PALS1 is a key regulator of the lateral distribution of tight junction proteins in renal epithelial cells.

The evolutionarily conserved apical Crumbs (CRB) complex, consisting of the core components CRB3a (an isoform of CRB3), PALS1 and PATJ, plays a key role in epithelial cell-cell contact formation and cell polarization. Recently, we observed that deletion of one Pals1 allele in mice results in functional haploinsufficiency characterized by renal cysts. Here, to address the role of PALS1 at the cellular level, we generated CRISPR/Cas9-mediated PALS1-knockout MDCKII cell lines. The loss of PALS1 resulted in increased paracellular permeability, indicating an epithelial barrier defect. This defect was associated with a redistribution of several tight junction-associated proteins from bicellular to tricellular contacts. PALS1-dependent localization of tight junction proteins at bicellular junctions required its interaction with PATJ. Importantly, reestablishment of the tight junction belt upon transient F-actin depolymerization or upon Ca2+ removal was strongly delayed in PALS1-deficient cells. Additionally, the cytoskeleton regulator RhoA was redistributed from junctions into the cytosol under PALS1 knockout. Together, our data uncover a critical role of PALS1 in the coupling of tight junction proteins to the F-actin cytoskeleton, which ensures their correct distribution along bicellular junctions and the formation of tight epithelial barrier.

PATJ inhibits histone deacetylase 7 to control tight junction formation and cell polarity.

The conserved multiple PDZ-domain containing protein PATJ stabilizes the Crumbs-Pals1 complex to regulate apical-basal polarity and tight junction formation in epithelial cells. However, the molecular mechanism of PATJ's function in these processes is still unclear. In this study, we demonstrate that knockout of PATJ in epithelial cells results in tight junction defects as well as in a disturbed apical-basal polarity and impaired lumen formation in three-dimensional cyst assays. Mechanistically, we found PATJ to associate with and inhibit histone deacetylase 7 (HDAC7). Inhibition or downregulation of HDAC7 restores polarity and lumen formation. Gene expression analysis of PATJ-deficient cells revealed an impaired expression of genes involved in cell junction assembly and membrane organization, which is rescued by the downregulation of HDAC7. Notably, the function of PATJ regulating HDAC7-dependent cilia formation does not depend on its canonical interaction partner, Pals1, indicating a new role of PATJ, which is distinct from its function in the Crumbs complex. By contrast, polarity and lumen phenotypes observed in Pals1- and PATJ-deficient epithelial cells can be rescued by inhibition of HDAC7, suggesting that the main function of this polarity complex in this process is to modulate the transcriptional profile of epithelial cells by inhibiting HDAC7.

Activation of Hippo Pathway Damages Slit Diaphragm by Deprivation of Ajuba Proteins.

SIGNIFICANCE STATEMENT: Nuclear exclusion of the cotranscription factor YAP, which is a consequence of activation of the Hippo signaling pathway, leads to FSGS and podocyte apoptosis. Ajuba proteins play an important role in the glomerular filtration barrier by keeping the Hippo pathway inactive. In nephrocytes from Drosophila melanogaster , a well-established model system for podocyte research, Ajuba proteins ensure slit diaphragm (SD) formation and function. Hippo pathway activation leads to mislocalization of Ajuba proteins, decreased SD formation, rearrangement of the actin cytoskeleton, and increased SD permeability. Targeting the kinases of the Hippo pathway with specific inhibitors in the glomerulus could, therefore, be a promising strategy for therapy of FSGS. BACKGROUND: The highly conserved Hippo pathway, which regulates organ growth and cell proliferation by inhibiting transcriptional cofactors YAP/TAZ, plays a special role in podocytes, where activation of the pathway leads to apoptosis. The Ajuba family proteins (Ajuba, LIM domain-containing protein 1 (LIMD1) and Wilms tumor protein 1-interacting protein [WTIP]) can bind and inactivate large tumor suppressor kinases 1 and 2, (LATS1/2) two of the Hippo pathway key kinases. WTIP, furthermore, connects the slit diaphragm (SD), the specialized cell-cell junction between podocytes, with the actin cytoskeleton. METHODS: We used garland cell nephrocytes of Drosophila melanogaster to monitor the role of Ajuba proteins in Hippo pathway regulation and structural integrity of the SD. Microscopy and functional assays analyzed the interplay between Ajuba proteins and LATS2 regarding expression, localization, interaction, and effects on the functionality of the SD. RESULTS: In nephrocytes, the Ajuba homolog Djub recruited Warts (LATS2 homolog) to the SD. Knockdown of Djub activated the Hippo pathway. Reciprocally, Hippo activation reduced the Djub level. Both Djub knockdown and Hippo activation led to morphological changes in the SD, rearrangement of the cortical actin cytoskeleton, and increased SD permeability. Knockdown of Warts or overexpression of constitutively active Yki prevented these effects. In podocytes, Hippo pathway activation or knockdown of YAP also decreased the level of Ajuba proteins. CONCLUSIONS: Ajuba proteins regulate the structure and function of the SD in nephrocytes, connecting the SD protein complex to the actin cytoskeleton and maintaining the Hippo pathway in an inactive state. Hippo pathway activation directly influencing Djub expression suggests a self-amplifying feedback mechanism.

