The canonical Wnt/β-catenin signalling pathway plays a crucial role in a variety of functions including cell proliferation and differentiation, tumorigenic processes and radioresistance in cancer cells. The Mre11–Rad50–Nbs1 (MRN) complex has a pivotal role in sensing and repairing DNA damage. However, it remains unclear whether a connection exists between Wnt/β-catenin signalling and the MRN complex in the repair of cisplatin-induced DNA interstrand cross-links (ICLs). Here, we report that (1) cisplatin exposure results in a significant increase in the levels of MRN complex subunits in human tumour cells; (2) cisplatin treatment stimulates Wnt/β-catenin signalling through increased β-catenin expression; (3) the functional perturbation of Wnt/β-catenin signalling results in aberrant cell cycle dynamics and the activation of DNA damage response and apoptosis; (4) a treatment with CHIR99021, a potent and selective GSK3β inhibitor, augments cisplatin-induced cell death in cancer cells. On the other hand, inactivation of the Wnt/β-catenin signalling with FH535 promotes cell survival. Consistently, the staining pattern of γH2AX-foci is significantly reduced in the cells exposed simultaneously to cisplatin and FH535; and (5) inhibition of Wnt/β-catenin signalling impedes cisplatin-induced phosphorylation of Chk1, abrogates the G2/M phase arrest and impairs recombination-based DNA repair. Our data further show that Wnt signalling positively regulates the expression of β-catenin, Mre11 and FANCD2 at early time points, but declining thereafter due to negative feedback regulation. These results support a model wherein Wnt/β-catenin signalling and MRN complex crosstalk during DNA ICL repair, thereby playing an important role in the maintenance of genome stability. Many chemicals with a mutagenic mode of action interact with DNA, causing changes in its structure; thereby blocking nuclear processes such as DNA replication, transcription, recombination and repair . The widXenopus laevis oocytes and mammalian cell lines [The insights into the formation of ICLs and their repair have been derived from studies on yeast cells, ll lines , 4. Specll lines . The celll lines –10. Regall lines , 12. A gll lines , 14.Among all the DNA lesions, interstrand cross-links are complex lesions that covalently link both strands of an undamaged DNA duplex . Thus, rMRE11 promoter contains a binding site for β-catenin/LEF heterodimer, the mediator of the canonical Wnt/β-catenin signalling pathway [LIG4, which in turn plays a key role in radioresistance in stem cells and cancer cells [MRE11 expression through the GSK3β/β-catenin/LEF pathway leads to enhanced DSB repair efficiency in cancer cells [Previous studies have provided evidence that the  pathway . This pa pathway , 23. Usi pathway , 23. Hower cells . Other ser cells . To dateAlthough cisplatin is effective against different types of cancers, the emergence of resistance to cisplatin has become a major challenge in successful cancer treatments . AberranThe Mre11 functions as a dimer and exhibits ATP-dependent 3′–5′ dsDNA exonuclease and ssDNA endonuclease activities, which resect the DNA ends to generate 3′ single-stranded DNA overhangs aided by CtIp1/Sae2 , 26, 27.The anticancer activities of cisplatin and its analogues correlate with their ability to cause DNA damage in cancer cells . A key mNext, a time course study was conducted of the expression of MRN complex subunits in soluble and chromatin-bound fractions in HeLa cells exposed to 50 μM cisplatin. After cisplatin treatment for 3 h, the cell culture media was changed to fresh media and the cells were allowed to grow. They were harvested at different time points over a 24 h period and the expression profiles of MRN subunits were determined. These time points were chosen because a significant number of cisplatin-treated cells undergo apoptosis at later time points. Interestingly, this analysis revealed that the relative levels of MRN complex subunits sharply increased in both soluble and chromatin-bound fractions in a time-dependent manner  and 1C. Furthermore, a similar expression pattern of MRN subunits was observed in cisplatin-treated U2OS cells . To test the generality of these results, the effect of interstrand cross-linking agent mitomycin C was explored. Remarkably, a significant rise in the levels of Mre11 and Nbs1 was found in HeLa cells treated with increasing concentrations of mitomycin C . These results from two different cell lines are consistent with the idea that DNA ICL agents act through a common mechanism to elicit the expression of MRN complex subunits.The increased abundance of MRN complex subunits in cisplatin and mitomycin C-treated HeLa and U2OS cells favour the notion that the MRN complex may play a key role in ICL-induced DNA double-strand breaks. Furthermore, accumulating evidence suggests a crosstalk between the cellular DNA repair machinery and DNA damage signalling pathways , 31. In 50 value of 17.1 μM  of CHIR or FH535. Our results show that exposure to cisplatin induced cytotoxicity in U2OS cells, in a concentration-dependent manner with an LC50 values of cisplatin . In the second approach, the cells were pre-treated with Wnt/β-catenin modulators or mirin for 24 h, before exposure to cisplatin. It was noticed that both mirin and CHIR hypersensitized the cells and, consequently reduced the LC50 values to 8.5 and 6.4 μM respectively (Supplementary Table 3). In parallel, it was seen that the Wnt/β-catenin modulators or mirin by themselves caused cytotoxicity, albeit at relatively high concentrations  and γH2AX in untreated cells  and 8B. A repair .MRE11, and upregulates their expression [A hallmark of the canonical Wnt/β-catenin signalling pathway is the activation of β-catenin. Consequently, the β-catenin/LEF-1 heterodimer binds to the promoter regions of specific target genes, including pression . Moreovepression . To bettAXIN2 and DKK1 [AXIN2 through the degradation of β-catenin. This premise was validated by testing the effect of CHIR on AXIN2 expression, which is directly responsible for formation of the β-catenin destruction complex. Remarkably, a time-dependent and robust up-regulation of AXIN2 mRNA was seen following CHIR treatment , indicating that the reduced levels of β-catenin at later time points is due to a negative feed-back mechanism.Our analysis revealed that the β-catenin levels rose by ~2.5-fold at 24 h in CHIR-treated cells compared to the untreated cells and gradually declined below its basal level at 72 h  and 9B. and DKK1 , 61–64. We next investigated the effect of FH535, which is a synthetic small molecule that antagonizes the action of β-catenin/TCF/LEF transcription factors , 65. OurMRE11, CTNNB1 (β-catenin) and CCND1  were assessed by real-time qPCR. The data reveal that MRE11 and CCND1 mRNA levels were significantly downregulated in the cell line LN229 compared to the cell line U87 , indicating a crosstalk between the Wnt/β-catenin signalling pathway and MRN complex.Previous studies have established that the deregulation of canonical Wnt/β-catenin pathway is associated with colorectal cancer, as well as other cancers, such as gliomas , 67. Acc2. The cell lines were verified through microscopic examination of their morphological properties and testing for mycoplasma by Hoechst staining.HeLa, U2OS and LN229 cells lines were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% FBS  at 37°C in humidified air containing 5% CO2. The cells after reaching 70% confluency were treated with the indicated concentrations of cisplatin (Sigma-Aldrich). After a wash, the cells were allowed to recover in the fresh medium. The cells were further cultured in DMEM containing 10% FBS with different concentrations of CHIR990021 (henceforth CHIR), mirin or FH535 (Sigma-Aldrich). In