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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. Here, we investigate Scrib using a 3D epithelial cell culture system. We show a novel association between the LRR domain of Scrib and the co-chaperone Sgt1 and demonstrate that these proteins are necessary for epithelial morphogenesis and tubulogenesis following 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- βPix-PAK complex. Inhibition of any member of this complex, Scrib, βPix or PAK, is sufficient to block HGF-mediated epithelial morphogenesis. The identification of Scrib as an Sgt1-HSP90 client protein required for 3D cell migration suggests that chaperonemediated regulation of polarity protein stability and homeostasis is an unappreciated mechanism underlying dynamic rearrangements during morphogenesis. © 2012.

A role for dynamin in clathrin-mediated endocytosis is now well established. However, mammals express three closely related, tissue-specific dynamin isoforms, each with multiple splice variants. Thus, an important question is whether these isoforms and splice variants function in vesicle formation from distinct intracellular organelles. There are conflicting data as to a role for dynamin-2 in vesicle budding from the TGN. To resolve this issue, we compared the effects of overexpression of dominant-negative mutants of dynamin-1 (the neuronal isoform) and dynamin-2 (the ubiquitously expressed isoform) on endocytic and biosynthetic membrane trafficking in HeLa cells and polarized MDCK cells. Both dyn1(K44A) and dyn2(K44A) were potent inhibitors of receptor-mediated endocytosis; however neither mutant directly affected other membrane trafficking events, including transport mediated by four distinct classes of vesicles budding from the TGN. Dyn2(K44A) more potently inhibited receptor-mediated endocytosis than dyn1(K44A) in HeLa cells and at the basolateral surface of MDCK cells. In contrast, dyn1(K44A) more potently inhibited endocytosis at the apical surface of MDCK cells. The two dynamin isoforms have redundant functions in endocytic vesicle formation, but can be targeted to and function differentially at subdomains of the plasma membrane.

We have investigated the controversial involvement of components of the SNARE (soluble N-ethyl maleimide-sensitive factor [NSF] attachment protein [SNAP] receptor) machinery in membrane traffic to the apical plasma membrane of polarized epithelial (MDCK) cells. Overexpression of syntaxin 3, but not of syntaxins 2 or 4, caused an inhibition of TGN to apical transport and apical recycling, and leads to an accumulation of small vesicles underneath the apical plasma membrane. All other tested transport steps were unaffected by syntaxin 3 overexpression. Botulinum neurotoxin E, which cleaves SNAP-23, and antibodies against alpha-SNAP inhibit both TGN to apical and basolateral transport in a reconstituted in vitro system. In contrast, we find no evidence for an involvement of N-ethyl maleimide-sensitive factor in TGN to apical transport, whereas basolateral transport is NSF-dependent. We conclude that syntaxin 3, SNAP-23, and alpha-SNAP are involved in apical membrane fusion. These results demonstrate that vesicle fusion with the apical plasma membrane does not use a mechanism that is entirely unrelated to other cellular membrane fusion events, but uses isoforms of components of the SNARE machinery, which suggests that they play a role in providing specificity to polarized membrane traffic.

beta-Catenin is essential for the function of cadherins, a family of Ca2+-dependent cell-cell adhesion molecules, by linking them to (alpha)-catenin and the actin cytoskeleton. beta-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant beta-catenins in MDCK epithelial cells to gain insights into the regulation of beta-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length beta-catenin, beta-catenin mutant proteins with NH2-terminal deletions before (deltaN90) or after (deltaN131, deltaN151) the alpha-catenin binding site, or a mutant beta-catenin with a COOH-terminal deletion (delta C) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All beta-catenin mutant proteins form complexes and colocalize with E-cadherin at cell-cell contacts; deltaN90, but neither deltaN131 nor deltaN151, bind alpha-catenin. However, beta-catenin mutant proteins containing NH2-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal-deleted beta-catenin poorly colocalize with APC protein. NH2-terminal deletions result in increased stability of beta-catenin bound to APC protein and E-cadherin, compared with full-length beta-catenin. At low density, MDCK cells expressing NH2-terminal-deleted beta-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH2 terminus, but not the COOH terminus of beta-catenin, regulates the dynamics of beta-catenin binding to APC protein and E-cadherin. Changes in beta-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of beta-catenin binding to alpha-catenin. These results demonstrate that regulation of beta-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.

