Genetic Variants in ARHGEF6 Cause Congenital Anomalies of the Kidneys and Urinary Tract in Humans, Mice, and Frogs.

BACKGROUND: About 40 disease genes have been described to date for isolated CAKUT, the most common cause of childhood CKD. However, these genes account for only 20% of cases. ARHGEF6, a guanine nucleotide exchange factor that is implicated in biologic processes such as cell migration and focal adhesion, acts downstream of integrin-linked kinase (ILK) and parvin proteins. A genetic variant of ILK that causes murine renal agenesis abrogates the interaction of ILK with a murine focal adhesion protein encoded by Parva , leading to CAKUT in mice with this variant. METHODS: To identify novel genes that, when mutated, result in CAKUT, we performed exome sequencing in an international cohort of 1265 families with CAKUT. We also assessed the effects in vitro of wild-type and mutant ARHGEF6 proteins, and the effects of Arhgef6 deficiency in mouse and frog models. RESULTS: We detected six different hemizygous variants in the gene ARHGEF6 (which is located on the X chromosome in humans) in eight individuals from six families with CAKUT. In kidney cells, overexpression of wild-type ARHGEF6 -but not proband-derived mutant ARHGEF6 -increased active levels of CDC42/RAC1, induced lamellipodia formation, and stimulated PARVA-dependent cell spreading. ARHGEF6-mutant proteins showed loss of interaction with PARVA. Three-dimensional Madin-Darby canine kidney cell cultures expressing ARHGEF6-mutant proteins exhibited reduced lumen formation and polarity defects. Arhgef6 deficiency in mouse and frog models recapitulated features of human CAKUT. CONCLUSIONS: Deleterious variants in ARHGEF6 may cause dysregulation of integrin-parvin-RAC1/CDC42 signaling, thereby leading to X-linked CAKUT.

P120 and E-cadherin: Double-edged swords in tumor metastasis.

Cell-cell adhesion by adherens junctions controls proliferation and cell polarization and is crucial to maintain epithelial architecture and homeostasis. Downregulation of two of the main components of adherens junctions, E-cadherin and p120, is an often recurring hallmark of carcinomas, causing loss of polarity and increased proliferation, survival and invasion of epithelial cells. On the other hand, tumor-promoting effects of both E-cadherin and p120 have been reported, substantiated by sustained, or even elevated expression of these molecules in many cancers. In this review, we will discuss how expression regulation by EMT, E-cadherin cleavage or p120 isoform expression can contribute to either tumor-supressing or tumor-promoting processes. Furthermore, we will focus on the contradictory functions of E-cadherin and p120 in the different phases of tumor progression, from carcinoma in situ up to the formation of distant metastasis. Finally, we will discuss the possibilities and challenges when using either protein as a biomarker.

L1 Cell Adhesion Molecule in Cancer, a Systematic Review on Domain-Specific Functions.

L1 cell adhesion molecule (L1CAM) is a glycoprotein involved in cancer development and is associated with metastases and poor prognosis. Cellular processing of L1CAM results in expression of either full-length or cleaved forms of the protein. The different forms of L1CAM may localize at the plasma membrane as a transmembrane protein, or in the intra- or extracellular environment as cleaved or exosomal forms. Here, we systematically analyze available literature that directly relates to L1CAM domains and associated signaling pathways in cancer. Specifically, we chart its domain-specific functions in relation to cancer progression, and outline pre-clinical assays used to assess L1CAM. It is found that full-length L1CAM has both intracellular and extracellular targets, including interactions with integrins, and linkage with ezrin. Cellular processing leading to proteolytic cleavage and/or exosome formation results in extracellular soluble forms of L1CAM that may act through similar mechanisms as compared to full-length L1CAM, such as integrin-dependent signals, but also through distinct mechanisms. We provide an algorithm to guide a step-wise analysis on L1CAM in clinical samples, to promote interpretation of domain-specific expression. This systematic review infers that L1CAM has an important role in cancer progression that can be attributed to domain-specific forms. Most studies focus on the full-length plasma membrane L1CAM, yet knowledge on the domain-specific forms is a prerequisite for selective targeting treatment.

