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Metrics details An Erratum to this article was published on 21 October 2016 This article has been updated Maturity Onset Diabetes of the Young type 3 (MODY3) linked to mutations in the transcription factor HNF1A is the most prevalent form of monogenic diabetes mellitus HNF1alpha-deficiency leads to defective insulin secretion via a molecular mechanism that is still not completely understood in MODY3 patients the severity of insulin secretion can be extremely variable even in the same kindred indicating that modifier genes may control the onset of the disease With the use of a mouse model for HNF1alpha-deficiency we show here that specific genetic backgrounds (C3H and CBA) carry a powerful genetic suppressor of diabetes A genome scan analysis led to the identification of a major suppressor locus on chromosome 3 (Moda1) Moda1 locus contains 11 genes with non-synonymous SNPs that significantly interacts with other loci on chromosomes 4 the absence of HNF1alpha in diabetic-prone (sensitive) strains leads to postnatal defective islets growth that is remarkably restored in resistant strains Our findings are relevant to human genetics since Moda1 is syntenic with a human locus identified by genome wide association studies of fasting glycemia in patients our results show that a single genetic locus can completely suppress diabetes in Hnf1a-deficiency the intimate molecular mechanisms responsible for the typical MODY3 insulin secretion defect as well as the reasons for the highly variable expressivity of MODY3 remain poorly understood Here we report the drastic effect of the naturally occurring genetic variations in different inbred mouse strains on the diabetic phenotype linked to Hnf1a-deficiency we identified and characterized a major locus that is able to suppress the diabetes linked to Hnf1a-deficiency Blood glucose in Hnf1a sensitive and resistant mutant strains (A) Blood glucose level in 129S2 Hnf1a−/− (129S2−/− n = 151) mice and wild-type control (black triangles 129S2−/− mice presented significant hyperglycemia compared to control animals (p-value < 0.0001) Hnf1a mutant CBA strain (dark green squares n = 41) remained normoglycemic and comparable to CBA wild-type mice (black squares (C) Intraperitoneal Glucose Tolerance Test (IPGTT) on sensitive (red n = 3) adult animals demonstrates that the resistant strain is glucose tolerant (D) Body weight affects the fasting blood glucose level in the sensitive mutant strain (red triangles n = 43) but has no impact in the two resistant mutant strains CBA (dark green squares The linear correlation between body weight and fasting glucose level indicated by solid lines is significant only in the sensitive mutant strain (Pearson R2 = 0.56) Error bars represent SEM for 3 to 7 fasting blood glucose measurements carried out on mice from 25 to 45 days old (P25 to P45) Another important consideration is that Hnf1a-deficiency typically leads to an impairment of renal glucose reabsorption8 A potentially more severe renal glucose waste could have normalized blood glucose levels of mutants in the resistant strains resistant mutant mice tended to be much less glucosuric compared to sensitive strains indicating that the blood glucose levels of resistant mutant mice were not corrected by an increased urinary glucose loss (data not shown) These results indicate that the specific genetic variability of CBA and C3H strains has an intrinsic and genuine ability to suppress the diabetic phenotype elicited by the inactivation of Hnf1a CBA and C3H genetic backgrounds suppress diabetes in F1 Hnf1a mutant mice (A) Hnf1a−/− mice carrying an F1 129B6 background (red triangles n = 21) presented hyperglycemia whereas mutant mice carrying F1 129C3H (green squares Fasting blood glucose measurements were carried out on P25-P45 mice (B) Intraperitoneal glucose tolerance test (IPGTT) on F1 sensitive mutant 129B6 strain (red curve F1 resistant mutant strains 129CBA and 129C3H (dark green and light green curves respectively) and F1 129B6 (n = 6) plus 129C3H (n = 6) wild-type mice (black curve) The time course of glucose clearance was comparable in both F1 resistant mutant strains and wild-type mice (C) Plasma insulin levels measured by ELISA at 15 min during IPGTT in F1 wild-type (n = 5) F1 sensitive mutant 129B6 (n = 5) and F1 resistant mutant 129C3H (n = 7) Insulin concentrations were not significantly different between F1 wild-type mice and F1 resistant mutant (A) Selected representative views of P15 pancreas histological sections labeled for beta cells (pink staining) and nuclei (blue staining) with insulin antibody and DAPI (B) Box plot representation of apparent islet size from each genotype at P15 (3 animals per genotype) The total number of islets analyzed were 1534 Nonparametric Wilcoxon test determined the significant differences between samples (**p-value < 0.005; ***p-value < 0.0001) the obtained progeny (referred to as “N2”) received a complete and uniform complement of B6 chromosomes a set of chromosomes represented by either CBA or 129S2 or a recombination between these two strains Blood glucose level segregation in N2 Hnf1a mutant animals Blood glucose of F1 sensitive mutant mice (red squares Identification of a major QTL for fasting blood glucose level on chromosome 3 Whole genome multipoint LOD score for the Moda1 was 32 and defined a critical interval on chromosome 3 of 6.5 Mb (99% confidence interval) The minimum significant threshold value for the LOD score is indicated by a red dashed line Genetic interaction between the major Moda1 locus and ancillary loci modulates glucose homeostasis in Hnf1a−/− mice (A) Interaction between Moda1 and Moda2 loci When Moda1 carries the sensitive allele 129S2 almost all mice are hyperglycemic whatever the status of the Moda2 locus (left part of the chart) in animals carrying the resistant CBA allele of Moda1 the concomitant presence of Moda2CBA leads to the suppression of diabetes (right part of the chart) Moda4 and Moda5 in mice carrying a Moda1CBA resistant allele The progressive decrease of blood glucose level is significantly correlated to the increase of ancillary modifier locus carrying CBA allele (linear regression of 24mg/dL per locus with a p-value < 0.0001) (C) The cumulative impact of all ancillary loci has no significant effect on mice that carry a sensitive Moda1 allele In all figures each dot represents a mouse the complementary B6 allele was not indicated All these results demonstrated that the interval identified by the genetic analysis is actually responsible for the suppression of the development of diabetes Expression of Ghrelin and Ghrelin receptor genes Ghrelin (Ghrl) and Ghrelin receptor (Ghsr) levels were determined by RT-qPCR in F1 wild-type 129B6 and 129C3H mice (n = 4 and n = 4 respectively) and F1 Hnf1a−/− 129B6 and 129C3H mice (n = 5 and n = 6 an increase in Ghrl transcripts is observed in both mutant strains compared to wild-type mice but no significant differences in expression were observed between the mutant strains Ghsr transcripts