The 17-juxtamembrane cytoplasmic residues of the polymeric immunoglobulin receptor contain an autonomous basolateral targeting signal that does not mediate rapid endocytosis (Casanova, J. E., G. Apodaca, and K. E. Mostov. Cell. 66:65-75). Alanine-scanning mutagenesis identifies three residues in this region, His656, Arg657, and Val660, that are most essential for basolateral sorting and two residues, Arg655 and Tyr668, that play a lesser role in this process. Progressive truncations suggested that Ser664 and Ile665 might also play a role in basolateral sorting. However, mutation of these residues to Ala or internal deletions of these residues did not affect basolateral sorting, indicating that these residues are probably not required for basolateral sorting. Two-dimensional NMR spectroscopy of a peptide corresponding to the 17-mer signal indicates that the sequence Arg658-Asn-Val-Asp661 has a propensity to adopt a beta-turn in solution. Residues COOH-terminal to the beta-turn (Arg662 to Arg669) seem to take up a nascent helix structure in solution. Substitution of Val660 with Ala destabilizes the turn, while mutation of Arg657 to Ala does not appear to affect the turn structure. Neither mutation detectably altered the stability of the nascent helix in the COOH-terminal portion of the peptide.

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic on each of these two pathways is misdirected to the basolateral surface. The apical recycling pathway is slowed in the presence of nocodazole but targeting is not disrupted. In contrast, the biosynthetic and recycling pathways to the basolateral surface are less affected by nocodazole and therefore appear to be more resistant to microtubule disruption.

The polymeric immunoglobulin receptor, a transmembrane protein, is made by a variety of polarized epithelial cells. After synthesis, the receptor is sent to the basolateral surface where it binds polymeric IgA and IgM. The receptor-ligand complex is endocytosed, transported across the cell in vesicles, and re-exocytosed at the apical surface. At some point the receptor is proteolytically cleaved so that its extracellular ligand binding portion (known as secretory component) is severed from the membrane and released together with the polymeric immunoglobulin at the apical surface. We have used a cDNA clone coding for the rabbit receptor and a retroviral expression system to express the receptor in a nonpolarized mouse fibroblast cell line, psi 2, that normally does not synthesize the receptor. The receptor is glycosylated and sent to the cell surface. The cell cleaves the receptor to a group of polypeptides that are released into the medium and co-migrate with authentic rabbit secretory component. Cleavage and release of secretory component do not depend on the presence of ligand. The cells express on their surface 9,600 binding sites for the ligand, dimeric IgA. The ligand can be rapidly endocytosed and then re-exocytosed, all within approximately 10 min. Very little ligand is degraded. At least some of the ligand that is released from the cells is bound to secretory component. The results presented indicate that we have established a powerful new system for analyzing the complex steps in the transport of poly-Ig and the general problem of membrane protein sorting.

During vascular development, endothelial cAMP-dependent protein kinase A (PKA) regulates angiogenesis by controlling the number of tip cells, and PKA inhibition leads to excessive angiogenesis. Whether this role of endothelial PKA is restricted to embryonic and neonatal development or is also required for vascular homeostasis later on is unknown. Here, we show that perinatal (postnatal days P1-P3) of later (P28-P32) inhibition of endothelial PKA using dominant-negative PKA expressed under the control of endothelial-specific Cdh5-CreERT2 recombinase (dnPKA iEC mice) leads to severe subcutaneous edema, hypoalbuminemia, hypoglycemia and premature death. These changes were accompanied by the local hypersprouting of blood vessels in fat pads and the secondary enlargement of subcutaneous lymphatic vessels. Most noticeably, endothelial PKA inhibition caused a dramatic disorganization of the liver vasculature. Hepatic changes correlated with decreased gluconeogenesis, while liver albumin production seems to be unaffected and hypoalbuminemia is rather a result of increased leakage into the interstitium. Interestingly, the expression of dnPKA only in lymphatics using Prox1-CreERT2 produced no phenotype. Likewise, the mosaic expression in only endothelial subpopulations using Vegfr3-CreERT2 was insufficient to induce edema or hypoglycemia. Increased expression of the tip cell marker ESM1 indicated that the inhibition of PKA induced an angiogenic response in the liver, although tissue derived pro- and anti-angiogenic factors were unchanged. These data indicate that endothelial PKA is a gatekeeper of endothelial cell activation not only in development but also in adult homeostasis, preventing the aberrant reactivation of the angiogenic program.

How tubular organs elongate is poorly understood. We found that attenuated ciliary Hedgehog signaling in the gut wall impaired patterning of the circumferential smooth muscle and inhibited proliferation and elongation of developing intestine and esophagus. Similarly, ablation of gut-wall smooth muscle cells reduced lengthening. Disruption of ciliary Hedgehog signaling or removal of smooth muscle reduced residual stress within the gut wall and decreased activity of the mechanotransductive effector YAP. Removing YAP in the mesenchyme also reduced proliferation and elongation, but without affecting smooth muscle formation, suggesting that YAP interprets the smooth muscle-generated force to promote longitudinal growth. Additionally, we developed an intestinal culture system that recapitulates the requirements for cilia and mechanical forces in elongation. Pharmacologically activating YAP in this system restored elongation of cilia-deficient intestines. Thus, our results reveal that ciliary Hedgehog signaling patterns the circumferential smooth muscle to generate radial mechanical forces that activate YAP and elongate the gut., (© 2021. The Author(s).)

