contacts.\n(**) The paper is also missing some key controls, and additional key studies are needed to rule\nout other proposed mechanisms. These points are described in more detail below.\nWe address all specific comments related to this concern below. However, we would like to\nemphasize that i) our experiments in cells unambiguously demonstrate a role for the IDRs of β-\ncatenin in organizing the β-catenin/E-cadherin complex into clusters and ii) this does not exclude\nthe possibility that other mechanisms also contribute to the organization of this complex. In fact,\nwe explicitly describe in our Discussion section (Page 13 line 15 – 27) that alternative mechanisms\ninvolved in clustering of the cadherin complex may be preceded by cadherin/β-catenin\ncondensation and further stabilize these clusters, or conversely, may promote condensate\nformation by locally concentrating cadherin/β-catenin complexes. As discussed below, these\nadditional mechanisms are not essential for the formation of IDR-dependent clusters. However,\nwe believe it is important to consider that these mechanisms are interconnected and all are\nexpected to influence cluster formation, as further outlined in our response to the specific\ncomments.\n1. If beta catenin undergoes liquid-liquid phase separation when associated with cadherin at the\nmembrane, the process should be controlled entirely by the local protein concentration on the\ncytoplasmic face of the membrane. Phase separation is driven by concentrations, solubility, and\nthe surface tension of the droplet. The physics should be independent of whether the cells are\nadhering or not. I did not see any evidence that these droplets form at free cell membranes. They\nseem to be restricted to adhesions, suggesting that that other mechanisms are more important.\nWe would like to note that trans-interactions of E-cadherin between cells will increase the retention\nof the E-cadherin/beta-catenin complex at the membrane (doi: 10.1083/jcb.142.4.1105) and\nthereby locally increase their concentration, which could thus promote condensation.\nNonetheless, we agree that cell-cell adhesion should not be a strict requirement for this process.\nTo support this, we have expanded our analysis to include β-catenin clusters at the free\nmembrane, which were not shown in the Figures of our initial submission. Imaging of\nendogenously tagged β-catenin shows it forms clusters at the free membrane (Fig. 2A). Moreover,\nco-expression with mScarlet-β-catenin demonstrate that this clustering at the free cell membrane\nis dependent on the IDRs, similar to clusters at cell-cell contacts (Fig. S2E). We hope these\nadditional data and discussion take away the Reviewer’s concern.\n2. A related experiment would be to test whether proposed condensates form in cells, if the\nadhesion complexes are decoupled from the cytoskeleton. Does force or reduced cadherin\nmobility have any effect? If clustering is due to beta catenin phase separation driven by the local\nconcentration, then force and mobility shouldn’t have much effect. Along these lines, mutations\nthat disrupt adhesion should not affect droplet formation.\nTo the best of our knowledge, it is technically not feasible to genetically decouple the cadherin\ncomplex from the actin cytoskeleton while preserving the integrity of the cadherin-catenin\ncomplex. Therefore, the only viable approach to addressing this question is through chemical\nmanipulation of the actin cytoskeleton. To this end, we treated HCT116 cells with Cytochalasin\nD, an inhibitor of actin polymerization. Under this condition, IDR-dependent clusters at the cell\ncortex remained present (Fig. S5C).\nHowever, we observed that under following inhibition of actin polymerization, individual β-\ncatenin clusters coalesced into larger structures. This suggests that the actin network, either\nthrough physical interactions or indirectly, is important for the dispersed distribution of β-catenin\nclusters.\nS5C\nMost importantly, these findings indicate that IDR-dependent clusters persist in the absence of\nactin. However, further understanding the functional interaction with the actin cytoskeleton\nprovides additional layers and we respectfully argue that unraveling this interconnection is out of\nthe scope of the current manuscript. Our primary conclusion remains that an IDR-dependent\nmechanism is essential for cluster formation.\nWe refer to our answer to comment #5 of this Reviewer for mutations that disrupt adhesion.\n3. Is the stoichiometry of components that form droplets in solution the same as at the\nmembrane in the cell? Conversely, if the stoichiometric amounts of the components at the\nmembrane are mixed in solution, do the proteins still form droplets in solution? This is needed to\nconfirm that the same physics in solution is also operating in the cell.