were decreased in mutant strains compared to the wild type counterparts This decrease was statistically significantly higher in sensitive mutant compared resistant mutant strains (*p < 0.05) Allelic expression (pyrosequencing) of the Ghsr gene is presented in F1 129C3H mice in (D) pancreas The results are represented as a percentage of expression from each allele Slc2a2 expression in sensitive and resistant mutant strains Slc2a2 expression was assessed by RT-qPCR in wild-type 129B6 and 129C3H mice and in Hnf1a−/− 129B6 and 129C3H mice in the pancreas at P6 (n = 5 for each genotype) A decrease of Slc2a2 transcripts is observed in sensitive and resistant mutant mice compared to wild-type mice Resistant mutant mice express statistically significant higher levels compared to sensitive mutant mice (***p-value < 0.005) GLUT2 may not necessarily be the limiting factor leading to diabetes in sensitive mutant mice it is unlikely to imagine that Slc2a2 is the diabetes suppressor none of the candidate genes identified within the locus has been clearly directly involved in pathways whose defect might lead to diabetes The novelty of our results stem from the fact that we demonstrate that the naturally occurring genetic variation in a single major locus is sufficient to suppress the series of events that lead to this loss of identity and insulin secretion function we observed that islet size in resistant mutant mice correlates with the ability to secrete insulin it is likely that morphologically preserved islet-size in resistant mutant mice may account for the restoration of the insulin secretion no difference of cell proliferation or apoptosis seems to account for larger beta islets in the resistant mice we observed that the preservation of islet growth leading to larger Langerhans islets in resistant mutant mice seems to occur during postnatal life since at birth mutant pups do not show any significant difference (data not shown) we propose that a defective aggregation process of beta cells after birth could explain smaller islet size in sensitive mutant mice This suggests that the reduced expression of Slc2a2 may not necessarily be the limiting step that leads to diabetes in Hnf1a deficiency Our results showed that resistant mutant mice suppressed diabetes in spite of the overexpression of Ghrl a hormone whose antagonist had been shown to be able to normalize insulin secretion and hyperglycemia in Hnf1a deficiency This suggests that in the resistant genetic background the effect of the overexpression of Ghrl is not sufficient to elicit insulin secretion defects located near Moda1 does not contain any non-synonymous SNP and its expression is decreased at the same extent both in sensitive and resistant mutants our results showed that in F1 resistant 129C3H mutant mice the Ghsr gene is equally expressed from both resistant and sensitive alleles the resistant phenotype cannot be ascribed to the Ghsr gene itself although we cannot rule out that post-translational modifications of the ghrelin receptor protein linked to the action of other genes may account for a potentially altered receptor activity The interest and the novelty of our study stems from the fact that we demonstrated that the mechanisms leading to MODY3 diabetes can be overcome by a single genetic locus even in the absence of the expression of HNF1alpha The corollary of this consideration is that the events that lead to the development of diabetes are not necessarily unconditionally triggered by the absence of Hnf1a all these considerations indicate that the pathogenic mechanisms of MODY3 are probably more subtle than what was initially thought Moda1 locus must contain gene variant(s) whose effect (in CBA or C3H) is able to prevent the disruption of a subtle mechanism triggers the defective growth and/or aggregation of islets This could generate a situation where beta-cells progressively lose their capacity to secrete insulin in response to glucose we cannot rule out the presence of non-coding mutations that might affect the expression of genes or modify the biological properties of non-coding RNAs The clarification of the nature of the modifier gene(s) responsible for the suppression will require further investigations These efforts could shed a novel light for better understanding of the mechanism that leads to diabetes caused by Hnf1a-deficiency In the last years several genome wide association studies have indicated that genes expressed in beta cells and potentially involved in insulin secretion might play an important role on the onset of type 2 diabetes mellitus a better comprehension of the gene networks that interact with HNF1A/MODY3 could be extremely useful in dissecting the molecular aspects underlying the variability of insulin secretion defects in type 2 diabetes The recombinant animals were then mated with 129 Hnf1a+/− mice and Hnf1a−/− progeny was challenged for glucose by IPGTT (see below) to determine their sensitive or resistant phenotype to diabetes Genomic DNA was isolated from mouse tail tips and subjected to PCR using GoTaq G2 polymerase (Promega). Primer sequences are listed in Supplementary Table S2 mouse tissues were snap-frozen in liquid nitrogen and stored at −80 °C RNAs were extracted from frozen samples with TRIzol as described by the manufacturer (Life Technologies) Since the pancreas is highly enriched in RNases the frozen tissue was ground to powder in liquid nitrogen before being resuspended in TRIzol and subjected to three rounds of TRIzol extraction The quality of the isolated RNA was assessed on a Bioanalyzer 2100 (Agilent) reverse transcription was performed on 2 μg of RNA with the High-capacity cDNA reverse transcription kit (Applied Biosystems) All experiments were performed on conscious mice Blood glucose level was measured on tail blood with Accu-Chek Performa glucose meter (Roche) in fasting and non-fasting conditions Intraperitoneal Glucose Tolerance Tests (IPGTTs) consisted injection of 2 g D-glucose/kg in fasting adult male mice 100 μL of blood was collected from the tail vein at 0 and 15 min and the insulin content was determined in the recovered plasma using the Mouse Ultrasensitive Insulin ELISA kit (Alpco) Glucosuria was monitored with Diabur-test 5000 strips (Roche) Statistics were calculated using the two-way two-tailed Student t test or the nonparametric Wilcoxon test and genetic interactions were validated using the Fischer’s exact test Differences were considered significant with a p-value < 0.05 A correction has been published and is appended to both the HTML and PDF versions of this paper Mutations in the hepatocyte nuclear factor-1alpha gene in maturity-onset diabetes of the young (MODY3) Diabetes mellitus and the beta cell: the last ten years Clinical characteristics and diagnostic criteria of maturity-onset diabetes of the young (MODY) due to molecular anomalies of the HNF1A gene The Journal of clinical endocrinology and metabolism 96 Genetic modifiers of the age at diagnosis of diabetes (MODY3) in carriers of hepatocyte nuclear factor-1alpha mutations map to chromosomes 