Angiogenesis supplies oxygen and nutrients to growing tumors. Inhibiting angiogenesis may stop tumor growth, but vascular endothelial growth factor inhibitors have limited effect in most tumors. This limited effect may be explained by an additional, less vascular endothelial growth factor-driven form of angiogenesis known as intussusceptive angiogenesis. The importance of intussusceptive angiogenesis in human tumors is not known. Epifluorescence and confocal microscopy was used to visualize intravascular pillars, the hallmark structure of intussusceptive angiogenesis, in tumors. Human malignant melanoma metastases, patient-derived melanoma xenografts in mice (PDX), and genetically engineered v-raf murine sarcoma viral oncogene homolog B1 (BRAF)-induced, phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)-deficient (BPT) mice (Braf CA/+ Pten f/f Tyr-Cre +/0 -mice) were analyzed for pillars. Gene expression in human melanoma metastases and PDXs was analyzed by RNA sequencing. Matrix metalloproteinase 9 (MMP9) protein expression and T-cell and macrophage infiltration in tumor sections were determined with multiplex immunostaining. Intravascular pillars were detected in human metastases but rarely in PDXs and not in BPT mice. The expression of MMP9 mRNA was higher in human metastases compared with PDXs. High expression of MMP9 protein as well as infiltration of macrophages and T-cells were detected in proximity to intravascular pillars. MMP inhibition blocked formation of pillars, but not tubes or tip cells, in vitro. In conclusion, intussusceptive angiogenesis may contribute to the growth of human melanoma metastases. MMP inhibition blocked pillar formation in vitro and should be further investigated as a potential anti-angiogenic drug target in metastatic melanoma., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Madin-Darby canine kidney II (MDCKII) cells are widely used to study epithelial morphogenesis. To better understand this process, we performed time course RNA-seq analysis of MDCKII 3D cystogenesis, along with polarized 2D cells for comparison. Our study reveals a biphasic change in the transcriptome that occurs after the first cell cycle and coincides with lumen establishment. This change appears to be linked to translocation of β-catenin, supported by analyses with AVL9 - and DENND5A -knockdown clones, and regulation by HNF1B, supported by ATAC-seq study. These findings indicate a qualitative change model for transcriptome remodeling during epithelial morphogenesis, leading to cell proliferation decrease and cell polarity establishment. Furthermore, our study reveals that active mitochondria are retained and chromatin accessibility decreases in 3D cysts but not in 2D polarized cells. This indicates that 3D culture is a better model than 2D culture for studying epithelial morphogenesis., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Epithelial-mesenchymal transition (EMT) encompasses dynamic changes in cellular organization from epithelial to mesenchymal phenotypes, which leads to functional changes in cell migration and invasion. EMT occurs in a diverse range of physiological and pathological conditions and is driven by a conserved set of inducing signals, transcriptional regulators and downstream effectors. With over 5,700 publications indexed by Web of Science in 2019 alone, research on EMT is expanding rapidly. This growing interest warrants the need for a consensus among researchers when referring to and undertaking research on EMT. This Consensus Statement, mediated by 'the EMT International Association' (TEMTIA), is the outcome of a 2-year-long discussion among EMT researchers and aims to both clarify the nomenclature and provide definitions and guidelines for EMT research in future publications. We trust that these guidelines will help to reduce misunderstanding and misinterpretation of research data generated in various experimental models and to promote cross-disciplinary collaboration to identify and address key open questions in this research field. While recognizing the importance of maintaining diversity in experimental approaches and conceptual frameworks, we emphasize that lasting contributions of EMT research to increasing our understanding of developmental processes and combatting cancer and other diseases depend on the adoption of a unified terminology to describe EMT.