3D in vitro cell culture models of tube formation.

Building the complex architecture of tubular organs is a highly dynamic process that involves cell migration, polarization, shape changes, adhesion to neighboring cells and the extracellular matrix, physicochemical characteristics of the extracellular matrix and reciprocal signaling with the mesenchyme. Understanding these processes in vivo has been challenging as they take place over extended time periods deep within the developing organism. Here, I will discuss 3D in vitro models that have been crucial to understand many of the molecular and cellular mechanisms and key concepts underlying branching morphogenesis in vivo.

Scrib regulates HGF-mediated epithelial morphogenesis and is stabilized by Sgt1-HSP90.

Scribble was originally identified as a Drosophila protein that regulates epithelial polarity and formation of the basolateral surface. The mammalian orthologue, Scrib, is evolutionarily conserved, but does not appear to be necessary for apical-basolateral epithelial polarity. Instead, it is implicated in the regulation of cell survival, protein trafficking, adhesion and migration. A key issue is to understand the molecular pathway by which Scrib participates in these processes. We have investigated Scrib using a three-dimensional epithelial cell culture system. We show a novel association between the leucine-rich repeat domain of Scrib and the co-chaperone Sgt1 and demonstrate that these proteins are necessary for epithelial morphogenesis and tubulogenesis following hepatocyte growth factor (HGF) stimulation. The molecular chaperone HSP90 is also required for Sgt1 association with Scrib, and both Sgt1 and HSP90 are needed to ensure proper Scrib protein levels. Furthermore, reduced Scrib stability, following inhibition of Sgt1-HSP90, lowers the cellular abundance of the Scrib-ßPix-PAK complex. Inhibition of any member of this complex, Scrib, ßPix or PAK, is sufficient to block HGF-mediated epithelial morphogenesis. The identification of Scrib as an Sgt1-HSP90 client protein required for three-dimensional cell migration suggests that chaperone-mediated regulation of polarity protein stability and homeostasis is an unappreciated mechanism underlying dynamic rearrangements during morphogenesis.

The syntaxin 4 N terminus regulates its basolateral targeting by munc18c-dependent and -independent mechanisms.

To generate and maintain epithelial cell polarity, specific sorting of proteins into vesicles destined for the apical and basolateral domain is required. Syntaxin 3 and 4 are apical and basolateral SNARE proteins important for the specificity of vesicle fusion at the apical and basolateral plasma membrane domains, respectively, but how these proteins are specifically targeted to these domains themselves is unclear. Munc18/SM proteins are potential regulators of this process. Like syntaxins, they are crucial for exocytosis and vesicle fusion. However, how munc18c and syntaxin 4 regulate the function of each other is unclear. Here, we investigated the requirement of syntaxin 4 in the delivery of basolateral membrane and secretory proteins, the basolateral targeting of syntaxin 4, and the role of munc18c in this targeting. Depletion of syntaxin 4 resulted in significant reduction of basolateral targeting, suggesting no compensation by other syntaxin forms. Mutational analysis identified amino acids Leu-25 and to a lesser extent Val-26 as essential for correct localization of syntaxin 4. Recently, it was shown that the N-terminal peptide of syntaxin 4 is involved in binding to munc18c. A mutation in this region that affects munc18c binding shows that munc18c binding is required for stabilization of syntaxin 4 at the plasma membrane but not for its correct targeting. We conclude that the N terminus serves two functions in membrane targeting. First, it harbors the sorting motif, which targets syntaxin 4 basolaterally in a munc18c-independent manner and second, it allows for munc18c binding, which stabilizes the protein in a munc18c-dependent manner.

