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1.  Apical targeting of the formin Diaphanous in Drosophila tubular epithelia 
eLife  2013;2:e00666.
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.
eLife digest
Many physiological processes are directional, which means that tissues and organs often need a sense of spatial orientation in order to function properly. In most tissues, this sense of direction relies on certain proteins and infrastructure components of the cell being located in specific subcellular regions, rather than being distributed in a more symmetrical fashion throughout the cell: the latter phenomenon is known as cell polarity.
Exocrine tissues (that is, glands) are composed of tubular epithelial cells organized around a central lumen: the cells in the gland secrete various products (such as enzymes) into the lumen, so that they can be carried to the target organ elsewhere in the body. Epithelial cells in these tissues are therefore polarized to enable directional transport to the lumen. An example of cell polarity is a network of actin filaments that lines the apical surface of these cells (the surface nearest the common lumen). This actin network helps to shuttle cargo to the lumen by assisting with directional, coordinated secretion, among other processes.
In fruitflies, the construction of the apical actin network depends on the presence of a protein called Diaphanous. However, the signals that lead to the localization of this protein near the apical membrane of the cells are not well understood. Now Rousso et al. report that a modified lipid, called PI(4,5)P2, is involved in this localization. However, they also show that this lipid does not govern the apical localization of Diaphanous on its own: rather, an enzyme called Rho1 must also be present to assist with the localization of Diaphanous and to ensure that actin is deposited in the correct place. Rousso et al. also demonstrate that PI(4,5)P2-mediated localization of Drosophila Diaphanous occurs in mammalian cells. Lipid-protein collaboration also targets other proteins to the apical membrane. A common mechanism may therefore underlie cell polarity in tubular organ tissues in flies and mammals.
PMCID: PMC3707080  PMID: 23853710
Diaphanous; formin; tubular epithelia; apical localization; PI(4,5)P2; Rho1; D. melanogaster
2.  Polarity, cell division, and out-of-equilibrium dynamics control the growth of epithelial structures 
The Journal of Cell Biology  2013;203(2):359-372.
Mathematical modeling and analysis of 3D epithelial structures indicate that epithelial growth can take place far from mechanical equilibrium, depending on cell–cell and cell–ECM contact, cell division, cortical contractility, and cell motility.
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.
PMCID: PMC3812962  PMID: 24145168
3.  Cell height: Tao rising 
The Journal of Cell Biology  2012;199(7):1023-1024.
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. 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.
PMCID: PMC3529524  PMID: 23266952
4.  Cyclic AMP regulates formation of mammary epithelial acini in vitro 
Molecular Biology of the Cell  2012;23(15):2973-2981.
Cyclic AMP–dependent protein kinase A (PKA) is required for MCF10A mammary epithelial acinus formation in vitro. PKA plays a dual role by facilitating polarization of cells attached to the extracellular matrix and apoptosis of detached cells.
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.
PMCID: PMC3408423  PMID: 22675028
5.  Transcriptional profiling identifies TNS4 function in epithelial tubulogenesis 
Current biology : CB  2011;21(2):161-166.
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 due to 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 tubulogensis. 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 TNS4-STAT3 axis in epithelial sheet invasion and tubulogenesis.
PMCID: PMC3031161  PMID: 21236678
6.  Liver Progenitor Cells Fold Up a Cell Monolayer into a Double-layered Structure during Tubular Morphogenesis 
Molecular Biology of the Cell  2009;20(9):2486-2494.