\nThe stoichiometry of E-cadherin:β-catenin:α-catenin at the plasma membrane has been\npreviously established as 1:1:1 (Ozawa and Kemler, 1992; Hinck et al., 1994). Based on this,\nwe combined these proteins in our in vitro experiments at the same ratio. We now describe this\nin the manuscript.\np5, line 15-17: “However, when combined at equimolar levels, similar to their stoichiometry at\nthe plasma membrane in cells 33,39, E-cadherincyto-mTagBFP2 incorporated into droplets of\nmEGFP-β-catenin (Figs. 1C and 1D).“\n4. Did they examine the effect of additives that disrupt phase segregated protein droplets, such\nas hexanediol?\nWe analyzed the effect of hexanediol and found that it disrupts the majority of β-catenin clusters\nin HCT116 cells (Rebuttal Figure 1). However, we would prefer not to include these data in the\nmanuscript. 1,6-Hexanediol has pleiotropic effects on condensates and more general on\nhydrophobic interactions throughout the cell, and is not very reliable to assess the presence of\nphase segregated proteins (PMID: 30682370). In our opinion, the IDR-mutated β-catenin\nproteins with reduced condensate-forming capacity represent a more precise experimental\nperturbation (Figure 2).\nRebuttal Figure 1\nWhat is the effect of extracellular domain cadherin mutants that disrupt lateral interactions? If\ntheir hypothesis that beta catenin plays a key role is correct, the intracellular phase segregation\nshould be independent of weak interactions between the extracellular regions.\nTo address this question, we introduced a truncated mutant of E-cadherin lacking almost the\nentirety of its extracellular domain (amino acids 166-708) into HCT116 cells. Despite this\ntruncation disrupting lateral cis interactions between E-cadherin proteins, as well as homotypic\ntrans interactions (thus disrupting the ability of E-cadherin to establish adhesion, see Comment\n#2), we found that this mutant still was able to cluster together with β-catenin at the free\nmembrane. We included these data in the revised manuscript (Fig. S4A).\nS4A\nThe proposed model is difficult to reconcile with the role of p120 catenin in clustering (Yap et al.\nstill bind, form droplets and cluster cadherins. This should be tested.\nTo the best of our knowledge, there is no experimental evidence supporting a defined role for\np120-catenin (or other members of this protein family) in the formation of larger clusters of the\ncadherin-catenin complex. Instead, the role of p120-catenin in the organization of the cadherin\ncomplex is primarily linked to its regulation of cadherin stability at the plasma membrane by\ncontrolling cadherin endocytosis (doi: 10.1083/jcb.200307111); which explains the absence of\ncadherin clusters at the plasma membrane following the disruption of p120-catenin binding\ndescribed in Yap et al., 1998). Thus, manipulating p120-catenin would primarily test how\nchanges of E-cadherin/β-catenin membrane levels, and thus local concentration, influence\ncluster formation. For this reason, we respectfully disagree on these experiments being\nessential to support our conclusion that β-catenin IDRs play a critical role in establishing E-\ncadherin/β-catenin clusters.\nReviewer #3 (Remarks to the Author):\nRemarks to the Author:\nThis paper by Monster et al. addresses whether protein condensation of β-catenin is a\nprerequisite for clustering of proteins of the E-cadherin complex and subsequent formation of\ncell-cell junctions that mediate adhesion between cells in culture. Specifically, the authors use\ndroplet formation assays and show that mutations in the intrinsically disordered N- and/or C-\nterminals of β-catenin inhibit the formation of phase separated droplets, that also can contain E-\ncadherin and α-catenin. By live-cell confocal microscopy they show that the same mutations that\ninhibit phase separation also inhibit the accumulation of cadherin/catenin complexes that form\nclusters along the cell-cell contact regions of the membrane in HCT116 cells. In addition, CLEM\nand FRAP analyses are used to determine the cluster´s ultrastructure and stability, respectively.\nLastly, live-cell confocal microscopy of sparsely seeded MDCK cells is used to visualize the\ndynamics of cluster organization during formation of cell-cell contact.\nIn general, I find this paper interesting, and most findings are both clear and well described. I\nhave, however, suggestions and comments that might improve the manuscript.