Many metazoan organs are comprised of branching trees of epithelial tubes; how patterning occurs in these trees is a fundamental problem of development. Commonly, branch tips fill the volume of the organ approximately uniformly, e.g., in mammalian lung, airway branch tips are dispersed roughly uniformly throughout the volume of the lung. In contrast, in the developing metanephric kidney, the tips of the ureteric bud tree are located close to the outer surface of the kidney rather than filling the kidney. Here, we describe a simple alteration in the branching rules that accounts for the difference between the kidney pattern that leads to tips near the organ surface versus previously known patterns that lead to the branch tips being dispersed throughout the organ. We further use a simple toy model to deduce from first principles how this rule change accounts for the differences in the two types of trees., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Apical-basal polarization is essential for epithelial tissue formation, segregating cortical domains to perform distinct physiological functions. Cortical lipid asymmetry has emerged as a determinant of cell polarization. We report a network of phosphatidylinositol phosphate (PIP)-modifying enzymes, some of which are transcriptionally induced upon embedding epithelial cells in extracellular matrix, and that are essential for apical-basal polarization. Unexpectedly, we find that PI(3,4)P 2 localization and function is distinct from the basolateral determinant PI(3,4,5)P 3 . PI(3,4)P 2 localizes to the apical surface, and Rab11a-positive apical recycling endosomes. PI(3,4)P 2 is produced by the 5-phosphatase SHIP1 and Class-II PI3-Kinases to recruit the endocytic regulatory protein SNX9 to basolateral domains that are being remodeled into apical surfaces. Perturbing PI(3,4)P 2 levels results in defective polarization through subcortical retention of apically destined vesicles at apical membrane initiation sites. We conclude that PI(3,4)P 2 is a determinant of apical membrane identity.

The formation of lumens in epithelial tissues requires apical-basal polarization of cells, and the co-ordination of this individual polarity collectively around a contiguous lumen. Signals from the Extracellular Matrix (ECM) instruct epithelia as to the orientation of where basal, and thus consequently apical, surfaces should be formed. We report that this pathway is normally absent in Calu-3 human lung adenocarcinoma cells in 3-Dimensional culture, but that paracrine signals from MRC5 lung fibroblasts can induce correct orientation of polarity and acinar morphogenesis. We identify HGF, acting through the c-Met receptor, as the key polarity-inducing morphogen, which acts to activate β1-integrin-dependent adhesion. HGF and ECM-derived integrin signals co-operate via a c-Src-dependent inhibition of the RhoA-ROCK1 signalling pathway via p190A RhoGAP. This occurred via controlling localization of these signalling pathways to the ECM-abutting surface of cells in 3-Dimensional culture. Thus, stromal derived signals can influence morphogenesis in epithelial cells by controlling activation and localization of cell polarity pathways., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In many types of tubules, continuity of the lumen is paramount to tubular function, yet how tubules generate lumen continuity in vivo is not known. We recently found that the F-actin-binding protein afadin is required for lumen continuity in developing renal tubules, though its mechanism of action remains unknown. Here, we demonstrate that afadin is required for lumen continuity by orienting the mitotic spindle during cell division. Using an in vitro 3D cyst model, we find that afadin localizes to the cell cortex adjacent to the spindle poles and orients the mitotic spindle. In tubules, cell division may be oriented relative to two axes: longitudinal and apical-basal. Unexpectedly, in vivo examination of early-stage developing nephron tubules reveals that cell division is not oriented in the longitudinal (or planar-polarized) axis. However, cell division is oriented perpendicular to the apical-basal axis. Absence of afadin in vivo leads to misorientation of apical-basal cell division in nephron tubules. Together, these results support a model whereby afadin determines lumen placement by directing apical-basal spindle orientation, resulting in a continuous lumen and normal tubule morphogenesis., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens., (© 2017 Ruch et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Exosomes, 40-150-nm extracellular vesicles, transport biological macromolecules that mediate intercellular communications. Although exosomes are known to originate from maturation of endosomes into multivesicular endosomes (also known as multivesicular bodies) with subsequent fusion of the multivesicular endosomes with the plasma membrane, it remains unclear how cargos are selected for exosomal release. Using an inducible expression system for the exosome cargo protein GPRC5B and following its trafficking trajectory, we show here that newly synthesized GPRC5B protein accumulates in the Golgi complex prior to its release into exosomes. The L-type lectin LMAN2 (also known as VIP36) appears to be specifically required for the accumulation of GPRC5B in the Golgi complex and restriction of GPRC5B transport along the exosomal pathway. This may occur due to interference with the adaptor protein GGA1-mediated trans Golgi network-to-endosome transport of GPRC5B. The adaptor protein CD2AP-mediated internalization following cell surface delivery appears to contribute to the Golgi accumulation of GPRC5B, possibly in parallel with biosynthetic/secretory trafficking from the endoplasmic reticulum. Our data thus reveal a Golgi-traversing pathway for exosomal release of the cargo protein GPRC5B in which CD2AP facilitates the entry and LMAN2 impedes the exit of the flux, respectively., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

