CCDC120 Phase Separation Contributes to Desmosomal Integrity and Cardiac Function

CCDC120 Phase Separation Contributes to Desmosomal Integrity and Cardiac Function | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CCDC120 Phase Separation Contributes to Desmosomal Integrity and Cardiac Function Ning Huang, Hui Meng, Wei Zhao, Yangyingzi Xi, Donghui Zhang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6141491/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Desmosomes are cell-cell adhesive junctions that provide structural integrity and mechanical resistance to tissues. Disruptions in desmosome organization lead to severe cardiac and dermatological disorders. Emerging evidence reveals that desmosomes exhibit dynamic behaviors during remodeling. However, the mechanisms that initiate and regulate these dynamics under physiological conditions, and their correlation with desmosome-related disorders remain elusive. Here, we uncovered the role of coiled-coil domain-containing 120 (CCDC120) in linking desmosomal organization to cardiac function via liquid-liquid phase separation (LLPS). CCDC120 localized to desmosomes and was required for desmosomal integrity. CCDC120 exhibited LLPS properties and co-condensed with the desmosomal component plakophilin-2 (PKP2) during desmosomal assembly, forming dynamic condensates crucial for preserving desmosomal structure and junction stability. Moreover, CCDC120 condensates were modulated by PKCα phosphorylation. Loss of CCDC120 or impaired CCDC120 phase separation led to intercalated disc structure impairment and cardiac dysfunction in mice. Our findings propose a model wherein CCDC120 phase separation orchestrates desmosomal integrity, thereby establishing connections between desmosomal dynamics and the molecular etiology of cardiac dysfunction. Biological sciences/Cell biology/Cell adhesion/Desmosomes Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights 1. CCDC120 is a desmosomal component essential for maintaining desmosomal integrity. 2. CCDC120 undergoes LLPS and co-condenses with PKP2 during desmosomal assembly. 3. CCDC120 condensates are modulated by PKCα phosphorylation. 4. Loss of CCDC120 or altered its phase separation leads to cardiac dysfunction in mice. Introduction Desmosomes are button-like intercellular junctions that anchor stress-bearing intermediate filaments (IFs) to the plasma membrane and are critical for tissues that experience high mechanical stress, such as cardiac muscle and the epidermis 1 , 2 . Desmosomes are precisely organized complexes assembled by a series of desmosomal components. Desmogleins (DSGs) and desmocollins (DSCs) are desmosomal cadherins that mediate cell-cell contact through their extracellular domains, while cytoplasmic plaque proteins link the intracellular regions of cadherins to IF networks. Plakophilins (PKPs) and plakoglobin (PG) are the main components of the outer dense plaque (ODP), whereas the inner dense plaque (IDP) is composed of the IF-binding protein desmoplakin (DP) 3 , 4 . Assembly of desmosomal components is a dynamic process, particularly during development, morphogenesis, and wound healing 5 – 7 . During desmosomal assembly, both desmosomal cadherins traffic to the plasma membrane through microtubule-based transport 8 . DP forms cytoplasmic particles that partially colocalize with PKP2 and exhibit fusion and transport events toward newly generated desmosomes 5 . Several signaling molecules, including protein kinase C α (PKCα) and Src kinase, modulate desmosomal dynamics 6 , 9 – 11 . Disruption of desmosomal structure impairs tissue integrity and physiological functions 12 , 13 . Emerging evidence suggests that impaired desmosomal organization is strongly associated with arrhythmogenic cardiomyopathy and skin disorders 14 . These findings raise a fundamental question of how desmosomal dynamics are regulated under physiological and pathological conditions. Liquid-liquid phase separation (LLPS) is an emerging mechanism for macromolecular organization that concentrates biomolecules into compartments and promotes biochemical reactions 15 . The spherical condensates formed by LLPS are characterized by fusion events and rapid molecular exchange with the surrounding solutions 16 . Mechanistically, phase separation is typically driven by proteins with intrinsically disordered regions (IDRs), where multivalent interactions among amino acid residues commonly occur 17 – 20 . Recent studies have shown that some scaffold proteins recruit client proteins into their condensates formed via phase separation to facilitate structural organization 21 – 24 . Condensation of the zonula occludens-1 (ZO-1) scaffolding protein facilitates the partitioning of other tight junction (TJ) proteins, thereby promoting TJ assembly and maintaining epithelial barrier function 21 . Furthermore, microtubule-induced LLPS of the actin nucleator Cordon-bleu (Cobl) is required for epithelial morphogenesis and the formation of paracellular barriers 25 . However, whether phase separation-related mechanisms contribute to desmosomal dynamics and cardiac function remains unclear. We previously reported that coiled-coil domain-containing 120 (CCDC120) is a centrosomal subdistal appendage (SDA) component. Loss of CCDC120 disrupts SDA assembly and microtubule anchoring in HeLa cells during interphase 26 . Here, we identified an unexpected role of CCDC120 in crosslinking desmosomal structure and maintaining cardiac function through phase separation. CCDC120 is enriched at the desmosomal plaque and is crucial for desmosomal integrity. CCDC120 undergoes LLPS and co-condenses with PKP2 to preserve functional desmosomal organization. Furthermore, CCDC120 condensation is regulated by PKCα phosphorylation. Loss of CCDC120 or altered CCDC120 phase separation impairs intercalated disc structure and leads to cardiac dysfunction. Results CCDC120 is a desmosomal component that maintains desmosomal integrity Coi led-coil domain-containing 120 (CCDC120) was previously identified as a subdistal appendage (SDA) component required for SDA assembly and microtubule anchoring at centrosomes in HeLa cells 26 . Unexpectedly, robust localization of GFP-tagged CCDC120 was observed at cell-cell junctions in Madin-Darby canine kidney (MDCK) cells (Fig. 1 A), a widely used model for studying cell junctions 27 . To confirm the localization of CCDC120 at cell-cell junctions, endogenous CCDC120 was co-labeled with the desmosomal adhesion marker DP I/II or the adhesion junction marker -catenin in MDCK cells. CCDC120 exhibited greater colocalization with DP I/II at cell borders when compared to that with -catenin (Fig. 1 B, C). Moreover, CCDC120 signaling was almost undetectable at cell junctions in CCDC120 knockout (KO) MDCK cell lines generated using CRISPR/Cas9 approaches (Supp Fig. 1 A-C), confirming the specificity of its localization. In addition, CCDC120 colocalized with the desmosomal protein PKP2 at sites where Keratin 19 filaments in adjacent cells anchor (Fig. 1 D), indicating that CCDC120 localizes to desmosome adhesions. Three-dimensional structured illumination microscopy (3D-SIM) was performed to characterize the detailed localization of CCDC120 within desmosomes. Compared with the distribution of DP I/II (Fig. 1 E), CCDC120 was localized closer to the cell membrane, where the ODP protein PKP2 was found (Fig. 1 F). Consistently, immunoprecipitation assay results showed that CCDC120 preferentially interacts with the desmosomal component PKP2 (Fig. 1 G), suggesting that CCDC120 localizes near the ODP of desmosomes in MDCK cells (Fig. 1 H). To determine the role of CCDC120 in desmosomes, we first examined the localization of different desmosomal proteins in both wild-type (WT) and CCDC120 KO MDCK cell lines. Depleting CCDC120 significantly disrupted the continuous distribution of DP I/II and PKP2 (Fig. 2 A-D), leading to a disorganized pattern. A previous study showed that most desmosomal components are insoluble in lysis buffer containing nonionic detergents, such as Triton X-100, because of their ability to associate with the desmosomal plaque 28 . To assess the solubility properties of desmosomal components, we next analyzed their insoluble fractions and found that, in CCDC120 KO MDCK cells, the insoluble fractions, but not total protein levels, of DP I/II and PKP2 were reduced (Fig. 2 E, F). This reduction further indicates impaired desmosomal organization after CCDC120 depletion. By contrast, β-catenin distribution at cell junctions remained unchanged in CCDC120 KO cells (Supp Fig. 1 D, E), confirming that CCDC120 specifically participates in desmosomal component assembly. However, the loss of PKP2, but not DP I/II, disrupted the proper localization of CCDC120 at desmosomes (Fig. 2 G, H and Supp Fig. 1 F), revealing a strong functional correlation between CCDC120 and PKP2. We then investigated whether CCDC120 loss alters desmosomal adhesion in MDCK cells. To assess this, we performed dispase intercellular adhesion assays. When constant shear stress was applied to the detached cell monolayer, CCDC120 KO cells exhibited a greater number of fragments than WT cells, indicating reduced intercellular adhesion following CCDC120 deletion (Fig. 2 I, J). Furthermore, because intermediate filaments and desmosomal structure contribute to the mechanical properties of cells 29 , 30 , we used atomic force microscopy (AFM) to measure the elasticity of the apical surface of the MDCK monolayers, from which the Young’s modulus was obtained (Fig. 1 K). AFM results showed that CCDC120 KO cells (Young’s modulus: 2.415 ± 0.027 kPa) exhibited a significant decline in cellular elasticity than WT cells (Young’s modulus: 4.186 ± 0.06 kPa, Fig. 2 L, M). Our results suggest that CCDC120 is enriched at desmosomes and is essential for maintaining desmosomal structural integrity and function. CCDC120 undergoes LLPS both in cells and in vitro To characterize CCDC120 dynamics in cells, we generated MDCK cell lines stably expressing GFP-tagged CCDC120. At low expression levels, GFP-CCDC120 localized predominantly to desmosomal adhesions (Fig. 3 A, left panel). Surprisingly, at high expression levels, round-shaped particles with varied sizes appeared in the cytoplasm of MDCK cells that highly expressed GFP-CCDC120 (Fig. 3 A, right panel). In fluorescence recovery after photobleaching (FRAP) assays, the fluorescent intensity of GFP-CCDC120 in cytoplasmic puncta was partially recovered after photobleaching (Fig. 3 B, C), indicating the exchange of GFP-CCDC120 between the particles and the cytoplasm. In addition, we transiently expressed GFP-CCDC120 in HEK293T cells, a cell line with high transfection efficiency but weakened desmosomal adhesion 21 . Our results showed that GFP-CCDC120 primarily formed one large particle in the cytoplasm (Supp Fig. 2 A) and exhibited exchange dynamics, as confirmed by FRAP assays (Supp Fig. 2 B, C). DP-GFP expression leads to the formation of cytosolic particles, and a series of DP-GFP dynamic events near the cell borders are triggered by cell-cell contact 5 . Building on this, we next examined the dynamic behavior of CCDC120 during desmosome assembly in MDCK cells. Live-cell imaging revealed that during cell-cell contact, some GFP-CCDC120 particles near the contacting borders appeared to fuse together, then moved towards the borders, and finally coalesced with assembled desmosomes at cell contact sites (Fig. 3 D). Consistently, similar fusion events of GFP-CCDC120-formed particles were observed at cell borders in HeLa cells (Supp Fig. 2 D), suggesting that these particles exhibit liquid-like properties. CCDC120 dynamics during cell-cell contact prompted us to examine whether CCDC120 undergoes LLPS. Since IDRs commonly play a key role in mediating phase separation 18 , 19 , 31 , we analyzed the CCDC120 protein sequence and identified two continuous IDRs on its C-terminus (IDR1: 210–430 aa and IDR2: 431–629 aa; Fig. 3 E). We first tested the LLPS properties of these two IDRs in cells. Depleting IDR1, but not IDR2, reduced the GFP-CCDC120 condensates in MDCK cells (Supp Fig. 2 E, F), indicating that IDR1 plays a predominant role in CCDC120 phase separation. To test whether CCDC120 condensates in the cells are directly generated via IDRs or associated with other cellular components, we performed a series of in vitro experiments. Purified His-GFP-tagged CCDC120 IDR1 or IDR1 + 2 formed spherical droplets, and droplet size changed in a protein concentration-dependent manner (Fig. 3 F and Supp Fig. 2 G, H). A FRAP assay revealed that IDR1-formed droplets could exchange proteins with in the surrounding solutions (Fig. 3 G, H). Furthermore, dynamic fusion events of the droplets formed by IDR1 were observed (Fig. 3 I), suggesting that these droplets possess liquid-like properties in vitro. Thus, CCDC120 undergoes LLPS, primarily mediated by IDR1. CCDC120 phase separation is required for desmosomal organization and function To determine whether the role of CCDC120 at desmosomes relies on phase separation, we first identified the amino acids within IDR1 responsible for LLPS of CCDC120. By generating and expressing a series of truncation mutations within CCDC120 IDR1, we found that the 210–250 aa region of CCDC120 is critical for CCDC120 condensation in HEK293T cells (Supp Fig. 3 A, B). By analyzing the protein sequence, we identified several evolutionary conserved acidic amino acids clustered within or near the 210–250 aa region of CCDC120 (Fig. 4 A). Electrostatic interactions are a type of multivalent interaction that mediate LLPS 32 , 33 , and clusters of negatively charged residues contribute to charge-driven phase separation formation 34 , 35 . To test this, we next neutralized these negative-charge residues by mutating ten aspartic acid and glutamate residues to alanine in IDR1 (DE10A), and examined its LLPS properties by performing phase separation assays under various protein and salt concentrations (Fig. 4 B, C). The IDR1-DE10A mutant displayed attenuated LLPS properties compared with IDR1-WT, forming only small droplets at low salt concentrations (Fig. 4 B, C). Finally, we assessed droplet formation of CCDC120 DE10A mutant in cells. The CCDC120 DE10A mutant exhibited fewer CCDC120 condensates in MDCK and HEK293T cells when compared to the droplets that formed by CCDC120 WT (Supp Fig. 3 C-F). Taken together, we concluded that the enriched aspartic acid and glutamate residues in IDR1 are essential for CCDC120 LLPS. To investigate the functional relevance of CCDC120 phase separation, GFP-tagged CCDC120 FL or the DE10A mutant were stably expressed in CCDC120 KO MDCK cells to evaluate their ability to support desmosomal assembly and function. First, we examined CCDC120 localization at cell junctions. In CCDC120 FL-rescued cells, a continuous distribution of CCDC120 FL was detected at cell-cell borders (Irregular score: 0.293 ± 0.009; Fig. 4 D, F). However, expressing the CCDC120 DE10A mutant resulted in a disorganized, bead-like localization around junction sites (Irregular score: 0.526 ± 0.015; Fig. 4 D, F). Moreover, the expression of CCDC120 FL, but not the DE10A mutant, rescued the mislocalization of endogenous PKP2 at desmosomes (Irregular score: FL, 0.385 ± 0.014; DE10A, 0.808 ± 0.027; Fig. 4 E, G), indicating that CCDC120 maintains desmosomal assembly through phase separation. Finally, we examined the correlation between CCDC120 LLPS ability and desmosomal function. A dispase assay showed that CCDC120-FL, but not the DE10A mutant, restored adhesion loss in CCDC120 KO cells (Fig. 4 H, I), supporting the role of CCDC120 phase separation in desmosomal adhesion. In addition, CCDC120 KO cells stably expressing CCDC120 FL, but not the DE10A mutant, exhibited a partial increase in Young’s modulus, as measured by AFM (FL: 3.288 ± 0.045; DE10A: 2.600 ± 0.036; Fig. 4 J, K). Together, these findings suggest that phase separation of CCDC120 is essential for desmosomal integrity, thus ensuring intercellular adhesion and cell stiffness. CCDC120 co-condenses with PKP2 Phase separation drives cellular compartmentalization 36 . Condensates of ZO proteins selectively partition TJ proteins during TJ assembly 37 . We asked whether CCDC120 LLPS similarly facilitates the selective enrichment of desmosomal components during desmosomal assembly. Due to the predominant interaction between CCDC120 and PKP2 (Fig. 1 G), we first examined whether CCDC120 