\nMajor points\n1. Since β-catenin is part of adherens junctions, it is appropriate to analyze if there is an\nassociation between this type of cell-cell junction and individual mEGFP-β-catenin clusters. The\nauthors address this by a CLEM analysis (Fig. 4G). Unfortunately, the fluorescence associated\nwith membrane region shown is weak. It is similar to the weak intensity in regions between\nclusters (Fig. 4B, E) or alternatively, the weak fluorescence intensity before onset of β-catenin\nenrichment (Fig. 5C). Moreover, the EM resolution is low and the cell membranes with relative\nhigh electron density are separated with cell membranes that appear not to be intact. Together\nthis makes it hard to agree with a conclusion that fluorescence intensity and electron density\nshow an accumulation of β-catenin to adherens junctions. I would recommend that the authors\nshow CLEM images of another region with intense mEGFP-β-catenin fluorescence. A suggestion is the intensely fluorescent cell-cell contact region located ~10 µm to the left of the\nregion boxed in yellow. The analyses should also include a statement about independent\nbiological replicates and if possible, a reference to a paper showing electron micrographs of\ncell-cell junctions between HCT116 cells.\nWe thank the reviewer for their suggestions to improve the manuscript and data presentation.\nRegarding the fluorescence signal intensity in Fig. 4G compared to other images, it is important\nto stress that this panel shows fluorescence images of endogenously expressed mEGFP-β-\ncatenin in 90-100 nm cryosections. The total amount of cell material is significantly less than\nthat of whole cells imaged in other figures. This difference limits direct comparison, and we have\nnow emphasized this in the Results section.\nTo preserve fluorescence, we also opted for mild fixation of the cells (0.2% gluteraldehyde + 2%\nformaldehyde) in contrast to traditional EM methods. This is why, as the reviewer noted, at some\nareas the plasma membrane and cell-cell junctions are not fully preserved. This was also the\ncase for the specific region suggested by the reviewer, of which we included a CLEM image\nhere for their interest (Rebuttal Figure 2). Importantly, we are convinced that this does not\ninfluence the interpretation of our data, as we still consistently observe sites of intimate cell-cell\ncontact and electron-dense regions akin to the zonula adherens at regions marked by intense\nfluorescent spots of mEGFP-β-catenin. To emphasize this, we have included several additional\nexamples from our CLEM analysis to further support our findings (Fig. S5A). Additionally, we\nnow explicitly state that this analysis is based on 4 individual samples from 2 biological\nreplicates.\nRebuttal figure 2\nAdditional CLEM figure of the area requested by the reviewer. Sample preparation as described\nin the main manuscript.\n2. The authors combine CLEM with electron microscopy detection of mEGFP-β-catenin by\nprotein-A-conjugated gold particles. The result of the gold particle detection does not show an\nassociation between β-catenin clusters or putative adherens junctions. This is due to the choice\nof analyzed region (commented above, pnt. 1) as well as an insufficient number of gold particles\ndetected. As little as 5 cold particles are detected and only 3 of these appear to be located\nintracellularly and at proximity to the cell membrane. Accordingly, no quantification of\nimmunogold staining is given that supports an increased accumulation β-catenin to the clusters\nor adherens junctions. This experiment should also be described in the result section and not\nonly in M&M and figure legend.\nWe acknowledge that the inclusion of protein-A-conjugated gold particles in our images required\nfurther clarification.\nIndeed, only few protein-A-conjugated gold particles are present in the example image. The low\namount of mEGFP-β-catenin that can be detected (as outlined in our answer to the previous\ncomment), combined with the relative inefficiency of gold labeling compared to fluorescence,\nexplains why not all fluorescent spots correspond to an abundance of gold particles in the EM\nimages. This indicates that gold labeling is not optimal for visualizing mEGFP-β-catenin clusters,\nreinforcing our rationale for using CLEM to localize mEGFP-β-catenin in a more sensitive manner.\nWhile gold particles remain present in our CLEM analysis, they are thus not essential for detecting\nβ-catenin clusters but merely provide localization at higher-resolution, albeit for only a subset of\nthe available mEGFP-β-catenin molecules.\nWe now describe the rationale of initially including gold particles and our interpretation more\nclearly in the Results section when introducing the EM analysis.\nMinor comments\n3. I interpret the term “ectopic expression” to mean that a gene product is present at an\nabnormal location. Not that a gene is transcribed from a genetically modified and abnormally\nlocated genomic locus. However, I notice that the same terminology is used by Zamudio et al.,\nwho generated the endogenously tagged mEGFP-β-catenin (ref. 36). I therefore leave it to the\neditor to decide if terms “ectopic” and “ectopically”, which are mentioned 22 times in the\nmanuscript, are used correctly.\nWe have now adjusted this wording and instead use the term ‘exogenous’ to indicate that these\nproteins were not endogenously expressed but exogenously introduced into the cells.