The transcription factor ATF4 mediates endoplasmic reticulum stress-related podocyte injury and slit diaphragm defects.

Mutations in OSGEP and four other genes that encode subunits of the KEOPS complex cause Galloway-Mowat syndrome, a severe, inherited kidney-neurological disease. The complex catalyzes an essential posttranscriptional modification of tRNA and its loss of function induces endoplasmic reticulum (ER) stress. Here, using Drosophila melanogaster garland nephrocytes and cultured human podocytes, we aimed to elucidate the molecular pathogenic mechanisms of KEOPS-related glomerular disease and to test pharmacological inhibition of ER stress-related signaling as a therapeutic principle. We found that ATF4, an ER stress-mediating transcription factor, or its fly orthologue Crc, were upregulated in both fly nephrocytes and human podocytes. Knockdown of Tcs3, a fly orthologue of OSGEP, caused slit diaphragm defects, recapitulating the human kidney phenotype. OSGEP cDNA with mutations found in patients lacked the capacity for rescue. Genetic interaction studies in Tcs3-deficient nephrocytes revealed that Crc mediates not only cell injury, but surprisingly also slit diaphragm defects, and that genetic or pharmacological inhibition of Crc activation attenuates both phenotypes. These findings are conserved in human podocytes where ATF4 inhibition improved the viability of podocytes with OSGEP knockdown, with chemically induced ER stress, and where ATF4 target genes and pro-apoptotic gene clusters are upregulated upon OSGEP knockdown. Thus, our data identify ATF4-mediated signaling as a molecular link among ER stress, slit diaphragm defects, and podocyte injury, and our data suggest that modulation of ATF4 signaling may be a potential therapeutic target for certain podocyte diseases.

A SEC61A1 variant is associated with autosomal dominant polycystic liver disease.

BACKGROUND AND AIMS: Autosomal dominant polycystic liver and kidney disease is a spectrum of hereditary diseases, which display disturbed function of primary cilia leading to cyst formation. In autosomal dominant polycystic kidney disease a genetic cause can be determined in almost all cases. However, in isolated polycystic liver disease (PLD) about half of all cases remain genetically unsolved, suggesting more, so far unidentified genes to be implicated in this disease. METHODS: Customized next-generation sequencing was used to identify the underlying pathogenesis in two related patients with PLD. A variant identified in SEC61A1 was further analysed in immortalized patients' urine sediment cells and in an epithelial cell model. RESULTS: In both patients, a heterozygous missense change (c.706C>T/p.Arg236Cys) was found in SEC61A1, which encodes for a subunit of the translocation machinery of protein biosynthesis at the endoplasmic reticulum (ER). While kidney disease is absent in the proposita, her mother displays an atypical polycystic kidney phenotype with severe renal failure. In immortalized urine sediment cells, mutant SEC61A1 is expressed at reduced levels, resulting in decreased levels of polycystin-2 (PC2). In an epithelial cell culture model, we found the proteasomal degradation of mutant SEC61A1 to be increased, whereas its localization to the ER is not affected. CONCLUSIONS: Our data expand the allelic and clinical spectrum for SEC61A1, adding PLD as a new and the major phenotypic trait in the family described. We further demonstrate that mutant SEC61A1 results in enhanced proteasomal degradation and impaired biosynthesis of PC2.