5p15 The type and the position of HNF1A mutation modulate age at diagnosis of diabetes in patients with maturity-onset diabetes of the young (MODY)-3 Defective insulin secretion in hepatocyte nuclear factor 1alpha-deficient mice Laron dwarfism and non-insulin-dependent diabetes mellitus in the Hnf-1alpha knockout mouse HNF1alpha controls renal glucose reabsorption in mouse and man Defective pancreatic beta-cell glycolytic signaling in hepatocyte nuclear factor-1alpha-deficient mice Mitochondrial dysfunction contributes to impaired insulin secretion in INS-1 cells with dominant-negative mutations of HNF-1alpha and in HNF-1alpha-deficient islets Loss of HNF-1alpha function in mice leads to abnormal expression of genes involved in pancreatic islet development and metabolism A transcription factor regulatory circuit in differentiated pancreatic cells Proceedings of the National Academy of Sciences of the United States of America 98 Hnf1alpha (MODY3) controls tissue-specific transcriptional programs and exerts opposed effects on cell growth in pancreatic islets and liver Hepatocyte nuclear factor 1 inactivation results in hepatic dysfunction R/qtl: QTL mapping in experimental crosses Quantitative trait locus analysis using J/qtl Ghrelin Inhibition Restores Glucose Homeostasis in Hepatocyte Nuclear Factor-1alpha (MODY3)-Deficient Mice New genetic loci implicated in fasting glucose homeostasis and their impact on type 2 diabetes risk Large-scale association analyses identify new loci influencing glycemic traits and provide insight into the underlying biological pathways Hepatic nuclear factor 1-alpha directs nucleosomal hyperacetylation to its tissue-specific transcriptional targets doi: 10.1128/MCB.21.9.3234-3243.2001 (2001) Transgenic reexpression of GLUT1 or GLUT2 in pancreatic beta cells rescues GLUT2-null mice from early death and restores normal glucose-stimulated insulin secretion Mouse genomic variation and its effect on phenotypes and gene regulation Epistasis of transcriptomes reveals synergism between transcriptional activators Hnf1alpha and Hnf4alpha How noise and coupling induce bursting action potentials in pancreatic {beta}-cells beta-cell turnover: its assessment and implications Download references We are grateful to Antonia Doyen for technical help We thank Camille Lebugle and Pierre Bourdoncle for help with beta cell mass determination (Cellular Imaging Platform We are grateful to Latif Rachdi for fruitful discussions and technical help with IPGTT and insulin measurements We thank Déborah Bourc’his for the access to the pyrosequencer Thanks are due to Luisa Dandolo and Fabiola Terzi for the critical reading of the manuscript This study was supported by funds from Agence Nationale de la Recherche ANR-09-GENO-020-01 MODYFIERS Fondation Bettencourt-Schueller (Prix coup d’Elan) and the Labex Who am I This research was also supported by grants from the Agence Nationale pour la Recherche ANR-10-IDEX-0002-02 ANR-10-LABX-0030-INRT and ANR-10-INBS-07- PHENOMIN to Y.H Present address: Laboratorio de Nefrología Complexo Hospitalario de Santiago de Compostela (CHUS) Instituto de Investigación Sanitaria (IDIS) Assistance Publique-Hôpitaux de Paris (APHP) Leibovici Michel and Pontoglio Marco jointly supervised this work Centre National de la Recherche Scientifique Institut de Génétique et de Biologie Moléculaire et Cellulaire Institut National de la Santé et de la Recherche Médicale provided the conceptual framework of the study and supervised the project The authors declare no competing financial interests Reprints and permissions Download citation Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign up for the Nature Briefing newsletter — what matters in science By subscribing, you agree to our Terms of Use and Privacy Policy Tokyo-based label WACKO MARIA has unveiled a new collaboration with PORTER just weeks after dropping a Spring/Summer 2021 collection lookbook linking with The Silence of the Lambs, Larry Clark, Jean-Michel Basquiat, Tecate and Dickies the brand once again delivers pattern-heavy motifs with the PORTER/Leopard velour shoulder pouch the shoulder bag is offered up in two colorways: brown and white The purse-like pouch is crafted with fabrics from the Italian mill PONTOGLIO and secured with a black drawcord strap and nylon lining Receive our weekly Newsletterand set tailored daily news alerts Knitwear, Knitted Outerwear, Knitted Accessories IVL acquires Brazil’s largest polyester fibres plant Metrics details mTOR activation is essential and sufficient to cause polycystic kidneys in Tuberous Sclerosis Complex (TSC) and other genetic disorders a sharp increase of proliferation and cyst formation correlates with a dramatic loss of oriented cell division (OCD) We find that OCD distortion is intrinsically due to S6 kinase 1 (S6K1) activation The concomitant loss of S6K1 in Tsc1-mutant mice restores OCD but does not decrease hyperproliferation leading to non-cystic harmonious hyper growth of kidneys Mass spectrometry-based phosphoproteomics for S6K1 substrates revealed Afadin a known component of cell-cell junctions required to couple intercellular adhesions and cortical cues to spindle orientation Afadin is directly phosphorylated by S6K1 and abnormally decorates the apical surface of Tsc1-mutant cells with E-cadherin and α-catenin Our data reveal that S6K1 hyperactivity alters centrosome positioning in mitotic cells affecting oriented cell division and promoting kidney cysts in conditions of mTOR hyperactivity Increased proliferation rates are accompanied by alterations in cell survival how the mTORC1 pathway promotes the various cellular responses in the context of polycystic kidneys remains largely unknown the molecular targets of mTORC1 that mediate cyst formation are unknown these evidences prompted the investigation of the role of S6K in TSC pathological lesions and in rapamycin-sensitive responses We show that mTORC1 controls two distinct pathways in kidney epithelia an S6K1-independent pathway regulating the rate of cell proliferation and an S6K1-dependent pathway regulating the orientation of cell division S6k1 deletion in the Tsc1-mutant background re-establishes the correct axis of cell division and rescues the cystic phenotype we show that the S6K1 phosphoproteome is enriched in proteins involved in the cell adhesion system and actomyosin cortex is a component of cell adhesion systems and contributes to the regulation of oriented cell division a Macroscopic photos of BrdU staining at post-natal day 90 of the different genotypes indicated b Kidney/body weight ratio of the indicated phenotypes at post-natal day 90 n = 17 Ksp-Cre; Tsc1 f/f and n = 12 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were quantified c Cystic index – (total cystic area/total kidney area) × 100 – of mice of indicated genotypes d Plasmatic urea concentrations (mmol/l) were determined in the indicated genotypes at post-natal day 20 n = 4 Ksp-Cre; Tsc1 f/f and n = 3 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were quantified e Representative pictures and f