co-condenses with PKP2. PKP2 associated with CCDC120 droplets in the cytoplasm of HeLa cells (Fig. 5 A). Furthermore, fusion events of CCDC120 droplets associated with PKP2 were observed at cell borders after cell-cell contact (Fig. 5 B). We then investigated the role of CCDC120 LLPS in PKP2 particle formation in MDCK cells. Interestingly, a robust decline in the percentage of cells with PKP2 particles was detected after CCDC120 depletion (Fig. 5 C, D). This phenotype was rescued by expressing CCDC120 FL, but not the DE10A mutant (Fig. 5 C, D), further demonstrating that CCDC120 phase separation is required for PKP2 particle formation. Since PKP2 colocalizes with cytoplasmic particles formed by DP-GFP overexpression 5 , we also examined the relationship between CCDC120 LLPS and DP particle formation. Although DP-GFP signaling was associated with CCDC120 condensates in HeLa cells (Supp Fig. 4 A), there was no significant change in DP particle formation between WT and CCDC120 KO cells (Supp Fig. 4 B, C), suggesting that DP may generate cytoplasmic particles in a CCDC120-independent manner. To further investigate the relationship between CCDC120 and PKP2, we mapped the domains responsible for their interaction. Immunoprecipitation assays revealed that the N-terminal region of PKP2, which mediates interactions with several desmosomal proteins such as DP and PG 38 , also binds to the C-terminal region of CCDC120 (Supp Fig. 4 D, E). Interestingly, an IDR domain (80–240 aa) within the PKP2 N-terminus was predicted (Supp Fig. 4 F). Consistent with the observed cellular phenotypes (Fig. 5 C, D), co-incubation with CCDC120 IDR1 + 2 WT, but not the DE10A mutant, increased the size of PKP2-IDR droplets compared to those formed by PKP2-IDR alone (Fig. 5 E, F and Supp Fig. 4 G). This suggests that PKP2 partitions into the condensed phase of CCDC120 through its IDR domain. Moreover, the impaired LLPS properties of CCDC120 did not affect its interaction with PKP2 (Supp Fig. 4 H). Together, these results indicate that CCDC120 specifically concentrate PKP2 through LLPS. PKCα-mediated phosphorylation promotes CCDC120 phase separation PKC signaling regulates various types of intercellular junction assembly 39 , 40 . In particular, PKCα governs desmosomal dynamics by forming a DP-PKP2-PKCα complex 41 . To test whether PKCα regulates the phase separation of CCDC120, we performed an in vitro phase separation assay by incubating purified CCDC120-IDR1 with PKCα. In the presence of ATP, liquid droplets formed by CCDC120-IDR1 were larger than those in the non-ATP groups (Fig. 6 A, B), indicating that PKCα activation increases CCDC120 droplet formation in vitro . We next examined the role of PKCα in CCDC120 droplet formation in cells. Treatment with the PKC activator PMA significantly increased CCDC120 LLPS in MDCK (Fig. 6 C, D) and HEK293T cells (Supp Fig. 5 A, B). Since phosphorylation may regulate LLPS via electrostatic modulation 37 , 42 , 43 , we asked whether PKCα modulates CCDC120 phase separation by phosphorylating IDR1. Using mass spectrometry analysis, we first screened phosphorylation sites of CCDC120 that could be targeted specifically by PKCα. Ten evolutionary conserved serine and threonine residues within IDR1 were identified (Fig. 6 E and Supp Fig. 5 C). We then constructed a non-phosphorylatable CCDC120 mutant by mutating all serine and threonine residues to alanine (ST10A), and performed an in vitro kinase assay. A strong thiophosphate-ester signal was detected after incubating PKCα with WT but not ST10A mutant CCDC120-IDR1 proteins (Fig. 6 F), further confirming the specificity of these phosphorylation sites. Finally, we investigated the role of PKCα in CCDC120 desmosomal localization and function by expressing the ST10A mutant in CCDC120 KO MDCK cells. Compared with CCDC120 FL signaling at the cell-cell borders (Irregular score: 0.186 ± 0.007; Fig. 6 G, I), CCDC120 ST10A mutants exhibited a disorganized distribution around the junction sites (Irregular score: 0.227 ± 0.007; Fig. 6 G, I), similar to the phenotypes observed in CCDC120 DE10A mutants (Fig. 4 D, F). Moreover, compared to CCDC120 FL expression, CCDC120 ST10A mutant expression provided minimal rescue of endogenous PKP2 at desmosomes (Irregular score: FL, 0.322 ± 0.020; DE10A, 0.456 ± 0.020; Fig. 6 H, J). This indicates that PKCα-mediated regulation of CCDC120 modulates its phase separation and desmosomal function primarily through these phosphorylation sites. Taken together, these results indicate that PKCα directly phosphorylates CCDC120-IDR1 and increases its droplet formation, thus maintaining desmosomal assembly. CCDC120 LLPS maintains cardiac function and ICD structure Recent studies show that mutations in genes encoding desmosomal proteins, such as DP and PKP2 , lead to an inherited heart disease known as arrhythmogenic right ventricular cardiomyopathy (ARVC) 44 , 45 . Since CCDC120 contributes to desmosome assembly and function (Figs. 1 and 2 ), we examined its role in cardiac function. We first generated Ccdc120 KO mice using CRISPR/Cas9. A 50-bp deletion in exon 4 of Ccdc120 was detected (Supp Fig. 6 A, B). Immunoblotting further confirmed the loss of CCDC120 protein in KO mice (Supp Fig. 6 C). Using echocardiography, we assessed left ventricular (LV) function in WT and Ccdc120 KO mice at 2 months of age (Fig. 7 A and Supp Table 1). We detected a significant decline in left ventricular ejection fraction (LVEF) and fraction shortening (FS) in Ccdc120 KO mice (Fig. 7 B). To explore whether CCDC120 maintains cardiac function through phase separation, we generated Ccdc120 DE10A mutant mice (Supp Fig. 6 D, E). Importantly, Ccdc120 DE10A mutant mice phenocopied the cardiac dysfunction observed in Ccdc120 KO mice (Fig. 7 A, B, Supp Table 1). Together, these results demonstrate that CCDC120 phase separation is essential for maintaining cardiac function in mice. Intercalated discs (ICDs) are specialized structures that connect adjacent cardiomyocytes. The area composita, the predominant junction type within ICDs, contains desmosomal and fascia adherens components 46 , 47 . To investigate the cause of cardiac dysfunction in Ccdc120 KO and DE10A mutant mice, we first examined the localization of desmosomal components in ICDs. Immunofluorescence analysis revealed a significant decline in DP and PKP2 signaling length on ICDs in Ccdc120 KO and DE10A mice compared to WT mice (Fig. 7 C, D). This mislocalization pattern was similar to the disrupted DP and PKP2 localization observed in CCDC120 KO and DE10A-rescued MDCK cells (Figs. 2 and 4 ). Transmission electron microscopy was performed to examine the ultra-structural changes in ICDs further. The length of the desmosomal structure was reduced in Ccdc120 KO and DE10A mice (Fig. 7 E, F). In addition, the area composita exhibited a widened gap in Ccdc120 KO mice, which was even more pronounced in Ccdc120 DE10A mice (Fig. 7 E, F). Finally, we analyzed the solubility properties of desmosomal components in the heart tissues of different mice. The insoluble fraction of CCDC120 in Ccdc120 DE10A was lower (Fig. 7 G) than in WT heart tissue, which is consistent with its irregular desmosomal localization in MDCK cells (Fig. 4 D). Moreover, the insoluble DP I/II and PKP2 protein levels were also decreased in Ccdc120 KO and DE10A mice (Fig. 7 G), further supporting the role of CCDC120 LLPS in the proper localization of desmosomal components within ICDs. These results suggest that CCDC120 phase separation is important for maintaining cardiac function by stabilizing ICD structure. Discussion In this study, we uncovered the role of CCDC120 in desmosome assembly and function. CCDC120 exhibits LLPS properties, which are further enhanced by PKCα phosphorylation. Moreover, CCDC120 specifically interacts with PKP2 and co-condenses with it during desmosomal formation. Impaired CCDC120 phase separation causes desmosomal protein mislocalization, weakened intercellular adhesion, and cell stiffness, ultimately resulting in cardiac dysfunction in mice. Our results demonstrate that CCDC120 colocalizes and interacts with PKP2. Loss of CCDC120 impaired both DP and PKP2 assembly on desmosomes. However, only PKP2 depletion reciprocally alters the desmosomal localization of CCDC120, suggesting that CCDC120 and PKP2 may function upstream of DP in desmosomal assembly. Although CCDC120 did not strongly interact with DP, GFP-CCDC120 colocalized with DP-formed particles in the cytoplasm. We speculate that this colocalization might be mediated by endogenous PKP2 or other desmosomal components. During desmosomal assembly, DP-GFP displays multiple dynamic phases triggered by cell-cell contact 48 .Some DP-GFP particles are co-transported with PKP2 and subsequently integrate into newly formed junctions 48 , 49 , a pattern similar to GFP-CCDC120 condensates formed via LLPS (Fig. 3 ). Since DP is predicted to exhibit LLPS properties ( www.predict.phasep.pro ), it would be interesting to determine whether cell contact-triggered DP-GFP dynamics are also dependent on phase separation. In addition, some DP-GFP particles at perinuclear sites colocalize with engulfed desmosomal cadherins 48 , 50 . Whether GFP-CCDC120 condensates also partition with desmosomal cadherins or PG requires further investigation. In our study, we found that PKCα directly phosphorylates multiple serine and threonine residues on CCDC120-IDR1, enhancing its LLPS properties (Fig. 6 ). Phosphorylation modulates phase separation in both positive and negative manners 21 , 51 , 52 . A recent study revealed that phosphorylation modification modulates LLPS by altering residue charges rather than by attaching phosphate groups at specific sites 51 . Since a negative charge block on IDR1 is essential for CCDC120 LLPS (Fig. 4 ), we speculate that PKCα-mediated phosphorylation further increases the negative charge on IDR, enhancing CCDC120 phase separation via charge interactions. Furthermore, PKCα regulates desmosomal protein dynamics. PKCα-mediated phosphorylation of DP at Ser2849 weakens its binding with IFs and is involved in desmosomal remodeling during both desmosome formation 48 , 49 , 53 and disassembly 54 , 55 . PKCα inhibition can also switch desmosomes from a calcium-dependent state to a more static conformation known as hyperadhesion 54 , 56 , thus reducing desmosomal protein turnover 57 . Future studies will focus on how PKCα modulates the dynamics of CCDC120 condensates in cells. ARVD/C is a complex myocardial disorder that leads to heart failure and sudden cardiac death 58 . Mutations in four genes encoding desmosome proteins ( DP , PKP2 , JUP , and DSG2 ) have been identified in ARVC/D patients 59 – 64 . Transgenic mouse models carrying these mutations exhibit cardiac dysfunction and even embryonic lethality 44 , 65 . This highlights the importance of desmosome proteins in maintaining the mechanical resilience of contracting cardiomyocytes. In our study, we show that loss of CCDC120 impairs the distribution of desmosomal components at ICDs and causes cardiac dysfunction in mice (Fig. 7 ), further supporting the concept that ARVD/C is a desmosomal disease 66 . However, whether CCDC120 abnormalities contribute to other ARVD/C phenotypes, such as arrhythmias and fibrosis 66 , as well as the correlation between CCDC120 mutations and ARVD/C, remains to be determined. Recent studies have highlighted the potential role of LLPS in cardiovascular diseases such as heart failure 67 , 68 , and cardiomyopathy 69 . Our findings demonstrate that CCDC120 phase separation is required for desmosomal assembly and cardiac function. Moreover, ZO-1, which is involved in ICD composition and atrioventricular node conduction at gap junctions 70 , 71 , also displays LLPS properties 72 . These findings suggest that LLPS properties and LLPS-related mutations in junctional proteins may provide new insights into the regulation of cardiovascular functions. In summary, we show that CCDC120 LLPS is critical for regulating desmosomal integrity, which is necessary for maintaining cardiac function. These findings shed light on the mechanistic interplay between LLPS-mediated desmosomal dynamics and cardiac function and may contribute to the understanding and diagnosis of desmosome-related cardiovascular disorders. Materials And Methods Animal models All animals were handled following the “Principles for the Utilization and Care of Vertebrate Animals” and the “Guide for the Care and Use of Laboratory Animals.” Animal studies were approved by the IACUC of the Center for Experimental Animal Research (China) and Peking University Laboratory Animal Center (IACUC No. LSC‐ChenJG‐3). Ccdc120 knockout mice were generated using a CRISPR/Cas9 approach on a C57BL/6J background by Shanghai BioModel Organisms Center, Inc. An sgRNA (5’-GTGAAGTCAGACCGTCTTCG-3’) was designed to target exon 4 of the ccdc120 gene. To generate Ccdc120 DE10A mutant mice, two sgRNAs targeting intron 6 and intron 9 (5’-TCTGTTTGCTGCATGCATAC-3’ and 5’-ACTATCGATTAGCAGAC-3’), respectively, along with one vector containing donor template to perform homologous-dependent-repair (Shanghai Model Organisms Center, Inc.). Detailed information on genotyping, sequencing primers, and donor templates is provided in Supplementary Table 2. Plasmid construction The full-length complementary DNAs (cDNAs) of Ccdc120 and Pkp2 were amplified from a cDNA library by PCR. The DP cDNA was a gift from Kathleen Green (Addgene plasmid #32227). The full-length and mutant cDNAs were subcloned into pEGFP-C2, pCMV7.1-3xFlag, pmApple-N1, or pET-28a. Cell culture, transfection, and treatment MDCK-II and HEK293T cells were purchased from ATCC and cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (CellMax) at 37 °C in 5% CO 2 . For cell transfection, Lipofectamine 3000 (Invitrogen) and jetPRIME (Polyplus-Sartorius) were used according to the manufacturer’s instructions. To activate PKCα kinase, MDCK-II cells were treated with PMA (15 nM, MCE) for 30 min. Generation of knockout and stable expressing MDCK cell lines All knockout cell lines were generated using a CRISPR/Cas9 approach. The sgRNAs targeting Ccdc120 , Pkp2 , and DP I/II were inserted into the LentiCRISPR-V2 vector. The vectors were then transfected into MDCK cells, and single-cell colonies were selected by puromycin treatment (3 μg/mL, Solarbio). The colonies were subjected to immunoblotting and genome sequencing to verify the knockout efficiency. Detailed information on the PCR primers used is provided in Supplementary Table 2. Full-length and mutant CCDC120 cDNAs were cloned into pEGFP-C2 vectors and transfected into wild-type (WT) or CCDC120 knockout MDCK cells for stable cell line selection. Single-cell colonies were selected by treatment with G418 (200 μg/mL, Invitrogen). Immunoblotting and Immunoprecipitation Total protein from cultured cells was extracted using SDS loading buffer and heated at 100 °C for 10 min. For mouse tissue protein, Triton X-100 soluble and insoluble fractions were extracted as previously described 73 . Briefly, tissues were homogenized in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) with protease inhibitor cocktail (Epizyme) and PMSF (1 mM, Solarbio). Triton X-100 soluble and insoluble fractions were separated by centrifugation at 12,000 × g for 30 min at 4 °C. The pellets were then solubilized for 30 min at room temperature in lysis buffer containing 8 M urea and 50 mM DTT. All protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Primary and secondary antibodies used for immunoblotting are listed in Supplementary Table 3. For immunoprecipitation, cultured cells or tissues were lysed on ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and 10% glycerol) containing protease inhibitor cocktail (Epizyme) for 30 min. After centrifugation at 12,000 × g for 15 min at 4 °C, the supernatants were incubated with the indicated antibodies for 2 h at 4 °C, followed by incubation with Protein A/G magnetic beads (Selleck) for an additional hour. Finally, the immunoprecipitated samples were mixed with SDS loading buffer, heated at 100 °C for 5 min, and analyzed by immunoblotting. Protein expression and purification All recombinant proteins were expressed in E. coli (BL21-DE3, Tsingke) and induced with 0.5 mM IPTG (Solarbio) for 16–18 h at 16 °C. To purify His-tagged recombinant proteins, E. coli cells were resuspended in binding buffer (50 mM NaH 2 PO 4 ,pH 8.0, 500 mM