\n4. I don´t understand why the authors make a distinction between stably integrated “ectopically”\nexpressed transgenes and the endogenously tagged mEGFP-β-catenin gene. The chimeric\nproteins generated by both types of transgenes are most likely not regulated or expressed in a\nmanner that mimics their corresponding endogenous proteins during the dynamic events that\nestablish cell-cell junctions. This issue is not addressed in the manuscript but is important for\nthe discussion about which biomolecular interactions that are the driving force of cadherin\ncluster formation under physiological conditions (e.g. page 13, line 312)\nWe make this distinction solely to accurately describe how the expression was technically\nachieved in each experiment.\nWe would like to highlight that the endogenously tagged mEGFP-β-catenin knock-in utilizes the\noriginal promoter and regulatory elements, meaning its transcriptional regulation will mirror the\nnative regulation of this protein. In contrast, exogenously expressed proteins are randomly\nintegrated and driven by a synthetic promoter. However, we do not make any further distinction\nor claim differences in the behavior or regulation of endogenously versus exogenously\nexpressed β-catenin.\n5. The experiments shown in Fig. S4A lack a positive control, i.e. a figure showing that\ntransiently expressed Axin1 is associated with clusters of β-catenin destruction complexes at the\ncell cortex. Perhaps the Axin1 expressed is sequestered into the cytosolic biomolecular\ncondensates shown.\nWe apologize for the interpretation of this experiment not being fully clear. The purpose of this\nexperiment was to rule out the possibility that β-catenin clusters at the cell cortex represent the\ndestruction complex, and thus represent Axin1-containing clusters. If this were the case, we\nwould expect exogenously expressed Axin1 to be present in these clusters as well. We do not\nobserve Axin1 in cortical clusters; despite the clear presence of β-catenin clusters at the\nmembrane, this does not overlap with the distribution of Axin1. Thus, thus indicates that beta-\ncatenin clusters do not represent clusters containing components of the destruction complex.\nThere is thus also no positive control for their combined presence at the membrane, as our\nresults show they do not reside together in membrane-associated clusters. We have rephrased\nthe text referring to these data for clarity.\n6. Page 7, line 161. ………… β-catenin with both IDRs mutated (Figs. 3G and H). Should be\n(Figs. 2G and H).\nWe thank the Reviewer for pointing out this mistake and have corrected this.\n7. Page 7, line 162-164. This sentence needs to be edited.\nWe have rephrased this sentence for clarity.\n8. Fig. 2H, The unit on the Y-axis should be “…. per cell-cell contact” and not “.... per junction”.\nWe have adjusted this throughout the Figures of the manuscript.\nWe thank all reviewers for their continued investment in our work and constructive feedback. We have made\ntextual adaptations in the manuscript and performed an additional EM experiment to address their\nremaining concerns, as detailed below in our point-to-point reply to their specific comments. We hope the\nReviewers share our enthusiasm for our revised manuscript and find it suitable for publication in Nature\nCommunications."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All the comments and concerns have been cleared in the revised manuscript and can be accepted to be\npublished.", "reviewer_2": "The authors have made several additions to the manuscript that strengthen their arguments. They have\naddressed many of the comments in the first review, and I commend them for carrying out several additional\nmeasurements that improve the data or address concerns. These additions improve the rigor of the study.\nThere are still remaining concerns that are described below.\n1. There is still one significant scientific weakness in this study. I have a scientific issue with the response\nto the role of p120ctn in clustering (Rev 2, comment 5). The authors state they are unaware of “experimental\nevidence supporting a defined role for p120-catenin (or other members of this protein family) in the\nformation of larger clusters of the cadherin-catenin complex”. In this context, they misinterpret Yap’s studies,\nwhich focused on the sequence N-terminal to the beta catenin binding region. Their comments suggest that\np120 merely regulates cadherin at the membrane and they appear to be unaware that a dileucine mutation\ndecouples internalization from p120ctn, protein surface expression and adhesion (Miyashita, Y., and\nOzawa, M. (2007) Increased internalization of p120-uncoupled E-cadherin and a requirement for a dileucine\nmotif in the cytoplasmic tail for endocytosis of the protein: https://doi.org/10.1074/jbc.M608351200). Also,\nsee Miyashita, Y., and Ozawa, M. (2007) J. Cell Sci. 120, 4395–4406. They also appear to be unaware of\nthe study by Vu et al Curr Biol (2022) that showed that p120ctn binding facilitates cadherin oligomerization,\nconsistent with the Yap et al study. This omission is a significant weakness of the study. Although b-catenin\nmay be involved in droplet formation (as their data suggest), the implication that the droplets direct larger\ncluster formation and the assembly of cadherin adhesions, independent of these other mechanisms, is not\nyet fully supported by their data.