relative quantifications of BrdU staining at post-natal day 90 of the indicated genotypes The percentage of BrdU-positive cells on the total number of tubular cells was counted in 10 different fields of each section Dotted circles show transversal cuts of normal tubules n = 3 Ksp-Cre; Tsc1 f/f and n = 4 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were quantified n = 3 mice/group g Representative pictures and h relative quantifications of pH3S10 staining at post-natal day 90 of the different genotypes indicated The percentage of pH3S10-positive cells on the total number of tubular cells was counted in 10 different fields of each section n = 3 Ksp-Cre; Tsc1 f/f and n = 4 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were quantified Source data are provided as a Source Data file S6K1 activity is required for robust cyst formation in mouse models of TSC a RT–QPCR for Tsc1 expression from mouse kidneys of indicated genotypes b Western blots of mouse whole-kidney extracts at pre-cystic age (post-natal day 20) of the indicated genotypes Antibodies used for the immunoblot analysis are indicated c Densitometry analysis and fold change over control of the indicated phospho-proteins relative to actin n = 4 Ksp-Cre; Tsc1 f/f and n = 4 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were quantified d Representative pictures and e relative measurements of tubular cell surface area at post-natal day 10 of the indicated genotypes Roughly n = 50 tubular sections from five different fields of each section were measured f Representative 3D reconstructed images of mitotic divisions (anaphase) of renal tubular cells longitudinally oriented in Tsc1fl/fl and Ksp-Cre; Tsc1fl/fl; S6k1−/− mice and aberrantly oriented in the Ksp-Cre; Tsc1fl/fl mice Mice were killed at precystic (post-natal day 20) stage The tubular axis orientation is marked with a cyan dotted line the mitosis orientation with a yellow dotted line g Quantification of the mitotic division orientation at post-natal day 20 in of Tsc1 f/f (n = 41) Ksp-Cre; Tsc1 f/f (n = 8059) and Ksp-Cre; Tsc1 f/f; S6k1 −/− (n = 49 = 43) distal tubules Median ± interquartile range and Min and Max values mTORC1 is likely to alter OCD in both Tsc1-mutant and neighboring wild-type cells consistent with the general efficacy of rapamycin to blunt polycystic disease a Cells were serum starved overnight and subsequently amino acid starved with EBSS (Earle’s Balanced Salt Solution) for 1.5 h They were then either collected in starvation or stimulated with complete medium for 0.5 h and collected or treated with 20 nM rapamycin in complete medium for 0.5 h and then collected Cell lysates were probed with the indicated antibodies to characterize the effects of the loss of Tsc1 and Tsc1/S6k1 on mTORC1 signaling pathways in the cell lines Densitometry analysis and relative quantification of the phosphorylated forms of the indicated proteins are reported under each blot plated as single cells in Matrigel and cultured for 5 days before being fixed with 4% PFA and stained with DAPI to quantify the number of cells per sphere derived from a single cell An average of 100 spheres pooled from three independent experiments were quantified for each genotype c Volume and d mass were analyzed by optical measurement at G1 (2.5 h after mitotic division) for the different genotypes An average of 100 cells pooled from three independent experiments were quantified for each genotype a Representative images of 5-day mIMCD3s spheres immunolabeled with γ-Tubulin and DAPI to quantify spheres with polarized centrosomes and primary cilia Only spheres with a visible lumen and with all the centrosomes and primary cilia correctly localized on the luminal (apical) side were considered as normal b Quantification of the percentage of spheres with polarized primary cilia on total number of spheres per genotype The ratio was calculated on n = 70 spheres per experiment The histograms are an average of three independent experiments c Representative images of 5-day mIMCD3s spheres immunolabeled with γ-tubulin and DAPI to measure spindle orientation Yellow dotted lines indicate the spindle vectors d Quantification of spindle angles relative to the apical-basal axis in 5-day spheres three independent experiments were analyzed This defect was rescued by the deletion of S6k1 suggesting a causal link between S6K1 hyperactivity and spindle pole mispositioning a Representative images of 5-day mIMCD3s spheres immunolabelled with γ-tubulin Par3 and DAPI to show the localization of the centrosomes compared to the apical markers b Representative images of 5-day mIMCD3s spheres immunolabelled with Arl13b and DAPI to show the localization of the primary cilia compared to the apical markers c Representative images of cells cultured on L shape patterns and recorded by time-lapse microscopy for the different genotypes d Distribution of mitotic spindle angles relative to pattern orientation (αb−αa) at anaphase onset An average of 120 angles from three independent experiments were quantified for each genotype a Volcano plots of AGC kinase-motif antibody enrichment and b IMAC enrichment The x-axis shows log2-ratio for phosphopeptides between samples and the y-axis shows the −log10 p-value The horizontal blue bar represents the statistical cut-off of significance (p value of 0.05) Red dots represent downregulated phospho-peptides in the Tsc1/S6k1 double knock-out genotype and green dots represent up-regulated phospho-peptides Afadin phospho-peptide is represented in yellow The numbers in the chart represent the number of statistically significant differentially enriched peptides Representative peptides and their sites of modification are shown showing statistically significant gene set enrichments comparing the IMAC statistically significant differentially phosphorylated proteins versus the entire IMAC enrichment set a Cells were serum starved overnight and subsequently amino acid starved with EBSS (Earle’s Balanced Salt Solution) for 1.5 h with or without the indicated treatments They were then collected and cell lysates were probed with the indicated antibodies Asterisk represents non-specific cross-reactive bands b Densitometry analysis and relative quantification of the phosphorylated forms of the indicated proteins n = 4 independent experiments were analyzed c Western blots of mouse whole-kidney extracts at pre-cystic age (post-natal day 20) d Densitometry analysis and relative quantification of the phosphorylated form of Afadin and n = 4 Ksp-Cre; Tsc1 f/f; S6k1 −/− mice were analyzed e Western blot of protein lysates extracted from patient samples Renal tumors (t) or normal tissue adjacent to renal tumor (n) were collected from TSC patients and non-TSC patients Protein lysates then probed with the indicated antibodies f Densitometry analysis and fold change of TSC patients over non-TSC patients of the indicated proteins relative to the indicated loading control g Analog-sensitive S6K1 was co-transfected in Tsc1/S6k1 dKO mIMCD3 cells with FLAG-Afadin-WT An in vivo kinase assay was performed in