NaCl, 1 mM DTT, 30 mM imidazole, 10% glycerol and 0.5% Triton X-100) containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by sonication on ice, and the lysates were centrifuged at 12,000 × g for 30 min at 4 °C. The supernatant was incubated with Ni-NTA agarose beads (CWBIO) and eluted using elution buffer (50 mM NaH 2 PO 4 , pH 8.0, 500 mM NaCl, 1 mM DTT, 300 mM imidazole, 10% glycerol and 0.5% Triton X-100). Finally, purified proteins were concentrated in P buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM DTT, and 10% glycerol) using centrifugal filtration (Millipore), aliquoted and stored at -80 °C. In vitro phase separation assays For most experiments, phase separation was induced by diluting purified proteins into phase separation buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, and 5% PEG 4000) at the indicated concentration, followed by incubation for 5 min at 25 °C. Droplet formation was observed by adding 4 μL protein mixture to a glass-bottom cell culture dish (NEST). This was followed by imaging using an Olympus FV3000 confocal microscope equipped with a 100×/1.45 NA oil immersion objective lens. To identify the role of PKC in CCDC120 droplet formation, 260 μM His-GFP-CCDC120-IDR1 proteins were diluted in kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl 2 , 500 mM KCl, 1 mM DTT and 0.01 mg/ml PKCα) with or without 200 μM ATP (Solarbio). After incubation at 30 °C for 1 h, the protein mixture was incubated with LLPS buffer (20 mM HEPES, pH 7.4, 150 mM KCl, and 5% PEG 4000), followed by imaging. For FRAP experiments, CCDC120 droplets were photobleached using a 488 nm laser at 100% laser power, followed by imaging at 5-s intervals for 2.5 min. Immunofluorescence and live-cell imaging Immunofluorescence staining of cell samples was performed as previously described 74 . Briefly, MDCK cells were fixed and permeabilized in cold methanol for 10 min at 4 °C. HEK293T cells were fixed in PBS containing 4% paraformaldehyde (PFA) and 4% sucrose for 20 min at RT, then permeabilized in PBS containing 0.5% Triton X-100 for 10 min. After blocking in PBS containing 4% BSA, samples were incubated with the indicated primary and secondary antibodies (Supplementary Table 3), and mounted for imaging. For confocal imaging, an Olympus FV3000 confocal microscopy equipped with a 100×/1.45 NA objective lens was used at RT. For desmosome super-resolution imaging, cell samples were imaged using a Zeiss Elyra 7 3D-SIM microscope equipped with a 63×/1.4 NA objective lens at RT. All images were processed by ImageJ software (NIH). To quantify the distribution of desmosomal proteins at cell borders, the mean fluorescence intensities of these proteins in different cells were measured using ImageJ. To further characterize the integrity of these proteins along cell-cell contacts, irregular score was calculated using the following equation 75 ： For immunostaining of tissue samples, mice were first anesthetized with tribromoethanol and perfused with 4% PFA. Heart tissues were then isolated and post-fixed in freshly prepared 4% PFA overnight. After dehydration in a graded series of alcohol and xylene, samples were embedded in paraffin and sectioned at a thickness of 4 μm. Sections were deparaffinized, rehydrated in water, heated in antigen retrieval buffer (10 mM citrate, pH 6.0) for 20 min in a microwave oven, and permeabilized in 0.25% Triton X-100. Samples were blocked, incubated with indicated primary and secondary antibodies (Supplementary Table 3), and then mounted for imaging. To quantify the localization of DP and PKP2 in the heart tissue sections, the length of DP and PKP2 signaling at the intercalated disc was measured using ImageJ software (NIH). For live-cell imaging, transfected cells were seeded in a glass-bottom dish and imaged using a spinning-disk confocal microscope (UltraView VoX, PerkinElmer) equipped with a 60×/1.4 NA objective lens or a Zeiss LSM 880 Airyscan confocal microscope equipped with a 40×/1.3 NA objective lens at 37 °C. Images were analyzed using Volocity software (PerkinElmer) or ZEN 3.8 (Zeiss). For FRAP assays, MDCK cells were transfected with a GFP-CCDC120 vector. FRAP assays were performed 24 h after transfection using a Zeiss LSM 880 Airyscan confocal microscope. GFP-CCDC120 droplets or desmosome-localized GFP-CCDC120 were photobleached using a 488 nm laser at 100% laser power, followed by imaging at 9-s intervals for 3 min. In vitro kinase assay The in vitro kinase assay was performed as described in a previous study 76 . Recombinant His-GFP-CCDC120-IDR1 WT or ST10A mutant protein, purified from E. coli , was used as a substrate and incubated with 0.05 μg PKCα (Abcam) for 1 h at 30 °C in kinase buffer (40 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 1 mM DTT, and 500 μM ATP-γS (Solarbio)) with protease inhibitor cocktail. A final concentration of 2.5 mM p -nitrobenzylmesylate (PNBM, Abcam) was added to the mixture for an alkylation reaction and incubated for 1 h at 25 °C. Reaction products were analyzed by immunoblotting using an anti-thiophosphate-ester antibody (SD2020, HUABIO). Atomic Force Microscopy (AFM) The AFM (BioScope Resolve, Bruker) indentation method was used to measure cell stiffness 77 . A pre-calibrated probe (MLCT-SPH-5UM, Bruker) with a hemispherical tip was used. Cantilever D was typically used with a spring constant of 0.028 N/m. The deflection sensitivity was measured before each experiment to ensure complete probe calibration. Cells were plated in a 35-mm cell-culture dish to form monolayers with greater than 80% confluency. AFM measurements were performed in cell culture medium at RT. The cantilever was brought into contact with the cell layer at a constant speed of 4 µm/s until reaching the force setpoint of 3 nN, at which point the maximum indentation depth of cells ranged from 0.5 to 1.0 µm. A force volume scan containing 256 spots in a 100 × 100 μm region was set, and 12–15 regions were randomly selected in one group of cells. Force-indentation curves were fit to the Hertz model for spherical tips using NanoScope Analysis software 1.8, with a Poisson’s ratio of 0.5 for all calculations, to determine Young’s modulus. Dispase assay The dispase assay was performed as described in a previous study 78 . Briefly, MDCK cells were plated in a 6-well plate. After 36 h, cells were treated with culture medium containing dispase II (2.4 U/mL) for 30 min at 37 °C. The detached monolayer was transferred into a 15-mL centrifuge tube and subjected to multiple inversions. The number of monolayer fragments was quantified using ImageJ software. Echocardiography Transthoracic echocardiography was performed using a Vevo 3100 Ultrasound Imaging System (VisualSonics Inc.) equipped with a 40-MHz probe. After anesthesia in an induction chamber containing 3% isoflurane in oxygen, the mouse was placed in a supine position on a warm table to maintain body temperature at approximate 37 °C. Anesthesia was maintained using 1% isoflurane in 500 mL O 2 /min via a nose mask. Images were acquired in parasternal short-axis views (M-mode), and cardiac function parameters were measured and calculated using Vevo3100 analytical software. Left ventricular ejection fraction (LVEF) and fractional shortening (FS) were calculated by at least five measurements of LV anterior (LVAW), LV internal diameter (LVID) and LV posterior wall (LVPW) in diastole and systole respectively. Transmission Electron Microscopy (TEM) Two-month-old mice were anesthetized with tribromoethanol and perfused with 2% PFA and 1% glutaraldehyde in 0.1 M phosphate buffer (PB) at RT. Heart tissues were isolated and fixed in 2% PFA and 2.5% glutaraldehyde in 0.1 M PB at RT for 1 h, then stained with 2% uranyl acetate. Samples were washed four times with 0.1 M PB and post-fixed in 1% OsO 4 and 0.8% K 4 Fe(CN) 6 for 1 h at RT. Following three washes with double-distilled H 2 O (ddH 2 O), cells were stained with 1% tannic acid for 1 h at RT, dehydrated through a graded ethanol series, and embedded in Embed 812 resin. Ultrathin sections (70 nm) were obtained using a Leica Microsystems UC7 ultramicrotome and mounted on single-slot copper grids. After counterstaining with uranyl acetate and lead citrate, samples were imaged using a Tecnai G2 Spirit BioTWIN (FEI) transmission electron microscope with a CCD camera (Orius 832, Gatan). At least 20 images per sample were acquired for measurements. The lengths and widths of desmosome-like segments of the area composita were measured in different groups, as described in a previous study 79 . Statistical analysis Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software). Data are presented as mean ± SEM. An unpaired two-tailed Student’s t -test was applied for comparisons between two groups. One-way ANOVA was applied for multiple group comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Declarations Author Contributions H.M. and W.Z. designed and performed most of the molecular and cellular biology experiments, Y.X., D.Z., R.C., Y.H., and Y.T. designed and performed animal experiments. X.H. analyzed the data, T.W. performed 3D-SIM imaging. J.T., J.C., and N.H. are the senior authors that designed the project. H.M., J.T., J.C., and N.H. wrote the manuscript. Acknowledgements The authors thank Siying Qin (Optical Imaging Core Facility at the National Center for Protein Sciences at Peking University) for AFM analysis, Jun Hu (the National Center for Protein Sciences at Peking University) for schematic model designation, and senior engineer Baochang Lai of Xi’an Jiaotong University for providing the technical support, Pengli Zheng and Qingzhou Chen for helpful reading. 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desmosomes.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eDual localization patterns of GFP-CCDC120 (green) in MDCK cells. Arrows indicate cell-cell junctions, while the arrowhead indicates the centrosomes. Scale bar, 10 μm. \u003cstrong\u003e(B, C)\u003c/strong\u003eImmunofluorescence staining of CCDC120 (green) and DP I/II (\u003cstrong\u003eB\u003c/strong\u003e, magenta) or β-catenin (\u003cstrong\u003eC\u003c/strong\u003e, magenta) in MDCK cells. Magnified views of the cell-cell boundary are shown on the right. Scale bars, 10 μm.\u003cstrong\u003e (D)\u003c/strong\u003e Immunostaining of Keratin 19 (gray) in HeLa cells coexpressing GFP-PKP2 (green) and mCherry-CCDC120 (magenta). Scale bar, 10 μm. \u003cstrong\u003e(E, F)\u003c/strong\u003e 3D-SIM images of MDCK cells double-immunostained with antibodies against CCDC120 (green) and DP I/II (\u003cstrong\u003eE\u003c/strong\u003e, magenta) or PKP2 (\u003cstrong\u003eF\u003c/strong\u003e, magenta). The intensity profiles of CCDC120 and PKP2 or DP I/II signals at cell borders are plotted on the right. Scale bars, 1 μm. \u003cstrong\u003e(G)\u003c/strong\u003e Immunoprecipitation (IP) showing the interaction between CCDC120 and PKP2. MDCK cell lysates were subjected to IP and immunoblotting, as indicated. CEP170 was detected as a positive control. \u003cstrong\u003e(H) \u003c/strong\u003eSchematic diagram illustrating the localization of CCDC120 at desmosomes.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/68c36617468b1dff92c7339f.png"},{"id":80783834,"identity":"d7a1ff43-0425-4428-8b58-5f8346a1364a","added_by":"auto","created_at":"2025-04-17 05:31:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":583991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCDC120 maintains functional desmosome assembly. (A, B) \u003c/strong\u003eImmunostaining of DP I/II (\u003cstrong\u003eA\u003c/strong\u003e, green) or PKP2 (\u003cstrong\u003eB\u003c/strong\u003e, green) in wild-type (WT) and CCDC120 knockout (KO) MDCK cells. DNA was stained with DAPI (gray). Magnified views of the cell-cell boundary are shown on the right. Scale bars, 10 μm.\u003cstrong\u003e (C, D) \u003c/strong\u003eQuantification of the irregular score of DP I/II \u003cstrong\u003e(C)\u003c/strong\u003e or PKP2 \u003cstrong\u003e(D)\u003c/strong\u003elocalization at borders from \u003cstrong\u003e(A)\u003c/strong\u003e or \u003cstrong\u003e(B)\u003c/strong\u003e, respectively. More than 60 cell-cell borders in different groups were analyzed from three independent experiments. \u003cstrong\u003e(E, F) \u003c/strong\u003eImmunoblotting analysis of the Triton X-100 insoluble fractions (\u003cstrong\u003eE\u003c/strong\u003e) and total protein levels (\u003cstrong\u003eF\u003c/strong\u003e) of the desmosome proteins in WT and CCDC120 KO MDCK cells. Keratin 19 was used as a loading control.\u003cstrong\u003e (G)\u003c/strong\u003e Desmosomal localization of GFP-CCDC120 in WT, PKP2 or DP I/II KO MDCK cells. DNA was stained with DAPI (gray). Magnified views of the cell-cell boundary are shown on the right. Scale bars, 10 μm. \u003cstrong\u003e(H)\u003c/strong\u003eQuantification of the irregular score of GFP-CCDC120 signaling at borders. More than 60 cell-cell borders in different groups were analyzed from three independent experiments. \u003cstrong\u003e(I, J)\u003c/strong\u003e Representative images \u003cstrong\u003e(I) \u003c/strong\u003eand quantifications\u003cstrong\u003e(J)\u003c/strong\u003e of the dispase mechanical dissociation assay in WT and CCDC120 KO MDCK cell lines. Fragment numbers in each group were quantified from four independent experiments. \u003cstrong\u003e(K)\u003c/strong\u003e Schematic diagram illustrating cell stiffness measurements using atomic force microscopy (AFM). \u003cstrong\u003e(L, M)\u003c/strong\u003eRepresentative heat maps \u003cstrong\u003e(L)\u003c/strong\u003e and quantifications\u003cstrong\u003e (M) \u003c/strong\u003eof Young’s modulus in WT and CCDC120 KO cell lines assessed by AFM. More than 1000 spots from indicated groups were analyzed. Data are mean ± SEM. Statistical significance was assessed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA with Dunnett’s multiple comparisons test. **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; n.s., not significant.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/3e2c66868c51ef875d4999d5.png"},{"id":80783830,"identity":"b9707bd5-ed7e-4599-af8c-299b3507b12a","added_by":"auto","created_at":"2025-04-17 05:31:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":412775,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCDC120 undergoes phase separation in cells and in vitro. (A)\u003c/strong\u003e Distinct expression patterns of GFP-CCDCC120 in MDCK cells. At low expression levels, GFP-CCDC120 localizes to desmosomes (left). At high expression levels, GFP-CCDC120 forms condensates within the cytoplasm (right). A magnified view highlights cytoplasmic condensates. Scale bar, 10 μm. \u003cstrong\u003e(B, C) \u003c/strong\u003eTime-lapse images \u003cstrong\u003e(B)\u003c/strong\u003e and quantification \u003cstrong\u003e(C)\u003c/strong\u003e of FRAP assays on GFP-CCDC120 condensates (arrowheads) before and after photobleaching in the cytoplasm of MDCK cells (\u003cem\u003en\u003c/em\u003e = 21 cells). Scale bar, 5 μm.\u003cstrong\u003e (D) \u003c/strong\u003eLive-cell imaging showing GFP-CCDC120 dynamics in MDCK cells triggered by cell-cell contact. Three colors of arrowheads mark three different dynamic events of GFP-CCDC120 particles, respectively. Scale bar, 5 μm. \u003cstrong\u003e(E)\u003c/strong\u003e Schematic representation of predicted internally disordered regions (IDRs) within CCDC120, based on PrDOS (https://prdos.hgc.jp). A score greater than 0.5 is considered intrinsically disordered.\u003cstrong\u003e (F) \u003c/strong\u003eConcentration-dependent phase separation assay of CCDC120 \u003cem\u003ein vitro\u003c/em\u003e. Scale bar, 5 μm. \u003cstrong\u003e(G, H)\u003c/strong\u003e Time-lapse images \u003cstrong\u003e(G) \u003c/strong\u003eand quantification \u003cstrong\u003e(H) \u003c/strong\u003eof FRAP analysis of CCDC120 condensates \u003cem\u003ein vitro \u003c/em\u003e(\u003cem\u003en\u003c/em\u003e= 31 condensates). Scale bar, 1 μm. \u003cstrong\u003e(I)\u003c/strong\u003e Representative time-lapse images showing fusion events of His-GFP-CCDC120-IDR1 droplets (arrowheads). Scale bar, 1 μm. Data are presented as mean ± SEM.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/5e76b61597fcc89524a81994.png"},{"id":80787111,"identity":"03391b93-9706-4ef8-a244-28d21438393d","added_by":"auto","created_at":"2025-04-17 06:04:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":618623,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCDC120 phase separation is required for desmosomal integrity and function.