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes.

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

Apical-basal polarity regulators are essential for slit diaphragm assembly and endocytosis in Drosophila nephrocytes.

Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.

Pals1 prevents Rac1-dependent colorectal cancer cell metastasis by inhibiting Arf6.

Loss of apical-basal polarity and downregulation of cell-cell contacts is a critical step during the pathogenesis of cancer. Both processes are regulated by the scaffolding protein Pals1, however, it is unclear whether the expression of Pals1 is affected in cancer cells and whether Pals1 is implicated in the pathogenesis of the disease.Using mRNA expression data and immunostainings of cancer specimen, we show that Pals1 is frequently downregulated in colorectal cancer, correlating with poorer survival of patients. We further found that Pals1 prevents cancer cell metastasis by controlling Rac1-dependent cell migration through inhibition of Arf6, which is independent of the canonical binding partners of Pals1. Loss of Pals1 in colorectal cancer cells results in increased Arf6 and Rac1 activity, enhanced cell migration and invasion in vitro and increased metastasis of transplanted tumor cells in mice. Thus, our data reveal a new function of Pals1 as a key inhibitor of cell migration and metastasis of colorectal cancer cells. Notably, this new function is independent of the known role of Pals1 in tight junction formation and apical-basal polarity.

Drosophila Sister-of-Sex-lethal reinforces a male-specific gene expression pattern by controlling Sex-lethal alternative splicing.

In Drosophila, female development is governed by a single RNA-binding protein, Sex-lethal (Sxl), that controls the expression of key factors involved in dosage compensation, germline homeostasis and the establishment of female morphology and behaviour. Sxl expression in female flies is maintained by an auto-regulatory, positive feedback loop with Sxl controlling splicing of its own mRNA. Until now, it remained unclear how males prevent accidental triggering of the Sxl expression cascade and protect themselves against runaway protein production. Here, we identify the protein Sister-of-Sex-lethal (Ssx) as an inhibitor of Sxl auto-regulatory splicing. Sxl and Ssx have a comparable RNA-binding specificity and compete for binding to RNA regulatory elements present in the Sxl transcript. In cultured Drosophila cells, Sxl-induced changes to alternative splicing can be reverted by the expression of Ssx. Moreover, in adult male flies ablation of the ssx gene results in a low level of productive Sxl mRNA splicing and Sxl protein production in isolated, clonal cell populations. In sum, this demonstrates that Ssx safeguards male animals against Sxl protein production to reinforce a stable, male-specific gene expression pattern.

A Deregulated Stress Response Underlies Distinct INF2-Associated Disease Profiles.

BACKGROUND: Monogenic diseases provide favorable opportunities to elucidate the molecular mechanisms of disease progression and improve medical diagnostics. However, the complex interplay between genetic and environmental factors in disease etiologies makes it difficult to discern the mechanistic links between different alleles of a single locus and their associated pathophysiologies. Inverted formin 2 (INF2), an actin regulator, mediates a stress response-calcium mediated actin reset, or CaAR-that reorganizes the actin cytoskeleton of mammalian cells in response to calcium influx. It has been linked to the podocytic kidney disease focal segemental glomerulosclerosis (FSGS), as well as to cases of the neurologic disorder Charcot-Marie-Tooth disease that are accompanied by nephropathy, mostly FSGS. METHODS: We used a combination of quantitative live cell imaging and validation in primary patient cells and Drosophila nephrocytes to systematically characterize a large panel of >50 autosomal dominant INF2 mutants that have been reported to cause either FSGS alone or with Charcot-Marie-Tooth disease. RESULTS: We found that INF2 mutations lead to deregulated activation of formin and a constitutive stress response in cultured cells, primary patient cells, and Drosophila nephrocytes. We were able to clearly distinguish between INF2 mutations that were linked exclusively to FSGS from those that caused a combination of FSGS and Charcot-Marie-Tooth disease. Furthermore, we were able to identify distinct subsets of INF2 variants that exhibit varying degrees of activation. CONCLUSIONS: Our results suggest that CaAR can be used as a sensitive assay for INF2 function and for robust evaluation of diseased-linked variants of formin. More broadly, these findings indicate that cellular profiling of disease-associated mutations has potential to contribute substantially to sequence-based phenotype predictions.