Tunable Hybrid Matrices Drive Epithelial Morphogenesis and YAP Translocation.

Morphogenesis is a tightly-regulated developmental process by which tissues acquire the morphology that is critical to their function. For example, epithelial cells exhibit different 2D and 3D morphologies, induced by distinct biochemical and biophysical cues from their environment. In this work, novel hybrid matrices composed of a Matrigel and synthetic oligo(ethylene glycol)-grafted polyisocyanides (PICs) hydrogels are used to form a highly tailorable environment. Through precise control of the stiffness and cell-matrix interactions, while keeping other properties constant, a broad range of morphologies induced in Madin-Darby Canine Kidney (MDCK) cells is observed. At relatively low matrix stiffness, a large morphological shift from round hollow cysts to 2D monolayers is observed, without concomitant translocation of the mechanotransduction protein Yes-associated protein (YAP). At higher stiffness levels and enhanced cell-matrix interactions, tuned by controlling the adhesive peptide density on PIC, the hybrid hydrogels induce a flattened cell morphology with simultaneous YAP translocation, suggesting activation. In 3D cultures, the latter matrices lead to the formation of tubular structures. Thus, mixed synthetic and natural gels, such as the hybrids presented here, are ideal platforms to dissect how external physical factors can be used to regulate morphogenesis in MDCK model system, and in the future, in more complex environments.

Pak1 regulates branching morphogenesis in 3D MDCK cell culture by a PIX and beta1-integrin-dependent mechanism.

Branching morphogenesis is a fundamental process in the development of the kidney. This process gives rise to a network of ducts, which form the collecting system. Defective branching can lead to a multitude of kidney disorders including agenesis and reduced nephron number. The formation of branching tubules involves changes in cell shape, cell motility, and reorganization of the cytoskeleton. However, the exact intracellular mechanisms involved are far from understood. We have used the three-dimensional (3D) Madin-Darby canine kidney (MDCK) cell culture system to study how p21-activated kinase 1 (Pak1), which is an important regulator of the cytoskeleton, modulates branching. Our data reveal that Pak1 plays a crucial role in regulating branching morphogenesis. Expression of a dominant-negative Pak1 mutant (DN-Pak1) in MDCK cysts resulted in the spontaneous formation of extensions and branching tubules. Cellular contractility and levels of phosphorylated myosin light chain (pMLC) were increased in DN-Pak1 cells in collagen. Expression of a DN-Pak1 mutant that does not bind to PIX (DN-Pak1-DeltaPIX) failed to form extensions in collagen and did not have increased contractility. This shows that the DN-Pak1 mutant requires PIX binding to generate extensions and increased contractility in 3D culture. Furthermore, a beta1-integrin function-blocking antibody (AIIB2) inhibited the formation of branches and blocked the increased contractility in DN-Pak1 cysts. Taken together, our work shows that DN-Pak1-induced branching morphogenesis requires PIX binding and beta1-integrin signaling.

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity.

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

Polyisocyanide Hydrogels as a Tunable Platform for Mammary Gland Organoid Formation.

In the last decade, organoid technology has developed as a primary research tool in basic biological and clinical research. The reliance on poorly defined animal-derived extracellular matrix, however, severely limits its application in regenerative and translational medicine. Here, a well-defined, synthetic biomimetic matrix based on polyisocyanide (PIC) hydrogels that support efficient and reproducible formation of mammary gland organoids (MGOs) in vitro is presented. Only decorated with the adhesive peptide RGD for cell binding, PIC hydrogels allow MGO formation from mammary fragments or from purified single mammary epithelial cells. The cystic organoids maintain their capacity to branch for over two months, which is a fundamental and complex feature during mammary gland development. It is found that small variations in the 3D matrix give rise to large changes in the MGO: the ratio of the main cell types in the MGO is controlled by the cell-gel interactions via the cell binding peptide density, whereas gel stiffness controls colony formation efficiency, which is indicative of the progenitor density. Simple hydrogel modifications will allow for future introduction and customization of new biophysical and biochemical parameters, making the PIC platform an ideal matrix for in depth studies into organ development and for application in disease models.