the presence of 6‐Bn‐ATP‐γ‐S both in presence and in absence of 20 nM rapamycin After immunoprecipitation using an anti-FLAG antibody the thio-phosphorylation of Afadin was revealed by western blot using an anti‐thiophosphate ester antibody FLAG and phospho-RPS6 were revealed by western blot on total extracts using the indicated antibody indicating the control of Afadin expression at different levels precluding a clear-cut determination of the effects on protein stability a Representative images of 5-day spheres from cells Tsc1/Afdn dKO transduced with lentivirus FLAG-Neon b Quantification of the percentage of spheres with polarized primary cilia on total number of spheres from cells WT and Tsc1/Afdn dKO transduced with lentivirus FLAG-Neon The ratio was calculated on n = 70 spheres per experiment per each genotype The histograms are an average of six independent experiments (for WT n = 3 independent experiments) c Representative images of 5-day spheres from WT mIMCD cells transduced with lentivirus FLAG-Neon FLAG-Afadin-S1795A (phospho-deficient) or FLAG-Afadin-S1795E (acidic phospho-mimetic) Arl13b and DAPI to quantify spheres with polarized centrosomes and primary cilia d Quantification of the percentage of spheres with polarized primary cilia on total number of spheres per genotype from WT mIMCD cells transduced with lentivirus FLAG-Neon a Representative images of 5-day mIMCD3s spheres of the indicated genotypes immunolabelled with Afadin (red) and DAPI to show the relative localization of Afadin and E-cadherin in interphase and relative to the mitotic spindle poles in 3D spheroids Insets show higher magnification of one mitotic cell per genotype to show the relative localization of Afadin and E-cadherin in mitotic cells yellow arrows indicate the localization of the Afadin spots White arrows indicate localization of the poles of the mitotic spindles A dotted yellow line indicates Afadin staining in the whole apical domain of mitotic cells b Percentage of mitosis showing one Afadin spot adjacent to each spindle pole (white) or perturbed afadin localization adjacent to a single spindle pole (dark gray) An average of 10 mitosis were analyzed per experiment per each genotype c Representative images of 5-day mIMCD3s spheres of the indicated genotypes immunolabelled with α-catenin ZO-1 and DAPI to show the loss of baso-lateral presence of α-catenin in the Tsc1 KO with a concomitant appearance of an apical mislocalization relative to the apico-lateral marker ZO-1 d Representative pictures of pre-cystic post-natal day 20 mice immunolabelled with Afadin antibody to show the localization of the protein in the indicated genotypes e Representative pictures of pre-cystic post-natal day 20 mice ZO-1 and DAPI to show the localization of the proteins in the indicated genotypes Here we identify S6K1 as a direct mTORC1 substrate required for cyst formation S6K1 deletion affects the orientation but not the rate of cell division in TSC mutant mice thus disconnecting the need for these two cell cycle alterations at the origin of polycystic kidneys Centrosome mispositioning in mitotic Tsc1-mutant cells accompanies the defect in oriented cell division where cells rely on cell-junctions to position the centrosome Performing an unbiased and comprehensive S6K1-phosphoproteome in the TSC background we highlight that peptides whose phosphorylation is S6K1-dependent are mainly regulators of intercellular junctions and actomyosin cortex Afadin is a direct S6K1 substrate involved in the control of oriented cell division possibly by coupling intercellular adhesions and cortical cues to the mitotic spindle We can speculate that the S6K1-dependent phosphoproteome orchestrates a pleiotropic response required for the intense tissue remodeling in polycystic kidneys S6K1 is known to regulate proteins involved in protein and nucleotide synthesis our MS data also suggest the existence of a class of S6K1 substrates with a role in intercellular adhesion and regulation of the actomyosin cortex Taking advantage of Afadin phospho-specific antibodies and mutant cells we demonstrated that Afadin is a direct S6K1 substrate participating in OCD control these studies did not reveal the underlying direct mTORC1-dependent mechanism indicating that the deregulation of the mTORC1-S6K1-Afadin axis may contribute to cystogenesis by altering the adhesion and spindle orientation program The definition of mTORC1/S6K1 action on OCD and cyst formation may suggest future therapeutic developments The R26R-Confetti mice were obtained from The Jackson Laboratory and crossed first with Tsc1fl/fl mice and then with Ksp-Cre mice All animal studies were approved by the Direction Départementale des Services Véterinaires (Prefecture de Police France; authorization number 75–1313) and the ethical committee of Paris Descartes University Mice were housed in a 12-h light/dark cycle and fed a standard chow Control mice were Cre-negative littermates of mutant mice Pharmacological inhibitors were added to cells for the time and at the concentration Rapamycin (#1292; Tocris) was dissolved in 100% ethanol (25 mg/ml) A 20-nM working solution was prepared in fresh media before use and cells were treated for the indicated time PF-4708671 (#S2163) and LY2584702 Tosylate (#S7704) were purchased by Selleckchem and were dissolved in 100% DMSO 10 µM and 1 µM working solution were prepared in fresh media before use and cells were treated for the indicated time Temsirolimus (LC Laboratories) was dissolved in 100% ethanol (25 mg/ml) 1 mg/ml working solution was prepared in 5% Tween-80 with vehicle or temsirolimus (5 mg/kg) every other day for 2 weeks before killing and anti-phospho Afadin (Ser1795) (#5485) were purchased from Cell Signaling Technology; anti-TSC1 (#sc- 377386) was purchased from Santa Cruz Biotechnology and anti-FLAG M2® (1/1000; #F1804) were purchased from Sigma-Aldrich and immunocytofluorescence: anti-Afadin (1/500; #A0224) and Phalloidin-TRITC (1/500; #P1951) were purchased from Sigma-Aldrich; anti-phospho Histone H3 (Ser10) (1/400; #ab5176) and anti-NuMA (1/200; #ab109262) were purchased from Abcam; rhodamine labeled Dolichos Biflorus Agglutinin (DBA) (1/200; #RL-1032) was purchased from Vector Laboratories; anti-Arl13b (1/400; #17711-1-AP) was purchased from Proteintech; anti-LGN (1/200; #ABT174) and anti-β-catenin (1/200; #06-734) were purchased from Merk Millipore; anti-aPKC (1/100; #sc-17781) and anti ZO-1 (1/200; #sc-33725) from Santa Cruz Biotechnology Inc; anti-FGFR1OP (FOP) (1/200; #H00011116-M01) was from Abnova; anti E-cadherin (1/400; # 13-1900) was purchased from Thermo Scientific; anti-Ezrin (1/200; P81) and anti-Integrin-β1 (1/200; P5D2) were purchased by DSHB For immunofluorescence after clearing: anti-phospho Histone H3 (Ser10) (1/200); anti AQP2-Alexa Fluor ® 488 conjugated (1/200; # sc-515770) was purchased from Santa Cruz Biotechnology Inc; FITC-labeled Wheat germ agglutinin (WGA) (1/100; #FL-1021) was purchased from Vector Laboratories; TO-PRO™-3 Iodide (1/1000; #T3605) from Thermo Fisher Scientific cells were trypsinized