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Multiple sequence alignment of CCDC120-IDR1. Acidic amino acids are highlighted. Mutagenesis of ten acidic amino acids to alanine residues (DE10A) in CCDC120 is listed at the bottom. \u003cstrong\u003e(B)\u003c/strong\u003e Schematic representation summarizing the \u003cem\u003ein vitro\u003c/em\u003e phase separation assay of CCDC120-IDR1-WT (left) and DE10A (right) under the indicated salt conditions and protein concentrations. The magenta circles indicate phase separation, while the gray circles indicate no phase separation. \u003cstrong\u003e(C)\u003c/strong\u003e Representative images from the \u003cem\u003ein vitro\u003c/em\u003e phase separation assay of CCDC120-IDR1-WT or DE10A mutant. Scale bar, 5 μm. \u003cstrong\u003e(D, E) \u003c/strong\u003eConfocal images showing GFP-CCDC120 FL or DE10A mutant \u003cstrong\u003e(D)\u003c/strong\u003e or endogenous PKP2 \u003cstrong\u003e(E)\u003c/strong\u003e localization in GFP-CCDC120 FL or DE10A-rescued CCDC120 KO MDCK cells. Magnified views of the cell-cell boundaries are shown on the right. Scale bars, 10 μm. \u003cstrong\u003e(F, G) \u003c/strong\u003eQuantification of the irregular score of GFP-CCDC120 \u003cstrong\u003e(F) \u003c/strong\u003eor PKP2 \u003cstrong\u003e(G)\u003c/strong\u003e signaling at borders. More than 60 cell-cell borders from three independent experiments were analyzed. \u003cstrong\u003e(H, I)\u003c/strong\u003e Representative images \u003cstrong\u003e(H) \u003c/strong\u003eand quantification\u003cstrong\u003e(I)\u003c/strong\u003e of the dispase mechanical dissociation assay in CCDC120 FL or DE10A-rescued MDCK cells. Fragment numbers in each group were quantified from four independent experiments. \u003cstrong\u003e(J, K)\u003c/strong\u003e Representative heat maps \u003cstrong\u003e(J)\u003c/strong\u003e and quantifications\u003cstrong\u003e (K) \u003c/strong\u003eof Young’s modulus in CCDC120 FL or DE10A-rescued MDCK cells assessed by AFM. More than 1000 spots from the indicated groups were analyzed. Data are presented as mean ± SEM. Statistical significance was assessed using a two-tailed Student’s \u003cem\u003et\u003c/em\u003e-test. **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/1f3c65cd66b22b0570175e0f.png"},{"id":80785129,"identity":"3a1543f5-e395-4ae2-adc8-927e3045d02f","added_by":"auto","created_at":"2025-04-17 05:39:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":329574,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCCDC120 recruits PKP2 into condensates formed by phase separation.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003eImmunostaining of Flag-PKP2 (magenta) in GFP-CCDC120-overexpressing HeLa cells. Arrows indicate colocalized condensates in the cytoplasm. Scale bar, 10 μm. \u003cstrong\u003e(B)\u003c/strong\u003eLive-cell imaging showing the dynamic assembly of PKP2-GFP (green) and mCherry-CCDC120 (gray) at cell-cell junctions in MDCK cells. A magnified view of the cell-cell boundary is shown on the right. Arrowheads indicate fusion events. Scale bar, 10 μm.\u003cstrong\u003e (C)\u003c/strong\u003e Immunostaining of Flag-PKP2 in WT, CCDC120 KO, CCDC120 FL-, or CCDC120 DE10A-rescued MDCK cells. Scale bar, 10 μm. \u003cstrong\u003e(D)\u003c/strong\u003eStatistical analysis of the percentage of cells with (gray) or without droplets (magenta) in \u003cstrong\u003e(C)\u003c/strong\u003e. More than 60 cells per group were analyzed from three independent experiments.\u003cstrong\u003e (E)\u003c/strong\u003e Representative images from the \u003cem\u003ein vitro\u003c/em\u003ephase separation assay of His-PKP2-IDR-mApple with His-GFP, His-GFP-CCDC120-IDR1 + 2-WT, or His-GFP-CCDC120-IDR1 + 2-DE10A mutant. Scale bar, 5 μm. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of PKP2 droplet numbers in different particle sizes from \u003cstrong\u003e(E)\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/5886bebffbdb7caf64a48f16.png"},{"id":80787109,"identity":"1cd4a5c6-f681-4896-9073-f806199962ca","added_by":"auto","created_at":"2025-04-17 06:04:14","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":334308,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePKC phosphorylates CCDC120 and promotes its LLPS.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eRepresentative images of His-GFP-CCDC120-IDR1 droplets \u003cem\u003ein vitro\u003c/em\u003e. Purified His-GFP-CCDC120-IDR1 proteins were incubated with PKCα and kinase buffer with or without ATP, followed by an \u003cem\u003ein vitro\u003c/em\u003e phase separation assay at the indicated time points. Scale bar, 5 μm. \u003cstrong\u003e(B)\u003c/strong\u003e Statistical analysis of particle numbers in different particle sizes in\u003cstrong\u003e (A)\u003c/strong\u003e. \u003cstrong\u003e(C, D)\u003c/strong\u003e Representative images\u003cstrong\u003e (C)\u003c/strong\u003e and quantification \u003cstrong\u003e(D)\u003c/strong\u003e of MDCK cells expressing GFP-CCDC120 WT, and treated with DMSO or 15 nM PMA for 30 min. Magnified views highlight GFP-CCDC120 condensates in the cytoplasm of MDCK cells. Scale bar, 10 μm. More than 60 cells were analyzed from three independent experiments.\u003cstrong\u003e (E)\u003c/strong\u003e Schematic representation showing PKCα phosphorylation sites on CCDC120-IDR1. \u003cstrong\u003e(F) \u003c/strong\u003ePhosphorylation of His-GFP-CCDC120-IDR1 WT or ST10A mutant by PKCα in an \u003cem\u003ein vitro\u003c/em\u003e kinase assay. Phosphorylated CCDC120 was detected using an anti-thiophosphate-ester antibody. CBB staining of purified His-GFP-CCDC120-IDR1 WT or ST10A mutant proteins are shown. \u003cstrong\u003e(G, H) \u003c/strong\u003eConfocal images showing the localization of GFP-CCDC120 FL or ST10A mutant (\u003cstrong\u003eG\u003c/strong\u003e) or endogenous PKP2 (\u003cstrong\u003eH\u003c/strong\u003e) in GFP-CCDC120 FL or ST10A-rescued CCDC120 KO MDCK cells. Magnified views of the cell-cell boundaries are shown on the right. Scale bars, 10 μm. \u003cstrong\u003e(I, J) \u003c/strong\u003eQuantification of the irregular score of GFP-CCDC120 \u003cstrong\u003e(I) \u003c/strong\u003eor PKP2 \u003cstrong\u003e(J)\u003c/strong\u003e signaling at cell borders. More than 35 cell-cell borders from three independent experiments were analyzed. Data are presented as the mean ± SEM. Statistical significance was assessed using a paired or unpaired Student’s \u003cem\u003et\u003c/em\u003e-test. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/8d27816f42bd24cb8c547edf.png"},{"id":80783846,"identity":"d29285d8-8c67-496c-828b-74bc4767ac36","added_by":"auto","created_at":"2025-04-17 05:31:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":712010,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlteration of CCDC120 phase separation impairs cardiac function and ICD integrity. (A)\u003c/strong\u003eRepresentative M-mode echocardiography of 2-month-old WT, \u003cem\u003eCcdc120\u003c/em\u003e KO, and DE10A mice. \u003cstrong\u003e(B) \u003c/strong\u003eQuantification of left ventricular ejection fraction (LVEF, left) and fractional shortening (FS, right) in \u003cstrong\u003e(A)\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003e\u003cem\u003en\u003c/em\u003e= 8–10 mice per group. \u003cstrong\u003e(C)\u003c/strong\u003e Immunostaining of DP I/II (green, top) or PKP2 (green, bottom) with actin (gray) in ventricular sections of 2-month-old WT, \u003cem\u003eCcdc120\u003c/em\u003e KO, and DE10A mice. Arrowheads indicate DP I/II or PKP2 signaling. Scale bar, 10 μm. \u003cstrong\u003e(D)\u003c/strong\u003e Quantification of the length of DP I/II (left) or PKP2 (right) signaling in \u003cstrong\u003e(C)\u003c/strong\u003e. More than 30 images from \u003cem\u003en\u003c/em\u003e = 3 mice per group were measured. \u003cstrong\u003e(E) \u003c/strong\u003eElectron micrographs of area composita at intercalated discs (ICDs) in the cardiac ventricles of 2-month-old WT, \u003cem\u003eCcdc120\u003c/em\u003eKO, or DE10A mice. Magnified views of desmosome-like segments of area composita are shown. \u003cstrong\u003e(F)\u003c/strong\u003e Quantification of the length (left) and width (right) of desmosome-like segments of the area composita in \u003cstrong\u003e(E)\u003c/strong\u003e. More than 30 images from \u003cem\u003en\u003c/em\u003e = 3 mice per group were analyzed. \u003cstrong\u003e(G) \u003c/strong\u003eTriton X-100 insoluble fractions of heart tissues from 2-month-old WT, \u003cem\u003eCcdc120\u003c/em\u003e KO, and DE10A mice were detected using the indicated antibodies. \u003cem\u003en\u003c/em\u003e = 2 mice per group. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparisons test. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/ba81bc50ffa8bb35ee07586b.png"},{"id":81695594,"identity":"25442024-827e-4582-8e9c-109ce3007059","added_by":"auto","created_at":"2025-04-30 12:00:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4636523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/7be94978-28eb-4dab-8ebd-569cdcb065f6.pdf"},{"id":80783839,"identity":"af29db27-52d4-403d-9360-046b8f9f3d4b","added_by":"auto","created_at":"2025-04-17 05:31:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3858914,"visible":true,"origin":"","legend":"Supplementary Informations","description":"","filename":"SupplementaryXXInformation250303.docx","url":"https://assets-eu.researchsquare.com/files/rs-6141491/v1/fc7d506e7413d75aa5d1a6bc.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"CCDC120 Phase Separation Contributes to Desmosomal Integrity and Cardiac Function","fulltext":[{"header":"Highlights","content":"\u003cp\u003e1. CCDC120 is a desmosomal component essential for maintaining desmosomal integrity.\u003c/p\u003e\u003cp\u003e2. CCDC120 undergoes LLPS and co-condenses with PKP2 during desmosomal assembly.\u003c/p\u003e\u003cp\u003e3. CCDC120 condensates are modulated by PKCα phosphorylation.\u003c/p\u003e\u003cp\u003e4. Loss of CCDC120 or altered its phase separation leads to cardiac dysfunction in mice.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eDesmosomes are button-like intercellular junctions that anchor stress-bearing intermediate filaments (IFs) to the plasma membrane and are critical for tissues that experience high mechanical stress, such as cardiac muscle and the epidermis \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Desmosomes are precisely organized complexes assembled by a series of desmosomal components. Desmogleins (DSGs) and desmocollins (DSCs) are desmosomal cadherins that mediate cell-cell contact through their extracellular domains, while cytoplasmic plaque proteins link the intracellular regions of cadherins to IF networks. Plakophilins (PKPs) and plakoglobin (PG) are the main components of the outer dense plaque (ODP), whereas the inner dense plaque (IDP) is composed of the IF-binding protein desmoplakin (DP) \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Assembly of desmosomal components is a dynamic process, particularly during development, morphogenesis, and wound healing \u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. During desmosomal assembly, both desmosomal cadherins traffic to the plasma membrane through microtubule-based transport \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. DP forms cytoplasmic particles that partially colocalize with PKP2 and exhibit fusion and transport events toward newly generated desmosomes \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Several signaling molecules, including protein kinase C α (PKCα) and Src kinase, modulate desmosomal dynamics \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Disruption of desmosomal structure impairs tissue integrity and physiological functions \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Emerging evidence suggests that impaired desmosomal organization is strongly associated with arrhythmogenic cardiomyopathy and skin disorders \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. These findings raise a fundamental question of how desmosomal dynamics are regulated under physiological and pathological conditions.\u003c/p\u003e \u003cp\u003eLiquid-liquid phase separation (LLPS) is an emerging mechanism for macromolecular organization that concentrates biomolecules into compartments and promotes biochemical reactions \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. The spherical condensates formed by LLPS are characterized by fusion events and rapid molecular exchange with the surrounding solutions \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Mechanistically, phase separation is typically driven by proteins with intrinsically disordered regions (IDRs), where multivalent interactions among amino acid residues commonly occur \u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Recent studies have shown that some scaffold proteins recruit client proteins into their condensates formed via phase separation to facilitate structural organization \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Condensation of the zonula occludens-1 (ZO-1) scaffolding protein facilitates the partitioning of other tight junction (TJ) proteins, thereby promoting TJ assembly and maintaining epithelial barrier function \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Furthermore, microtubule-induced LLPS of the actin nucleator Cordon-bleu (Cobl) is required for epithelial morphogenesis and the formation of paracellular barriers \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, whether phase separation-related mechanisms contribute to desmosomal dynamics and cardiac function remains unclear.\u003c/p\u003e \u003cp\u003eWe previously reported that coiled-coil domain-containing 120 (CCDC120) is a centrosomal subdistal appendage (SDA) component. Loss of CCDC120 disrupts SDA assembly and microtubule anchoring in HeLa cells during interphase \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Here, we identified an unexpected role of CCDC120 in crosslinking desmosomal structure and maintaining cardiac function through phase separation. CCDC120 is enriched at the desmosomal plaque and is crucial for desmosomal integrity. CCDC120 undergoes LLPS and co-condenses with PKP2 to preserve functional desmosomal organization. Furthermore, CCDC120 condensation is regulated by PKCα phosphorylation. Loss of CCDC120 or altered CCDC120 phase separation impairs intercalated disc structure and leads to cardiac dysfunction.