other assays, the cells were pre-treated with Wnt modulators for 24 h before cisplatin treatment. After 3 h with cisplatin, the cells were recovered in a fresh medium and incubated with CHIR or FH535. The cell viability after short-term (3 days) and long-term (10 days) exposures to cisplatin were determined by an MTT assay as described [570 of treated samples/A570 of untreated samples) × 100.Typically, ~2000 cells were grown in a 96-well plate in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% FBS at 37°C in humidified air containing 5% COescribed . The relThe Wnt modulators  or dimethyl sulfoxide were added to the cultures, which had been exposed to cisplatin for 3 h and recovered in a fresh medium. After incubation for 36 h, the cells were harvested by well-trypsinization and fixed with 70% ethanol for 12 h at –20°C. After filtration through a 40 micron-cell strainer, the cells were centrifuged and incubated with 0.1 mg/ml RNase A in phosphate buffered saline (PBS) for 3 h at 42°C. The cells were stained with propidium iodide solution (50 μg/ml) in the dark on ice for 5 min. The DNA content of each cell preparation (~10000 cells) was analysed by a FACSVerse flow cytometer  as described . The aggThe cells from the exponential growth phase were seeded onto round glass coverslips (diameter of 18 mm). After 24 h, the cells were exposed to cisplatin (10 μM) for 5 h at 37°C; then, they were treated for 24 h with 25 μM of CHIR, FH535 or mirin at 37°C. The cells were fixed in PBS containing 4% formaldehyde and permeabilized with 90% methanol for 10 min at 24°C. The cells were washed with PBS and blocked for 30 min in PBS containing 0.5% bovine serum albumin and 0.5% Triton X-100. After incubation with γH2AX antibody (1:2000) for 2 h, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (1:100). In the case of Mre11, cells were incubated with Mre11 antibody (1:1000) for 2 h and incubated with TRITC-conjugated secondary antibody (1:100). After 1 h at 24°C, the cells were stained with DAPI  for 5 min at 24°C. The cells were washed four times with PBS and examined using a Leica TCS SP5 Confocal Imaging System . The images were processed using the LAS-AF-Lite 2.4.1 software (Leica Biosystems) and quantified using the imageJ software. A minimum of 100 cells were used for quantification of γH2AX foci per sample.MRE11 cDNA (5ʹ-TAGCATCTCAGCAGCAACCA-3ʹ and 5ʹ-TTTAAAGGCTCTTCCTCTTTGAGAC -3ʹ) or AXIN2 cDNA (5ʹ-CTTGACCCTGGGCCACTTTA-3ʹ and 5ʹ-ATCTCCTCAAACACCGCTCC-3ʹ), respectively. The following reference genes were used to normalize the qPCR data: GAPDH, HPRT, and TBP. Their cDNA was amplified using the forward and reverse primers, respectively; GAPDH (5ʹ-GTCATCCCTGAGCTGAACGG-3ʹ and 5ʹ-GG CAGGTTTTTCTAGACGGC-3ʹ); HPRT (5ʹ-ACACTG GCAAAACAATGCAGA-3ʹ and 5ʹ-ATCCAACAC TTCGTGGGGTC-3ʹ) or TBP (5ʹ-AAAATGGTG TGCACAGGAGC-3ʹ and 5ʹ-CTTCACATCACAGC TCCCCA-3ʹ), respectively. A real-time qPCR was performed using the BioRad iQ5 multicolor PCR detection system.Total RNA was isolated from lysed cells using TRI reagent (Sigma-Aldrich). One mL of cell homogenate was mixed with 0.2 ml chloroform. After incubation for 10 min at 24°C, the suspension was centrifuged at 13000 rpm for 10 min at 4°C. The aqueous phase was transferred to a clean tube and the RNA was precipitated with 2-propanol. The pellet was washed with 70% ethanol and resuspended in 20 μl of RNAase free water. The samples were then treated with DNase I to remove any genomic DNA contamination. The RNA (1 μg) was reverse transcribed using the iScript cDNA synthesis kit . The cDNA was quantified using the iTaq universal SYBR green supermix kit . The forward and reverse primers were used to amplify The chromatin fractionation was performed as previously described  with somThe cells were harvested and lysed using RIPA buffer . The protein samples (15 μg) were subjected to electrophoresis on denaturing 8% polyacrylamide gels and transferred to polyvinylidene difluoride membranes . The blots were blocked with 3% bovine serum albumin in TBST buffer  for 3 h at 4°C. Subsequently, the blots were separately incubated with a primary antibody  at the indicated dilutions: Rad50 (1:500), Mre11 (1:2000), Nbs1 (1:1000), Rad51 (1:500), α-tubulin (1:6000), Orc2 (1:500), β-catenin (1:5000), FANCD2 (1:100), CHK1 (1:100), GSK3β (1:1000), CHK1-p (1:1000) and γ-H2AX (1:1000) for 12 h at 4°C. The blots were washed 3 times with TBST buffer for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibody (1:6000 dilution) for 4 h at 4°C. After washing the blots with TBST buffer thrice for 5 min each, the signals were visualized with the ImageQuant LAS 4000 system .+ cells through fluorescence-activated cell sorting (FACS) analysis. In these assays, the efficiency of transfection of plasmid DNA was about 65–80%. The data obtained from the spontaneous GFP+ cells was subtracted from the frequency of I-SceI induced GFP+ cells.The green fluorescent protein (GFP)-based assay for HR was performed as described . Typicalp values were calculated by the Student’s unpaired t-test as mentioned in the figure legends: *p < 0.05; **p < 0.01; ***p < 0.001. ns, non-significant (p ≥ 0.05). Graphical display and statistical analyses of data were performed using GraphPad Prism software ver. 6.0. The group data are reported as mean ± S. E. M. of n = 3 independent experiments.The In this study, we sought to elucidate the potential relationship between Wnt/β-catenin signalling and the MRN complex in ICL repair. Consistent with the established roles of the MRN complex subunits in sensing, detecting and processing of DSBs during HR-directed repair, a marked increase in the abundance of MRN complex subunits occurred following exposure to cisplatin. Our studies also show that promoting Wnt/β-catenin signalling, after cells were exposed to cisplatin, reduces the cell survival due to cell cycle progression of cells bearing unresolved DNA damage, increases the number of γH2AX foci-positive cells, and inhibits HR. Conversely, inhibition of Wnt/β-catenin signalling resulted in increased cell viability. Although inhibition of Wnt/β-catenin signalling led to an unexpected decrease in HR efficiency, it resulted in increased cell survival, enhanced cell cycle progression, and reduced the number of cells having γ-H2AX-positive foci. These results suggest that Wnt/β-catenin signalling plays a pivotal role in cisplatin-induced DNA damage and repair, indicating a crosstalk between Wnt/β-catenin signalling and MRN mediated repair of ICL DNA damage.What do these new insights convey about the functional relationship between Wnt/β-catenin signalling and the MRN complex in the regulation of ICL DNA repair? This study demonstrates that the MRN complex subunits are targets of the Wnt/β-catenin signalling pathway. Although the MRN complex is key for the repair of DSBs, the observed repair was dependent on the Wnt/β-catenin signalling pathway. The perturbation of Wnt/β-catenin signalling by loss or gain of function has been linked to the progression of various diseases, including cancer. Several strategies are currently being used to enhance the sensitivity of tumour cells to cisplatin. Clinically, the functional characterization of the critical Wnt/β-catenin signalling axis in ICL DNA repair may shed light on novel therapeutic modalities. In that scenario, a combinatorial treatment of chemotherapeutic agents along with small molecule inhibitors of Wnt signalling seems very promising. However, it is possible that there exist additional downstream targets of Wnt/β-catenin signalling that may be functionally important in conferring cisplatin resistance; this remains to be determined. Overall, this study demonstrates a novel mechanism whereby Wnt/β-catenin signalling regulates ICL repair and, consequently, genome stability and cell proliferation.