Tubulogenesis is fundamental to the development of many epithelial organs. Although lumen formation in cysts has received considerable attention, less is known about lumenogenesis in tubes. Here, we utilized tubulogenesis induced by hepatocyte growth factor (HGF) in MDCK cells, which form tubes enclosing a single lumen. We report the mechanism that controls tubular lumenogenesis and limits each tube to a single lumen. Knockdown of p114RhoGEF (also known as ARHGEF18), a guanine nucleotide exchange factor for RhoA, did not perturb the early stages of tubulogenesis induced by HGF. However, this knockdown impaired later stages of tubulogenesis, resulting in multiple lumens in a tube. Inhibition of Rho kinase (ROCK) or myosin IIA, which are downstream of RhoA, led to formation of multiple lumens. We studied lumen formation by live-cell imaging, which revealed that inhibition of this pathway blocked cell movement, suggesting that cell movement is necessary for consolidating multiple lumens into a single lumen. Lumen formation in tubules is mechanistically quite different from lumenogenesis in cysts. Thus, we demonstrate a new pathway that regulates directed cell migration and formation of a single lumen during epithelial tube morphogenesis., (© 2015. Published by The Company of Biologists Ltd.)

The formation of epithelial tissues containing lumens requires not only the apical-basolateral polarization of cells, but also the coordinated orientation of this polarity such that the apical surfaces of neighboring cells all point toward the central lumen. Defects in extracellular matrix (ECM) signaling lead to inverted polarity so that the apical surfaces face the surrounding ECM. We report a molecular switch mechanism controlling polarity orientation. ECM signals through a β1-integrin/FAK/p190RhoGAP complex to downregulate a RhoA/ROCK/Ezrin pathway at the ECM interface. PKCβII phosphorylates the apical identity-promoting Podocalyxin/NHERF1/Ezrin complex, removing Podocalyxin from the ECM-abutting cell surface and initiating its transcytosis to an apical membrane initiation site for lumen formation. Inhibition of this switch mechanism results in the retention of Podocalyxin at the ECM interface and the development instead of collective front-rear polarization and motility. Thus, ECM-derived signals control the morphogenesis of epithelial tissues by controlling the collective orientation of epithelial polarization.

A fundamental question in development is how cells assemble to form a tubular network during organ formation. In glandular organs, tubulogenesis is a multistep process requiring coordinated proliferation, polarization and reorganization of epithelial cells to form a lumen, and lumen expansion. Although it is clear that epithelial cells possess an intrinsic ability to organize into polarized structures, the mechanisms coordinating morphogenetic processes during tubulogenesis are poorly understood. Here, we demonstrate that parasympathetic nerves regulate tubulogenesis in the developing salivary gland. We show that vasoactive intestinal peptide (VIP) secreted by the innervating ganglia promotes ductal growth, leads to the formation of a contiguous lumen, and facilitates lumen expansion through a cyclic AMP/protein kinase A (cAMP/PKA)-dependent pathway. Furthermore, we provide evidence that lumen expansion is independent of apoptosis and involves the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-regulated Cl(-) channel. Thus, parasympathetic innervation coordinates multiple steps in tubulogenesis during organogenesis., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The mucosal epithelium consists of polarized cells with distinct apical and basolateral membranes that serve as functional and physical barriers to external pathogens. The apical surface of the epithelium constitutes the first point of contact between mucosal pathogens, such as Pseudomonas aeruginosa, and their host. We observed that binding of P. aeruginosa aggregates to the apical surface of polarized cells led to the striking formation of an actin-rich membrane protrusion with inverted polarity, containing basolateral lipids and membrane components. Such protrusions were associated with a spatially localized host immune response to P. aeruginosa aggregates that required bacterial flagella and a type III secretion system apparatus. Host protrusions formed de novo underneath bacterial aggregates and involved the apical recruitment of a Par3/Par6α/aPKC/Rac1 signaling module for a robust, spatially localized host NF-κB response. Our data reveal a role for spatiotemporal epithelial polarity changes in the activation of innate immune responses., (Copyright © 2014 Elsevier Inc. All rights reserved.)