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCCDC120 is a desmosomal component that maintains desmosomal integrity\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003eCoi\u003c/strong\u003e \u003cp\u003eled-coil domain-containing 120 (CCDC120) was previously identified as a subdistal appendage (SDA) component required for SDA assembly and microtubule anchoring at centrosomes in HeLa cells \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Unexpectedly, robust localization of GFP-tagged CCDC120 was observed at cell-cell junctions in Madin-Darby canine kidney (MDCK) cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), a widely used model for studying cell junctions \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. To confirm the localization of CCDC120 at cell-cell junctions, endogenous CCDC120 was co-labeled with the desmosomal adhesion marker DP I/II or the adhesion junction marker -catenin in MDCK cells. CCDC120 exhibited greater colocalization with DP I/II at cell borders when compared to that with -catenin (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C). Moreover, CCDC120 signaling was almost undetectable at cell junctions in CCDC120 knockout (KO) MDCK cell lines generated using CRISPR/Cas9 approaches (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C), confirming the specificity of its localization. In addition, CCDC120 colocalized with the desmosomal protein PKP2 at sites where Keratin 19 filaments in adjacent cells anchor (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), indicating that CCDC120 localizes to desmosome adhesions. Three-dimensional structured illumination microscopy (3D-SIM) was performed to characterize the detailed localization of CCDC120 within desmosomes. Compared with the distribution of DP I/II (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE), CCDC120 was localized closer to the cell membrane, where the ODP protein PKP2 was found (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Consistently, immunoprecipitation assay results showed that CCDC120 preferentially interacts with the desmosomal component PKP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), suggesting that CCDC120 localizes near the ODP of desmosomes in MDCK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003eTo determine the role of CCDC120 in desmosomes, we first examined the localization of different desmosomal proteins in both wild-type (WT) and CCDC120 KO MDCK cell lines. Depleting CCDC120 significantly disrupted the continuous distribution of DP I/II and PKP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D), leading to a disorganized pattern. A previous study showed that most desmosomal components are insoluble in lysis buffer containing nonionic detergents, such as Triton X-100, because of their ability to associate with the desmosomal plaque \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To assess the solubility properties of desmosomal components, we next analyzed their insoluble fractions and found that, in CCDC120 KO MDCK cells, the insoluble fractions, but not total protein levels, of DP I/II and PKP2 were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F). This reduction further indicates impaired desmosomal organization after CCDC120 depletion. By contrast, β-catenin distribution at cell junctions remained unchanged in CCDC120 KO cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E), confirming that CCDC120 specifically participates in desmosomal component assembly. However, the loss of PKP2, but not DP I/II, disrupted the proper localization of CCDC120 at desmosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H and Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF), revealing a strong functional correlation between CCDC120 and PKP2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then investigated whether CCDC120 loss alters desmosomal adhesion in MDCK cells. To assess this, we performed dispase intercellular adhesion assays. When constant shear stress was applied to the detached cell monolayer, CCDC120 KO cells exhibited a greater number of fragments than WT cells, indicating reduced intercellular adhesion following CCDC120 deletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI, J). Furthermore, because intermediate filaments and desmosomal structure contribute to the mechanical properties of cells \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e, we used atomic force microscopy (AFM) to measure the elasticity of the apical surface of the MDCK monolayers, from which the Young\u0026rsquo;s modulus was obtained (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). AFM results showed that CCDC120 KO cells (Young\u0026rsquo;s modulus: 2.415\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027 kPa) exhibited a significant decline in cellular elasticity than WT cells (Young\u0026rsquo;s modulus: 4.186\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 kPa, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL, M). Our results suggest that CCDC120 is enriched at desmosomes and is essential for maintaining desmosomal structural integrity and function.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCCDC120 undergoes LLPS both in cells and\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo characterize CCDC120 dynamics in cells, we generated MDCK cell lines stably expressing GFP-tagged CCDC120. At low expression levels, GFP-CCDC120 localized predominantly to desmosomal adhesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, left panel). Surprisingly, at high expression levels, round-shaped particles with varied sizes appeared in the cytoplasm of MDCK cells that highly expressed GFP-CCDC120 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, right panel). In fluorescence recovery after photobleaching (FRAP) assays, the fluorescent intensity of GFP-CCDC120 in cytoplasmic puncta was partially recovered after photobleaching (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C), indicating the exchange of GFP-CCDC120 between the particles and the cytoplasm. In addition, we transiently expressed GFP-CCDC120 in HEK293T cells, a cell line with high transfection efficiency but weakened desmosomal adhesion \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Our results showed that GFP-CCDC120 primarily formed one large particle in the cytoplasm (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and exhibited exchange dynamics, as confirmed by FRAP assays (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDP-GFP expression leads to the formation of cytosolic particles, and a series of DP-GFP dynamic events near the cell borders are triggered by cell-cell contact \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Building on this, we next examined the dynamic behavior of CCDC120 during desmosome assembly in MDCK cells. Live-cell imaging revealed that during cell-cell contact, some GFP-CCDC120 particles near the contacting borders appeared to fuse together, then moved towards the borders, and finally coalesced with assembled desmosomes at cell contact sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Consistently, similar fusion events of GFP-CCDC120-formed particles were observed at cell borders in HeLa cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), suggesting that these particles exhibit liquid-like properties.\u003c/p\u003e \u003cp\u003eCCDC120 dynamics during cell-cell contact prompted us to examine whether CCDC120 undergoes LLPS. Since IDRs commonly play a key role in mediating phase separation \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, we analyzed the CCDC120 protein sequence and identified two continuous IDRs on its C-terminus (IDR1: 210\u0026ndash;430 aa and IDR2: 431\u0026ndash;629 aa; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). We first tested the LLPS properties of these two IDRs in cells. Depleting IDR1, but not IDR2, reduced the GFP-CCDC120 condensates in MDCK cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, F), indicating that IDR1 plays a predominant role in CCDC120 phase separation. To test whether CCDC120 condensates in the cells are directly generated via IDRs or associated with other cellular components, we performed a series of \u003cem\u003ein vitro\u003c/em\u003e experiments. Purified His-GFP-tagged CCDC120 IDR1 or IDR1\u0026thinsp;+\u0026thinsp;2 formed spherical droplets, and droplet size changed in a protein concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, H). A FRAP assay revealed that IDR1-formed droplets could exchange proteins with in the surrounding solutions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). Furthermore, dynamic fusion events of the droplets formed by IDR1 were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI), suggesting that these droplets possess liquid-like properties in vitro. Thus, CCDC120 undergoes LLPS, primarily mediated by IDR1.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCCDC120 phase separation is required for desmosomal organization and function\u003c/h3\u003e\n\u003cp\u003eTo determine whether the role of CCDC120 at desmosomes relies on phase separation, we first identified the amino acids within IDR1 responsible for LLPS of CCDC120. By generating and expressing a series of truncation mutations within CCDC120 IDR1, we found that the 210\u0026ndash;250 aa region of CCDC120 is critical for CCDC120 condensation in HEK293T cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). By analyzing the protein sequence, we identified several evolutionary conserved acidic amino acids clustered within or near the 210\u0026ndash;250 aa region of CCDC120 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Electrostatic interactions are a type of multivalent interaction that mediate LLPS \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, and clusters of negatively charged residues contribute to charge-driven phase separation formation \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. To test this, we next neutralized these negative-charge residues by mutating ten aspartic acid and glutamate residues to alanine in IDR1 (DE10A), and examined its LLPS properties by performing phase separation assays under various protein and salt concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). The IDR1-DE10A mutant displayed attenuated LLPS properties compared with IDR1-WT, forming only small droplets at low salt concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Finally, we assessed droplet formation of CCDC120 DE10A mutant in cells. The CCDC120 DE10A mutant exhibited fewer CCDC120 condensates in MDCK and HEK293T cells when compared to the droplets that formed by CCDC120 WT (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-F). Taken together, we concluded that the enriched aspartic acid and glutamate residues in IDR1 are essential for CCDC120 LLPS.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the functional relevance of CCDC120 phase separation, GFP-tagged CCDC120 FL or the DE10A mutant were stably expressed in CCDC120 KO MDCK cells to evaluate their ability to support desmosomal assembly and function. First, we examined CCDC120 localization at cell junctions. In CCDC120 FL-rescued cells, a continuous distribution of CCDC120 FL was detected at cell-cell borders (Irregular score: 0.293\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F). However, expressing the CCDC120 DE10A mutant resulted in a disorganized, bead-like localization around junction sites (Irregular score: 0.526\u0026thinsp;\u0026plusmn;\u0026thinsp;0.015; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F). Moreover, the expression of CCDC120 FL, but not the DE10A mutant, rescued the mislocalization of endogenous PKP2 at desmosomes (Irregular score: FL, 0.385\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014; DE10A, 0.808\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, G), indicating that CCDC120 maintains desmosomal assembly through phase separation. Finally, we examined the correlation between CCDC120 LLPS ability and desmosomal function. A dispase assay showed that CCDC120-FL, but not the DE10A mutant, restored adhesion loss in CCDC120 KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I), supporting the role of CCDC120 phase separation in desmosomal adhesion. In addition, CCDC120 KO cells stably expressing CCDC120 FL, but not the DE10A mutant, exhibited a partial increase in Young\u0026rsquo;s modulus, as measured by AFM (FL: 3.288\u0026thinsp;\u0026plusmn;\u0026thinsp;0.045; DE10A: 2.600\u0026thinsp;\u0026plusmn;\u0026thinsp;0.036; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ, K). Together, these findings suggest that phase separation of CCDC120 is essential for desmosomal integrity, thus ensuring intercellular adhesion and cell stiffness.\u003c/p\u003e\n\u003ch3\u003eCCDC120 co-condenses with PKP2\u003c/h3\u003e\n\u003cp\u003ePhase separation drives cellular compartmentalization \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Condensates of ZO proteins selectively partition TJ proteins during TJ assembly \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We asked whether CCDC120 LLPS similarly facilitates the selective enrichment of desmosomal components during desmosomal assembly. Due to the predominant interaction between CCDC120 and PKP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), we first examined whether CCDC120 co-condenses with PKP2. PKP2 associated with CCDC120 droplets in the cytoplasm of HeLa cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Furthermore, fusion events of CCDC120 droplets associated with PKP2 were observed at cell borders after cell-cell contact (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We then investigated the role of CCDC120 LLPS in PKP2 particle formation in MDCK cells. Interestingly, a robust decline in the percentage of cells with PKP2 particles was detected after CCDC120 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). This phenotype was rescued by expressing CCDC120 FL, but not the DE10A mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D), further demonstrating that CCDC120 phase separation is required for PKP2 particle formation. Since PKP2 colocalizes with cytoplasmic particles formed by DP-GFP overexpression \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e, we also examined the relationship between CCDC120 LLPS and DP particle formation. Although DP-GFP signaling was associated with CCDC120 condensates in HeLa cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), there was no significant change in DP particle formation between WT and CCDC120 KO cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C), suggesting that DP may generate cytoplasmic particles in a CCDC120-independent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the relationship between CCDC120 and PKP2, we mapped the domains responsible for their interaction. Immunoprecipitation assays revealed that the N-terminal region of PKP2, which mediates interactions with several desmosomal proteins such as DP and PG \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e, also binds to the C-terminal region of CCDC120 (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E). Interestingly, an IDR domain (80\u0026ndash;240 aa) within the PKP2 N-terminus was predicted (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Consistent with the observed cellular phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D), co-incubation with CCDC120 IDR1\u0026thinsp;+\u0026thinsp;2 WT, but not the DE10A mutant, increased the size of PKP2-IDR droplets compared to those formed by PKP2-IDR alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F and Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). This suggests that PKP2 partitions into the condensed phase of CCDC120 through its IDR domain. Moreover, the impaired LLPS properties of CCDC120 did not affect its interaction with PKP2 (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Together, these results indicate that CCDC120 specifically concentrate PKP2 through LLPS.\u003c/p\u003e\n\u003ch3\u003ePKCα-mediated phosphorylation promotes CCDC120 phase separation\u003c/h3\u003e\n\u003cp\u003ePKC signaling regulates various types of intercellular junction assembly \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. In particular, PKCα governs desmosomal dynamics by forming a DP-PKP2-PKCα complex \u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. To test whether PKCα regulates the phase separation of CCDC120, we performed an \u003cem\u003ein vitro\u003c/em\u003e phase separation assay by incubating purified CCDC120-IDR1 with PKCα. In the presence of ATP, liquid droplets formed by CCDC120-IDR1 were larger than those in the non-ATP groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B), indicating that PKCα activation increases CCDC120 droplet formation \u003cem\u003ein vitro\u003c/em\u003e. We next examined the role of PKCα in CCDC120 droplet formation in cells. Treatment with the PKC activator PMA significantly increased CCDC120 LLPS in MDCK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D) and HEK293T cells (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Since phosphorylation may regulate LLPS via electrostatic modulation \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, we asked whether PKCα modulates CCDC120 phase separation by phosphorylating IDR1. Using mass spectrometry analysis, we first screened phosphorylation sites of CCDC120 that could be targeted specifically by PKCα. Ten evolutionary conserved serine and threonine residues within IDR1 were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). We then constructed a non-phosphorylatable CCDC120 mutant by mutating all serine and threonine residues to alanine (ST10A), and performed an \u003cem\u003ein vitro\u003c/em\u003e kinase assay. A strong thiophosphate-ester signal was detected after incubating PKCα with WT but not ST10A mutant CCDC120-IDR1 proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), further confirming the specificity of these phosphorylation sites. Finally, we investigated the role of PKCα in CCDC120 desmosomal localization and function by expressing the ST10A mutant in CCDC120 KO MDCK cells. Compared with CCDC120 FL signaling at the cell-cell borders (Irregular score: 0.186\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, I), CCDC120 ST10A mutants exhibited a disorganized distribution around the junction sites (Irregular score: 0.227\u0026thinsp;\u0026plusmn;\u0026thinsp;0.007; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, I), similar to the phenotypes observed in CCDC120 DE10A mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F). Moreover, compared to CCDC120 FL expression, CCDC120 ST10A mutant expression provided minimal rescue of endogenous PKP2 at desmosomes (Irregular score: FL, 0.322\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020; DE10A, 0.456\u0026thinsp;\u0026plusmn;\u0026thinsp;0.020; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, J). This indicates that PKCα-mediated regulation of CCDC120 modulates its phase separation and desmosomal function primarily through these phosphorylation sites. Taken together, these results indicate that PKCα directly phosphorylates CCDC120-IDR1 and increases its droplet formation, thus maintaining desmosomal assembly.