and dissociated into single cells by mechanically pipetting in trypsin and resuspending in medium Cells were then diluted to 50,000/ml in 50% normal medium and 50% growth factor-reduced Matrigel (#356230; BD Biosciences) and plated on 8-well slides (LabTek-II #155409; Thermo Scientific) with normal medium added on top the medium was changed to FBS-free DMEM and left until fixation For the generation of Tsc1 and S6k1 knockout (KO) lines sgRNA oligos were cloned into pX459 (Addgene #62988) which contains the Cas9 nuclease sequence and a puromycin resistance cassette sgRNA oligos were similarly cloned into a pGuide vector (containing the human U6 promoter and with a modified scaffold sequence to improve stability) mIMCD3 cells were transfected with 2 µg of plasmid using TransIT-X2® (MIR 6003; Mirus Bio) cells were trypsinized into puromycin (1 μg/ml) selection medium for 2 days and in regular medium thereafter The colonies were allowed to grow until it was possible to pick them with a filtered tip and were transferred individually into a well of a 96-well-plate Clones were expanded and verified for the loss of Tsc1 The sgRNAs sequences used to generate the clones for this study are the following: Tsc1 5′-TCT TTG GCC GTC TCT CGT CA-3′(PAM: TGG); S6k1 5′-TTT GAG CTA CTT CGG GTA CT-3′ (PAM: TGG); and Afdn 5′- CTG AAG TTG TCA TGA AAC GG-3′ (PAM: CGG) The 3×-Flag tagged human Afadin (pCMV-3xAF6) construct was obtained from Dundee University The mutation of serine 1795 to alanine was performed by overlapping PCR using the following primers: Forward (5′-CAG AGA CTG TTC gCC CAG GGC CAA GAC GTG TCC AAC AAA G-3′) and Reverse (5′-TGG GcG AAC AGT CTC TGC CGC TCT TTA AAG GTC AGG TTC TCT GG-3′) and pCMV-3xAF6 as template The mutation of serine 1795 to glutamic acid was performed by overlapping PCR using the following primers: forward (5′-AGC GGC AGA GAC TGT TCg aaC AGG GCC AAG ACG TG-3′) and reverse (5′- CAC GTC TTG GCC CTG ttc GAA CAG TCT CTG CCG CT-3′) and pLJM1-3xAF6 as template the resulting PCR reaction was transformed into competent E Clones were verified for the presence of the amino acid substitution by sequencing The pLJM1-Rheb (Addgene) construct was used as the backbone to create the Flag-Neon and Flag-Afadin constructs The pLJM1-Rheb construct was digested with Nhe1/EcoR1 and a 3×-flag mNeon cassette amplified by PCR to contain the same restriction sites Amplification of the wild type and S1795A Afadin constructs was performed by PCR to include Nhe1 sites at both the 5′ and 3′ ends The pLJM1-mNeon construct was digested with Nhe1/Xba1 and replaced with the Afadin PCR constructs or Afadin S1795E-FLAG were produced by the VVTG platform (SFR Necker France) or generated by transient transfection of the lentiviral vector (plasmid) together with packaging plasmids (pBA.Rev The supernatant containing lentiviruses was collected and filtered through 0.45 µm filter and stored in aliquots at −80 °C or immediately used to infect recipient cells cells were selected in puromycin (1 µg/ml) and further passaged in culture as a bulk preparation for functional assays Total RNA was extracted from cells or renal tissue using the RNAeasy Mini Kit (QIAGEN) Single‐strand cDNA was synthesized from 1 μg of total RNA with SuperScript II (Invitrogen) and 125 ng random hexamer primers Real‐time quantitative PCR was performed on MX3005P instrument (Agilent) using a Brilliant III SYBR Green QPCR Master Mix (Agilent) Relative amounts of the indicated mRNAs were determined by means of the 2−ΔΔCT method The primers used for Tsc1 mRNA were the following: forward (5′-AGC TCC GGA CCC TCC GAG AC-3′) and reverse (5′-AGC CGC TGC TCG GAT CAC CT-3′) The primer used for Afadin mRNA were the following: forward (5′-ACT CCC TCT ATG AAG TGC ATG T-3′) and reverse (5′-TAA GGA CGA ATC GAC CCT CTC-3′) Cells and tissues were lysed in NP-40 lysis buffer (20 mM Tris pH 8.8; 138 mM NaCl; 5 mM EDTA; 2.7 mM KCl; 20 mM NaF; 5% Glycerol; 1% NP-40- IGEPAL®; #CA-630; Sigma-Aldrich) supplemented with complete protease inhibitor and phosSTOP phosphatase inhibitor cocktails (Roche) Protein extracts were resolved by SDS-PAGE Uncropped and unprocessed scans of the most important blots are provided in the Source Data file Human biopsies were obtained from TSC and non-TSC patients undergoing partial or total nephrectomy normal tissue adjacent to tumoral tissue was resected and analyzed separately This study was approved by the French national ethics committee (Comité de Protection des Personnes Number 19.05.27.61541; national identification 2018-A03147-48) Uncropped western blots are provided in the Source Data file Mouse tissues were fixed in 4% paraformaldehyde (PFA) embedded in paraffin and sectioned at 4 μm Sections were deparaffinized and hydrated before being incubated in citrate buffer (pH6) then boiling for 3 min and then sub-boiling for 10 min Sections were blocked in 3% normal goat serum 1% BSA and 0.1% Triton X-100 for 30 min-1h and then incubated with the primary antibodies (or lectins) overnight at 4 °C sections were incubated with biotinylated secondary antibodies and detected with the Vectastain Elite ABC kit (Vector) and DAB chromogen system (DAKO) The percentage of BrdU incorporation was determined by counting BrdU-positive nuclei on the total number of nuclei among 10 distinct fields in three different animals for each condition For Confetti-reporter detection and immunofluorescence of Afadin tissues were fixed in 4% paraformaldehyde (PFA) for 10′ followed by two washes in PBS and cryopreservation in 30% sucrose at 4° over-night The sections were then placed on super frost slides and post-fixed for 104 in 4% PFA Sections were blocked and permeabilized with 7 mg/ml porcine skin gelatin 0.5% Triton X-100 in PBS and incubated with the primary antibodies in the same solution cells were grown on coverslips and fixed in 4% PFA (unless otherwise stated) The fixed cells were permeabilized with 0.3% Triton in PBS; blocked with 5% normal goat serum 3% BSA in PBS; and incubated with the primary antibodies Spheroids in chamber slides were washed twice with PBS containing calcium and magnesium and fixed in ice-cold 4% PFA for 30-45 min at 4 °C under agitation spheres were blocked and permeabilized in 7 mg/ml gelatin (porcine skin #G1890; Sigma-Aldrich) and 0.5% Triton in PBS with calcium and magnesium at RT for 1 h and then incubated with the primary antibodies diluted in the same blocking/permeabilization buffer overnight at 4 °C spheres were incubated with Alexa Fluor conjugated secondary antibodies or Phalloidin-TRITC for 4 h at RT and then DNA was stained by DAPI within the mounting medium (Vector Labs) Immunofluorescence slides were analyzed using an ApoTome2 microscope (Zeiss) or a Confocal TCS SP5 (Leica) ImageJ software (National Institutes of Health) or Imaris v 9.2 and Bitplane AG was used for quantification Tissue clearing with CUBIC reagents was performed as described72 mice were fixed by transcardial perfusion with 4% PFA post-fixed at 4 °C for 6 h and included in agarose to be sectioned with a vibratome (Leica) sagittally