\n2. In the rebuttal to Rev 2, the authors describe studies of droplets on free cell membranes. This is a positive\naddition to the paper and reveals differences between free and adhering complexes, presumably due to\nother factors contributing to adhesion assembly.\n3. On pg 9, there is a suggestion that IDR clusters by themselves may enable the formation of stable\nstructures at cell-cell contacts. However, once cells bind, cadherin self-organization and actin\npolymerization start kicking in and some of these processes can lead to stable oligomers in the absence of\ncytoplasmic domain interactions. A more physically plausible argument (based on these and prior data) is\nthat the clusters form and then are further immobilized through actin anchorage and cis E-cadherin\nclustering at the contacts. In this context, the droplets aid the nucleation of cadherin clusters but are one of\nmany participants in steps leading to “immobile” structures. Clarifying these points would strengthen the\npaper.\n4. Also, “stable” could imply thermodynamic equilibrium, especially in this context of LLPS. The authors\nshould discriminate between mechanical stability and thermodynamic stability.\n5. The results showing the droplet coalescence after actin disruption is important and a nice addition.\n6. In response to Rev 1 comments regarding comparisons of the physical properties (viscosity) of in vitro\nand in vitro (cells), they describe fusion studies. This does not actually address the reviewer. Fusion relates\nto surface tension, but viscosity reflects molecular organization in the droplets. The authors—to their\ncredit—acknowledge that other proteins in cells may alter the behavior. However, this still begs the question\nregarding whether in vitro droplets are a model for what happens at the plasma membrane.\n7. In response to Rev 1, comment 5, the authors altered their statement, but they still don’t meet the mark\nbecause they haven’t shown that the IDRs are driving the assembly of highly organized cadherin\naggregates at cell adhesions. Here a broader, clearer model of what b-catenin is doing at different stages\nof adhesion assembly, in the context of other factors would be useful.", "reviewer_3": "My main critique was that it is difficult to agree that the CLEM analysis shows β-catenin fluorescence in\nadherens junctions between HCT116 cells. Since the β-catenin-associated plasma membrane is damaged,\nthe β-catenin fluorescence could instead correspond to β-catenin fluorescence associated with plasma\nmembrane fragments. The image from the same preparation (rebuttal Fig. 2) unfortunately shows that my\nconcern was justified. To the right of inset 1 there is intense β-catenin staining in continuous plasma\nmembrane regions that lack cell-cell contact. It is noteworthy that authors do not remove the initial image\n(Fig. 4G, now 5A) even though they agree that the plasma membrane is damaged (they also agree that the\nmembranes are damaged in rebuttal Fig. 2). Instead, the fluorescence contrast in the original image now\nappears overly adjusted, which also is the case for an additional supplementary image with low EM\nresolution and damaged plasma membrane (Fig. S5A). The authors claim that the reason for the\ndiscontinuous plasma membrane is that a milder fixation is used in CLEM compared to the fixation used in\ntraditional EM methods. While it is true that the antibody fluorescence is preserved by mild fixation, the\nfixation used (2% PFA, 0.2% glutaraldehyde) should not result in damage plasma membranes. Moreover,\na putative explanation for poor membrane quality does not make the results more credible. The CLEM\nanalysis was done with the aim to examine the ultrastructure of the β-catenin clusters. However, the results\ndo not even reveal the ultrastructure of the electron-dense regions. The authors “mild fixation” explanation\nopens for a straightforward and simple experiment, which is to use a stronger fixation and perform a\ntraditional EM. This should reveal the ultrastructure of electron-dense cell-cell contacts and if adherens\njunctions exist between HCT116 cells under the same culture conditions as for the CLEM analysis.