Epithelial polarity and tubulogenesis in vitro.

The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.

Differential expression of p120-catenin 1 and 3 isoforms in epithelial tissues.

P120 catenin (p120) is a non-redundant master regulatory protein of cadherin-based cell-cell junctions, intracellular signaling, and tissue homeostasis and repair. Alternative splicing can generate p120 isoforms 1 and 3 (p120-1 and p120-3), which are implicated in non-overlapping functions by differential expression regulation and unique interactions in different cell types, with often predominant expression of p120-1 in mesenchymal cells, and p120-3 generally prevalent in epithelial cells. However, the lack of specific p120-3 protein detection has precluded analysis of their relative abundance in tissues. Here, we have developed a p120-3 isoform-specific antibody and analyzed the p120-3 localization relative to p120-1 in human tissues. p120-3 but not p120-1 is highly expressed in cell-cell junctions of simple gastrointestinal epithelia such as colon and stomach, and the acini of salivary glands and the pancreas. Conversely, the basal layer of the epidermis and hair follicles expressed p120-1 with reduced p120-3, whereas most other epithelia co-expressed p120-3 and p120-1, including bronchial epithelia and mammary luminal epithelial cells. These data provide an inventory of tissue-specific p120 isoform expression and suggest a link between p120 isoform expression and epithelial differentiation.

P120 Catenin Isoforms Differentially Associate with Breast Cancer Invasion and Metastasis.

Tumor metastasis is the endpoint of tumor progression and depends on the ability of tumor cells to locally invade tissue, transit through the bloodstream and ultimately to colonize secondary organs at distant sites. P120 catenin (P120) has been implicated as an important regulator of metastatic dissemination because of its roles in cell-cell junctional stability, cytoskeletal dynamics, growth and survival. However, conflicting roles for P120 in different tumor models and steps of metastasis have been reported, and the understanding of P120 functions is confounded by the differential expression of P120 isoforms, which differ in N-terminal length, tissue localization and, likely, function. Here, we used in silico exon expression analyses, in vitro invasion assays and both RT-PCR and immunofluorescence of human tumors. We show that alternative exon usage favors expression of short isoform P120-3 in 1098 breast tumors and correlates with poor prognosis. P120-3 is upregulated at the invasive front of breast cancer cells migrating as collective groups in vitro. Furthermore, we demonstrate in histological sections of 54 human breast cancer patients that P120-3 expression is maintained throughout the metastatic cascade, whereas P120-1 is differentially expressed and diminished during invasion and in metastases. These data suggest specific regulation and functions of P120-3 in breast cancer invasion and metastasis.

Beta1-integrin orients epithelial polarity via Rac1 and laminin.

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.

Hepatocyte growth factor switches orientation of polarity and mode of movement during morphogenesis of multicellular epithelial structures.

Epithelial cells form monolayers of polarized cells with apical and basolateral surfaces. Madin-Darby canine kidney epithelial cells transiently lose their apico-basolateral polarity and become motile by treatment with hepatocyte growth factor (HGF), which causes the monolayer to remodel into tubules. HGF induces cells to produce basolateral extensions. Cells then migrate out of the monolayer to produce chains of cells, which go on to form tubules. Herein, we have analyzed the molecular mechanisms underlying the production of extensions and chains. We find that cells switch from an apico-basolateral polarization in the extension stage to a migratory cell polarization when in chains. Extension formation requires phosphatidyl-inositol 3-kinase activity, whereas Rho kinase controls their number and length. Microtubule dynamics and cell division are required for the formation of chains, but not for extension formation. Cells in the monolayer divide with their spindle axis parallel to the monolayer. HGF causes the spindle axis to undergo a variable "seesaw" motion, so that a daughter cells can apparently leave the monolayer to initiate a chain. Our results demonstrate the power of direct observation in investigating how individual cell behaviors, such as polarization, movement, and division are coordinated in the very complex process of producing multicellular structures.