Tripartite motif protein 36 (TRIM36) plays a tumor-suppressive role in prostate cancer. However, there is little information on the clinical relevance of TRIM36 expression in esophageal cancer (ESCA).  TRIM36 expression was analyzed by using The Cancer Genome Atlas (TCGA) ESCA dataset as well as by quantitative real-time polymerase chain reaction (PCR) and immunohistochemical (IHC) staining on samples from our hospital. β-catenin pathway. IHC staining data showed that 66.25% (53/80) and 51.25% (41/80) of ESCA cases had a low expression of TRIM36 and a high expression of β-catenin, respectively. By Fisher's exact test, we found that TRIM36 protein expression was significantly correlated with tumor size (P = 0.0104), tumor stage (P = 0.0169), lymph node metastasis (P = 0.0021), vital status (P = 0.0443), and β-catenin expression (P = 0.0329). These findings suggest the potential clinical significance of TRIM36 in ESCA. Kaplan–Meier and log-rank test demonstrated that a low expression of TRIM6 and a high expression of β-catenin were associated with poor overall survival of ESCA patients.  In the current study, the analysis of TCGA ESCA dataset suggested the decreased expression of TRIM36 in ESCA tissues. Further analyses on samples from our hospital demonstrated that TRIM36 was significantly downregulated in ESCA tissues than in the noncancerous controls at both the mRNA and protein levels. Moreover, gene set enrichment analysis on TCGA ESCA dataset suggested that TRIM36 expression was inversely correlated with the  Our study provides evidence for the prognostic value of TRIM36 in ESCA. Esophageal cancer (ESCA) is one of the most common malignancies worldwide . Esophagβ-Catenin is the key control protein in the canonical Wnt pathway. The Wnt signaling pathway transmits signals by inducing the accumulation of β-catenin in the cytoplasm and nuclei [β-catenin pathway is deregulated, abnormal cell growth and cancer are promoted [β-catenin pathway is frequently detected in various human cancers, including ESCA [The Wnt signaling pathway, a homolog of the Wingless (Wg) signaling pathway in Drosophila, regulates embryonic development, cell proliferation, differentiation, etc. via a wide spectrum of target genes . β-Catend nuclei . When thpromoted . The abning ESCA , 8.β-catenin pathway. The interactions of the Wnt/β-catenin pathway and other members of the TRIM protein family, such as TRIM29 [β-catenin has not yet been studied.Tripartite motif (TRIM) proteins are characterized by a common domain structure consisting of a RING finger, one or two zinc-binding motifs known as B-boxes, and a coiled-coil motif . TRIM fas TRIM29 , 19, TRIs TRIM29 , TRIM32 s TRIM29 , TRIM33 s TRIM29 , and TRIs TRIM29 , may plaβ-catenin pathway. Subsequently, immunohistochemical staining was performed to assess the expression of TRIM36 and β-catenin in ESCA. Finally, we investigated the possible prognostic value of TRIM36 and β-catenin expression in ESCA by analyzing the relationship between TRIM36 protein expression and the clinicopathological parameters.In the present study, we analyzed a public ESCA dataset and found a significant downregulation of TRIM36 in ESCA samples and a negative correlation between TRIM36 expression and the https://tcga-data.nci.nih.gov/tcga/) for ESCA. Gene set enrichment analysis (GSEA) was performed based on TCGA ESCA dataset using the GSEA version 2.0 according to Subramanian et al. [The gene expression data were obtained from The Cancer Genome Atlas website  between February 2009 and May 2010 (48 men and 32 women) were enrolled in this study after written informed consents were obtained. No patients received any preoperative chemotherapy or radiotherapy. ESCA and normal esophageal mucosa tissues collected from all the patients were formalin fixed, paraffin embedded, cut into 4 m thick sections, and used for immunohistochemical (IHC) staining of TRIM36 and ‐△△CT. The primers are as follows: TRIM36, 5′-CGTCGGTCCTCCAGAGTTTGTG-3′ and 5′-GTGGCAAGTTCCGTCGTCTTCC-3′; GAPDH, 5′-AATCCCATCACCATCTTC-3′ and 5′-AGGCTGTTGTCATACTTC-3′.Total RNA was isolated from the tissue samples with TRIzol  following the manufacturer's protocol. qRT-PCR was performed to examine the expression levels of TRIM36 mRNA with the SYBR® Green kit (Thermo Fisher Scientific) on ABI 7300 instrument (Applied Biosystems). GAPDH was amplified as an internal control. Dissociation curve analysis was performed at the end of PCR cycles to verify specific product amplification. The relative expression of TRIM36 was calculated by the formula 2β-catenin in tissue samples. After deparaffinization and rehydration, antigen retrieval was performed with 0.01 M citrate buffer (pH 6.0) in a microwave for 15 min. Following the inactivation of endogenous peroxidases and the blockage of nonspecific antigens, the sections were incubated with anti-TRIM36  or anti-β-catenin  overnight at 4°C. After washing, the slides were probed with horseradish peroxidase- (HRP-) conjugated secondary antibody for 1 h at room temperature, treated with the 3,3-diaminobenzidine (DAB) solution, and counterstained with hematoxylin. The slides were reviewed independently by two pathologists. The immunoreactive score (IS) was assessed by using the following formula: IS = percentage  × staining intensity . The target protein was classified as high expression when IS ranged from 3 to 9, otherwise classified as low expression.A standard IHC technique was performed to determine the expression of TRIM36 and t-test was performed to examine the differences between groups. Fisher's exact test was used to determine the correlation between TRIM36 protein expression and clinicopathological features, such as gender, age, tumor size, TNM stage, lymph node metastasis, and vital status. Kaplan–Meier survival plots and log-rank analysis were carried out to determine the difference in survival. A P value <0.05 was deemed significant.Statistical analysis was performed using the GraphPad Prism software . Student's P < 0.05, P < 0.001, By analyzing data from TCGA ESCA, we found that TRIM36 mRNA expression was significantly decreased in ESCA samples as compared with normal esophageal mucosa specimens  and 51.25% (41 cases) of the tested ESCA specimens, respectively.Furthermore, GSEA analysis showed that TRIM36 expression was negatively correlated with the  pathway . Thus, td nuclei  and highP = 0.0104), tumor stage (P = 0.0169), lymph node metastasis (P = 0.0021), vital status (P = 0.0443), and β-catenin expression (P = 0.0329). These findings suggest the potential clinical significance of TRIM36 in ESCA.In addition, we analyzed the correlation between TRIM36 expression and the clinical parameters of ESCA using Fisher's exact test. As shown in P = 0.0235; β-catenin expressions (P = 0.0088; β-catenin, respectively. Furthermore, patients with low expressions of TRIM36 and high expressions of β-catenin had the shortest overall survival, whereas those with high expressions of TRIM36 and low expressions of β-catenin had the longest overall survival time (P = 0.0028; The correlation between the expression of both proteins and the overall survival of patients with ESCA was also explored. Kaplan–Meier survival curves showed that patients with low TRIM36 expressions (It has been reported that negative TRIM36 expression could be used as a prognostic indicator of the biochemical recurrence-free survival in prostate cancer . In the β-catenin is associated with esophageal tumorigenesis [β-catenin expression, suggesting the prognostic values of β-catenin in ESCA. Several studies have suggested the interactions of other members of TRIM proteins and the Wnt/β-catenin pathway in human cancers. For example, Zhou et al. found that TRIM29 positively correlates with the abnormal expression of β-catenin in lung squamous-cell carcinoma, but not in lung adenocarcinoma [β-catenin levels in pancreatic cancer, and their study also indicated that the oncogenic effects of TRIM29 is dependent on β-catenin function [β-catenin in glioblastoma samples and that TRIM33 plays a tumor-suppressive role by degrading nuclear β-catenin [β-catenin signaling. In this study, GSEA analysis suggested a negative correlation between TRIM36 expression and the β-catenin pathway in TCGA ESCA dataset. We then provided evidence for the negative correlation in protein levels by immunohistochemical staining. Furthermore, patients with high expressions of TRIM36 and low expressions of β-catenin showed the best overall survival, whereas patients with low expressions of TRIM36 and high expressions of β-catenin displayed the worst overall survival. Therefore, the current study suggests that TRIM36 and β-catenin may cooperate in ESCA. TRIM36 has ubiquitin E3 ligase activity [β-catenin are to be investigated in the future.Currently, studies have reported that the abnormal expression of igenesis , 8. In carcinoma . Wang etfunction . Xue et -catenin . TRIM32 -catenin , TRIM28 -catenin , and TRI-catenin  promote activity . The mecβ-catenin expression in ESCA. TRIM36 expression may be used as a novel prognostic indicator for ESCA. The biological functions of TRIM36 in the development of ESCA still warrant further investigation.In summary, we have shown for the first time the correlation between TRIM36 and