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCCDC120 LLPS maintains cardiac function and ICD structure\u003c/h3\u003e\n\u003cp\u003eRecent studies show that mutations in genes encoding desmosomal proteins, such as \u003cem\u003eDP\u003c/em\u003e and \u003cem\u003ePKP2\u003c/em\u003e, lead to an inherited heart disease known as arrhythmogenic right ventricular cardiomyopathy (ARVC) \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Since CCDC120 contributes to desmosome assembly and function (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), we examined its role in cardiac function. We first generated \u003cem\u003eCcdc120\u003c/em\u003e KO mice using CRISPR/Cas9. A 50-bp deletion in exon 4 of \u003cem\u003eCcdc120\u003c/em\u003e was detected (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B). Immunoblotting further confirmed the loss of CCDC120 protein in KO mice (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Using echocardiography, we assessed left ventricular (LV) function in WT and \u003cem\u003eCcdc120\u003c/em\u003e KO mice at 2 months of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and Supp Table\u0026nbsp;1). We detected a significant decline in left ventricular ejection fraction (LVEF) and fraction shortening (FS) in \u003cem\u003eCcdc120\u003c/em\u003e KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). To explore whether CCDC120 maintains cardiac function through phase separation, we generated \u003cem\u003eCcdc120\u003c/em\u003e DE10A mutant mice (Supp Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E). Importantly, \u003cem\u003eCcdc120\u003c/em\u003e DE10A mutant mice phenocopied the cardiac dysfunction observed in \u003cem\u003eCcdc120\u003c/em\u003e KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B, Supp Table\u0026nbsp;1). Together, these results demonstrate that CCDC120 phase separation is essential for maintaining cardiac function in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIntercalated discs (ICDs) are specialized structures that connect adjacent cardiomyocytes. The area composita, the predominant junction type within ICDs, contains desmosomal and fascia adherens components \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. To investigate the cause of cardiac dysfunction in \u003cem\u003eCcdc120\u003c/em\u003e KO and DE10A mutant mice, we first examined the localization of desmosomal components in ICDs. Immunofluorescence analysis revealed a significant decline in DP and PKP2 signaling length on ICDs in \u003cem\u003eCcdc120\u003c/em\u003e KO and DE10A mice compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D). This mislocalization pattern was similar to the disrupted DP and PKP2 localization observed in CCDC120 KO and DE10A-rescued MDCK cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Transmission electron microscopy was performed to examine the ultra-structural changes in ICDs further. The length of the desmosomal structure was reduced in \u003cem\u003eCcdc120\u003c/em\u003e KO and DE10A mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). In addition, the area composita exhibited a widened gap in \u003cem\u003eCcdc120\u003c/em\u003e KO mice, which was even more pronounced in \u003cem\u003eCcdc120\u003c/em\u003e DE10A mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003eFinally, we analyzed the solubility properties of desmosomal components in the heart tissues of different mice. The insoluble fraction of CCDC120 in \u003cem\u003eCcdc120\u003c/em\u003e DE10A was lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG) than in WT heart tissue, which is consistent with its irregular desmosomal localization in MDCK cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Moreover, the insoluble DP I/II and PKP2 protein levels were also decreased in \u003cem\u003eCcdc120\u003c/em\u003e KO and DE10A mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG), further supporting the role of CCDC120 LLPS in the proper localization of desmosomal components within ICDs. These results suggest that CCDC120 phase separation is important for maintaining cardiac function by stabilizing ICD structure.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we uncovered the role of CCDC120 in desmosome assembly and function. CCDC120 exhibits LLPS properties, which are further enhanced by PKCα phosphorylation. Moreover, CCDC120 specifically interacts with PKP2 and co-condenses with it during desmosomal formation. Impaired CCDC120 phase separation causes desmosomal protein mislocalization, weakened intercellular adhesion, and cell stiffness, ultimately resulting in cardiac dysfunction in mice.\u003c/p\u003e \u003cp\u003eOur results demonstrate that CCDC120 colocalizes and interacts with PKP2. Loss of CCDC120 impaired both DP and PKP2 assembly on desmosomes. However, only PKP2 depletion reciprocally alters the desmosomal localization of CCDC120, suggesting that CCDC120 and PKP2 may function upstream of DP in desmosomal assembly. Although CCDC120 did not strongly interact with DP, GFP-CCDC120 colocalized with DP-formed particles in the cytoplasm. We speculate that this colocalization might be mediated by endogenous PKP2 or other desmosomal components. During desmosomal assembly, DP-GFP displays multiple dynamic phases triggered by cell-cell contact \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.Some DP-GFP particles are co-transported with PKP2 and subsequently integrate into newly formed junctions \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e, a pattern similar to GFP-CCDC120 condensates formed via LLPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Since DP is predicted to exhibit LLPS properties (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"https://prdos.hgc.jp\" target=\"_blank\"\u003ewww.predict.phasep.pro\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.predict.phasep.pro\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), it would be interesting to determine whether cell contact-triggered DP-GFP dynamics are also dependent on phase separation. In addition, some DP-GFP particles at perinuclear sites colocalize with engulfed desmosomal cadherins \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Whether GFP-CCDC120 condensates also partition with desmosomal cadherins or PG requires further investigation.\u003c/p\u003e \u003cp\u003eIn our study, we found that PKCα directly phosphorylates multiple serine and threonine residues on CCDC120-IDR1, enhancing its LLPS properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Phosphorylation modulates phase separation in both positive and negative manners \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. A recent study revealed that phosphorylation modification modulates LLPS by altering residue charges rather than by attaching phosphate groups at specific sites \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Since a negative charge block on IDR1 is essential for CCDC120 LLPS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), we speculate that PKCα-mediated phosphorylation further increases the negative charge on IDR, enhancing CCDC120 phase separation via charge interactions. Furthermore, PKCα regulates desmosomal protein dynamics. PKCα-mediated phosphorylation of DP at Ser2849 weakens its binding with IFs and is involved in desmosomal remodeling during both desmosome formation \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e and disassembly \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. PKCα inhibition can also switch desmosomes from a calcium-dependent state to a more static conformation known as hyperadhesion \u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, thus reducing desmosomal protein turnover \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Future studies will focus on how PKCα modulates the dynamics of CCDC120 condensates in cells.\u003c/p\u003e \u003cp\u003eARVD/C is a complex myocardial disorder that leads to heart failure and sudden cardiac death \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Mutations in four genes encoding desmosome proteins (\u003cem\u003eDP\u003c/em\u003e, \u003cem\u003ePKP2\u003c/em\u003e, \u003cem\u003eJUP\u003c/em\u003e, and \u003cem\u003eDSG2\u003c/em\u003e) have been identified in ARVC/D patients \u003csup\u003e\u003cspan additionalcitationids=\"CR60 CR61 CR62 CR63\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. Transgenic mouse models carrying these mutations exhibit cardiac dysfunction and even embryonic lethality \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. This highlights the importance of desmosome proteins in maintaining the mechanical resilience of contracting cardiomyocytes. In our study, we show that loss of CCDC120 impairs the distribution of desmosomal components at ICDs and causes cardiac dysfunction in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), further supporting the concept that ARVD/C is a desmosomal disease \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. However, whether CCDC120 abnormalities contribute to other ARVD/C phenotypes, such as arrhythmias and fibrosis \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e, as well as the correlation between CCDC120 mutations and ARVD/C, remains to be determined. Recent studies have highlighted the potential role of LLPS in cardiovascular diseases such as heart failure \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, and cardiomyopathy \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. Our findings demonstrate that CCDC120 phase separation is required for desmosomal assembly and cardiac function. Moreover, ZO-1, which is involved in ICD composition and atrioventricular node conduction at gap junctions \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, also displays LLPS properties \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. These findings suggest that LLPS properties and LLPS-related mutations in junctional proteins may provide new insights into the regulation of cardiovascular functions.\u003c/p\u003e \u003cp\u003eIn summary, we show that CCDC120 LLPS is critical for regulating desmosomal integrity, which is necessary for maintaining cardiac function. These findings shed light on the mechanistic interplay between LLPS-mediated desmosomal dynamics and cardiac function and may contribute to the understanding and diagnosis of desmosome-related cardiovascular disorders.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals were handled following the \u0026ldquo;Principles for the Utilization and Care of Vertebrate Animals\u0026rdquo; and the \u0026ldquo;Guide for the Care and Use of Laboratory Animals.\u0026rdquo; Animal studies were approved by the IACUC of the Center for Experimental Animal Research (China) and Peking University Laboratory Animal Center (IACUC No. LSC‐ChenJG‐3). \u003cem\u003eCcdc120\u003c/em\u003e knockout mice were generated using a CRISPR/Cas9 approach on a C57BL/6J background by Shanghai BioModel Organisms Center, Inc. An sgRNA (5\u0026rsquo;-GTGAAGTCAGACCGTCTTCG-3\u0026rsquo;) was designed to target exon 4 of the \u003cem\u003eccdc120\u003c/em\u003e gene. To generate \u003cem\u003eCcdc120\u0026nbsp;\u003c/em\u003eDE10A mutant mice, two sgRNAs targeting intron 6 and intron 9 (5\u0026rsquo;-TCTGTTTGCTGCATGCATAC-3\u0026rsquo; and 5\u0026rsquo;-ACTATCGATTAGCAGAC-3\u0026rsquo;), respectively, along with one vector containing donor template to perform homologous-dependent-repair (Shanghai Model Organisms Center, Inc.). Detailed information on genotyping, sequencing primers, and donor templates is provided in Supplementary Table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe full-length complementary DNAs (cDNAs) of \u003cem\u003eCcdc120\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePkp2\u0026nbsp;\u003c/em\u003ewere amplified from a cDNA library by PCR. The DP cDNA was a gift from Kathleen Green (Addgene plasmid #32227). The full-length and mutant cDNAs were subcloned into pEGFP-C2, pCMV7.1-3xFlag, pmApple-N1, or pET-28a.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture, transfection, and treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMDCK-II and HEK293T cells were purchased from ATCC and cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (CellMax) at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e. For cell transfection, Lipofectamine 3000 (Invitrogen) and jetPRIME (Polyplus-Sartorius) were used according to the manufacturer\u0026rsquo;s instructions. To activate PKC\u0026alpha; kinase, MDCK-II cells were treated with PMA (15 nM, MCE) for 30 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGeneration of knockout and stable expressing MDCK cell lines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll knockout cell lines were generated using a CRISPR/Cas9 approach. The sgRNAs targeting \u003cem\u003eCcdc120\u003c/em\u003e, \u003cem\u003ePkp2\u003c/em\u003e, and \u003cem\u003eDP I/II\u003c/em\u003e were inserted into the LentiCRISPR-V2 vector. The vectors were then transfected into MDCK cells, and single-cell colonies were selected by puromycin treatment (3 \u0026mu;g/mL, Solarbio). The colonies were subjected to immunoblotting and genome sequencing to verify the knockout efficiency. Detailed information on the PCR primers used is provided in Supplementary Table 2. Full-length and mutant CCDC120 cDNAs were cloned into pEGFP-C2 vectors and transfected into wild-type (WT) or CCDC120 knockout MDCK cells for stable cell line selection. Single-cell colonies were selected by treatment with G418 (200 \u0026mu;g/mL, Invitrogen).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunoblotting and Immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal protein from cultured cells was extracted using SDS loading buffer and heated at 100 \u0026deg;C for 10 min. For mouse tissue protein, Triton X-100 soluble and insoluble fractions were extracted as previously described \u003csup\u003e73\u003c/sup\u003e. Briefly, tissues were homogenized in lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) with protease inhibitor cocktail (Epizyme) and PMSF (1 mM, Solarbio). Triton X-100 soluble and insoluble fractions were separated by centrifugation at 12,000 \u0026times; \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 30 min at 4 \u0026deg;C. The pellets were then solubilized for 30 min at room temperature in lysis buffer containing 8 M urea and 50 mM DTT. All protein samples were separated by SDS-PAGE and transferred to PVDF membranes (Millipore). Primary and secondary antibodies used for immunoblotting are listed in Supplementary Table 3.