\nIn accordance with my concern the conclusion about the immunogold staining is toned down in the revised\nversion. The authors have also dealt with my minor comments appropriately except that they now get\nentangled in a reasoning about endogenous and exogenous gene expression that is confusing and as\nwritten, misses what is important. This is exemplified in the rebuttal letter where they write “endogenously\ntagged mEGFP-β-catenin knock-in utilizes the original promoter and regulatory elements, meaning its\ntranscriptional regulation will mirror the native regulation of this protein”. Of course, the\ntranscriptional regulation will be mirrored, but not the regulation of the protein. It is not uncommon that a transcript\nand/or protein with a sequence corresponding to three consecutive copies of the green fluorescent proteins will\ninterfere with translation and/or protein regulation (i.e. modifications, interactions, location and stability). Thus, it was the distinction\nbetween the endogenous (β-catenin) and exogenous (mEGFP-β-catenin) proteins I wanted the authors to pay attention to in\ntheir interpretation of the results, not the difference between exogenous gene transcription from knock-in loci versus stably\ntransfected plasmids.", "reviewer_4": "Many key cell biological machines assemble from multivalent interactions, leading to formation of\nbiomolecular condensates. Evidence now suggests that the cadherin-catenin complex at the adherens\njunction is one of them. One key component, beta-catenin, can phase separate in vitro, and evidence\nsupports a role for this in its other cellular job, as part of the Wnt signaling pathway. Here the authors explore\nthe role of beta-catenin phase separation in cell-cell adhesion. They find that the intrinsically disordered\nregions (IDRs) of beta-catenin play an important role in condensate formation in vitro and are important for\nefficient formation of junctional condensates with E-cadherin and alpha-catenin in cells, and they confirm a\nrole for aromatic amino acids in this region. Condensates can form on non-contacting cell membranes, but\ntrans-adhesion mediated by the E-cadherin extracellular domain increases their stability. Actin plays an\ninteresting role, acting to prevent excessive condensate mergers. Finally, they provide evidence that the\nIDRs are important for timely and high-fidelity initiation of cell-cell adhesion.\nSince I was asked to come into this process after the original reviews and revision, I have confined my\nefforts to assessing whether the work is important, and the data support the conclusions, rather than\nsuggesting new areas of investigation. I also have examined whether the authors have effectively\naddressed the concerns of the previous reviewers, particularly Reviewer 2 who was unavailable. I think\nthey have effectively addressed all of the issues raised by Reviewer 2, some of which appear to have\nresulted from some confusion about points made in the original manuscript. They also appear to have\naddressed the issues raised by the other reviewers, though I leave it to them to comment on this. In\nsummary, I think the authors work is exciting and well-supported and will be broadly read by cell,\ndevelopmental and cancer biologists.\nMinor issues that do not require re-review\n1. I found the description of what is meant in Fig 1C by “normalized condensed fraction” incomplete. The\ndata support the conclusion but a clearer explanation of what this metric precisely measures would be\nhelpful. I also thought it worth mentioning that while the IDR** mutants reduced condensate formation,\nmany of the condensates that did form in Fig 1F contained all three proteins.\n2. The authors might note that cadherin puncta have been observed and molecules within them counted in\nvivo in Drosophila embryos. https://pubmed.ncbi.nlm.nih.gov/19468069/\nhttps://pubmed.ncbi.nlm.nih.gov/37163320/\n3. They might also note that in Drosophila the C-terminal IDR is important for Wnt signaling but is not\nessential for adhesion. Likewise, individually deleting the protein of the N-terminal IDR that is not involved\nin alpha-catenin binding also does not impair the role of beta-catenin in adhesion, suggesting potential\nredundancy in vivo.\nhttps://pubmed.ncbi.nlm.nih.gov/10471715/\nhttps://pubmed.ncbi.nlm.nih.gov/9187151/"}, "author_response": "Response:\nWe thank the reviewer for highlighting these additional studies. While, to our understanding, Vu et al. focus\non dimerization rather than the formation of larger clusters per se, we agree that these findings related to\np120-catenin are relevant and important to incorporate in our manuscript. Accordingly, we have made the\nfollowing revisions:\n1. In the final paragraph of the Discussion, where we had already noted a role for p120-catenin in clustering,\nwe have now included all references suggested by the reviewer:\nLines 351-353: “For instance, p120-catenin and Afadin, which are both implicated in clustering and\nadherens junction formation67-71, both have predicted IDRs72.”