Bile ducts are hepatic tubular structures that are lined by cholangiocytes, a type of liver epithelial cell. Cholangiocytes first form a single layer of cells, termed the ductal plate, surrounding the portal vein, which eventually remodels into the branching tubular network of bile ducts. The process of bile duct morphogenesis is not yet clear: a conventional model where cholangiocytes proliferate to duplicate a single layer of the ductal plate before lumen formation seems inconsistent with the observation that proliferation is dramatically reduced when hepatoblasts, liver progenitor cells, differentiate into cholangiocytes. Here, we developed a new culture system in which a liver progenitor cell line, HPPL, reorganizes from a monolayer to tubular structures in response to being overlaid with a gel containing type I collagen and Matrigel. We found that some of the HPPL in the monolayer depolarized and migrated to fold up the monolayer into a double-cell layer. These morphogenetic processes occurred without cell proliferation and required phosphatidylinositol 3-kinase and Akt activity. Later in morphogenesis, luminal space was generated between the two cell layers. This process, in particular enlargement of the apical lumen, involved transcriptional activity of HNF1β. Thus, using this sandwich culture system, we could segregate tubulogenesis of bile ducts into distinct steps and found that the PI3K/Akt pathway and HNF1β regulated different steps of the morphogenesis. Although the process of tubulogenesis in culture specifically resembled early bile duct formation, involvement of these two key players suggests that the sandwich culture might help us to find common principles of tubulogenesis in general.
PMCID: PMC2675627  PMID: 19297530
7.  Liver Progenitor Cells Develop Cholangiocyte-Type Epithelial Polarity in Three-dimensional Culture 
Molecular Biology of the Cell  2007;18(4):1472-1479.
Cholangiocytes are cellular components of the bile duct system of the liver, which originate from hepatoblasts during embryonic liver development. Although several transcription factors and signaling molecules have been implicated in bile duct development, its molecular mechanism has not been studied in detail. Here, we applied a three-dimensional (3D) culture technique to a liver progenitor cell line, HPPL, to establish an in vitro culture system in which HPPL acquire differentiated cholangiocyte characteristics. When HPPL were grown in a gel containing Matrigel, which contains extracellular matrix components of basement membrane, HPPL developed apicobasal polarity and formed cysts, which had luminal space inside. In the cysts, F-actin bundles and atypical protein kinase C were at the apical membrane, E-cadherin was localized at the lateral membrane, and β-catenin and integrin α6 were located at the basolateral membrane. HPPL in cysts expressed cholangiocyte markers, including cytokeratin 19, integrin β4, and aquaporin-1, but not a hepatocyte marker, albumin. Furthermore, HPPL transported rhodamine 123, a substrate for multidrug resistance gene products, from the basal side to the central lumen. These data indicate that HPPL develop cholangiocyte-type epithelial polarity in 3D culture. Phosphatidylinositol 3-kinase signaling was essential for proliferation and survival of HPPL in culture, whereas laminin-1 was a crucial component of Matrigel for inducing epithelial polarization of HPPL. Because HPPL cysts display structural and functional similarities with bile ducts, the 3D culture of HPPL recapitulates in vivo cholangiocyte differentiation and is useful to study the molecular mechanism of bile duct development in vitro.
PMCID: PMC1838984  PMID: 17314404
8.  The Role of Syntaxins in the Specificity of Vesicle Targeting in Polarized Epithelial CellsD⃞ 
Molecular Biology of the Cell  2005;16(12):5784-5792.
In polarized epithelial cells syntaxin 3 is at the apical plasma membrane and is involved in delivery of proteins from the trans-Golgi network to the apical surface. The highly related syntaxin 4 is at the basolateral surface. The complementary distribution of these syntaxins suggests that they play a role in the specificity of membrane traffic to the two surfaces. We constructed a chimeric syntaxin where we removed the N-terminal 29 residues of syntaxin 3 and replaced it with the corresponding portion of syntaxin 4. When expressed in polarized epithelial cells, this chimera was exclusively localized to the basolateral surface. This indicates that the N-terminal domain of syntaxin 3 contains information for its polarized localization. In contrast to the apical localization of syntaxin 3, the basolateral localization of syntaxin 4 was not dependent on its N-terminal domain. Syntaxin 3 normally binds to Munc18b, but not to the related Munc18c. Overexpression of the chimera together with overexpression of Munc18b caused membrane and secretory proteins that are normally sent primarily to the apical surface to exhibit increased delivery to the basolateral surface. We suggest that syntaxins may play a role in determining the specificity of membrane targeting by permitting fusion with only certain target membranes.