LEF1 supports metastatic brain colonization by regulating glutathione metabolism and increasing ROS resistance in breast cancer.

More than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma.

Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Reactivation of Notch signaling in kidneys of animal models and patients with chronic kidney disease (CKD) has been shown to contribute to epithelial injury and fibrosis development. Here, we investigated the mechanisms of Notch-induced injury in renal epithelial cells. We performed genome-wide transcriptome analysis to identify Notch target genes using an in vitro system of cultured tubular epithelial cells expressing the intracellular domain of Notch1. One of the top downregulated genes was Disabled-2 ( Dab2). With the use of Drosophila nephrocytes as a model system, we found that Dab (the Drosophila homolog of Dab2) knockdown resulted in a significant filtration defect, indicating that loss of Dab2 plays a functional role in kidney disease development. We showed that Dab2 expression in cultured tubular epithelial cells is involved in endocytic regulation and that it also protects cells from TGF-ß-induced epithelial-to-mesenchymal transition. In vivo correlation studies indicated its additional role in renal ischemia-induced injury. Together, these data suggest that Dab2 plays a versatile role in the kidney and may impact on acute and CKDs.-Schütte-Nütgen, K., Edeling, M., Mendl, G., Krahn, M. P., Edemir, B., Weide, T., Kremerskothen, J., Michgehl, U., Pavenstädt, H. Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Redundant regulation of localization and protein stability of DmPar3.

Apical-basal polarity is an important characteristic of epithelia and Drosophila neural stem cells. The conserved Par complex, which consists of the atypical protein kinase C and the scaffold proteins Baz and Par6, is a key player in the establishment of apical-basal cell polarity. Membrane recruitment of Baz has been reported to be accomplished by several mechanisms, which might function in redundancy, to ensure the correct localization of the complex. However, none of the described interactions was sufficient to displace the protein from the apical junctions. Here, we dissected the role of the oligomerization domain and the lipid-binding motif of Baz in vivo in the Drosophila embryo. We found that these domains function in redundancy to ensure the apical junctional localization of Baz: inactivation of only one domain is not sufficient to disrupt the function of Baz during apical-basal polarization of epithelial cells and neural stem cells. In contrast, mutation of both domains results in a strongly impaired protein stability and a phenotype characterized by embryonic lethality and an impaired apical-basal polarity in the embryonic epithelium and neural stem cells, resembling a baz-loss of function allele. Strikingly, the binding of Baz to the transmembrane proteins E-Cadherin, Echinoid, and Starry Night was not affected in this mutant protein. Our findings reveal a redundant function of the oligomerization and the lipid-binding domain, which is required for protein stability, correct subcellular localization, and apical-basal cell polarization.

Electron microscopy of Drosophila garland cell nephrocytes: Optimal preparation, immunostaining and STEM tomography.

Due to its structural and molecular similarities to mammalian podocytes, the Drosophila nephrocyte emerged as a model system to study podocyte development and associated diseases. Similar to podocytes, nephrocytes establish a slit diaphragm between foot process-like structures in order to filter the hemolymph. One major obstacle in nephrocyte research is the distinct visualization of this subcellular structure to assess its integrity. Therefore, we developed a specialized dissection and fixation protocol, including high pressure freezing and freeze substitution techniques, to improve the preservation of the intricate ultrastructural details necessary for electron microscopic assessment. By means of scanning transmission electron microscopy (STEM) tomography, a three-dimensional dataset was generated to further understand the complex architecture of the nephrocyte channel system. Moreover, a staining protocol for immunolabeling of ultrathin sections of Epon-embedded nephrocytes is discussed, which allows the reliable detection of GFP-tagged fusion proteins combined with superior sample preservation. Due to the growing number of available GFP-trap fly lines, this approach is widely applicable for high resolution localization studies in wild type and mutant nephrocytes.