Canonical Wnt/β-catenin signaling is a well-studied endogenous regulator of mesenchymal cell fate determination, promoting osteoblastogenesis and inhibiting adipogenesis. However, emerging genetic evidence in humans links a number of Wnt pathway members to body fat distribution, obesity, and metabolic dysfunction, suggesting that this pathway also functions in adipocytes. Recent studies in mice have uncovered compelling evidence that the Wnt signaling pathway plays important roles in adipocyte metabolism, particularly under obesogenic conditions. However, complexities in Wnt signaling and differences in experimental models and approaches have thus far limited our understanding of its specific roles in this context.β-cat) knockout mouse and cultured cell models. We used RNA sequencing, ChIP sequencing, and molecular approaches to assess expression of Wnt targets and lipogenic genes. We then used functional assays to evaluate effects of β-catenin deficiency on adipocyte metabolism, including lipid and carbohydrate handling. In mice maintained on normal chow and high-fat diets, we assessed the cellular and functional consequences of adipocyte-specific β-catenin deletion on adipose tissues and systemic metabolism.To investigate roles of the canonical Wnt pathway in the regulation of adipocyte metabolism, we generated adipocyte-specific β-catenin (de novo lipogenesis (DNL) and fatty acid monounsaturation. Further, β-catenin mediates effects of Wnt signaling on lipid metabolism in part by transcriptional regulation of Mlxipl and Srebf1. Intriguingly, adipocyte-specific loss of β-catenin is sensed and defended by CD45-/CD31- stromal cells to maintain tissue-wide Wnt signaling homeostasis in chow-fed mice. With long-term high-fat diet, this compensatory mechanism is overridden, revealing that β-catenin deletion promotes resistance to diet-induced obesity and adipocyte hypertrophy and subsequent protection from metabolic dysfunction.We report that in adipocytes, the canonical Wnt/β-catenin pathway regulates de novo lipogenesis, and lipid desaturation. In addition, adipose tissues rigorously defend Wnt signaling homeostasis under standard nutritional conditions, such that stromal-vascular cells sense and compensate for adipocyte-specific loss. These findings underscore the critical importance of this pathway in adipocyte lipid metabolism and adipose tissue function.Taken together, our studies demonstrate that Wnt signaling in adipocytes is required for lipogenic gene expression,   •Wnt/β-catenin signaling regulates lipogenesis and lipid unsaturation in adipocytes.•Effects on lipogenesis are mediated in part by the regulation of Mlxipl and Srebf1.•-/CD31- cells in chow-fed mice.Loss of adipocyte β-catenin is defended by CD45•β-cat−/− mice are protected from diet-induced obesity and metabolic dysfunction. BindiWnt signaling through the β-catenin-dependent pathway has been shown to have profound effects on mesenchymal cell fate determination and differentiation. Although this pathway is also involved in myogenesis and chondrogenesis , its difWNT10B correlate with increased risk of type 2 diabetes -acetate  for 2, 4, or 8 h at 37 °C. Following the indicated incubation times, cells were harvested and lipids extracted for analysis by scintillation counting and thin-layer chromatography.Prior to evaluation of DNL, cultured adipocytes were incubated in fresh serum-free DMEM:F12 medium overnight. To measure DNL, cells were then incubated in fresh DMEM:F12 medium  according to the manufacturer's instructions. M-MLV Reverse Transcriptase  was used to reverse-transcribe 1 μg RNA to cDNA. qRT-PCR was performed using qPCRBIO SyGreen Mix  on a StepOnePlus System . All primers were validated with cDNA titration curves prior to use; qPCR product specificities were confirmed by melting curve analysis and gel electrophoresis. Gene expression was calculated using a cDNA titration curve within each plate and subsequently normalized to peptidylprolyl isomerase A (PPIA) mRNA expression. The qPCR primer sequences are included in 2.16β-catfl/fl and β-cat−/− preadipocytes and terminally-differentiated adipocytes were treated with 20 ng/ml recombinant Wnt3a  or vehicle for 4 h prior to lysis and RNA isolation (n = 4 per group). After DNase treatment, samples were submitted to the University of Michigan Advanced Genomics Core for quality control, library preparation, and sequencing on an Illumina Hi-Seq platform. Read files for each sample were subsequently downloaded and collated into a single FASTQ file. FastQC (version 0.11.30) was used to evaluate the quality of raw read data and identify problematic features . Alignment, differential expression analyses, and post-analysis diagnostics were conducted using the Tuxedo Suite software package. Briefly, TopHat (version 2.0.13) and Bowtie2 (version 2.2.1) were used to align reads to the UCSC reference genome. A second quality control round was performed post-alignment using FastQC to ensure that only high-quality data were used for gene expression quantitation and differential expression analyses. Differential expression analyses were performed using two independent methods, Cufflinks/CuffDiff and HTSeq/DESeq2, using UCSC build mm10 as the reference genome sequence. Pathway analyses were conducted on ranked lists of log2 fold change using Gene Set Enrichment Analysis (GSEA) software v4.0.3 and the Broad Institute Molecular Signature Database (MSigDB) -acetate to evaluate whether lipogenesis is functionally impaired in β-cat−/− adipocytes. Differentiated adipocytes were incubated in medium containing [14C]-acetate for 2, 4, or 8 h. Cells were then lysed and lipids extracted to measure incorporation of radiolabel into TAG, diacylglycerol (DAG), and phospholipid (PL) fractions. Conditioned media , an acetate transporter, and Acss2, the cytosolic enzyme that catalyzes activation of acetate for use in lipid synthesis, were suppressed in β-cat−/− adipocytes . Thus, we interrogated a recently published chromatin immunoprecipitation sequencing (ChIP-seq) dataset of Tcf7l2 binding in cultured Tcf7l2fl/fl and Tcf7l2−/− adipocytes [Mlxipl, Srebf1, Fasn, and Scd1; binding occupancy of Tcf7l2 was subsequently lost in the Tcf7l2−/− cells  to mediate transcription of downstream targets, we analyzed the promoter regions of ipocytes  and foun/− cells K. Taken 3.6in vivo and thus generated adipocyte-specific β-catenin knockout mice by crossing β-catfl/fl mice with adiponectin-Cre mice. Genetic recombination was specifically observed in various adipose depots, but not in other tissues, including the liver, muscle, pancreas, lung, or heart  but did not observe differences in growth over time , Cd68, and Cd11c, were elevated, whereas markers for endothelial and stromal cells did not change  or indirectly by stimulating production in neighboring cells. Although macrophage markers were increased, expression of classical inflammatory markers were either decreased or unchanged in whole WAT of β-cat−/− mice  mRNA levels were increased in eWAT of β-cat−/− mice, whereas Lep expression was decreased and Adipoq and Retn (resistin) remained unchanged; similar trends were observed in iWAT of knockout mice . These data are strongly supported by recently published work from our group showing that blocking secretion and downstream signaling of adipocyte-derived Wnts also inhibits DNL and lipid unsaturation [Tcf7l2 deletion in precursor cells stimulates adipogenesis and increased expression of genes related to lipid metabolism, providing further support for the presumptive role of β-catenin-dependent signaling in regulation of this process in adipocytes [Consistent with repressed expression of DNL enzymes, turation . Additioipocytes .Srebf1 and Mlxipl, encoding SREBP1c and ChREBP, respectively, as key upstream transcriptional regulators of DNL enzymes [Srebf1 and Mlxipl was significantly decreased in β-cat−/− adipocytes, and that ectopic expression of ChREBP or SREBP1c partially rescued expression of DNL genes in adipocytes lacking β-catenin. These data are consistent with genome-wide ChIP-seq analysis of ChREBP binding sites in WAT, which identified binding sites on many genes related to metabolism, including Acaca, Fasn, and Scd1 [Srebf1 and Mlxipl. Indeed, ChIP-seq analyses of Tcf7l2 binding sites in cultured adipocytes, used as a surrogate for β-catenin/TCF/LEF activity, identified specific Tcf7l2 occupancies in regions surrounding the transcriptional start sites of Mlxipl and Srebf1, but also downstream lipogenic genes such as Fasn and Scd1. Although these data suggest that β-catenin regulates transcription of lipogenic genes through a combination of indirect (via ChREBP and SREBP1c) and direct mechanisms, ChIP-seq analyses of β-catenin binding sites in adipocytes will be required to further elucidate the direct versus indirect effects of β-catenin on transcription of lipogenic genes.Extensive studies in the liver have identified  enzymes . In thisand Scd1 . Thus, βSfrp5 deletion or adipocyte-specific deletion of Tcf7l2, Wntless, or Ctnnb1, also did not reveal an overt metabolic phenotype in chow-fed mice [Wntless deletion [β-cat−/− mice and were surprised to find that although β-catenin was efficiently ablated at the genomic level in knockout adipocytes, the mRNA and protein were still detectable at much higher levels than expected. These data are consistent with those recently published by Chen et al., who reported a ∼50% reduction in β-catenin mRNA expression in adipocytes isolated from iWAT or eWAT of knockout mice [β-cat−/− mice exhibited significantly up-regulated expression of Ctnnb1 and downstream Wnt targets, including Axin2, Nkd1, Tcf7l2, Wnt10b, and Wnt16. Although Chen et al. did not report elevated Ctnnb1 expression in the SVF of knockout mice, their data suggest a trend toward increased expression in SVF isolated from eWAT [Adipocyte-specific β-catenin deletion does not appear to influence global metabolism in chow-fed mice. Indeed, consistent with our studies, recent investigations into roles of Wnt signaling in mature adipocytes, including global fed mice . Howeverdeletion . Thus, wout mice . Perhapsrom eWAT . These d+, CD31+, and CD45-/CD31- populations isolated by FACS demonstrated that Ctnnb1 and downstream targets Axin2 and Nkd1 are significantly upregulated in CD45-/CD31- stromal cells of knockout mice. These data suggest that a sub-population of CD45-/CD31- cells, which include adipose stem cells, committed preadipocytes, and pericytes [An important question raised by our studies is the underlying mechanism by which β-catenin expression is up-regulated in SVF of knockout animals. Gene expression analysis of CD45ericytes , is ableericytes ,77,78.F4/80, Cd68, and Cd11c, were increased in chow-fed β-cat−/− mice. Of