\u003c/p\u003e\n\u003cp\u003eFor immunoprecipitation, cultured cells or tissues were lysed on ice in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, and 10% glycerol) containing protease inhibitor cocktail (Epizyme) for 30 min. After centrifugation at 12,000 \u0026times; \u003cem\u003eg\u0026nbsp;\u003c/em\u003efor 15 min at 4 \u0026deg;C, the supernatants were incubated with the indicated antibodies for 2 h at 4 \u0026deg;C, followed by incubation with Protein A/G magnetic beads (Selleck) for an additional hour. Finally, the immunoprecipitated samples were mixed with SDS loading buffer, heated at 100 \u0026deg;C for 5 min, and analyzed by immunoblotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein expression and purification\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll recombinant proteins were expressed in \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003e(BL21-DE3, Tsingke) and induced with 0.5 mM IPTG (Solarbio) for 16\u0026ndash;18 h at 16 \u0026deg;C. To purify His-tagged recombinant proteins, \u003cem\u003eE. coli\u003c/em\u003e cells were resuspended in binding buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e,pH 8.0, 500 mM NaCl, 1 mM DTT, 30 mM imidazole, 10% glycerol and 0.5% Triton X-100) containing protease inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by sonication on ice, and the lysates were centrifuged at 12,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 30 min at 4 \u0026deg;C. The supernatant was incubated with Ni-NTA agarose beads (CWBIO) and eluted using elution buffer (50 mM NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, pH 8.0, 500 mM NaCl, 1 mM DTT, 300 mM imidazole, 10% glycerol and 0.5% Triton X-100). Finally, purified proteins were concentrated in P buffer (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1 mM DTT, and 10% glycerol) using centrifugal filtration (Millipore), aliquoted and stored at -80 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;phase separation assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor most experiments, phase separation was induced by diluting purified proteins into phase separation buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol, and 5% PEG 4000) at the indicated concentration, followed by incubation for 5 min at 25\u0026nbsp;\u0026deg;C. Droplet formation was observed by adding 4 \u0026mu;L protein mixture to a glass-bottom cell culture dish (NEST). This was followed by imaging using an Olympus FV3000 confocal microscope equipped with a 100\u0026times;/1.45 NA oil immersion objective lens. To identify the role of PKC in CCDC120 droplet formation, 260 \u0026mu;M His-GFP-CCDC120-IDR1 proteins were diluted in kinase buffer (20 mM HEPES, pH 7.4, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 500 mM KCl, 1 mM DTT and 0.01 mg/ml PKC\u0026alpha;) with or without 200 \u0026mu;M ATP (Solarbio). After incubation at 30\u0026nbsp;\u0026deg;C for 1 h, the protein mixture was incubated with LLPS buffer (20 mM HEPES, pH 7.4, 150 mM KCl, and 5% PEG 4000), followed by imaging. For FRAP experiments, CCDC120 droplets were photobleached using a 488 nm laser at 100% laser power, followed by imaging at 5-s intervals for 2.5 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence and live-cell imaging\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining of cell samples was performed as previously described \u003csup\u003e74\u003c/sup\u003e. Briefly, MDCK cells were fixed and permeabilized in cold methanol for 10 min at 4 \u0026deg;C. HEK293T cells were fixed in PBS containing 4% paraformaldehyde (PFA) and 4% sucrose for 20 min at RT, then permeabilized in PBS containing 0.5% Triton X-100 for 10 min. After blocking in PBS containing 4% BSA, samples were incubated with the indicated primary and secondary antibodies (Supplementary Table 3), and mounted for imaging. For confocal imaging, an Olympus FV3000 confocal microscopy equipped with a 100\u0026times;/1.45 NA objective lens was used at RT. For desmosome super-resolution imaging, cell samples were imaged using a Zeiss Elyra 7 3D-SIM microscope equipped with a 63\u0026times;/1.4 NA objective lens at RT. All images were processed by ImageJ software (NIH).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo quantify the distribution of desmosomal proteins at cell borders, the mean fluorescence intensities of these proteins in different cells were measured using ImageJ. To further characterize the integrity of these proteins along cell-cell contacts, irregular score was calculated using the following equation \u003csup\u003e75\u003c/sup\u003e：\u003c/p\u003e\n\u003cp\u003e\u003cimg 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FX/M69Dnx0icm8bMOWPBNtoyXXxP/vd//7eM3z/7sz+b/OEf/mFvDC98AdoFQabJM6Y2B24Gn332Wdlygct2EFgMgqFogtYFU3pi4A6BBSMLBNAAuvXWW8u2j76BX0Pvi+RF2Lzo46DoU9LQBQu+n6U9i0ZxSFzmthC/8l8bDHwtArlYq+yy3re57LLL1urEBr7CnP4qbs6O4+DBg5Mzzzyz6KItL7zwQlmULhomKtK8vh9CnJhrUEfXhX1eNA6I9dxG0DiqMc+cEy94mou54MYLHouWuMC45ZZb1upgfBJLjzzySHN2FexBB38JQbJxIdXHeeed1+z1o/gYU6YN+UA2Ky3zeiQYU3wxzQJGdujdytOBxx9/fK1dxMlbb71V0lg2u8/65m7NP4u6dsabXow92kHSDST9IBSz+nGeOWco3/zmNydnnXVWScwbGRameZ5a6gIUmNiZxHKi07XKXwZcSGs2jL24ctGknHQwERO4Y16K1mKTMtwJ1R2IWekLJuluk5v34os/cuKCyZ/R6oLB0fUD4Jxzzmn2lgODv9aOIb9yiQvaonJcXJgwuhYK+nE0D+inXtXNtu9XaBdMGtiudnA8j76hzOP7eWF8cJFro2/xo4tUHzE+Yhv7/qQV/kdOZZhzuCsyZM7hhwgXPO6IMM5pqy54/Pil3XE+wt/qC+qk7tz/HCMnm/bv31+O9+7d20iMo8t/8n3bQmPsvIkP5MeYaEu8Hs06Nofagw/pd/mQ6wDxwV2leRgai5sJNhCfxIV8y523HEcw5KlKX58xB9Z0D4WycWzGuXvR106eQDD28JF+EOouLOORG1N6WjvGj23MOucM4be//W2zV+fFF1/csAZY6gKUX9Y4+eOPPy5OU9Jdlnlvnw9BdwGOHDmyzgYSd45qK/c+og4eI9KhYx5PEtQsOpnoY8ANQb+UIvyS6nvMwnnkMuTNehdSv0JzP3LML62+C6TiI0+a+AXGXlxmRRNA7bEE7ZjlTiaPsZmcahOX6ssX1Vkei0h/9lWtr/tQP+Rf+3qcMwvESK2P8Sl3fDbD95m2uGchB21xLJu6fgiho21MCj0Kz37EJ7Sx60dKDeYcGDLnaLHJmFJ75XP+5q8WpSB9ub19j7m5yDC/5D5uI/e1/CI/RTR/a04Q1EVcjbkz1TbfME/RD/R/WzzOMwag7yYBCyl+LLBgmBX6t1a+Fp+biRbvF1xwQdmK6FNirjY/5jE4pM9UTy3+ZrmG5Ll7kddOxRd/o1ZjT75gPIJkhvixjUXPOTXuvffeErc131AHaQPTxdJgai+3Cp3LkKcXamHq2HUvC6OLY8r3kV8kngbChpeBOYeMXtrNx6AXyKNdfMVF3nTAluO2l5apT7bqy6+Iyuml3mwjx6QM9lGONBT5PL74nNuh9se2AvaRj7zIZQHbo725n/N59vFJ9BvHtTbXQPd0EDdHX/mFPhNtsVYD2ej/Nn+o3wQvVGN3jBv8jMz0V2OTUwdd2Qdqh/TlNnAcy9A/HCOjWGprN3lqj9oR7Va/xrLso68P5LL/ZJfqlE9jnV2gL/axYlF9PMT3aqf8leOwDWQoV4v72K/owwah/sA2QZnsQ3SQXxuT0T71t/oW8En0NfLxGNAV7QL5IurqAllS9C/1kKc+Bc7nPNVFwvfI5HaoP2O5GoqbWF7xFf2a454+4Djan30n2xUfgJ7sT46jP2v1Kx6zjegf4nP6MdYh29QmdHA+tke+kR0qo/qyTwR58rt0xFjXOBoyVrINEPULdGV9ihOQniijNpOvduu6HP0gmVjnkD6jTDzO59ugHmTRKeR72YXtHNfmkHjtzDZIT9QN+CXLsk9e9NlQP6pshnxkFUOQx82s0P6f//zna755//33S57a+h//8R8bYgQ2WtmBGpYdCG2NJi8GDxM0huAIJQZ430UdFNSqHz3ZeeqkGMRyPElMf0Gvs4Ey0YZcl6A+9AHyUbcSukW2UR2EXLQRyK91UhvULXtUd26H/BH7QDBYZEutLKA/2kRdyIl8vuYTjrPeNpBDZywf/QnZhi7UfvTggzZ/qL8jTIjRDuwa2g5dtGLqWkTX/KYJWRNGW7tze7IejrMu9snvI8cIibxaXOh8H7SVslFn9A30+V79pfGZ7WkDGSXpRk/u1yE2tvmQsjGG0YMcW4FMjhHORzs4Rk+E89HXSnmMdIHe6DtAJ3nx4gQ1G6kLWc1fua9IlOtDYzHXwXH0g2yL5Dqz70B9oB8DeS4AylA26sr1Q66P45q/aqArxwN1xDbl6xGJdssOLV7GzgV5/FKO+kl9yF+UEVk/1PRpfArFTGwfbcv62/wc6xzSZ7ndyMfzXeR4JGFHZMi1M7cNckyCfBXXA+rv7OshfmTLcQa53LbsF+KL/FxvG9iJfLT9iiuuWDn77LPX5aGvprNcLabGDkKi0wrLNtJ2jvxaXqSmr42ob2idsb62fOjTA7nOrANqdfTlcSt8GpzlPYmh78JOf2GU2935vdWazTlPZPtrZUH5fceQdUJb/TXG2tRHzeZa2Vp+ny1ddJXNNkGW59UU3rmcXgjKY5laGSC/Sw9wPsq16aqR9UmX9iHKzKoz0yfD+Vx/X91Zp5i1fhhSVrTpEPF8m/6a7poNbdT0ttUFNRvJq5UXQ+yJc97tt9/e5G4s22ZbX53xvM7VdA21PevjuE02U6sjl88ykM/rWLK5/qwTsl5edeHx/JD3/nI9Xfr7bMl2cK5WNsrxul7tuljTlRki08Yi9Ot8W9tyfq380HpjXXG/Rq18pK3eNrJ8rf7pgnvy0EMPrb1CI0a9A4rCNsPazrXlxTSGKN9WPudJris/n4O2vJiv45giQ/NefvnlMtCGLj4j0lfTC7U8MaRszO87BuXFNIa+sm35bWT5trK1fJVVGkNX2Zind2RZcEZ5/pfw9Nfs2ntLsUwk50kuJuWLmN+HZGOZuA/5fB9Rvq1Mn0zMa5PJSC6nGn0ybfkQy8WU6Tpfy4Mo3ybTRa1Mlx6dizJZtiYzhq6yfflDzouabJTL5yJZpks2E8u2lc8ytfOidh5iHn+JgMWmZEnMMfyg/d73vtdIdaNyIu6LLCNynuSifNxnDuQd7yijdyPzdTHKkGoMkWljSNk+mVp+m3w+hloeRB2SaduvofNtcl1la2T5ml7eO/7d3/3dyaFDh5qcVZb+FbxZDy//MvD4cn7ZL4eb7QG/CvlTGvlPg/3FX/zF2t/gNMaYMXCxZ/EW5xTmmJMnv/o7wdsJ/ooMd2Wjvdwl34w/+2aWyxVXXFH+ukb+u+qjHsGbzaF2y3oIs5Yz25M8FN2v5nSHmHecbx61y/t29vepZq8ZRttaxQtQY4wxxhizVPwI3hhjjDHGLBUvQI0xxhhjzFLxAtQYY4wxxiwVL0CNMcYYY8xS8QLUGGOMMcYsFS9AjTHGGGPMUvEC1BhjjDHGLBUvQI0xxhhjzFLxAnQE/F9d/jXYPLz00kvl/90KjvmXY/w/8EWj/zFOHWPINi4T/WvS6GeOlwF10sfbBfpgs9q+WXGHznljZzPHxKzkMbGZNnrcLhfGPLaSfvjDH67zfa1d25lFzBnEH/9HXswaj6c7s/oa327V3Easx75d5pyhOFKCU2OG2AbQUceOHWuOZuf73//+hn83tln/jIr/Mc7/1929e3eTM4yajcti3759xWb9r2IuAMuy5YknntiydtfAls2yh5jAz8TIIlmUzdupH8DjtputHLfzoHmd/zeO/T/4wQ82+H6Z7dCCN6e4aOjiySefLPbPw8MPP7y0WD+VmcXXLMJeffXV5mj5fPDBB5NDhw41R8udM4irnTt3Fp/Jb16ALpllD+RZ/o/uVk82/t+/y8F+Ho7HbT+nYjydOHGibLFd9m9lO7SQzykuGrqI7TCby6no62zzMucM3cBbZ8PUgE4uueSSlUcffXRlWqCkm266qTmzUvaVT3rxxRebMysrx48fX8uLMrE87N27t+RFXSLWS0I2E8+TKJOhDVHmyJEjzZlVVC63R8gO3MVWdqAnypOy7gjn0SE9+Aj/cDykreRFmVpbI7JP/aI6st1RD8fZRhHL5HPSmXXn/lZcxBTjRjYip/bKFs7Rl6QMsrX8SO7fKM+52O5oU6bWhho57rIvQO1Vin1xzz33FHuyTJdtQL34oy2eAR3k0V/az/bJ/tjPWWe2BXuxu4scI1kP++hBTvT5fGj8QW5DjLkanIuxgZxszH3jcbu4cRv9wFYp+5jyqp8Ube+KV52TnRzXfM/56LNsT/bVViLfi75+y+DLGEf4g8Rx9DGp1u4sk/sq09fH2KO8Wn1qrxL1Z7JuEvUK2SxblGq6ItnX2EpetkmxIztivIm+mEIn+rMcx5Hsf1IEHdLNudjXBw8eLNtau9vyI8jEpHYrBmNdsrt3Abpjx45S4PDhwysnT55cma5iS74ClTwS55GTkVSqyg4cOLAmR150brzIRv3qRNVbK8v5/fv3r52XDbFTOKYNksGWLCM7yZMcZWijoBxy2Md5BWu0D1vIk+Mz0kub2QfqVP3Sg075RNDuWD9byuCnNjRxqK3o45h2qa7sD/KyjQqgXbt2rZVTWzWQVRd50UbyZKP0xHhQ/dIjG5FVPRyzD+oHyQvyONcGbY72k2Ifc0ybFSttUC/2yj8qR17s9yxDwsYYv4rx3Kfqd8pTpsvvNWgD5WL96KCcUNxJj/yaY4VYFH1jHlRvG+inTBw3igHVnW2Tz+NYzz5HhjLktcUfaBzlusmL/RdBzuN2+eM22qZ4qfUp7c4ygL80nkk5XsmTnWq76sy+V2xwnH1BHZpH5oE60X3llVc2OSsrDz74YMlri80M/Y+9MKTfMsgQL4oZiP3QFiPAPjJxbCMT57xM1K0yipWoS/Wp72DI2FJ/SYaUx4D6uGt81Yi+BuJANkpPnndpDzJqFwyJKdXVNZalR+dJyEc96CUPOM9xnDM4R14EH8R2ZhRn0kPKtqku2S8GLUDbDMKJEVUKGIUMRkUUXDJMjo0DTGXRF1HnUVYy0iNi4xRYBF+EOmOnIKNOEbJTdqnN+TgT66+hDhfSQ30R7JFNtLEmk23MqBx1gPyR+y3blI/VR7ltNRtzn1FW8SOZbG+tzySj4wjH5IvcNxnVyzaSdUdb26C9yESb2Y8+a7Mn6uccMrlP4xiS33P8khfbn6GObCM6YjnZGH1C2zRpaEIRku8a84BMLJeJMRPpsk0+j8jnuripj4fEX26D/NwWP4COmj88bleRjZLRcYRj9THIh31+yLZxHMthp2wVffGa7dRxm+91Po8L0NhSmVlAP3VhL7ref//9cozO3H9dqM9hSL/ViPEBQ2KEOmoyag86arTpJi/7uRa3fWMrjwmQrbmP+8ZXJvoaanEoO9X+fAxDYkp1dV0LkIn9BvR17G/sizK5jdSHzhgz0e81ZFuOK8q0LX7FoHdAp0qavVWmHTWZKptcf/31Tc4q1157Lb2x7guvG264odlb5Y477ijbo0ePlq3Qy+vw7rvvlu1tt91WtkL1URZ5bEB/fEF72pnN3mTyxhtvTKYNLnIRykw7ct2XjbF+iHpqXHXVVWXLC+K8OC76yrVx/vnnN3sbka/kO3H11VeXrfw1lHPPPbfZG8brr78+mQbZhrbdeOONk9dee605WuXCCy9s9jaiD16Ip/jl3VifYcuzzz7bHE0mP/3pT0s/5z4U1DsdHGv166vX+++/vxyP+cKV9t59993rbGafl6vxE/ABA/XJHn1I8Mwzz0ymA7vkqc/Uh+K+++4r73xFcvwOAXuijejAR4yJNl555ZUyfhkf2P7UU081Z8aN+S6ogwR8ACLfwCeffFK2GXxOeyLyeX6hvyv+NI5yG/IcNQaP2+GMHbeCGIvouhD9V7tGQVu8jvU9EOOUvfPOO5ucVRgXJNU5C/iA9z+x96yzzpo88sgjky+++KLMJWP9LBbVb6IrRuTPrmt2F7mP4aKLLmr2NjJ0bPFxmeYxfeClWNH7v2Ls+KqR47CPsTFFXhvMY4xr2qj5mL4e09/6+O7ll18uW66N6LzrrrvKcQ3mGcZxrod5Jq+zMjN9hIRCLqS6eChpIv/000/LFs4777xmbzgKDDoz1wG6UBFce/bsKRchnY9/RofGq0NiUmd/9tlnZTsLTBhaZLCQke7N+JMGam9sA6ltIC0a+prFU65/lgUcPmPh8PTTT6/pGfqFp6D/sEmDrG+AwC233LJWH3HBIo9JfgxqZ+xvpbi4hMsuu2ztHDFCfVyAhzDrBSLSNXm3Qb3YyISYF9ljxnwX77zzzuTMM88sZelH+iAvuCPy+Szt2Wo8btczy7iFc845p9kbDm3ruljPgmKcmI/+JNGuMf7sAj//8pe/LF8Nz8si+m0IQ6/Zi2Lo2Dp48ODafEPcv/DCC2XdsF1YZEyxeOTaxvwd9Y3981nxh6IW8vGvQmSws+9HZBszLUCBwc2FIycCvsvYMQ7Vn8bI+vWLhgsk++RxjuCiA+IAY2WedZAoo1+Is0L90UYWwwz0eEd0kUT7lWgHv5w3G4Kyrf4xwYfPuAOmPmMBwg+IMQt3+o34O3DgwNrg6oo5ZKjjrbfeWrMZO+ICawxaNMWEThLQ/8ShYoPtrHUtEmzqgoUBCxZgss5jddYxH/nOd75TLogqyx3fLt8ottouYLMsMha1UBhK9JUSPvO47R63bQy5cUDbuDC2McuNEaF5JPtTd/bn5fPPPy/bRcwZi+i3MfRdsxdNrkv1aWzxo4frss5xx3Q7zMWZRcUU40t9TWIc4AP96BtC/KHIU4q+GyfU2TWnds0zMy1AubWKgR9//HHpTCX92oiNzbfetaK+5ppryraGzr333nvr9FMf+h9//PFyHuJ5ggtnMQiACx2/sqMMifLoWcSFKOplkNEZi/61p7tM3D2K9elu0thfOGNhEc9t9lg36d577y31jyXqYAGC/q6LRQ3u0GHTkAGiX8M8YlG90PU4ugYDif59880317WBxB1W7nqC+h9ZnQc9oge9wpEfBdKX/GqdNzZz29CHjxm7bbA41ARGW+NFasyY74JFMLqjX/rKKv4y5OmOxxA0r+QFzDyPTrvwuN3ImHEr8hjRNUVjqIZ8n2NLZWe5q6r6/vM//3OdL0j4E7/OCz9eL7/88uLXRf1QinbO2m99jLlmL4IhYwv/cTeQpyc6DzmetpLNiKmog/UPPhj6hAriD0XKsyDtQuusDNcg9HQx0wKUXxcojpM/na1fG/HOIndSNAlIhomn6+4j55DJK3cGDhcv6ic/315GP3dwcAjoV1d8LE85bOKXYNfKvA+9TxInCS7GDGwNjkXBXQLajt5YH8e0dZa7CGPAj7Qr3lnG79zt5a7zULS4in3KPsHbtTCqceuttxabhgyQCy64oGz1XgvQf7VB08fzzz9fysXFGbq4q/Dcc8+VYz0ujrGJfJz0iT3FeOxTjaF5YhOwEbsE8cOYbbvrhn0sDmkfExdb2iQdY8Z8F+jQXVbRN14eeuih4rvoc/lzzJ0VbNQ4EvRRtmdReNxuZMy4FXGMaP7ev39/5xjBt8Ra7OuhZdugDGXREcc2/uUizx8mnwd08t6nHr3/4he/KHmxrjEsst/6GHLNXiRDxpb6OL53rDlru7DImKJMXOvAj3/847Lt+rFWgx+KzIuMob65/Xvf+17ZxnlG11euI51MG9nJ1IDqF1AnT371JwaU+JKNfJhOMuXLKPLaZGAatEUug0wuS32x7OHDq39WIyb0RRn2aUOUyV/OUT/lIrWvwaRHX95m+2q6M5xHL7J8Cad64ldxQFuj32kHNsa6clsz+uIuf+0X2wS0K7Y/2wjUozwl/C9yXQK96BfojjpI+FHUbFS98c9rAP7h3BBqsYT90WbFLOf1ZWGN6UJtTU6JvEitr+TX6NNsV+xT9mvtI6/LPvVnjPs8dmLcyeexH0BtkH8ojx7pJFEm6kUP5dpAFpmog35Ar+Ik2iayz9mP9Q6Nv1ob1C95XEQ8bpc/bmVbHiPYEsHO6HPR1teRbPcQ32dfUH/sT2TJz+1uQ/IxLh544IGSx3YM6Ii+7eu3GvIBsvhhaIxge+4r/B99k5FuthHy8H0EXbGf0YsNsb7oQ2ibq2PMDB1fmezrWhzW2ocMdkx/zDc5/TGV6xLkyU/IS7cS5+O1KdtIvcggG23EF+R3tT9C3dKjlK+JNf+Uv58yLdiKTk8Vlm0kF40y/NKYVjiZBu3k9ttvb3I36unSD111QM38PpmhNpAf86Ie5ffprpHry/VAloFcF/TVl3WPraur/pqeMbpFn54oH/O5G8evXe7oDKFWb1td2aZMTVckn4dafdCmq82Wmo6I/JLvDnbp6bIr5itPDCmTqenI5Thu0y1q59vKxPysh8eDPJrM+Zmsa9b6IJfLZN1j6+qqv6ZnjG7RpyfKx/yh45Y7aXxFPb0wrvtKeoitos9mqNnddQx9enW+Vl+NLB/1D9UBtXr7bK1RsyeXq9UFY+tr0z2kvlwXDLEn6xpaX6SmA2p6Yp7koC0faufG6BZ9emp5cewNfbqV667ZCjG/9xE8wlmR0LkuGeiSGVO2JpfPD5HJdOVHJBfzY17M7yLL1srV9Ckvpj6yTK1MTVdXXu0ctOXlfOXVzkGXvGCATH+lrd3+H0LUI11RJ8RzXUiuTT6fl4y2kZoc5GNRy6uh8kP0tJ3P+cqrnYO2/EgsL9m4D3FfSCbLiq4yoEeS+b0xPZbtI+qCuC+yDCgvpj6yTK1MTVdXXu0ctOXlfOXVzkGXvJhl3EJNl2jLh1iuSybSdwzSp5Rpy28jy+t4jA6olYm68rk2smytXJs+5bedz7TpyNT0KS+mTO183Ie4L7JMpqajJp/zJNeV33Yu0yYXU2Ro3oEDB8orDkMXnyA9NX1Qy5/5K3hjtgIuYCwieNeHQTLLu1zm6wnvhfHuI7FDDCmRxx1Qs3l43Bqz/eHdesYp738u6q86dNH7CH4eUF1bCRszD7Vb+eYr7J9u8pRnPy2HWeLS1xBjlkecG5cx7jZ1AWqMMcYYY0zGj+CNMcYYY8xS8QLUGGOMMcYsFS9AjTHGGGPMUvEC1BhjjDHGLBUvQI0xxhhjzFLxAtQYY4wxxiwVL0CNMeuIf6R93sQfIDfGGGMy/jugxph1LHJK8B8RN8YYU2Nb3AG99NJLJx999FFzNAz9y6jtyHa2bZHQRtoK+ld7/L/tIej/co/t93nZqnq3CsaW+mgoLBoXlcaArTfffHNztBjQF+/CbkYdbcTxsZ1hLGDrY4891uSYPvAZsSTGzn99zHJN3I4Q/9EnjL3oN8BvSj/84Q/L9nR6cqJrztg2MR7jmNzO64pZbdvy1tA5x44da46G8+STT06+/PLL5mh7wR2kr8ON5dzOsW3eKh99HfpG5D6ahTvuuGPDRWMz+OCDDyaHDh1qjuaHCf/VV19tjlZZhD+GwvzEPHUq8HUaE4vgiSee2OCzRflw1mvidoMF9NNPP90crcL4ZpwLFlj4jbFC+sEPflC21113XSNx6rN79+6Z2nT//fc3e6ts5zXPrLadsrfpZrnDYowZBxcRXRB/9atfNbmbw+k2pj1HGbOePCY++eSTso35p+OYmaVN+QdN9NF2Y1bbeheg3FblVwp3QNgn5bshOq/bsCQ9PuCc8kjxkRTn7rzzzrJ/ySWXbDjXVg7yLV/Vr3yl/BgjtoOU9Q4h10HqQ7fhlfJjQHRiW5bjOBPPq91sx4Bfsp4MNkaZmi3z8u67766rg37PDPEdedFekfuqRvZFllP9MSb1OEXHSjX7+8h+zn2htuUxkR/p5HbMYkvm5ZdfnjzwwAOTs846a/Kzn/2syd0caDftFLnfSXk8t0HZG264oeyzjXoh+7ymN55vk+mCMsQf0Fccxz7Exq7Yzf0tXZEso2N0g3yYY0V1dhH1knL95MX6a/bVGNKv8pdSzVbiJcrENmosRPvYF8pTGtO32PLMM89Mjh8/XsrK13DixIl1emt25z4jqX7OtV0T+8g+wy58JB1jYoEyURcpojK5LdLNFvuB9mhOo4z22eJHoCx11mxUX8aUqfl0KCrbFoOxf3PMZb9xPtrC+VqbpDsm1aP2AndBta8+iUi3UraHMtiU5WKbYB7/QbaNOslTvtKGcTZdZXeCyHRlu/Loo4+unDx5sqQdO3asTIOrkVgp55C75557yvljx46V/L1795ayHKsscjfddFM5DwcOHCh5kgGVO3z4cGs56iJPYBPH0c5du3YVPQKbZSOJOlVmKNhAXdJRqyfbhv7cnuxDlUGXZPbv31/KHTlypJFaKcdRRuXQNxT0oQf90oPOaA/76NV5bKdM9BXnqR/QyfGLL75YjvtADnl0qu9rdYzxHbKcV/zRV7U2kDe9eBQZ+QIdkmOfPMnIVvld+pGJPpT+oT4A2hH7k5Tbx3nq77KxrU9VblawhTrQUYux999/f+Xss88uKfqL4zF+APRjM1CW9ig2YpuHQn/QfsUOUAd5ihX5KeqlHRzHfqmNxT6i7zU+qB99iiHOR3uUP2QOzGODshwjJ9+z5TjbTdvka9orG3SMnhhL6EYGuwTHyKmP1P9dDOlXyXBtkAx+i23nfOwfZMlTu9Um8iRDnto2T99SRuOBfVD/okdtq/lM/aq6pYs8+a92Teyj5jOO0aMYHBILMOQax7F0Sya3Q+2XTUA5dIPKICMd2Ua26OyqR+NA50nUE+fQPqhT8S9km8h2kDgf45K2IRfHZG6T+iqO7Ry/0q0xCNmePP5J1F+7NuIPyeR4X4T/sm3YwTG6o07qiXxVogUZH6HB5MtZVMJxDGyCgzwaG8ll1TkKpr5yqqPWYAW2QDaWYV/1ChwzFNlGsERyG7JtOJ28iAangl5lyI9EGfk52sx+re1d0J+5T2UPbVF78HlEgSuQUbvk6+zfNlRH9mWuY4zv5H+QPbU2RFn8gO9qPlW9sjW2TbGg2BJj4inHp1A/C2zkOOpW/bKp1qfII5P9N5Sf//znK1dccUXZlw9YcEbQ/T//8z8rZ5111spPfvKT0hbyxvhB4HO1AR/kmEbnvP5FZ9ab5RRPua6aj7uIvlcdilmhumLsqm/75kDGRpZhPCGjuFC/RR9AbIvqk205/kRuPzJj/AFD+pULn/wmYttlX54rKaO5Q23KeuTvefsWPbEd6t/cH1muJqOy6iP1WYyJPvBZtl8+kw/GxELfNQ55jqMfVVaxl4+BctEf6g+RbZR8rId98tQutrJfIJP7uIvcTxDryO0XuRz7WU+tTdleQCbOD/mYusgTY66NXesKZBbhv2hbzQ85zmHQfdYbb7yx2Vvl+uuvn0yVT6YB3uSscs455zR7q49W4dprry1bQVnIZYXK3XbbbWUrVO7o0aNlW2M6CJu9OlMnr3scANNObPb6ufjiiyfTTikfZQC3u7mtrEcmn332WdlGuNU+9fPa40CB/0hvvPFGk7MKeW0gSxuizezv3LmzORrGa6+9tqFPqZe28cI0fcOxfC7oS9oSHyXMy9VXX93srRLrGOs7+kcoTmptiOCLu+++u+rT119/vclZ5bzzzmv2VuvChvx4d0w88VI6PtfL6Xq8o5fP4+MK6urSXetT5ImXWfnHf/zHyXe/+92y/61vfats82P4p556avIHf/AHxV//9m//Nnn44YdL3hg/1Ljmmms2POJE57x6oW+eoN+nE+qGuvAvfp6HCy64oNlbT4zdIXOgxkaO5zyeZmHfvn0lLiE+/qTt0wtZyRfR7iH09Sv1UUce87RdY4VxrzkgQpk8P1100UXN3iqb2bdw4YUXNnt1aMN9991X9vV4Um399NNPy3YW8FntOj2WMde4vjlpEQyZn/EfctipvkdmjG20j7hUebYcq91cF/GL4p26SHoVI9I3v7zyyislQXw0Dnontg+N/zxONC7GrCsW4b8afX6AQQvQtkmzC96FgbgoHYLKYbw6ZpYOqkGnP/LII2WwZr1Duffee9fK8YXthx9+OHnhhReasxvRpBIXL9sVBRwXAV0kYlrERJnpuoBttu+0wNN7NjHVJpYMfb9nz54SByoXf9wM4ZZbblkrS1zyJSExutXgGxZCWnj+/u///uT222+fHDx4sBwLxcyf/MmflElsEYtP0OKcSZaLgHzEu0qbDf1O/6tOpdoPg81gM+fAoZx55pmlPmzgYk9czvNjRszTr4orxsmQi1uNre7bxx9/fK1OvhB/6623SpqHLpu7Fh5tjL3GbRZD52cWh8SEFmSSye85dkFc4qsDBw6UY7Yc6+YAXHbZZWu6uW4xJvgxM5Z33nlnbXwxBpjvx35Bvshr4yL8NyvjVl+BvouzFq21u4KQf5lmCHo6JSYmLi5ws8IExq9P9KCPgY/Thy4a+HXApMFglD0EadcFVwHStmgbeweBQFkGtCv6Xok2E7DLYBG+G3JB0QSQ20nqgn4nHhVPxAX9k18Eb4MBzgRPHKpOdHbFUxeLXJj88z//c+lnFp7i29/+dhn3ta/hv/jii7Kd1fYa6JJvSSyEuBgtY2LkwqJ6Y8KesWN2VobMgX0/Bme5QHFnjnlRdfLDfTv1K4vPrnmw76bHVvUtcxHt1HxDfdylnNe3Q20eEguzXOM2myHzMzZyrPP4hMWd7ugNgTutLGyBLceCH0jEnMYk21l98p3vfGftRx2JNclYXYteVyzCf7MwaAGaH5fLqHz7N3LVVVeVbX5krrJtd1V5RAPvvfde6RSljz/+uPxq4BfkvEgng3///v2DF3Xq7PPPP39NB7S9TgD6BZVlmIy4mPctxCM8Yqkt/POj4j64k5Fv0QO/ehhoqgefq50k7n7RB4sMyqxL8YLf5vGd4kiPM0WMRwYZA+/NN99c104Sdyb5xdtHLMNjKy5uTE5D0J2uc889d00H1PqmD/q0FgezPlZkAmYCirQ9hqcPtQDN/p6X6F8WQiyM5LfNQr6MdZO4M0T8bzZD5kCNjeyLof7vmvMYW1p0KMGssVQj6o79qjFZG/O6K6P5Kf+4VJmui+9W9i03Y2gnj+lVL+Rr5CzU5nTNk33EWJjlGrdZjJ2f43liFV/3/UCL3HrrrWXLDzDQMejHfRwXMPbaC/ibtkU9Y6+pi1xXCNlDmsV/szBoAcrFKP46ZeFJwHfdCcPBXIz5xRed21cWx1Iur74pg07eT5oFTWD5Uc/f/u3fFt1D0K8O3aYH/KJfTW2wyM0+pE6CeUx7JBvvsDFYhkwykYceeqgEWPSF7npQBwnb4mMu/Eef8MhZwb8I4o8Y+pt4if6d1XcxjnShkv7I888/X3yhSQfwC3cmn3vuuSZnI+jKjymoB1v5dTsE/QjjTx0J6p7lQs9dMeIgtmPondgMbTr77LM39HPtMTx3Q3nvk0S//Pu//3vJI8bmgXbg34h+fGqBtlnIl9F/+ER3hjaboXMgYyPOrxqjEfUh/SP65gzq4Hyse9ZYygzp17//+7/fMOYph11cN9T+OG8jS5k4d9TYyr7Vndmf/vSnZQu1OWkWanN69A8MiYVZr3GbxZD5mb7MTzF//OMfl61uhA2B+MJnxILGmtCCLsdk1zhqg2tX9mfXzbw2FrWuWJT/ZmK66OgEkWlDyxdXStPJcd0XUvoqcdoZTc4qyPSVhanDyjn9OYNaualj15VDTzQfHchE8ldX018e63TW9PYxHZhFp8pPF2mtX55FKNdVb60MRL1AmVg/eihL+8eQfYHOaA/76I4y9EmUoQx1g3wdv3jsQl8G5n7GT5lZfYdMTT+yMVbVf1GOPJG/YhTTX5/rypCwJdrWRy3O0Ut98iV5uX+xP8oA9cZ2YIviYwzTReYGfyiRT+JreGL/yiuvLF/BA1/Nc45x/Jvf/KbkjSGOYdqC3bl+fDMG9FEOW2HIPAHZl7PUTXn5vm18cJ78TC12sTvHVlt8x3pyfDOfoEt+UCxpnqEO+S2WoS7kNHbYHxtbQ/s1jy36LbadffKiTNSR2xShbPRHLjsE6acsdXT1bxy71JPrlj3Rl2pb/BM/feQ5Hbuy3r5YAMWQZGrXOORju6A2J8W+hlyO85QRlOU4jsVsM4k8gf9yvCIfZYai+nM/QmwLiWP5SvbStuhLyG1qi7/smzi28W32Fcx6bSRPfbkI/+V6an7QGIl9W6JimtnK1JjJ1NDJ3/zN3zQ5q5AfQU3OE7mKWlkRz3WV0znl5WNBfpdOyGX6qNkV6+myJVKzq1amT49+zfOrcAxd9sDQ88qv2dqF5GM9beW7bMl2ZLJ+1Rvp0g+1MpDLQZsdbdTqjvXp/BCbavbAGJvadERko/ZFm61DyGVrdozVG3W02Qzk1/Iis9atcl11tOkeYkOU4QMHXi2aTvLr7mBHGXTkejmOutvqjXJZx1CybqjpaLNBDDnfZltf2SFIh8rW6ssyUKu7pktknV3kcqTp4qDc1RNZJtcNUQYkJ5laGYgyEPVIh/ahpifrgKgH+s5DlhlKrX5oqyPKS6ZmX8zLuqRH+yLmdemOtJWPkF+Ti+QyXeR6htY7+CMkCsWUqeWJIWVr52J+2zlRk4G2cjGNpVY+6on5EeXXztfyIObp8ZVkSVxweEQxyyPPqIeUGXpe1GS6kLz0dJWPMlmulhfJ5WqyUabtfI1crk2ui1r5qCfmR9ryamkMtfI5RblILW8ouayOYxpLLtumpy0vprHkcjUdfbp1PsvFV4rieb0KkV+fiDLxWMR90PkuuXxuKCoXU40+mSHn2+grO4Rctqanpl958Vzch3x+KEPKZZmafJTRuSgT8yM5T3LKj/uQjyEfg+Rq8pDP12SG0lY265ecthDzIzlPclE+7ou+86D82vlaHrTJxTSGXKZNR84bvAA1Ww//8J93MLnwKPHuyLFjxzZccIwxpy+8n8bHbrw/GOcD8vU3Bo0xZjvT+wg+3zI1W0utu9w/xnw98XxgulB8OCbMdqR3AWqMMcYYY8wi8SN4Y4wxxhizVLwANcYYY4wxS8ULUGOMMcYYs1S8ADXGGGOMMUvFC1BjjDHGGLNUvAA1xhhjjDFLxQtQY4wxxhizVLwANcYYY4wxS6V3AXrppZdObr755uZoGPyfYsptBfF/JAv+h/pLL73UHG0e1EHdb7/9dpOz9WBL7L/taGMm26j/gd+G5On7ZbJV9RpjjDGnOr0L0A8++GBy6NCh5mgYTzzxRPVfxC0D/hfyl19+Odm3b185ZnHw9NNPl/1lsN3+sdTDDz+8waZT4Z9fRRvZ77N5q9p0KvjSGGOM2W70LkD5H7Kn2v+R9f+9NcYYY4zZvox6BK/H2zx6JJ99UnzcjuwzzzwzOX78+Jqs0KNUpfiYHNCDTJbLjzipI56Pj5hlI7p5hHvJJZeU/DvvvLPoJ5/z+RE0x7X8iB65xtQlD7InpkisN8rENoncbrUl+0fQ3tdee60k5KKtR48eXacLn2ekv0umRi7HcaTPJ7Py7rvvrtOZ64Xch9nPlFEcSkb+zXFZY2h/Rx9pjOhYqWa/McYYc1qw0sOOHTtWdu3aVfani0qeN66cccYZK4cPH145efJkSeTddNNNRYbje+65p5RjX3AeuWPHjpV8tujZu3dvI7FaFzKPPvromm7qRk6ghzydJ1FGemQjOoB6OD5w4ECRBY6xMcKx2lkDfdgh+0n79+8veUeOHCkyL774YtGtY7acR05lqIc87JQMZaJu2Rx9I/9JD+2hDHnSlUGONslfIBspK130JXnyGVA3Mm393IbKxbZwLN3yCX6Q3uyT7EfOc9xGbJPqxe5YL6gPY5uIuemPlEZiVQZdsg99kP0v/eTFviSvq23qb40P6Ucmxon00zZjjDHmdGOmBSgX1QgXTvIF5ykndNFl0RThOF7AKRPLgcpqMcL5uKgAXbRBNkpGx/FCnu2DWKYG8rndWXdeOOG3XA92kqeFnNqHDyPRRsmwKIkgQ778V0MLUCEbc19EObUr20T95Kt9GZXLutHDYgrkE/UXyCfyb/aj2tlGW5tivcB+7kMWgJRV32sBGtvY5n/Fvfw/pG3SFWNNfst+jXqMMcaY04mZnn1edNFFzd4q0wt7s1eHx71wxx13lK24+uqry5ZHp0KPzNvYuXPn5P777y+PKAX199kQ4XH89KK/9khaj0BvvfXWsq3x4YcfTp566qmyr8ensvXEiRNlm+HRN/ZGsJO8V199tclZ5cILL2z2NiL/XX/99WUrbrjhhmZvPOeff36ztxH1x2233Va2QvXLnozKqV/FfffdVz4MA3xy9913r+sv+eT1119vcmYj13vttdeyai39TGI/+2y6OCzpjTfeaHJWOeecc5q9dv+jPzKmbRdccEGzt/rhHDZgW3wlIOoxxhhjTicW8/JdD5988knZxvfbhizgarAIfOGFF8piIupqew+yxnXXXVcu+AcOHCjHhw8fnuzatassBNo4ePDg5Mwzzyx1sQDGBhalbcievFg/FVB/0D/RxyRQf46BxZR8oh8QMem94Xno6r9PP/20bM8777yyXTTzto1Y2rNnT/lhonLx3WpjjDHmdGIpC1DBXbCcTp48ufYnk4bAQoY7qZSjPBduFqN9d04z3KliYQBs77rrrrLfBndNWSDIbmzoukOlxVDbYo0F8FjGLLIXAb5Ve5Xwu+4Ez8ojjzxS1UvaLLTw1EI007V4FUP8P2vbiCX8ihxl+IFz7Nix6sdoxhhjzKnOUhageuz5zjvvlAutEsfcVYxfyg9FOnQnk0XomAWaHrfry+7du3eXbQ30op+7maoX4qsDNbirWnusTN6YBfM111xTtp999lnZCu7cbgaq77333ltrL+njjz8u/fX444+X85mrrrqqbLNf9OU50F9vvvnmOr2kW265ZXLZZZcVmVmJX/mDHp1zx5sE2Wf0LXcnu+5Uyx+5XfFVBD1Gn6dtsQw/cO65557Ou+zGGGPMqcpSFqAs7liMsRCNi0SOeT+ua/GXYSGT/xzQ3/3d35WL/5C7WAJZbOKP1HOh70J6n3322bIF2sFd0S4eeuihsriJ9rJP3pi7iCye5D/Bok53cBcN9eET2hcXdXpNoe2ONedULvaz7h5z/vnnny/vSkaf8E4tj56fe+65Jmc2on+wm8fhes0C9u/fX3wWf/DQJmKn6y589IfaJf2RWduGLuI62kU92JrfITbGGGNOC1Z6mF6cN3wdnb8W15fDQnJnnPHVn8E5efKrP0mjxDH5ItYl9NWwvhBGXrqVKCc9NRtjvRF9Pc22D30tHeslL9qcv96GXI792Ga1L9uAzegWlKGeWD9ffVOWNrch/chTR81GQHf0PfXpK3Ilzkfba9TK5X5u86XINlKe4zYkn+vNX8UDeVEmt0mxnH1aa1fN/31ta+tvvrCPZUjZb+T1/RksY4wx5lSgrMimF8RWdHp68StbjrUfyfm5HNSq6isDbboj+XybfMzn7hRfaNf01chy6FKe9Oa6oVYu0lUm5mc9PAofYn/WNbQ+yLrz+Tb6yg05r7w22yKSj3rb5MfUncn6a7Kz6s/lIMrpfK2sMcYYcyrR+wiei1284LVd/HJ+LgfKiylSy4M2uZgiteOaHI/UeSw7lKhHuuI+xH0hmSwrusqA3qHM79DyCHiI/VEXxH2RZYTy28630VduyHnRJhPRecl2yUeZmtzQsjrORJm28zVyuSxXyzPGGGNOPSaT/weYoUbbhPRgBgAAAABJRU5ErkJggg==\" height=\"149\" width=\"584\"\u003e\u003c/p\u003e\n\u003cp\u003eFor immunostaining of tissue samples, mice were first anesthetized with tribromoethanol and perfused with 4% PFA. Heart tissues were then isolated and post-fixed in freshly prepared 4% PFA overnight. After dehydration in a graded series of alcohol and xylene, samples were embedded in paraffin and sectioned at a thickness of 4 \u0026mu;m. Sections were deparaffinized, rehydrated in water, heated in antigen retrieval buffer (10 mM citrate, pH 6.0) for 20 min in a microwave oven, and permeabilized in 0.25% Triton X-100. Samples were blocked, incubated with indicated primary and secondary antibodies (Supplementary Table 3), and then mounted for imaging. To quantify the localization of DP and PKP2 in the heart tissue sections, the length of DP and PKP2 signaling at the intercalated disc was measured using ImageJ software (NIH).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor live-cell imaging, transfected cells were seeded in a glass-bottom dish and imaged using a spinning-disk confocal microscope (UltraView VoX, PerkinElmer) equipped with a 60\u0026times;/1.4 NA objective lens or a Zeiss LSM 880 Airyscan confocal microscope equipped with a 40\u0026times;/1.3 NA objective lens at 37 \u0026deg;C. Images were analyzed using Volocity software (PerkinElmer) or ZEN 3.8 (Zeiss).\u003c/p\u003e\n\u003cp\u003eFor FRAP assays, MDCK cells were transfected with a GFP-CCDC120 vector. FRAP assays were performed 24 h after transfection using a Zeiss LSM 880 Airyscan confocal microscope. GFP-CCDC120 droplets or desmosome-localized GFP-CCDC120 were photobleached using a 488 nm laser at 100% laser power, followed by imaging at 9-s intervals for 3 min.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eIn vitro\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;kinase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe in vitro kinase assay was performed as described in a previous study \u003csup\u003e76\u003c/sup\u003e. Recombinant His-GFP-CCDC120-IDR1 WT or ST10A mutant protein, purified from\u003cem\u003e\u0026nbsp;E. coli\u003c/em\u003e, was used as a substrate and incubated with 0.05 \u0026mu;g PKC\u0026alpha; (Abcam) for 1 h at 30 \u0026deg;C in kinase buffer (40 mM Tris-HCl pH 7.5, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM DTT, and 500 \u0026mu;M ATP-\u0026gamma;S (Solarbio)) with protease inhibitor cocktail. A final concentration of 2.5 mM \u003cem\u003ep\u003c/em\u003e-nitrobenzylmesylate (PNBM, Abcam) was added to the mixture for an alkylation reaction and incubated for 1 h at 25 \u0026deg;C. Reaction products were analyzed by immunoblotting using an anti-thiophosphate-ester antibody (SD2020, HUABIO).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAtomic Force Microscopy (AFM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AFM (BioScope Resolve, Bruker) indentation method was used to measure cell stiffness \u003csup\u003e77\u003c/sup\u003e. A pre-calibrated probe (MLCT-SPH-5UM, Bruker) with a hemispherical tip was used. Cantilever D was typically used with a spring constant of 0.028 N/m. The deflection sensitivity was measured before each experiment to ensure complete probe calibration.\u003c/p\u003e\n\u003cp\u003eCells were plated in a 35-mm cell-culture dish to form monolayers with greater than 80% confluency. AFM measurements were performed in cell culture medium at RT. The cantilever was brought into contact with the cell layer at a constant speed of 4 \u0026micro;m/s until reaching the force setpoint of 3 nN, at which point the maximum indentation depth of cells ranged from 0.5 to 1.0 \u0026micro;m. A force volume scan containing 256 spots in a 100 \u0026times; 100 \u0026mu;m region was set, and 12\u0026ndash;15 regions were randomly selected in one group of cells.\u003c/p\u003e\n\u003cp\u003eForce-indentation curves were fit to the Hertz model for spherical tips using NanoScope Analysis software 1.8, with a Poisson\u0026rsquo;s ratio of 0.5 for all calculations, to determine Young\u0026rsquo;s modulus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDispase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe dispase assay was performed as described in a previous study \u003csup\u003e78\u003c/sup\u003e. Briefly, MDCK cells were plated in a 6-well plate. After 36 h, cells were treated with culture medium containing dispase II (2.4 U/mL) for 30 min at 37 \u0026deg;C. The detached monolayer was transferred into a 15-mL centrifuge tube and subjected to multiple inversions. The number of monolayer fragments was quantified using ImageJ software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEchocardiography\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTransthoracic echocardiography was performed using a Vevo 3100 Ultrasound Imaging System (VisualSonics Inc.) equipped with a 40-MHz probe. After anesthesia in an induction chamber containing 3% isoflurane in oxygen, the mouse was placed in a supine position on a warm table to maintain body temperature at approximate 37 \u0026deg;C. Anesthesia was maintained using 1% isoflurane in 500 mL O\u003csub\u003e2\u003c/sub\u003e/min via a nose mask. Images were acquired in parasternal short-axis views (M-mode), and cardiac function parameters were measured and calculated using Vevo3100 analytical software. Left ventricular ejection fraction (LVEF) and fractional shortening (FS) were calculated by at least five measurements of LV anterior (LVAW), LV internal diameter (LVID) and LV posterior wall (LVPW) in diastole and systole respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission Electron Microscopy (TEM)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo-month-old mice were anesthetized with tribromoethanol and perfused with 2% PFA and 1% glutaraldehyde in 0.1 M phosphate buffer (PB) at RT. Heart tissues were isolated and fixed in 2% PFA and 2.5% glutaraldehyde in 0.1 M PB at RT for 1 h, then stained with 2% uranyl acetate. Samples were washed four times with 0.1 M PB and post-fixed in 1% OsO\u003csub\u003e4\u003c/sub\u003e and 0.8% K\u003csub\u003e4\u003c/sub\u003eFe(CN)\u003csub\u003e6\u003c/sub\u003e for 1 h at RT. Following three washes with double-distilled H\u003csub\u003e2\u003c/sub\u003eO (ddH\u003csub\u003e2\u003c/sub\u003eO), cells were stained with 1% tannic acid for 1 h at RT, dehydrated through a graded ethanol series, and embedded in Embed 812 resin. Ultrathin sections (70 nm) were obtained using a Leica Microsystems UC7 ultramicrotome and mounted on single-slot copper grids. After counterstaining with uranyl acetate and lead citrate, samples were imaged using a Tecnai G2 Spirit BioTWIN (FEI) transmission electron microscope with a CCD camera (Orius 832, Gatan). At least 20 images per sample were acquired for measurements. The lengths and widths of desmosome-like segments of the area composita were measured in different groups, as described in a previous study \u003csup\u003e79\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using GraphPad Prism 9 (GraphPad Software). Data are presented as mean \u0026plusmn; SEM. An unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was applied for comparisons between two groups. One-way ANOVA was applied for multiple group comparisons. *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; n.s., not significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.M. and W.Z. designed and performed most of the molecular and cellular biology experiments, Y.X., D.Z., R.C., Y.H., and Y.T. designed and performed animal experiments. X.H. analyzed the data, T.W. performed 3D-SIM imaging. J.T., J.C., and N.H. are the senior authors that designed the project. H.M., J.T., J.C., and N.H. wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Siying Qin (Optical Imaging Core Facility at the National Center for Protein Sciences at Peking University) for AFM analysis, Jun Hu (the National Center for Protein Sciences at Peking University) for schematic model designation, and senior engineer Baochang Lai of Xi’an Jiaotong University for providing the technical support, Pengli Zheng and Qingzhou Chen for helpful reading. The authors thank all members of the laboratory for their help and advice. The work was supported by the National Natural Science Foundation of China (32270733, 32130024) and Natural Science Basic Research Program of Shaanxi (2022JQ831).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWaschke J (2008) The desmosome and pemphigus. Histochem Cell Biol 130:21\u0026ndash;54\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBharathan NK, Mattheyses AL, Kowalczyk AP (2024) The desmosome comes into focus. J Cell Biol 223\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePasani S, Menon KS, Viswanath S (2024) The molecular architecture of the desmosomal outer dense plaque by integrative structural modeling. 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Cell Rep 41\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6141491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6141491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDesmosomes are cell-cell adhesive junctions that provide structural integrity and mechanical resistance to tissues. Disruptions in desmosome organization lead to severe cardiac and dermatological disorders. Emerging evidence reveals that desmosomes exhibit dynamic behaviors during remodeling. However, the mechanisms that initiate and regulate these dynamics under physiological conditions, and their correlation with desmosome-related disorders remain elusive. Here, we uncovered the role of coiled-coil domain-containing 120 (CCDC120) in linking desmosomal organization to cardiac function via liquid-liquid phase separation (LLPS). CCDC120 localized to desmosomes and was required for desmosomal integrity. CCDC120 exhibited LLPS properties and co-condensed with the desmosomal component plakophilin-2 (PKP2) during desmosomal assembly, forming dynamic condensates crucial for preserving desmosomal structure and junction stability. Moreover, CCDC120 condensates were modulated by PKCα phosphorylation. Loss of CCDC120 or impaired CCDC120 phase separation led to intercalated disc structure impairment and cardiac dysfunction in mice. Our findings propose a model wherein CCDC120 phase separation orchestrates desmosomal integrity, thereby establishing connections between desmosomal dynamics and the molecular etiology of cardiac dysfunction.\u003c/p\u003e","manuscriptTitle":"CCDC120 Phase Separation Contributes to Desmosomal Integrity and Cardiac Function","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-17 05:31:25","doi":"10.21203/rs.3.rs-6141491/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"97bf2ca9-628a-44b2-8141-e5d65e06147d","owner":[],"postedDate":"April 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":47234380,"name":"Biological sciences/Cell biology/Cell adhesion/Desmosomes"},{"id":47234381,"name":"Health sciences/Diseases/Cardiovascular diseases/Cardiomyopathies/Cardiac hypertrophy"}],"tags":[],"updatedAt":"2026-04-21T09:51:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-17 05:31:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6141491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6141491","identity":"rs-6141491","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}