How cells communicate during development and regeneration is a critical question. One mechanism of intercellular communication is via exosomes, extracellular vesicles that originate by the fusion of multivesicular endosomes with the plasma membrane [1-8]. To model exosome-based intercellular communication, we used Madin-Darby canine kidney (MDCK) cell cysts grown in 3D gels of extracellular matrix, which form tubules in response to hepatocyte growth factor (HGF). We report that GPRC5B, an orphan G protein coupled receptor, is in exosomes produced by HGF-treated cysts and released into the cyst lumen. Exosomal GPRC5B is taken up by nearby cells and together with HGF promotes extracellular signal-regulated kinase 1/2 (ERK1/2) activation and tubulogenesis, even under conditions where tubulogenesis would otherwise not occur. Recovery from injury, such as acute kidney injury (AKI), often recapitulates developmental processes. Here, we show that GPRC5B is elevated in urinary exosomes from patients with AKI. Our results elucidate how GPRC5B is carried by exosomes and augments HGF-induced morphogenesis. The unexpected role of exosomes in transporting GPRC5B between cells during morphogenesis and the ability of GPRC5B to predict the disease state of AKI elucidate a novel mechanism for intercellular communication during development and repair., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The growth of a well-formed epithelial structure is governed by mechanical constraints, cellular apico-basal polarity, and spatially controlled cell division. Here we compared the predictions of a mathematical model of epithelial growth with the morphological analysis of 3D epithelial structures. In both in vitro cyst models and in developing epithelial structures in vivo, epithelial growth could take place close to or far from mechanical equilibrium, and was determined by the hierarchy of time-scales of cell division, cell-cell rearrangements, and lumen dynamics. Equilibrium properties could be inferred by the analysis of cell-cell contact topologies, and the nonequilibrium phenotype was altered by inhibiting ROCK activity. The occurrence of an aberrant multilumen phenotype was linked to fast nonequilibrium growth, even when geometric control of cell division was correctly enforced. We predicted and verified experimentally that slowing down cell division partially rescued a multilumen phenotype induced by altered polarity. These results improve our understanding of the development of epithelial organs and, ultimately, of carcinogenesis.

Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface. PI(4,5)P2 levels at the apical membrane regulate Dia localization in both the MDCK cyst model and in Drosophila tubular epithelia. An N-terminal basic domain of Dia is crucial for apical localization, implying direct binding to PI(4,5)P2. Dia apical targeting also depends on binding to Rho1, which is critical for activation-induced conformational change, as well as physically anchoring Dia to the apical membrane. We demonstrate that binding to Rho1 facilitates interaction with PI(4,5)P2 at the plane of the membrane. Together these cues ensure efficient and distinct restriction of Dia to the apical membrane. DOI:http://dx.doi.org/10.7554/eLife.00666.001.

During oogenesis in Drosophila melanogaster, the cells in the follicular epithelium of the ovary undergo a transition from a cuboidal to a squamous shape. In this issue, Gomez et al. (2012. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201207150) show that the kinase Tao promotes the endocytosis of the cell adhesion molecule Fasciclin 2 from the lateral surface of the cell and is critical for the cuboidal to squamous cell shape transition. Their results indicate that Tao is rising as a regulator of cell height.

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-βPix-PAK complex. Inhibition of any member of this complex, Scrib, βPix 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.

During development, epithelial progenitors establish intercellular junctions, including tight junctions (TJs), and form three-dimensional (3D) tissue structures, which are often associated with luminal structures. Here we identify grainyhead-like 2 (Grhl2) as a transcription factor that regulates the size of luminal space surrounded by polarized epithelial cells. We show that HPPL, a liver progenitor cell line, transfected with Grhl2 cDNA forms remarkably larger cysts than the control cells in 3D cultures. We find that Grhl2 up-regulates claudin (Cldn) 3 and Cldn4, and their functions are necessary for the formation of large cysts. Overexpression of Cldn3 alone induces the cyst expansion. In contrast, expression of Cldn4 alone does not induce expansion, as it is not localized at TJs. Of interest, Rab25, another Grhl2 target, not only increases the Cldn4 protein, but also enhances its localization at TJs. Taken together, the results indicate that Grhl2 regulates epithelial morphogenesis through transcriptional up-regulation of Cldn3 and Cldn4, as well as of Rab25, which increases the Cldn4 protein and its localization at TJs. The results reveal a molecular network regulating epithelial lumen formation organized by Grhl2.

Epithelial cells form tubular and acinar structures notable for a hollow lumen. In three-dimensional culture utilizing MCF10A mammary epithelial cells, acini form due to integrin-dependent polarization and survival of cells contacting extracellular matrix (ECM), and the apoptosis of inner cells of acini lacking contact with the ECM. In this paper, we report that cyclic AMP (cAMP)-dependent protein kinase A (PKA) promotes acinus formation via two mechanisms. First, cAMP accelerates redistribution of α6-integrin to the periphery of the acinus and thus facilitates the polarization of outer acinar cells. Blocking of α6-integrin function by inhibitory antibody prevents cAMP-dependent polarization. Second, cAMP promotes the death of inner cells occupying the lumen. In the absence of cAMP, apoptosis is delayed, resulting in perturbed luminal clearance. cAMP-dependent apoptosis is accompanied by a posttranscriptional PKA-dependent increase in the proapoptotic protein Bcl-2 interacting mediator of cell death. These data demonstrate that cAMP regulates lumen formation in mammary epithelial cells in vitro, both through acceleration of polarization of outer cells and apoptosis of inner cells of the acinus.