note, various isoforms of CD45 are present on almost all differentiated hematopoietic cells, and although Cd68 and Cd11c are commonly used as macrophage markers, they are also expressed by non-hematopoietic cell types, including endothelial and stromal cells [β-catfl/fl and β-cat−/− mice may help clarify specific sub-population changes following adipocyte-specific loss of β-catenin.Gene expression analysis of whole SVF indicated that several macrophage markers, including al cells ,80. ThusCtnnb1 mRNA and β-catenin protein in knockout adipocytes also suggests the intriguing possibility that β-catenin is delivered back to deficient cells, perhaps via SVF-derived sEVs. Many different cell types are known to secrete sEVs containing proteins, lipids, and genetic material; these sEVs serve as a unique mechanism for intercellular communication and have varied and intricate effects on receiving cells [β-cat−/− mice: despite efficient genetic ablation of Cav1 (caveolin-1), cav1 protein was readily detectable in deficient adipocytes. Ultimately, this was found to be the result of sEV-mediated trafficking of cav1 protein from surrounding endothelial cells back to Cav1−/− adipocytes. In addition to cav1, proteomic analysis of isolated endothelial sEVs identified members of the Wnt signaling pathway, including β-catenin [The relatively high levels of ng cells ,82. Thisng cells ,84, fromng cells  to promong cells . sEVs arng cells , endotheng cells , and strng cells , and canng cells . Indeed,-catenin . Thus, aβ-cat−/− mice fed HFD demonstrated decreased weight gain and fat mass, significantly improved glucose homeostasis, decreased circulating TAG and glucose-stimulated insulin release, and protection from hepatosteatosis. Of note, the metabolic effects observed in our mice were remarkably consistent with data recently reported by Chen et al.; in their study deleting β-catenin from adipocytes, obese β-cat−/− mice also exhibited decreased body weight and adiposity, accompanied by improved glucose tolerance, insulin sensitivity, and hepatosteatosis [Wntless−/− mice fed HFD were also characterized by decreased fat mass and protection from glucose intolerance and hepatosteatosis [Tcf7l2−/− mice demonstrated increased WAT mass, impaired glucose tolerance, and insulin insensitivity [Scd1. There are many potential reasons for these discordant results, including the generation of alternative Tcf7l2 splice variants [To date, studies of Wnt signaling in mature adipocytes have consistently reported that metabolic phenotypes are revealed with long-term HFD. However, conflicting results have emerged: our work in teatosis . Additioteatosis . In contsitivity . Furthervariants , compensvariants ,19, or dvariants . Neverthβ-cat−/− mice contained fewer PDGFRα+ preadipocytes, indicating that reduced fat mass is caused by decreased hyperplasia [β-cat−/− mice leads to less macrophage recruitment and activation and subsequently less PDGFRα+ cell proliferation. Indeed, global Saa3-deficient mice are resistant to diet-induced obesity, adipose tissue inflammation, and dyslipidemia [Saa3 was mildly higher in β-cat−/− mice maintained on chow diet and was subsequently suppressed in knockout mice with HFD feeding. Thus, it is possible that β-catenin mediates cross-talk between mature adipocytes and surrounding cells through Saa3, but future studies are required to directly answer this question.One final point of interest is the functional protection from diet-induced obesity and subsequent metabolic dysfunction that loss of adipocyte-specific β-catenin signaling appears to afford mice. Chen et al. found that WAT of HFD-fed erplasia . Furthererplasia . Chen etipidemia . Our stuSrebf1 and Mlxipl. Perhaps most interestingly, in chow-fed mice, CD45-/CD31- stromal cells respond to adipocyte-specific β-catenin depletion by up-regulating β-catenin and downstream target gene expression to defend canonical Wnt signaling homeostasis within WAT. We contend that this compensatory mechanism may explain the lack of observable phenotypes under standard nutritional conditions in virtually all mouse models that have been developed to interrogate the function of Wnt signaling in adipocytes. Finally, HFD feeding obesity overrides this compensatory mechanism, revealing that β-cat−/− mice are protected from diet-induced obesity and metabolic dysfunction. Together, these novel findings underscore the critical importance of Wnt signaling in regulation of glucose and lipid metabolism in mature adipocytes.In summary, we report that β-catenin-dependent canonical Wnt signaling regulates various metabolic pathways in mature adipocytes, including lipid metabolism. Indeed, β-catenin is required for coordinate regulation of DNL and fatty acid desaturation, partly mediated through the key transcription factors D.P.B. and O.A.M. conceived the project, designed the experiments, and wrote the manuscript. D.P.B performed the majority of the experiments, data analyses, and manuscript preparation. A.N. conducted analyses of the RNA-seq and ChIP-seq data. Z.L. and J. B. D. assisted with the experiments. Z.L., C.A.C., J.H., and B.S.L. assisted with the large-scale animal studies. H.M. and C.N.L. contributed intellectually to the experiments and provided key feedback. O.A.M. supervised the research.


Previous research on interlimb coordination has shown that some coordination patterns are more stable than others, and function as attractors in the space of possible phase relations between different rhythmic movements. The canonical coordination patterns, i.e. the two most stable phase relations, are in-phase (0 degree) and anti-phase (180 degrees). Yet, musicians are able to perform other coordination patterns in intrapersonal as well as in interpersonal coordination with remarkable precision. This raises the question of how music experts manage to produce these unstable patterns of movement coordination. In the current study, we invited participants with at least five years of training on a musical instrument. We used an adaptation paradigm to address two factors that may facilitate producing unstable coordination patterns. First, we investigated adaptation in different coordination settings, to test the hypothesis that the lower coupling strength between individuals during joint performance makes it easier to achieve stability outside of the canonical patterns than the stronger coupling during individual bimanual performance. Second, we investigated whether adding to the structure of action effects may support achieving unstable coordination patterns, both intra- and inter-individually. The structure of action effects was strengthened by adding a melodic contour to the action effects, a measure that has been shown to improve the acquisition of bimanual coordination skills. Adaptation performance was measured both in terms of asynchrony and variability thereof. As predicted, we found that producing unstable patterns benefitted from the weaker coupling during joint performance. Surprisingly, the structure of action effects did not help with achieving unstable coordination patterns. The only chance to come in is by ‘thinking’ this pattern as a gestalt in its own right” . FormingWhereas all of the experimental studies reviewed so far used bimanual coordination in individuals, the Amadinda example and some studies of joint action suggest that there may be similar effects of action effect structure for joint performances. In particular, there is evidence that joint action is often based on representing joint action outcomes that combine individual action effects into a pattern that is more than the sum of its parts. Loehr, Kourtis, Vesper, Sebanz and Knoblich  invited Based on previous literature, we investigated two potential factors that may affect how able people are to produce unstable temporal patterns during rhythmic performances. First, we considered the inherent coupling strength characterizing different coordination settings. While the coupling between two limbs of the same person is strong, coupling between the two limbs of two people is weaker and the coupling between the limb of a person and a computer produced sounds is basically absent . Coupling strength is one of the factors that determines how difficult it is to maintain unstable phase relations . To inveThe second factor, structure of action effects, was tested in Experiment 2. Based on previous literature we examined the role of adding melodic structure to action effects. Experiments 1 provided participants with concurrent auditory feedback realized in percussive sounds to highlight the rhythmic pattern. In Experiment 2, we tested whether adding structure in the pitch dimension to the action effects helps to retain complex target patterns and thereby skill acquisition and performance . To inveParticipants were recruited through flyers distributed in music schools in Budapest, on Facebook sites related to music-making in Budapest and on the CEU campus. Informed consent was obtained from all participants. Participants received gift vouchers as compensation. This study was approved by the United Ethical Review Committee for Research in Psychology (EPKEB) in Hungary. We invited 16 participants, of which 4 participants were not able to fulfill the minimal task requirements, i.e. they completed less than 80% of all trials successfully. The remaining 12 participants were included in the data analysis: 4 women, 8 men, mean age = 25 years, SD = 4 years. All participants in Experiment 1 had completed at least 5 years of private lessons on a musical instrument . Sample size for both experiments was constrained by our ability to recruit musical experts with sufficient experience. Because this constraint implied a small sample size, we provide detailed information about statistical power to obtain large effects (Cohen’s f = 0.4), medium effects, (Cohen’s f = 0.25) and small effects (Cohen’s f = 0.1). We used G*Power (version 3.1)  to perfoParticipants were tapping on iPad Pros that were connected via the iOS app