\n2. In the 3rd paragraph of the Discussion, we discussed how β-catenin condensation likely acts in concert\nwith other clustering mechanisms of the cadherin complex. We now highlight that besides cis-interactions\nbetween E-cadherin ectodomains and delimited diffusion by the actin cytoskeleton, also the involvement of\nother cadherin-associated proteins represents another mechanism of clustering:\nLines 317-320: “β-Catenin-dependent condensation likely acts in concert with other clustering mechanisms\nof the cadherin complex, including cis-interactions between E-cadherin ectodomains, and delimited\ndiffusion by the actin cytoskeleton, and the involvement of other cadherin-associated proteins.”\nFinally, we would like to reiterate that we do not conclude from our data that β-catenin condensation alone\ndrives the formation of larger clusters or the assembly of cadherin adhesions. We elaborately discuss this\nin paragraph 3 and 5 of the Discussion, and further refer to our answer to Comment #3 of this Reviewer for\nthis.\nResponse:\nWe thank the Reviewer for positively evaluating these newly added data.\nResponse:\nWe fully agree with the reviewer's interpretation of our data. In line with this, in the 3rd paragraph of the\nDiscussion, we discuss that β-catenin condensation may nucleate clusters, but that other mechanisms of\nclustering will likely contribute to the immobilization and further organization of these clusters. However, we\nacknowledge that this model may not have been described well enough throughout the manuscript. To\nensure this point is unambiguous, we have also clarified it in the legend of our model figure (Figure 7), and\nnow explicitly address it at the first point of data interpretation in the Results section:\nLines 233–237: “Altogether, our findings support a model in which IDR-dependent clustering drives the\nassembly of dynamic E-cadherin/β-catenin clusters at the membrane. These clusters can mature into stable\nstructures upon the formation of cell-cell contacts, potentially involving additional anchoring interactions\nthat render the complex immobile.”\nFinally, in line with the reviewer's suggestion, we now use the term nucleation to describe how β-catenin\ncondensation contributes to catenin/cadherin cluster formation.\nResponse:\nWhen first introducing the term ‘stable clusters’, we now clarify that we mean immobile clusters. Where\nappropriate, we have also replaced the term stable with immobile to avoid confusion that we aim to\ndiscriminate between mechanical and thermodynamic stability.\nLines 226–229: “This showed that mEGFP-β-catenin clusters at cell-cell contacts did not recover beyond\nthe fluorescence levels of the surrounding membrane outside of the clusters, indicating that the observed\nIDR-dependent clusters at cell-cell contacts are immobile, stable structures (Fig. 5D, S5C and Movie S9).”\nResponse:\nWe thank the Reviewer for positively evaluating these newly added data\nResponse:\nReviewer 1 asked us to test material properties, specifically requesting an analysis of droplet fusion events.\nWe conducted these experiments accordingly to the satisfaction of this reviewer. However, we remain\ncareful in our interpretation of these data in the manuscript and do not make any claims regarding material\nproperties or viscosity.\nRegarding the validity of the in vitro model, as stated in our original response to this comment, we fully\nagree that direct comparisons between the properties of in vitro condensates and those formed in vivo are\nnot possible. While in vitro droplets cannot fully recapitulate cluster formation in cells, we maintain that this\nmethod represents an effective and appropriate approach for studying protein condensation properties,\nprecisely because it offers a minimalistic and controlled system.\nResponse:\nPlease see our answer to comment #3 of this Reviewer\nResponse:\nWe acknowledge that the plasma membrane quality of our previously included Supplementary Figure was\nsuboptimal, we therefore have taken additional images from our CLEM samples and included novel\nexamples (Fig. S5A). Furthermore, as requested, we have performed traditional Resin-embedded EM to\nbenchmark the structure of cell-cell junctions in HCT116 cells and included these data in the manuscript\n(Fig. S5B).\nImportantly, the overall morphology of cell-cell contacts between these cells appears identical in the Resin-\nembedded sections and similar to other studies (PMIDs: 30863155, 26316041, 31215349). The cells are\nspaced relatively far apart and form only sparse cell-cell adhesions of a focal nature. Both datasets contain\ntwo morphologically distinct adhesions in HCT116 cells (Fig. S5A,B). One is characterized by prominent,\nelectron-dense bands below the plasma membrane, typical of desmosomes, and where no fluorescent β-\ncatenin enrichment is detected in the CLEM experiments. The other represents clear intercellular\nattachments with less pronounced intracellular protein density, resembling adherens junctions (albeit\npredominantly focal instead of linear) and representing the β-catenin-positive sites observed in CLEM.