The formation of epithelial tissues requires both the generation of apical-basal polarity and the coordination of this polarity between neighbouring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to the formation of a singular apical membrane, resulting in the contribution of each cell to only a single lumen. Here, from a functional screen for genes required for three-dimensional epithelial architecture, we identify key roles for synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in the generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PtdIns(4,5)P(2)-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE syntaxin-3. Together, Slp2-a/4-a coordinate the spatiotemporal organization of vectorial apical transport to ensure that only a single apical surface, and thus the formation of a single lumen, occurs per cell.

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Polymeric IgA (pIgA) is transcytosed by the pIgA receptor (pIgR) across mucosal epithelial cells. After transcytosis to the apical surface, the extracellular, ligand-binding portion of the pIgR is proteolytically cleaved. A missense mutation in human pIgR, A580V, is associated with IgA nephropathy and nasopharyngeal carcinoma. We report that this mutation reduces the rate of transcytosis of pIgR and pIgA, and seemingly the rate of pIgR cleavage. We propose that the defects in pIgR trafficking caused by the A580V mutation may underlie the pathogenesis of both diseases.

Growing evidence is pointing to the importance of multicellular bacterial structures in the interaction of pathogenic bacteria with their host. Transition from planktonic to host cell-associated multicellular structures is an essential infection step that has not been described for the opportunistic human pathogen Pseudomonas aeruginosa. In this study we show that P. aeruginosa interacts with the surface of epithelial cells mainly forming aggregates. Dynamics of aggregate formation typically follow a sigmoidal curve. First, a single bacterium attaches at cell-cell junctions. This is followed by rapid recruitment of free-swimming bacteria and association of bacterial cells resulting in the formation of an aggregate on the order of minutes. Aggregates are associated with phosphatidylinositol 3,4,5-trisphosphate (PIP3)-enriched host cell membrane protrusions. We further show that aggregates can be rapidly internalized into epithelial cells. Lyn, a member of the Src family tyrosine kinases previously implicated in P. aeruginosa infection, mediates both PIP3-enriched protrusion formation and aggregate internalization. Our results establish the first framework of principles that define P. aeruginosa transition to multicellular structures during interaction with host cells., (© 2011 Blackwell Publishing Ltd.)

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.

The study of epithelial morphogenesis is fundamental to increasing our understanding of organ function and disease. Great progress has been made through study of culture systems such as Madin-Darby canine kidney (MDCK) cells, but many aspects of even simple morphogenesis remain unclear. For example, are specific cell actions tightly coupled to the characteristics of the cell's environment or are they more often cell state dependent? How does the single lumen, single cell layer cyst consistently emerge from a variety of cell actions? To improve insight, we instantiated in silico analogues that used hypothesized cell behavior mechanisms to mimic MDCK cystogenesis. We tested them through in vitro experimentation and quantitative validation. We observed novel growth patterns, including a cell behavior shift that began around day five of growth. We created agent-oriented analogues that used the cellular Potts model along with an Iterative Refinement protocol. Following several refinements, we achieved a degree of validation for two separate mechanisms. Both survived falsification and achieved prespecified measures of similarity to cell culture properties. In silico components and mechanisms mapped to in vitro counterparts. In silico, the axis of cell division significantly affects lumen number without changing cell number or cyst size. Reducing the amount of in silico luminal cell death had limited effect on cystogenesis. Simulations provide an observable theory for cystogenesis based on hypothesized, cell-level operating principles.

The Rab GTPases are the largest family of proteins regulating membrane traffic. Rab proteins form a nidus for the assembly of multiprotein complexes on distinct vesicle membranes to regulate particular membrane trafficking pathways. Recent investigations have demonstrated that Myosin Vb (Myo5B) is an effector for Rab8a, Rab10, and Rab11a, all of which are implicated in regulating different pathways for recycling of proteins to the plasma membrane. It remains unclear how specific interactions of Myo5B with individual Rab proteins can lead to specificity in the regulation of alternate trafficking pathways. We examined the relative contributions of Rab/Myo5B interactions with specific pathways using Myo5B mutants lacking binding to either Rab11a or Rab8a. Myo5B Q1300L and Y1307C mutations abolished Rab8a association, whereas Myo5B Y1714E and Q1748R mutations uncoupled association with Rab11a. Expression of Myo5B tails containing these mutants demonstrated that Rab11a, but not Rab8a, was required for recycling of transferrin in nonpolarized cells. In contrast, in polarized epithelial cyst cultures, Myo5B was required for apical membrane trafficking and de novo lumen formation, dependent on association with both Rab8a and Rab11a. These data demonstrate that different combinations of Rab GTPase association with Myo5B control distinct membrane trafficking pathways.