PMCID: PMC1289421  PMID: 16207812
9.  Pseudomonas aeruginosa interacts with epithelial cells rapidly forming aggregates that are internalized by a Lyn-dependent mechanism 
Cellular microbiology  2011;13(8):10.1111/j.1462-5822.2011.01611.x.
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.
PMCID: PMC3813436  PMID: 21615664
10.  Molecular Regulation of Lumen Morphogenesis Review 
Current biology : CB  2011;21(3):R126-R136.
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.
PMCID: PMC3771703  PMID: 21300279
11.  Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells 
Nature cell biology  2012;14(8):838-849.
The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-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 co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell.
PMCID: PMC3433678  PMID: 22820376
Cell polarity; epithelial morphogenesis; Rab GTPases; phosphoinositides; vesicle trafficking; lumen formation; synaptotagmin-like proteins
12.  Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b 
Developmental biology  2011;361(1):68-78.
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.
PMCID: PMC3235368  PMID: 22020048
Claudin; liver development; zebrafish; biliary duct remodeling; biliary cells; biliary duct morphogenesis; tight junctions; cholestasis
13.  Grainyhead-like 2 regulates epithelial morphogenesis by establishing functional tight junctions through the organization of a molecular network among claudin3, claudin4, and Rab25 
Molecular Biology of the Cell  2012;23(15):2845-2855.
Grainyhead-like 2 (Grhl2) is a transcription factor that regulates the size of the luminal space surrounded by polarized epithelial cells. Grhl2 promotes epithelial barrier function and the formation of large lumen by up-regulating Cldn3, Cldn4, and Rab25. The results reveal a molecular network regulating epithelial lumen formation.
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.
PMCID: PMC3408412  PMID: 22696678
14.  A kinase cascade leading to Rab11-FIP5 controls transcytosis of the polymeric immunoglobulin receptor 
Nature cell biology  2010;12(12):1143-1153.
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.
PMCID: PMC3072784  PMID: 21037565
15.  MDCK Cystogenesis Driven by Cell Stabilization within Computational Analogues 
PLoS Computational Biology  2011;7(4):e1002030.
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.
Author Summary
Epithelial cells perform essential functions throughout the body, acting as both barrier and transporter and allowing an organism to survive and thrive in varied environments. Although the details of many processes that occur within individual cells are well understood, we still lack a thorough understanding of how cells coordinate their behaviors to create complex tissues. In order to achieve deeper insight, we created a list of targeted attributes and plausible rules for the growth of multicellular cysts formed by Madin-Darby canine kidney (MDCK) cells grown in vitro. We then designed in silico analogues of MDCK cystogenesis using object-oriented programming. In silico components (such as the cells and lumens) and their behaviors directly mapped to in vitro components and mechanisms. We conducted in vitro experiments to generate data that would validate or falsify the in silico analogues and then iteratively refined the analogues to mimic that data. Cells in vitro begin to stabilize at around the fifth day even as cysts continue to expand. The in silico system mirrored that behavior and others, achieving new insights. For example, luminal cell death is not strictly required for cystogenesis, and cell division orientation is very important for normal cyst growth.
PMCID: PMC3072361  PMID: 21490722
16.  STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis 
Molecular Biology of the Cell  2010;21(22):3926-3933.
Signal transducers and activators of transcription (STAT)1 is the key to the sequential control of Madin-Darby canine kidney tubulogenesis. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 is sufficient to restore cord formation but not mature lumens. These data suggest that STAT1 is necessary for the redifferentiation phase of tubulogenesis and that mature lumenogenesis requires a distinct signal.
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.
PMCID: PMC2982126  PMID: 20861313
17.  A molecular network for de novo generation of the apical surface and lumen 
Nature cell biology  2010;12(11):1035-1045.