at 1 mm The sections were then incubated in CUBIC-reagent 1 at 37 °C for 1 week washed thoroughly in PBS and then incubated at 4 °C for 30 days in primary antibody and DNA staining described in the text diluted in PBS containing 2% Triton X100 (PBS-2T) sections were incubated with Alexa Fluor ® secondary antibody 488 (1/200) and DNA staining in PBS-T for 2 weeks at 4° After staining the samples were cross-linked in PBS 4% PFA for 3 h then washed thoroughly in PBS before being transfer in Cubic-reagent 2 and acquisition Sections were imaged using a Lightsheet Z.1 (Carl Zeiss) with ×20 objective For analysis of the tissues after clearing the spindle vector Vs and the tubular axis vector Vt were generated from the coordinates (x z) measured in ImageJ of the nuclei in anaphase and of the tubular staining respectively Then the angle between the 2 vectors was measured in Excel and plotted using Prism 6 for Windows Confocal images of entire mIMCD3’s spheroids stained with γ-Tubulin and DAPI were collected and opened with ImageJ the spindle vector Vs was generated using the 3D coordinates of γ-Tubulin staining (x z) of metaphase or anaphase mitosis measured in ImageJ zm) of Vs was calculated and the centroid (xc zc) of each spheroid in the middle z-plane of the sphere was determined using ImageJ The projected angle between Vs and Vc was eventually calculated using Excel The cyst formation was quantified from sagittal sections of whole kidneys75 whole kidney sections were scanned using a Nanozoomer 2.0 HT (Hamamatsu) after H&E staining; 4x images for each kidney section were selected We used a cut-off of 2000 pixels as the minimum surface that a cyst should have to be included in the analysis We next calculated the ratio between the obtained cystic area and the area of the entire kidney section and expressed it as the percentage of cysts for the given kidney 4-μm kidney sections were stained with rhodamine labeled DBA and an average of 80 tubular sections were measured for each genotype on three different mice using ImageJ the area of external profile of the tubule and the area of the lumen were measured and epithelial surface was calculated as the difference between the two areas and resuspended in medium supplemented with 1 mg/ml Alexa Fluor 488 Dextran (MW 10 kD; Life Technologies) at a concentration of 106 cells/ml before being injected in PDMS chips coated with fibronectin with a known height of 23 µm the chamber was immersed in medium to prevent evaporation Acquisitions were performed on a Ti inverted (Nikon) or an Axio Observer microscope (Carl Zeiss) at 37 °C or a SFluor 20× objective (NA 0.75 without phase ring) for the mass measurements Images were acquired using MetaMorph (Molecular Devices) Images were acquired with a CoolSNAP HQ2 camera (Photometrics) for fluorescence or a PHA SICS camera (PHA SICS) for mass measurements Images were analyzed with a custom-made MatLab program (MathWorks) Enriched peptides were subjected to secondary digest with trypsin and second STAGE tip prior to LC-MS/MS analysis Files were searched against the most recent update of the UniProt mus musculus FASTA database A mass accuracy of ±5 ppm was used for precursor ions and 0.02 Da for product ions with at least one tryptic (K- or R-containing) terminus required per peptide and up to four mis-cleavages allowed Cysteine carboxamidomethylation was specified as a static modification oxidation of methionine and phosphorylation on serine or tyrosine residues were allowed as variable modifications Reverse decoy databases were included for all searches to estimate false discovery rates and filtered using a 2.5% FDR in the Linear Discriminant module of Core Peptides were also manually filtered using a ±5 ppm mass error range and presence of a phosphorylated residue (IMAC) or phosphorylated residue within an RXX(s/t) motif (Akt Substrate Motif antibodies) All quantitative results were generated using Skyline (MacLean) to extract the integrated peak area of the corresponding peptide assignments in the MS1 channel Accuracy of quantitative data was ensured by manual review in Skyline or in the ion chromatogram files All experiments were repeated at least three times Standard two-tailed Student’s t test (when two groups were compared) or one-way ANOVA with Turkey correction for multiple comparisons (where three or more groups were compared) were used in the experiments unless otherwise stated The relative level of the p-value is expressed as asterisks Further information on research design is available in the Nature Research Reporting Summary linked to this article The mTOR pathway is regulated by polycystin-1 and its inhibition reverses renal cystogenesis in polycystic kidney disease Kidney-specific inactivation of Ofd1 leads to renal cystic disease associated with upregulation of the mTOR pathway The role of the Birt-Hogg-Dube protein in mTOR activation and renal tumorigenesis Emerging targeted strategies for the treatment of autosomal dominant polycystic kidney disease A study of sirolimus and mTOR kinase inhibitor in a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling Phosphoproteomic analysis identifies Grb10 as an mTORC1 substrate that negatively regulates insulin signaling mTORC1 phosphorylation sites encode their sensitivity to starvation and rapamycin Tsc tumour suppressor proteins antagonize amino-acid-TOR signalling S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5’-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway Disruption of the p70(s6k)/p85(s6k) gene reveals a small mouse phenotype and a new functional S6 kinase Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex mTORC1-mediated inhibition of polycystin-1 expression drives renal cyst formation in tuberous sclerosis complex Epithelial-specific Cre/lox recombination in the developing kidney and genitourinary tract Cystogenesis and elongated primary cilia in Tsc1-deficient distal convoluted tubules in renal tubular cells causes polycystic kidney disease by activating mTORC1 Regulation of YAP by mTOR and autophagy reveals a therapeutic target of tuberous sclerosis complex Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1 Rictor is a novel target of p70 S6 kinase-1 glucose intolerance and diminished beta-cell size in S6K1-deficient mice Atrophy of S6K1(−/−) skeletal muscle cells reveals distinct mTOR effectors for cell cycle and size control Defective planar cell polarity in polycystic kidney disease Loss of Fat4 disrupts PCP signaling and oriented cell division and leads to cystic kidney disease Acute kidney injury and aberrant planar cell polarity induce cyst formation in mice lacking renal cilia Defects in cell polarity underlie TSC and ADPKD-associated cystogenesis Loss of oriented cell division does not initiate cyst formation Disruption of core planar cell polarity signaling regulates renal tubule morphogenesis but is not cystogenic Exploring the function of cell shape and size during mitosis 3D spheroid model of mIMCD3 cells for studying ciliopathies and renal epithelial disorders Regulation of mitotic spindle