The asymmetric polarization of cells allows specialized functions to be performed at discrete subcellular locales. Spatiotemporal coordination of polarization between groups of cells allowed the evolution of metazoa. For instance, coordinated apical-basal polarization of epithelial and endothelial cells allows transport of nutrients and metabolites across cell barriers and tissue microenvironments. The defining feature of such tissues is the presence of a central, interconnected luminal network. Although tubular networks are present in seemingly different organ systems, such as the kidney, lung, and blood vessels, common underlying principles govern their formation. Recent studies using in vivo and in vitro models of lumen formation have shed new light on the molecular networks regulating this fundamental process. We here discuss progress in understanding common design principles underpinning de novo lumen formation and expansion., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Hepatocyte growth factor (HGF) plays central roles in tubulogenesis and metastasis [1-4]. HGF treatment of Madin-Darby canine kidney (MDCK) cells grown as cysts in three-dimensional culture induces tubulogenesis [5, 6], which like most tubulogenic processes proceeds through distinct intermediate phases. Identification of genes associated with these phases is central to understanding the molecular mechanisms of tubulogensis; however, because of inefficient, asynchronous tubule formation, isolating such genes has been unfeasible. Here we developed a synchronous, efficient tubulogenesis system and used time-course transcriptional profiling to identify genes temporally regulated in developmental intermediates. Knockdown (KD) of tensin 4 (TNS4), a particularly highly upregulated gene, leads to a decrease in formation of extensions and tubules, two sequential intermediates in tubulogenesis. Exogenous expression of TNS4 marks invasive cells in an epithelial sheet. A mutation in the SH2 domain of TNS4 prevents the transition from extension formation to invasive migration during tubule formation and leads to increased basal activation of STAT3. Exogenous expression of a constitutively active STAT3 mimics the defect by the mutation. Our study highlights the role of the TNS4-STAT3 axis in epithelial sheet invasion and tubulogenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Polymeric immunoglobulin A (pIgA) transcytosis, mediated by the polymeric immunoglobulin receptor (pIgR), is a central component of mucosal immunity and a model for regulation of polarized epithelial membrane traffic. Binding of pIgA to pIgR stimulates transcytosis in a process requiring Yes, a Src family tyrosine kinase (SFK). We show that Yes directly phosphorylates EGF receptor (EGFR) on liver endosomes. Injection of pIgA into rats induced EGFR phosphorylation. Similarly, in MDCK cells, pIgA treatment significantly increased phosphorylation of EGFR on various sites, subsequently activating extracellular signal-regulated protein kinase (ERK). Furthermore, we find that the Rab11 effector Rab11-FIP5 is a substrate of ERK. Knocking down Yes or Rab11-FIP5, or inhibition of the Yes-EGFR-ERK cascade, decreased pIgA-pIgR transcytosis. Finally, we demonstrate that Rab11-FIP5 phosphorylation by ERK controls Rab11a endosome distribution and pIgA-pIgR transcytosis. Our results reveal a novel Yes-EGFR-ERK-FIP5 signalling network for regulation of pIgA-pIgR transcytosis.

Tubule formation in vitro using Madin-Darby canine kidney (MDCK) epithelial cells consists mainly of two processes. First, the cells undergo a partial epithelial-mesenchymal transition (pEMT), losing polarity and migrating. Second, the cells redifferentiate, forming cords and then tubules with continuous lumens. We have shown previously that extracellular signal-regulated kinase activation is required for pEMT. However, the mechanism of how the pEMT phase is turned off and the redifferentiation phase is initiated is largely unknown. To address the central question of the sequential control of these two phases, we used MDCK cells grown as cysts and treated with hepatocyte growth factor to model tubulogenesis. We show that signal transducer and activator of transcription (STAT)1 controls the sequential progression from the pEMT phase to the redifferentiation phase. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 allows redifferentiation to occur even when cells are otherwise prevented from progressing beyond the pEMT phase by exogenous activation of Raf. Moreover, tyrosine phosphorylation defective STAT1 partially restored cord formation in such cells, suggesting that STAT1 functions in part as nonnuclear protein mediating signal transduction in this process. Constitutively active or inactive forms of STAT1 did not promote lumen maturation, suggesting this requires a distinct signal.

To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is regulated by polarity complexes that are hypothesized to direct downstream events, such as polarized membrane traffic, although this interconnection is not well understood. We have found that Rab11a regulates apical traffic and lumen formation through the Rab guanine nucleotide exchange factor (GEF), Rabin8, and its target, Rab8a. Rab8a and Rab11a function through the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation through the Cdc42 GEF, Tuba. These components assemble at a transient apical membrane initiation site to form the lumen. This Rab11a-directed network directs Cdc42-dependent apical exocytosis during lumen formation, revealing an interaction between the machineries of vesicular transport and polarization.