MIRA 1.2.2 to a custom patch created in Max MSP 7.3.1 running on a Mac computer. The Max patch created tap contingent sounds that were played back to the participants via headphones. An occluder prevented both participants from seeing their own iPad and their partner’s iPad in the Interpersonal condition.Participants were instructed to tap out the rhythm depicted in In test trials, we introduced an artificial delay of 89 ms to the production of the sounds notated in staff A see . Hence, Both experiments consisted of four segments see  –Panel AEach segment consisted of 24 baseline trials and 60 delay trials in the following arrangement see  –Panel BPerformance measures were computed from the timestamped midi data. First, we checked whether participants followed the task of producing the required rhythm. The interspersed metronome clicks allowed us to calculate target times for the tone onsets produced by the participants. We computed the temporal deviation from these target times. In the Intrapersonal condition this measure was computed for each hand, in the Interpersonal condition, for each participant and in the Computer condition only for the participant’s right hand. If a tap fell outside a target window of +- 300 ms around the target time, it was considered an error. A missing tap or too many taps between two metronome clicks were also considered to be errors.The main dependent variables were absolute asynchrony and its variability. Absolute asynchrony between tone onsets in milliseconds is a direct measure of how well participants managed to align two sounds in time. Variability of absolute asynchrony, calculated as the standard deviation of absolute asynchrony also in milliseconds, describes how stable this temporal alignment is. We expected participants to show different baseline performances in different coordination settings. To account for this, we computed baseline corrected values for all asynchrony measures. For this calculation we took the asynchrony values and subtracted the condition-specific baseline performance for each participant. A baseline corrected value of around zero milliseconds therefor implied that the performance in the delay trials was equal to the performance in the non-delay trials.To investigate whether participants’ group average improved over time, we computed 3 x 3 ANOVAs, with the two within-subjects factors Condition  and Block . Significant results for Block imply a significant change of performance over the course of three blocks of delay trials. ANOVAs were computed for asynchronies and variability thereof in absolute terms and relative to baseline. Although the latter is more relevant for our study, we also included the former to establish comparability of our results with the results of previous studies addressing temporal coordination.To assess whether individual participants managed to reach their baseline performance, we split each of the three blocks of delay trials in each condition into two halves which results in six bins spanning ten trials each. We then calculated 95% confidence intervals for mean absolute asynchrony and its variability for each of the 6 bins. Using both measures, asynchrony and variability, we determined in how many bins the non-delay baseline was encompassed by the confidence interval, i.e. in how many bins the performance on delay trials was not significantly different from baseline performance.Intrapersonal = 93.35%, SDIntrapersonal = 4.85%, MInterpersonal = 90.58%, SDInterpersonal = 8.24%, MComputer = 96.53%, SDComputer = 4.69%) revealed that the main effect of Condition fell just above a significance level of .05, F = 3.707, p = .056, η2 = .146. Hence, there was no significant difference among the conditions in terms of accuracy.Trials that included missing, additional or misplaced taps were marked as errors. These trials were excluded from further analysis. The mean accuracy for the participants in Experiment 1 was 93.49% (SD = 4.32%). A Greenhouse-Geiser corrected one-way ANOVA with the factor Condition  = 18.963, p < .001, η2 = .520. Post-hoc paired-samples t-tests with Holm-Bonferroni corrected p-values [t(11) = 6.613, p < .001, d = 1.909, and the Computer condition, t(11) = 5.277, p = .001, d = 1.523. There was no significant difference between the Interpersonal and the Computer condition t(11) = .955, p = .360, d = .276. To summarize, participants exhibited the best baseline performance in the Intrapersonal condition.For the performance in non-delay baseline trials we observed the lowest absolute mean asynchrony for the Intrapersonal condition , with Joint  and Computer  showing comparable levels of asynchrony see  –Panel Ap-values  showed tF = 5.531, p = .027, η2 = .159, and Block, F = 18.124, p < .001, η2 = .062, but no significant interaction F = 2.516, p = .102, η2 = .018. Post-hoc comparisons with Holm-Bonferroni corrected p-values revealed that the main effect of condition was driven by the lower absolute asynchrony in the Computer condition. There was no significant difference between the Intra- and the Interpersonal condition, t(11) = .500, p = .627, d = .144, nor between the Intrapersonal and the Computer condition, t(11) = 2.013, p = .208, d = .581. Compared to the Computer condition the asynchrony was higher in the Interpersonal condition, t(11) = 5.043, p = .002, d = 1.456. Post-hoc comparisons further revealed that the performance in the Intrapersonal condition in the first block  and in the last block  did not differ significantly, t(11) = 1.557, p = .295, d = .449, whereas the performance in the Interpersonal condition  improved significantly, t(11) = 4.747, p = .003, d = 1.370. There was also a significant improvement in the Computer condition , t(11) = 3.148, p = .037, d = .909. To, summarize, overall participants exhibited the lowest asynchronies in the Computer condition and showed significant improvement in the Computer and the Interpersonal condition.To asses participants’ improvements across delay trials, we computed a 3 x 3 ANOVA with the factors Condition  and Block . The corresponding plot for absolute asynchronies can be seen in F = 8.386, p = .006, η2 = .289, and Block, F = 18.124, p < .001, η2 = .043, but no significant interaction F = 2.516, p = .102, η2 = .013. Post-hoc comparisons with Holm-Bonferroni corrected p-values revealed that in the first block, the baseline-corrected performance in the Interpersonal condition  was not significantly different from the performance in the Intrapersonal condition , t(11) = 2.074, p = .125, d = .599, and not different from the performance in the Computer condition , t(11) = 2.402, p = .105, d = .693. Due to the improvement of the performance in the Interpersonal condition, this pattern was different for the last block, where the baseline-corrected performance in the Interpersonal condition  was significantly lower than the performance in the Intrapersonal condition , t(11) = 3.626, p = .016, d = 1.047, but not statistically different from the performance in the Computer condition , t(11) = 1.022, p = .329, d = .295. When tested against 0 , a one-sample t-test revealed that in the Computer condition participants performance was not significantly different from baseline performance already in the first block, t(11) = .983, p = .347, d = .284. In the Interpersonal condition, participants’ performance was significantly different from baseline in the first block, t(11) = 3.306, p = .007, d = .954, but not in the last block, t(11) = 1.008, p = .335, d = .291. To summarize, due to significant improvement participants reached their baseline performance in the Interpersonal and the Computer condition, but not in the Intrapersonal condition.A similar ANOVA was computed for baseline-corrected absolute asynchronies see  –Panel BF = 38.107, p < .001, η2 = .690. Post-hoc paired-samples t-tests with Holm-Bonferroni corrected p-values showed that variability in the Intrapersonal condition was significantly lower than in the Computer condition, t(11) = 11.416, p < .001, d = 3.296 and variability in the Computer condition was significantly lower than in the Interpersonal condition, t(11) = 3.584, p = .004, d = 1.035. To summarize, participants exhibited the lowest variability in the Intrapersonal condition.As we turn to the variability of absolute asynchrony, we see a slightly different picture. In non-delay baseline trials we observed the lowest variability in the Intrapersonal condition , with Interpersonal  showing the highest variability and Computer  falling in between see  –Panel BF = 35.871, p < .001, η2 = .544, and Block, F = 11.853, p = .001, η2 = .104, but no significant interaction, F = 1.810, p = .189, η2 = .028. Post-hoc comparisons with Holm-Bonferroni corrected p-values showed that variability in the Intrapersonal condition was significantly lower than in the Computer condition, t(11) = 3.557, p = .013, d = 1.027, which was in turn significantly lower than the variability in the Interpersonal condition, t(11) = 6.401, p < .001, d = 1.848. Post-hoc comparisons furthermore revealed that the performance in the Intrapersonal condition in the first block  and in the last block  differed significantly, t(11) = 2.333, p = .04, d = .674, as did the performance in the Interpersonal condition , t(11) = 4.350, p = .005, d = 1.256. There was also a significant reduction of variability in the Computer condition , t(11) = 3.130, p = .019, d = .904. To summarize, participants exhibited the lowest variability in the Intrapersonal condition and showed significant improvement in all conditions.To asses participants’ reduction of variability across delay trials, we computed a 3 x 3 ANOVA with the factors Condition  and Block . The corresponding plot for variability can be seen in F = 11.853, p = .001, η2 = .070. Neither the main effect of Condition, F = 1.130, p = .323, η2 = .041, nor the interaction, F = 1.810, p = .189, η2 = .018, were significant. Comparing the baseline-corrected variability measures to zero in one-sample t-tests, showed that in the