To form epithelial organs cells must polarize and generate de novo an apical domain and lumen. Epithelial polarization is masterminded by polarity complexes, which are thought to direct downstream events such as polarized membrane traffic, though this interconnection is not well understood. We report that Rab11a regulates apical traffic and lumen formation via the Rab GEF Rabin8, and its target Rab8a. Rab8a/11a act via the exocyst to target Par3 to the apical surface, and control apical Cdc42 activation via 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 a novel interplay of the machineries of vesicular transport and polarization.
PMCID: PMC2975675  PMID: 20890297
18.  A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma 
Cancer cell  2006;9(5):341-349.
The PI3 kinase family of lipid kinases promotes cell growth and survival by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate. To define targets critical for cancers driven by activation of PI3 kinase, we screened a panel of potent and structurally diverse drug-like molecules that target this enzyme family. Surprisingly, a single agent (PI-103) effected proliferative arrest in glioma cells, despite the ability of many compounds to block PI3 kinase signaling through its downstream effector, Akt. The unique cellular activity of PI-103 was traced directly to its ability to inhibit both PI3 kinase α and mTOR. PI-103 showed significant activity in xenografted tumors with no observable toxicity. These data demonstrate an emergent efficacy due to combinatorial inhibition of mTOR and PI3 kinase α in malignant glioma.
PMCID: PMC2925230  PMID: 16697955
19.  From cells to organs: building polarized tissue 
How do animal cells assemble into tissues and organs? A diverse array of tissue structures and shapes can be formed by organizing groups of cells into different polarized arrangements and by coordinating their polarity in space and time. Conserved design principles underlying this diversity are emerging from studies of model organisms and tissues. We discuss how conserved polarity complexes, signalling networks, transcription factors, membrane-trafficking pathways, mechanisms for forming lumens in tubes and other hollow structures, and transitions between different types of polarity, such as between epithelial and mesenchymal cells, are used in similar and iterative manners to build all tissues.
PMCID: PMC2921794  PMID: 18946477
20.  Identification of a cytoplasmic signal for apical transcytosis 
Traffic (Copenhagen, Denmark)  2009;10(8):1128-1142.
Polarized epithelial cells contain apical and basolateral surfaces with distinct protein compositions. To establish and maintain this asymmetry, newly made plasma membrane proteins are sorted in the trans-Golgi network for delivery to apical or basolateral surfaces. Signals for basolateral sorting are generally located in the cytoplasmic domain of the protein, while signals for apical sorting can be in any part of the protein and can depend on N-linked glycosylation of the protein. Signals for constitutive transcytosis to the apical surface have not been reported. Here we utilized the polymeric immunoglobulin receptor (pIgR), which is biosynthetically delivered to the basolateral surface. There the pIgR can bind a ligand and, with or without bound ligand, the pIgR can then be transcytosed to the apical surface. We found that the glycosylation of the pIgR did not affect the biosynthetic transport of the pIgR. However, glycosylation had an effect on pIgR apical transcytosis. Importantly, analysis of the cytoplasmic tail of the pIgR suggested that a short peptide segment was sufficient to transcytose the pIgR or a neutral reporter from the basolateral to the apical surface. This apical transcytosis sorting signal was not involved in polarized biosynthetic traffic of the pIgR.
PMCID: PMC2920487  PMID: 19522755
Epithelial polarity; apical sorting; transcytosis; glycosylation; polymeric immunoglobulin receptor
Experimental cell research  2006;313(4):707-718.
Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer’s role in pIgR-pIgA transcytosis.
PMCID: PMC1810347  PMID: 17184770
MDCK; receptor; pIgR; transcytosis; retromer; Vps35; sorting-nexin; phosphoinositide; phosphatidylinositol; LY294002
22.  Slug Is Required for Cell Survival during Partial Epithelial-Mesenchymal Transition of HGF-induced Tubulogenesis 
Molecular Biology of the Cell  2007;18(5):1943-1952.