orientation: an integrated view The extracellular matrix guides the orientation of the cell division axis External forces control mitotic spindle positioning Classical cadherins control nucleus and centrosome position and cell polarity Immunoaffinity profiling of tyrosine phosphorylation in cancer cells Nectin spot: a novel type of nectin-mediated cell adhesion apparatus Cofilin phosphatases and regulation of actin dynamics The CAMSAP3-ACF7 complex couples noncentrosomal microtubules with actin filaments to coordinate their dynamics Coordination of Golgi functions by phosphatidylinositol 4-kinases MIM and cortactin antagonism regulates ciliogenesis and hedgehog signaling Afadin orients cell division to position the tubule lumen in developing renal tubules ZRF1 is a novel S6 kinase substrate that drives the senescence programme The adherens junction protein afadin is an AKT substrate that regulates breast cancer cell migration Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells Primary-cilium-dependent autophagy controls epithelial cell volume in response to fluid flow 4E-BP1 and 4E-BP2 double knockout mice are protected from aging-associated sarcopenia Concomitant binding of Afadin to LGN and F-actin directs planar spindle orientation Regulation of epithelial wound closure and intercellular adhesion by interaction of AF6 with actin cytoskeleton The TSC1 tumour suppressor hamartin regulates cell adhesion through ERM proteins and the GTPase Rho the tuberous sclerosis complex 2 tumor suppressor gene product Retention of membrane-localized beta-catenin in cells lacking functional polycystin-1 and tuberin Tuberin regulates E-cadherin localization: implications in epithelial-mesenchymal transition The loss of tuberin promotes cell invasion through the ss-catenin pathway A mitotic transcriptional switch in polycystic kidney disease Aberrant regulation of planar cell polarity in polycystic kidney disease Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis Aberrant planar cell polarity induced by urinary tract obstruction Proximal tubule proliferation is insufficient to induce rapid cyst formation after cilia disruption Comparison of folate-conjugated rapamycin versus unconjugated rapamycin in an orthologous mouse model of polycystic kidney disease Everolimus for angiomyolipoma associated with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2): a multicentre Everolimus in patients with autosomal dominant polycystic kidney disease Sirolimus and kidney growth in autosomal dominant polycystic kidney disease Sirolimus therapy to halt the progression of ADPKD Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis LGN regulates mitotic spindle orientation during epithelial morphogenesis Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1 Optical volume and mass measurements show that mammalian cells swell during mitosis Complementary PTM profiling of drug response in human gastric carcinoma by immunoaffinity and IMAC methods with total proteome analysis Stop and go extraction tips for matrix-assisted laser desorption/ionization and LC/MS sample pretreatment in proteomics Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database A tissue-specific atlas of mouse protein phosphorylation and expression Large-scale phosphorylation analysis of mouse liver Download references and the members of INSERM-U1151 for support critically reading the manuscript and helpful discussions and viral vector facilities at the SFR Necker for the technical help We thank Marine Largeau and Veronique Cussin from the Biological Resources Center and Tumor Bank Platform of Georges Pompidou European Hospital for technical support (BB-0033-00063) for providing biological samples This work was supported by grants from the European Research Council These authors contributed equally: Martina Bonucci Equipe Labellisée par la Ligue contre le Cancer Assistance Publique-Hôpitaux de Paris (AP-HP centre) designed and conducted most of the experiments performed and interpreted micropattern and cell size experiments performed and interpreted phospho-proteomics experiments set-up the kidney clearing/3D reconstruction and interpreted the polycystic kidney phenotype provided the human biopsy samples and interpretation performed and interpreted analysis of cell–cell adhesion markers All authors discussed the results and commented on the manuscript The authors declare no competing interests Peer review information Nature Communications Elizabeth Henske reviewers for their contributions to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-020-16978-z Cellular & Molecular Biology Letters (2024) has expanded its retail store network into Southeast Asia with the region’s first standalone Moleskine store opening in Singapore.  Occupying a 500sqft space of Paragon Shopping Centre’s second floor Moleskine’s new flagship houses a curated collection of products writing accessories and digitally connected notebooks The store design features clean aesthetics and contemporary neutral colours that “mirror the elegance and simplicity of the legendary notebook” The store also presents a dedicated space for Moleskine’s capsule collection An “experience table” is designed for customers to try out different products and use interactive customisation tools to personalise their purchases The launch coincides with the release of several limited-edition collections in partnership with Kaweco Missoni and illustrator Lok Ng for a special 2022 Year of the Tiger edition Moleskine has more than 80 stores across the world By continuing, you agree to Octomedia Terms And Conditions and Privacy Policy Coconuts Jot this down somewhere convenient: A popular Italian luxury stationery company has just launched its first retail store in Singapore Moleskine yesterday opened a flagship location – the first in Southeast Asia – at Paragon Shopping Centre in Orchard they have dropped exclusive collections from collaborations with German pen company Kaweco and Chinese illustrator Lok Ng.The 500 square foot space on the second floor of the mall is decorated with their curated collection of journals and digital notebooks laid out across shelves and tables A space also lets customers try out the products and use their tools to personalize them The Moleskine x Missoni collection features four notebooks with the Italian fashion brand’s iconic zig-zag patterns from its famous knitwear in blue The Velvet Collection with Pontoglio 1833 showcases another four notebooks in jet black and ruby red with “2022” debossed on the silk-blend velvet front To celebrate next year’s Year of the Tiger according to the Chinese zodiac Moleskine brought in Chinese-born illustrator Lok Ng to design three notebooks splashed with the vivid markings of tiger stripes Each comes with red packets and retails at S$48 the brand launched three sleek writing sets with German pen manufacturer Kaweco featuring a fountain pen or red and are enclosed in a vintage-style metal tin Get the best of Coconuts delivered to your inbox Copyright © 2025 Coconuts Media Limited Terms Of Service Privacy Policy