\nIn addition, in both experiments we find multiple examples of ruptured junctions, indicating that this is a\nbiological phenomenon rather than a technical artifact. In line with this, we also observed breakage of cell-\ncell junctions between HCT116 cells with live-cell imaging. We occasionally also observed somewhat\ndiscontinuous plasma membranes, which likely originate from non-orthogonal sectioning as is more\ncommonly observed with EM preparations, however these membranes were not associated with mEGFP\nenrichment. While we find more technical artifacts in CLEM (e.g tissue folds as in the original Rebuttal Fig.\n2), these regions have been excluded from further analysis.\nImportantly, in well-preserved regions, we consistently find that fluorescent β-catenin clusters overlap with\nelectron-dense regions with adjacent membranes in close proximity. We therefore remain confident that our\nconclusion that fluorescent β-catenin clusters correspond to regions of cell-cell adhesion is well supported,\nand not confounded by technical limitations of CLEM. Furthermore, this interpretation that β-catenin puncta\ncorrespond to adhesion sites does not rely solely on the CLEM data (Fig. 5A), as this is further supported\nby the subsequent mosaic culture experiments (Fig. 5B–D).\nNonetheless, we acknowledge that it would be good to moderate the description of the CLEM findings, i.e.,\navoiding language that implies we are characterizing the ultrastructure of all beta-catenin puncta, removing\nthe direct comparison to the zonula adherens, and describing the focal nature of the observed adhesions.\nAccordingly, we have made the following revisions:\nLines 201, 209-214: “To further examine IDR-dependent β-catenin clusters, we applied electron microscopy\n(EM). … By overlaying the two images we discerned that clusters of mEGFP-β-catenin identified by light\nmicroscopy represent cell-cell junctions, typically of a focal nature, with high protein densities at the\nadjacent cytosolic sites (Figs. 5A and S5A). The overall cell morphology and of the identified cell-cell\nadhesions was similar in classical resin-embedded EM, validating our CLEM findings (Fig. S5B)48–50. These\nresults indicate that the IDR-dependent β-catenin clusters at cell-cell contacts represent sites of intercellular\nadhesion.”\nResponse:\nWe apologize for misinterpreting the initial Reviewer’s comment about the difference between endogenous\nand exogenous gene tagging. We agree that we cannot exclude that adding a tag to a protein may impact\nits properties, which is an unavoidable limitation of all techniques involving live-imaging of proteins. We\nwant to stress that we attempted to minimize this risk by using long linkers between β-catenin and the\nfluorescent tags, and that in all our experimental conditions we compare conditions where in both situations\nthe same tag is present (e.g., cluster formation is only observed in wildtype beta-catenin and not IDR\nmutants, despite having the same tag). Altogether we are confident that the presence of fluorescent tags\ndid not affect our overall conclusions.\nResponse:\nWe apologize for the lack of clarity regarding the normalized condensed fraction measurement. Although\nwe had briefly explained this measure in the Figure legends and described in the Methods section how we\ncalculated and normalized this condensed fraction, we have now expanded the explanation in the Methods\nto provide greater clarity.\nResponse:\nWe thank the Reviewer for this point out, and we now included a statement about this when describing\nthese results:\nLines 126-128: “The small fraction of β-cateninIDRs* droplets that did form contained all three proteins,\nindicating that the mutations impair the ability of β-catenin to form droplets but not its interactions with E-\ncadherin and α-catenin (Fig. 1G).”\nResponse:\nWe thank the reviewer for referring us to these articles. However, as we cite general Reviews when\ndiscussing the various types of cadherin puncta observed across model systems, we have not incorporated\nthese specific references into the manuscript.\nResponse\nWe thank the Reviewer for referring us to these articles, we now cite this work when discussing the\nmutations introduced in the IDRs:\nLines 121-124: “We previously identified that condensate formation of β-catenin relies on weak\nintermolecular interactions through aromatic amino acids in its N- and C-terminal IDRs36–38, which do not\noverlap with the established high-affinity binding sites in β-catenin for E-cadherin (Fig. S1D)40–44.”"}, {"round": 3, "reviewer_comments": {"reviewer_3": "The revised manuscript now includes additional CLEM images and, as requested, high resolution EM images that\ncomplement the CLEM analysis (Fig. S5b). This together with additional citations and adequate description of the CLEM\nresults have improved the conclusion made about the correlation between β-catenin clusters and the sites of intercellular\nadhesion. Thus, I consider that the revised version is suitable for publication in Nature communications."}, "author_response": ""}]

Decision letter