Transcription factors of the Snail family are key regulators of epithelial-mesenchymal transition (EMT). In many processes during development or disease, cells do not acquire all the characteristics associated with EMT, leading to what we refer to as partial EMT (p-EMT). However, little is known of the implications of the Snail transcription factors in processes that only involve a p-EMT. To assess this, we used the hepatocyte growth factor (HGF)-induced Madin-Darby canine kidney tubulogenesis system, which provides a three-dimensional culture model of a morphogenetic process including a p-EMT. We found that although Slug (Snail2) is highly and transitory up-regulated during the p-EMT phase of tubulogenesis, it is not a repressor of E-cadherin during this process. Using inducible knockdown of Slug, we demonstrate that Slug is not an inducer of cell movement and instead is required for survival during p-EMT. We conclude that in epithelial cells, promoting cell survival can be a primary function of Slug, rather than being acquired concomitantly with EMT.
PMCID: PMC1855025  PMID: 17344479
23.  Simulating Properties of In Vitro Epithelial Cell Morphogenesis  
PLoS Computational Biology  2006;2(10):e129.
How do individual epithelial cells (ECs) organize into multicellular structures? ECs are studied in vitro to help answer that question. Characteristic growth features include stable cyst formation in embedded culture, inverted cyst formation in suspension culture, and lumen formation in overlay culture. Formation of these characteristic structures is believed to be a consequence of an intrinsic program of differentiation and de-differentiation. To help discover how such a program may function, we developed an in silico analogue in which space, events, and time are discretized. Software agents and objects represent cells and components of the environment. “Cells” act independently. The “program” governing their behavior is embedded within each in the form of axioms and an inflexible decisional process. Relationships between the axioms and recognized cell functions are specified. Interactions between “cells” and environment components during simulation give rise to a complex in silico phenotype characterized by context-dependent structures that mimic counterparts observed in four different in vitro culture conditions: a targeted set of in vitro phenotypic attributes was matched by in silico attributes. However, for a particular growth condition, the analogue failed to exhibit behaviors characteristic of functionally polarized ECs. We solved this problem by following an iterative refinement method that improved the first analogue and led to a second: it exhibited characteristic differentiation and growth properties in all simulated growth conditions. It is the first model to simultaneously provide a representation of nonpolarized and structurally polarized cell types, and a mechanism for their interconversion. The second analogue also uses an inflexible axiomatic program. When specific axioms are relaxed, growths strikingly characteristic of cancerous and precancerous lesions are observed. In one case, the simulated cause is aberrant matrix production. Analogue design facilitates gaining deeper insight into such phenomena by making it easy to replace low-resolution components with increasingly detailed and realistic components.
To gain new insights into how normal and abnormal epithelial cell (EC) morphogenesis occurs, Grant and colleagues designed, built, and studied a series of discrete event analogues capable of mimicking epithelial growth characteristics in four different culture conditions. The analogues use independent software agents and objects to represent cells and the two environment components. “Cells” interact with local components using an axiomatic decisional process deduced from experimental in vitro observations. During simulations, “cells” form stable structures that mimic counterparts in cell cultures: a set of targeted in vitro phenotypic attributes is matched by the analogue's phenotype. However, the foundational analogue failed to exhibit a behavior characteristic of functionally polarized ECs in stable structures. Iterative refinement solved the problem: the improved analogue is the first model to simultaneously provide a representation of nonpolarized and structurally polarized cell types, and a mechanism for their interconversion. Inflexible axiom application is essential to simulate normal attributes. Selectively changing an axiom or relaxing its application caused growths strikingly characteristic of cancerous and precancerous lesions. Gaining deeper insight into such phenomena can be achieved by replacing low-resolution components with increasingly detailed and realistic components.
PMCID: PMC1592363  PMID: 17029556
24.  β1-Integrin Orients Epithelial Polarity via Rac1 and LamininD⃞ 
Molecular Biology of the Cell  2005;16(2):433-445.
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 β1-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 β1-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 β1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.
PMCID: PMC545874  PMID: 15574881
25.  Hepatocyte Growth Factor Switches Orientation of Polarity and Mode of Movement during Morphogenesis of Multicellular Epithelial Structures 
Molecular Biology of the Cell  2003;14(2):748-763.
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.
PMCID: PMC150005  PMID: 12589067

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