Making Heads or Tails of It: Cell-Cell Adhesion in Cellular and Supracellular Polarity in Collective Migration.

Collective cell migration is paramount to morphogenesis and contributes to the pathogenesis of cancer. To migrate directionally and reach their site of destination, migrating cells must distinguish a front and a rear. In addition to polarizing individually, cell-cell interactions in collectively migrating cells give rise to a higher order of polarity, which allows them to move as a supracellular unit. Rather than just conferring adhesion, emerging evidence indicates that cadherin-based adherens junctions intrinsically polarize the cluster and relay mechanical signals to establish both intracellular and supracellular polarity. In this review, we discuss the various functions of adherens junctions in polarity of migrating cohorts.

Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.

Rho GTPases are critical regulators of epithelial morphogenesis. A powerful means to investigate their function is three-dimensional (3D) cell culture, which mimics the architecture of epithelia in vivo. However, the nature of 3D culture requires specialized techniques for morphological and biochemical analyses. Here, we describe protocols for 3D culture studies with Madin-Darby Canine Kidney (MDCK) epithelial cells: establishing cultures, immunostaining, and expressing, detecting, and assaying Rho proteins. These protocols enable the regulation of epithelial morphogenesis to be explored at a detailed molecular level.

Translating Membrane Tension into Cytoskeletal Action by FBP17.

A recent article by Tsujita et al. (2015) in Nature Cell Biology provides insight into how cells sense and translate plasma membrane tension toward polarized actin polymerization and migration. They identify FBP17 as a multifunctional adaptor that senses membrane curvature and delivers feedback to actin dynamics and directed cell migration.

Collective cell migration: guidance principles and hierarchies.

Collective cell migration results from the establishment and maintenance of collective polarization, mechanocoupling, and cytoskeletal kinetics. The guidance of collective cell migration depends on a reciprocal process between cell-intrinsic multicellular organization with leader-follower cell behavior and results in mechanosensory integration of extracellular guidance cues. Important guidance mechanisms include chemotaxis, haptotaxis, durotaxis, and strain-induced mechanosensing to move cell groups along interfaces and paths of least resistance. Additional guidance mechanisms steering cell groups during specialized conditions comprise electrotaxis and passive drift. To form higher-order cell and tissue structures during morphogenesis and cancer invasion, these guidance principles act in parallel and are integrated for collective adaptation to and shaping of varying tissue environments. We review mechanochemical and electrical inputs and multiparameter signal integration underlying collective guidance, decision making, and outcome.

Rho GTPases in collective cell migration.

The family of Rho GTPases are intracellular signal transducers that link cell surface signals to multiple intracellular responses. They are best known for their role in regulating actin dynamics required for cell migration, but in addition control cell-cell adhesion, polarization, vesicle trafficking, and the cell cycle. The roles of Rho GTPases in single mesenchymal cell migration are well established and rely on Cdc42- and Rac-dependent cell protrusion of a leading edge, coupled to Rho-dependent contractility required to move the cell body forward. In cells migrating collectively, cell-cell junctions are maintained, and migrating leader cells are mechanically coupled to, and coordinate, migration with follower cells. Recent evidence suggests that Rho GTPases provide multifunctional input to collective cell polarization, cell-cell interaction, and migration. Here, we discuss the role of Rho GTPases in initiating and maintaining front-rear, apical-basal cell polarization, mechanotransduction, and cell-cell junction stability between leader and follower cells, and how these roles are integrated in collective migration. Thereby, spatiotemporal fine-tuning of Rho GTPases within the same cell and among cells in the cell group are crucial in controlling potentially conflicting, divergent cell adhesion and cytoskeletal functions to achieve supracellular coordination and mechanocoupling.