Intrapersonal condition even in the last block participants did not reach the baseline performance, t(11) = 7.146, p < .001, d = 2.063. There was also a significant difference between the performance in delay trials and the baseline in the Computer condition, t(11) = 2.423, p = 0.034, d = .699. However, the performance in the last block of the Interpersonal condition was not significantly different from baseline, t(11) = .783, p = 0.450, d = .226, indicating that baseline performance was reached in the Interpersonal condition. Hence, in terms of variability of asynchrony participants reached levels comparable to baseline performance only in the Interpersonal condition.A similar ANOVA was computed for baseline-corrected variability see  –Panel DFor the second experiment, we invited another 12 musicians to participate in the experiment . All 12 participants managed to produce 80% of the trials successfully. All participants in Experiment 2 had completed at least 5 years of private lessons on a musical instrument . As in Experiment 1, information about statistical power was computed with G*Power . As the For the between comparison of Experiment 1 and 2, we used data from all 24 participants and entered them into a 2x3 mixed ANOVA. Based on the total sample size of 24, power for the 3-level within-subjects factor in the mixed design corresponds to 0.99, 0.74 and 0.16, for large, medium and small effect sizes. Power for the 2-level between-subjects factor in the mixed design for the comparison across experiments corresponds to 0.63, 0.30 and 0.09 for large, medium and small effect sizes.Procedure and design were almost identical to Experiment 1. As in Experiment 1, the 12 musicians were grouped into 6 pairs for the Interpersonal condition. In Experiment 2, the actions participants had to perform were exactly the same as in Experiment 1. Only the outcomes that were produced by their actions were different. In Experiment 2 all participants’ taps, as well as the computer metronome, produced piano sounds of different pitches that taken together generated a short polyphonic melody of ten beats see . Hence, F = 4.753, p = .042, η2 = .22. However, post-hoc comparisons with Holm-Bonferroni corrected p-values revealed no significant differences. The accuracy was highest in the Computer condition , but not significantly different from both the accuracy in the Intrapersonal condition , t(11) = 2.623, p = .058, d = .757, and the accuracy in the Interpersonal condition , t(11) = 2.737, p = .058, d = .79. Furthermore, there was no significant difference between Intrapersonal and Interpersonal condition, t(11) = 1.546, p = .150, d = .446. Hence, as in Experiment 1, there was no significant difference among the conditions in terms of accuracy.The mean accuracy for the participants in Experiment 2 was 94.91% (SD = 3.50%). A Greenhouse-Geiser corrected one-way ANOVA with the Factor Condition revealed a significant main effect, F = 29.94, p < .001, η2 = .603. Post-hoc paired-samples t-tests with Holm-Bonferroni corrected p-values showed that this was mainly due to the lower asynchrony in the Intrapersonal condition, as compared to the Interpersonal t(11) = 7.563, p < .001, d = 2.183, and the Computer condition, t(11) = 6.205, p < .001, d = 1.791. There was no significant difference between the Interpersonal and the Computer condition, t(11) = .828, p = .425, d = .239. Hence, as in Experiment 1, when there was no delay participants exhibited lowest asynchronies in the Intrapersonal condition.In non-delay baseline trials in Experiment 2, we observed the lowest absolute mean asynchrony for the Intrapersonal condition , with comparable levels of asynchrony for Interpersonal  and Computer condition  see  –Panel CF = 6.71, p = .017, η2 = .046, but no effect of Condition, F = 2.894, p = .087, η2 = .12, and no significant interaction effect, F = 2.165, p = .116, η2 = .018. Post-hoc comparisons with Holm-Bonferroni corrected p-values revealed that in contrast to the results in Experiment 1, in Experiment 2 there was no significant improvement. Asynchronies in the Intrapersonal condition was higher in the first block  than in the last block , t(11) = 2.325, p = .080, d = .671. The performance in the Interpersonal condition also improved, albeit not significantly , t(11) = 2.679, p = .064, d = .773 and there was no significant improvement in the Computer condition , t(11) = 1.448, p = .176, d = .418. This suggests that there was an overall effect of Block, which did not manifest in separate improvement for any one of the conditions.Absolute asynchronies are displayed in F = 6.998, p = .008, η2 = .252, and a significant main effect of Block, F = 6.71, p = .017, η2 = .030. The interaction, however, was not significant, F = 2.165, p = .116, η2 = .012. Post-hoc t-tests with Holm-Bonferroni corrected p-values showed that the baseline-corrected performance in the Interpersonal condition was neither significantly different from the Intrapersonal condition, t(11) = 2.043, p = .122, d = .59, nor from the Computer condition, t(11) = 2.083, p = .122, d = .601. Asynchronies in the Intrapersonal condition, however, were significantly higher than in the Computer condition, t(11) = 3.224, p = .024, d = .931. Comparing the baseline-corrected values to zero in one-sample t-tests, showed that, as in Experiment 1, participants reached baseline performance in the last block of the Interpersonal condition, t(11) = 1.643, p = 0.129, d = .474, and already in the first block in the Computer condition, t(11) = .071, p = 0.945, d = .020. However, in the last block of the Intrapersonal condition, performance was still significantly higher than baseline, t(11) = 5.232, p < .001, d = 1.51. This means that in the Computer and the Interpersonal condition, but not in the Intrapersonal condition, participants reached levels of asynchrony which are comparable to their baseline performance.A similar ANOVA was computed for baseline-corrected absolute asynchronies see  –Panel BF = 37.596, p < .001, η2 = .658. Post-hoc paired-samples t-tests with Holm-Bonferroni corrected p-values showed that this was due to the significant difference between the Intrapersonal condition and both the Interpersonal condition, t(11) = 8.145, p < .001, d = 2.351, and the Computer condition, t(11) = 5.892, p < .001, d = 1.701, and a significant difference between the Interpersonal condition and the Computer condition, t(11) = 2.273, p = .044, d = .656. Hence, as in Experiment 1, participants exhibited their lowest asynchronies in non-delay trials in the Intrapersonal condition.In non-delay baseline trials we observed the lowest variability in the Intrapersonal condition , with higher variability in the Interpersonal condition  and in the Computer condition  see  –Panel DF = 21.282, p < .001, η2 = .450, and a significant main effect of block, F = 5.737, p = .011, η2 = .038. The interaction was not significant, F = .757, p = .491, η2 = .012. Post-hoc t-tests with Holm-Bonferroni corrected p-values revealed that the variability in the Intrapersonal condition was not significantly lower than the variability in the Computer condition, t(11) = 2.601, p = .099, d = .751, which in turn was significantly lower than the variability in the Interpersonal condition, t(11) = 7.18, p < .001, d = 2.073. Post-hoc comparisons revealed furthermore that the performance in the Intrapersonal condition in the first block  and in the last block  did not differ significantly, t(11) = 2.020, p = .137, d = .583., nor was there a significant reduction of variability in the Interpersonal condition , t(11) = 2.480, p = .099, d = .716, nor in the Computer condition , t(11) = .619, p = .548, d = .179. This means, that the significant main effect of Block was not due to any individual condition in particular.We computed a 3 x 3 ANOVA with the factors Condition  and Block . The corresponding plot can be seen in F = 5.737, p = .011, η2 = .024, but not of condition, F = 2.810, p = .091, η2 = .099. The interaction was also not significant, F = .757, p = .491, η2 = .008. Post-hoc comparisons with Holm-Bonferroni corrected p-values revealed no significant difference between the Block One and Block Three in the Interpersonal condition, t(11) = 2.48, p = .092, d = .716, nor in the Intrapersonal condition, t(11) = 2.020, p = .137, d = .583, nor in the Computer condition, t(11) = .619, p = .548, d = .179. Comparing the baseline-corrected variability measures to zero in one-sample t-tests, showed that even in the last block participants had a performance significantly worse than baseline, in the Intrapersonal condition, t(11) = 5.461, p < .001, d = 1.576. However, the performance in the last block of the Interpersonal condition, t(11) = 2.179, p = 0.052, d = .629 and of the Computer condition, t(11) = 1.190, p = 0.259, d = .344, was not significantly different from baseline performance. As in Experiment 1, participants reached their baseline performance in terms of variability of asynchronies in the Interpersonal condition.A similar ANOVA was computed for baseline-corrected variability see  –Panel DIn Experiment 2, there was no participant who reached their baseline performance in all three conditions see . Nine paF = 24.507, p < .001, η2 = .357, but no effects for Experiment, F = .020, p = .888, η2 = .000, and no significant interaction, F = .288, p = 0.742, η2 = .006.In Experiment 1 participants reached their baseline performance on average in 2.5 of 6 bins in the Interpersonal condition and on average 3.6 of 6 bins in the Computer condition. In Experiment 2, these numbers are 2.9 of 6 bins and 3.8 of 6 bins respectively. A Greenhouse-Geiser corrected 3 x 2 ANOVA on this data with the within-subjects factor Condition  and the between-subjects factor Experiment (1 and 2) revealed a main effect for Condition, To establish whether an effect of Experiment would only manifest itself in either asynchronies or variability thereof, we also calculated 3x3x2 ANOVAs with the factors Condition, Block and Experiment for all