Distinct functions of Crumbs regulating slit diaphragms and endocytosis in Drosophila nephrocytes.

Mammalian podocytes, the key determinants of the kidney's filtration barrier, differentiate from columnar epithelial cells and several key determinants of apical-basal polarity in the conventional epithelia have been shown to regulate podocyte morphogenesis and function. However, little is known about the role of Crumbs, a conserved polarity regulator in many epithelia, for slit-diaphragm formation and podocyte function. In this study, we used Drosophila nephrocytes as model system for mammalian podocytes and identified a conserved function of Crumbs proteins for cellular morphogenesis, nephrocyte diaphragm assembly/maintenance, and endocytosis. Nephrocyte-specific knock-down of Crumbs results in disturbed nephrocyte diaphragm assembly/maintenance and decreased endocytosis, which can be rescued by Drosophila Crumbs as well as human Crumbs2 and Crumbs3, which were both expressed in human podocytes. In contrast to the extracellular domain, which facilitates nephrocyte diaphragm assembly/maintenance, the intracellular FERM-interaction motif of Crumbs is essential for regulating endocytosis. Moreover, Moesin, which binds to the FERM-binding domain of Crumbs, is essential for efficient endocytosis. Thus, we describe here a new mechanism of nephrocyte development and function, which is likely to be conserved in mammalian podocytes.

Pals1 Haploinsufficiency Results in Proteinuria and Cyst Formation.

The nephron is the basic physiologic subunit of the mammalian kidney and is made up of several apicobasally polarized epithelial cell types. The process of apicobasal polarization in animal cells is controlled by the evolutionarily conserved Crumbs (CRB), Partitioning-defective, and Scribble protein complexes. Here, we investigated the role of protein associated with LIN-7 1 (Pals1, also known as Mpp5), a core component of the apical membrane-determining CRB complex in the nephron. Pals1 interacting proteins, including Crb3 and Wwtr1/Taz, have been linked to renal cyst formation in mice before. Immunohistologic analysis revealed Pals1 expression in renal tubular cells and podocytes of human kidneys. Mice lacking one Pals1 allele (functionally haploid for Pals1) in nephrons developed a fully penetrant phenotype, characterized by cyst formation and severe defects in renal barrier function, which led to death within 6-8 weeks. In Drosophila nephrocytes, deficiency of the Pals1 ortholog caused alterations in slit-diaphragm-like structures. Additional studies in epithelial cell culture models revealed that Pals1 functions as a dose-dependent upstream regulator of the crosstalk between Hippo- and TGF-ß-mediated signaling. Furthermore, Pals1 haploinsufficiency in mouse kidneys associated with the upregulation of Hippo pathway target genes and marker genes of TGF-ß signaling, including biomarkers of renal diseases. These findings support a link between apical polarity proteins and renal diseases, especially renal cyst diseases. Further investigation of the Pals1-linked networks is required to decipher the mechanisms underlying the pathogenesis of these diseases.

Localization and Function of Pals1-associated Tight Junction Protein in Drosophila Is Regulated by Two Distinct Apical Complexes.

The transmembrane protein Crumbs (Crb) and its intracellular adaptor protein Pals1 (Stardust, Sdt in Drosophila) play a crucial role in the establishment and maintenance of apical-basal polarity in epithelial cells in various organisms. In contrast, the multiple PDZ domain-containing protein Pals1-associated tight junction protein (PATJ), which has been described to form a complex with Crb/Sdt, is not essential for apical basal polarity or for the stability of the Crb/Sdt complex in the Drosophila epidermis. Here we show that, in the embryonic epidermis, Sdt is essential for the correct subcellular localization of PATJ in differentiated epithelial cells but not during cellularization. Consistently, the L27 domain of PATJ is crucial for the correct localization and function of the protein. Our data further indicate that the four PDZ domains of PATJ function, to a large extent, in redundancy, regulating the function of the protein. Interestingly, the PATJ-Sdt heterodimer is not only recruited to the apical cell-cell contacts by binding to Crb but depends on functional Bazooka (Baz). However, biochemical experiments show that PATJ associates with both complexes, the Baz-Sdt and the Crb-Sdt complex, in the mature epithelium of the embryonic epidermis, suggesting a role of these two complexes for the function of PATJ during the development of Drosophila.

Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes.

Podocytes constitute the outer layer of the glomerular filtration barrier, where they form an intricate network of interdigitating foot processes which are connected by slit diaphragms. A hitherto unanswered puzzle concerns the question of whether slit diaphragms are established between foot processes of the same podocyte or between foot processes of different podocytes. By employing focused ion beam-scanning electron microscopy (FIB-SEM), we provide unequivocal evidence that slit diaphragms are formed between foot processes of different podocytes. We extended our investigations of the filtration slit by using dual-axis electron tomography of human and mouse podocytes as well as of Drosophila melanogaster nephrocytes. Using this technique, we not only find a single slit diaphragm which spans the filtration slit around the whole periphery of the foot processes but additional punctate filamentous contacts between adjacent foot processes. Future work will be necessary to determine the proteins constituting the two types of cell-cell contacts.

Evolutionary and molecular facts link the WWC protein family to Hippo signaling.

The scaffolding protein KIBRA (also called WWC1) is involved in the regulation of important intracellular transport processes and the establishment of cell polarity. Furthermore, KIBRA/WWC1 is an upstream regulator of the Hippo signaling pathway that controls cell proliferation and organ size in animals. KIBRA/WWC1 represents only one member of the WWC protein family that also includes the highly similar proteins WWC2 and WWC3. Although the function of KIBRA/WWC1 was studied intensively in cells and animal models, the importance of WWC2 and WWC3 was not yet elucidated. Here, we describe evolutionary, molecular, and functional aspects of the WWC family. We show that the WWC genes arose in the ancestor of bilateral animals (clades such as insects and vertebrates) from a single founder gene most similar to the present KIBRA/WWC1-like sequence of Drosophila. This situation was still maintained until the common ancestor of lancelet and vertebrates. In fish, a progenitor-like sequence of mammalian KIBRA/WWC1 and WWC2 is expressed together with WWC3. Finally, in all tetrapods, the three family members, KIBRA/WWC1, WWC2, and WWC3, are found, except for a large genomic deletion including WWC3 in Mus musculus. At the molecular level, the highly conserved WWC proteins share a similar primary structure, the ability to form homo- and heterodimers and the interaction with a common set of binding proteins. Furthermore, all WWC proteins negatively regulate cell proliferation and organ growth due to a suppression of the transcriptional activity of YAP, the major effector of the Hippo pathway.

Elucidating the Membrane Binding Process of a Disordered Protein: Dynamic Interplay of Anionic Lipids and the Polybasic Region.

Intrinsically disordered regions of proteins are responsible for many biological processes such as in the case of liver kinase B1 (LKB1)-a serine/threonine kinase relevant for cell proliferation and cell polarity. LKB1 becomes fully activated upon recruitment to the plasma membrane by binding of its disordered C-terminal polybasic motif consisting of eight lysines/arginines to phospholipids. Here, we present extensive molecular dynamics (MD) simulations of the polybasic motif interacting with a model membrane composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleyl phosphatidic acid (PA) and cell culture experiments. Protein-membrane binding effects are due to the electrostatic interactions between the polybasic amino acids and PAs. For significant binding, the first three lysines turn out to be dispensable, which was also recapitulated in cell culture using transfected GFP-LKB1 variants. LKB1-membrane binding results in nonmonotonous changes in the structure of the protein as well as the membrane, in particular, accumulation of PAs and reduced thickness at the protein-membrane contact area. The protein-lipid binding turns out to be highly dynamic due to an interplay of PA-PA repulsion and protein-PA attraction. The thermodynamics of this interplay is captured by a statistical fluctuation model, which allows the estimation of both energies. Quantification of the significance of each polar amino acid in the polybasic provides detailed insights into the molecular mechanism of protein-membrane binding of LKB1. These results can likely be transferred to other proteins, which interact by intrinsically disordered polybasic regions with anionic membranes.