four dependent variables separately. While the main effects for Condition and Block were significant  the main effects for Experiment were not .age, main training (the number of years they had received training on their main instrument), total training  and practice per week (in hours). We systematically removed the factor with the highest p-value until only significant predictors were left [age and main training. Whereas an increase in main training predicted better performance, an increase in age predicted worse performance (see age and main training.All participants provided us with some biographic data in a post-experiment questionnaire. Besides standard items such as age and handedness, we asked which instruments they played, for how long they had training on them, whether they had experience playing in ensembles or teaching their instrument. To see whether any of these items are good predictors for their performance in the adaptation task, we entered them in a linear regression model as predictors for how many delayed bins where performed at baseline levels. We started with a model that contained the following four predictors: ere left . This lesuccessful bins ~ age + main training + error. For the bootstrapping procedure we used 2000 iterations. Age was coded as the age of participants at the time of the experiment. Main training was coded as the amount of years each participant had on their main instrument.Results for the linear model In this study we investigated the effects of coordination setting and structure of action outcomes on musicians’ ability to adapt to unstable coordination patterns. First, we tested conditions implying various degrees of coupling strength between two movements. For non-delay baseline performance, we found a clear pattern that repeated across both experiments. Absolute asynchrony was significantly lower and more stable in the Intrapersonal condition than in the Interpersonal condition, which is in line with findings from studies that compare intrapersonal and interpersonal coordination . This paWhereas participants’ baseline performance was best in the Intrapersonal condition, we predicted that the weaker coupling in the Interpersonal condition and the Computer condition should be advantageous for the performance of unstable coordination patterns. In terms of absolute asynchrony, for which we saw a clear advantage of stronger coupling during in-phase coordination, we find that this advantage disappeared during the performance of an unstable coordination pattern. In Experiment 1, asynchrony was lowest in the Computer condition and equal in the Intrapersonal and Interpersonal condition. In Experiment 2, we found no differences between the three coordination conditions. In Experiment 1, the improvement of asynchrony performance from the first to the last block was not significant in the Intrapersonal condition, whereas it was significant for the Interpersonal condition. For asynchrony in the Interpersonal condition, on the other hand, we see significant improvement across blocks in Experiment 1. In Experiment 2 there was a general effect of Block. While none of the individual conditions showed significant improvement after p-value corrections, participants reached their baseline performance only in the Interpersonal and the Computer condition. The effects of an unstable coordination pattern in the different coordination conditions was clearly visible in comparison to the baseline. In the Computer condition, where the coupling was just unidirectional, we saw that participants managed to reach the same performance as in their baseline already in Block 1 in both experiments. In the Interpersonal condition, participants initially performed significantly worse than their baseline. However, thanks to continuous adaptation across the three blocks, participants eventually reached their baseline performance in Experiment 1 and 2. In the Intrapersonal condition participants never reached their baseline performance. Hence, in terms of absolute asynchrony, we conclude that stronger intrapersonal coupling impedes coordination of, and adaptation to, unstable patterns.While in absolute terms the variability of asynchrony in delay trials was lowest in the Intrapersonal condition, baseline corrected variability showed that participants were on average not able to reach their baseline performance. In contrast, in the Interpersonal condition baseline performance was reached in Experiment 1 and Experiment 2. Taking together asynchrony and its variability, we found that during the Intrapersonal Condition, the condition with the strongest coupling, the group average never reached the baseline performance, even though we saw significant reduction in variability for Experiment 1. It is interesting to note that the Intrapersonal condition was also the condition with the consistently lowest variability. Individual motor learning studies have shown that variability is advantageous for motor learning ,43. ThisTaken together, the results discussed so far show that reducing the coupling strength between interacting limbs by the means of splitting a task across two people might facilitate the production of otherwise unstable coordination patterns. This principle is reminiscent of Ugandan xylophone players who manage to produce unstable coordination patterns at incredibly high tempi by distributing them across two musicians while keeping in-phase actions within musicians.In cultural evolution, where intending patterns, retaining them and reproducing them is limited by cognitive constraints ,46 and pAs a second factor we investigated the effects of structure realized along the harmonic pitch dimension on the adaptation to unstable coordination patterns. We predicted better adaptation in Experiment 2, where the auditory outcome was supplemented by a melodic structure. In terms of how often participants accomplished performance in delay trials that was comparable to their baseline, the data exhibited the effect of coordination setting, but no differences between the results of the two experiments. Hence, the melodic enhancement of the auditory outcome did not lead to significantly better adaptation. Because we needed to recruit experienced musical experts for our study our small sample size was relatively small. Therefore, we cannot exclude the possibility of having missed effects with small to medium effect sizes. Thus, further studies might be needed to investigate the effects of added structure in the production of unstable coordination patterns and its relation to sonification effects in bimanual motor skill learning .Given that musicians frequently encounter unstable coordination patterns, we had expected the invited musicians to more readily adapt to the pattern than the data suggested. As mentioned above, our subjects had on average around 11 years of musical training. Maybe this is not enough training to build the necessary skills to deploy similar strategies like the third player in Amadinda music, who has to be able to use structured action effects on which to map the necessary motor commands. However, the data show that at least some participants were in fact able to adapt to the strange phase relation in all conditions, especially in the Computer and the Interpersonal condition, but also in the Intrapersonal condition, where coupling was strongest.In the Intrapersonal condition of Experiment 1, three participants reached a delay performance that was not significantly different from their non-delay baseline, both in terms of absolute asynchrony as well as its variability. In the Intrapersonal condition in Experiment 2, only one participant managed to reach their baseline performance. Across both experiments, there were six participants who reached the baseline performance within the 3 blocks in all three condition, including the Intrapersonal condition for which the group average never reached the baseline. One participant did not manage to reach their baseline performance in any of the coordination conditions.Years of training was positively correlated with success, which suggests that experience as a musician improves the ability to adapt to unstable phase relations. Age, however, was negatively correlated with success. The fact that the model accounted for less than 20% of the variability suggests that there are other factors that contribute to the ability to adapt to unstable coordination patterns. The effect of years of training could, for example, be mediated by instrument type and type of training. Some instruments might require more temporal flexibility than other instruments, such as drums and piano, on which playing different rhythmic patterns at the same time is frequent. Experience in mixed ensembles with instruments that exhibit different rise times might also train the ability to adapt to unstable coordination patterns.To identify possible predictors of success in terms of reaching one’s baseline performance, we used a linear model procedure on collected questionnaire data, from which two significant predictors emerged: age and years of training on the main instrument. It might also be noteworthy that participant 9 in Experiment 1, who adapted well to all three conditions, had eight years of experience as an organ player. Gould and Helder  reportedFor further research on unstable coordination patterns in joint actions, it would be interesting to identify the skills or strategies that allowed some participants to quickly adapt to the phase shift. Following up on this it would also be interesting to investigate whether these skills or strategies are transferable to other unstable coordination patterns and also whether non-musicians would be able to learn these skills and/or strategies isolated from musical training. Another possible avenue for further research in this direction would lead towards an understanding of the influence of joint music-making as a tool to avoid certain attractor states, such as in-phase coordination in the transmission of culture.