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1.  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.
doi:10.1083/jcb.201305044
PMCID: PMC3812962  PMID: 24145168
2.  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.
Summary
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.
doi:10.1111/j.1462-5822.2011.01611.x
PMCID: PMC3813436  PMID: 21615664
3.  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.
doi:10.1016/j.cub.2010.12.003
PMCID: PMC3771703  PMID: 21300279
4.  Phosphoinositides in Cell Architecture 
Inositol phospholipids have been implicated in almost all aspects of cellular physiology including spatiotemporal regulation of cellular signaling, acquisition of cellular polarity, specification of membrane identity, cytoskeletal dynamics, and regulation of cellular adhesion, motility, and cytokinesis. In this review, we examine the critical role phosphoinositides play in these processes to execute the establishment and maintenance of cellular architecture. Epithelial tissues perform essential barrier and transport functions in almost all major organs. Key to their development and function is the establishment of epithelial cell polarity. We place a special emphasis on highlighting recent studies demonstrating phosphoinositide regulation of epithelial cell polarity and how individual cells use phosphoinositides to further organize into epithelial tissues.
PtdIns(3,4,5)P3 specifies the basolateral surface of polarized epithelial cells, whereas PtdIns(4,5)P2 specifies the apical domain. Individual cells also use phosphoinositides to further organize into epithelial tissues.
doi:10.1101/cshperspect.a004796
PMCID: PMC3140688  PMID: 21576256
5.  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.
DOI: http://dx.doi.org/10.7554/eLife.00666.001
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.
DOI: http://dx.doi.org/10.7554/eLife.00666.002
doi:10.7554/eLife.00666
PMCID: PMC3707080  PMID: 23853710
Diaphanous; formin; tubular epithelia; apical localization; PI(4,5)P2; Rho1; D. melanogaster
6.  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. 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.
doi:10.1083/jcb.201211015
PMCID: PMC3529524  PMID: 23266952
7.  Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells 
Nature cell biology  2012;14(8):838-849.
SUMMARY
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.
doi:10.1038/ncb2541
PMCID: PMC3433678  PMID: 22820376
Cell polarity; epithelial morphogenesis; Rab GTPases; phosphoinositides; vesicle trafficking; lumen formation; synaptotagmin-like proteins
8.  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.
doi:10.1016/j.ydbio.2011.10.004
PMCID: PMC3235368  PMID: 22020048
Claudin; liver development; zebrafish; biliary duct remodeling; biliary cells; biliary duct morphogenesis; tight junctions; cholestasis
9.  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.
doi:10.1091/mbc.E12-02-0097
PMCID: PMC3408412  PMID: 22696678
10.  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.
doi:10.1091/mbc.E12-02-0078
PMCID: PMC3408423  PMID: 22675028
11.  Transcriptional profiling identifies TNS4 function in epithelial tubulogenesis 
Current biology : CB  2011;21(2):161-166.
Summary
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.
doi:10.1016/j.cub.2010.12.037
PMCID: PMC3031161  PMID: 21236678
12.  Cse1l is a negative regulator of CFTR-dependent fluid secretion 
Current biology : CB  2010;20(20):1840-1845.
Summary
Transport of chloride through the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates[1, 2]. Loss of CFTR function leads to cystic fibrosis (CF)[1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine and vas deferens. Conversely, un-controlled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera[5, 6] and other secretory diarrheas [7] as well as Polycystic Kidney Disease (PKD)[8–10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the un-controlled activation of the CFTR channel. Analyses in zebrafish embryos and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much needed model system to study CFTR regulation in vivo.
doi:10.1016/j.cub.2010.09.012
PMCID: PMC2963654  PMID: 20933420
13.  Simulation of lung alveolar epithelial wound healing in vitro 
The mechanisms that enable and regulate alveolar type II (AT II) epithelial cell wound healing in vitro and in vivo remain largely unknown and need further elucidation. We used an in silico AT II cell-mimetic analogue to explore and better understand plausible wound healing mechanisms for two conditions: cyst repair in three-dimensional cultures and monolayer wound healing. Starting with the analogue that validated for key features of AT II cystogenesis in vitro, we devised an additional cell rearrangement action enabling cyst repair. Monolayer repair was enabled by providing ‘cells’ a control mechanism to switch automatically to a repair mode in the presence of a distress signal. In cyst wound simulations, the revised analogue closed wounds by adhering to essentially the same axioms available for alveolar-like cystogenesis. In silico cell proliferation was not needed. The analogue recovered within a few simulation cycles but required a longer recovery time for larger or multiple wounds. In simulated monolayer wound repair, diffusive factor-mediated ‘cell’ migration led to repair patterns comparable to those of in vitro cultures exposed to different growth factors. Simulations predicted directional cell locomotion to be critical for successful in vitro wound repair. We anticipate that with further use and refinement, the methods used will develop as a rigorous, extensible means of unravelling mechanisms of lung alveolar repair and regeneration.
doi:10.1098/rsif.2010.0041
PMCID: PMC2894878  PMID: 20236957
multi-agent model; discrete event simulation; alveolar wound repair; acute lung injury
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.
doi:10.1038/ncb2118
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.
doi:10.1371/journal.pcbi.1002030
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.
doi:10.1091/mbc.E10-02-0112
PMCID: PMC2982126  PMID: 20861313
17.  The Cdc42 GEF Intersectin 2 controls mitotic spindle orientation to form the lumen during epithelial morphogenesis 
The Journal of Cell Biology  2010;189(4):725-738.
Intersectin 2 localizes to centrosomes, where it regulates Cdc42 and helps to orient the apical surface correctly during cyst formation. (See also companion paper from Qin et al., in this issue.)
Epithelial organs are made of tubes and cavities lined by a monolayer of polarized cells that enclose the central lumen. Lumen formation is a crucial step in the formation of epithelial organs. The Rho guanosine triphosphatase (GTPase) Cdc42, which is a master regulator of cell polarity, regulates the formation of the central lumen in epithelial morphogenesis. However, how Cdc42 is regulated during this process is still poorly understood. Guanine nucleotide exchange factors (GEFs) control the activation of small GTPases. Using the three-dimensional Madin–Darby canine kidney model, we have identified a Cdc42-specific GEF, Intersectin 2 (ITSN2), which localizes to the centrosomes and regulates Cdc42 activation during epithelial morphogenesis. Silencing of either Cdc42 or ITSN2 disrupts the correct orientation of the mitotic spindle and normal lumen formation, suggesting a direct relationship between these processes. Furthermore, we demonstrated this direct relationship using LGN, a component of the machinery for mitotic spindle positioning, whose disruption also results in lumen formation defects.
doi:10.1083/jcb.201002047
PMCID: PMC2872911  PMID: 20479469
18.  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.
doi:10.1038/ncb2106
PMCID: PMC2975675  PMID: 20890297
19.  Pseudomonas aeruginosa-Mediated Damage Requires Distinct Receptors at the Apical and Basolateral Surfaces of the Polarized Epithelium ▿ †  
Infection and Immunity  2009;78(3):939-953.
Pseudomonas aeruginosa, an important opportunistic pathogen of humans, exploits epithelial damage to establish infection. We have rigorously explored the role of N-glycoproteins and heparan sulfate proteoglycans (HSPGs) in P. aeruginosa-mediated attachment and subsequent downstream events at the apical (AP) and basolateral (BL) surfaces of polarized epithelium. We demonstrate that the N-glycan chains at the AP surface are necessary and sufficient for binding, invasion, and cytotoxicity to kidney (MDCK) and airway (Calu-3) cells grown at various states of polarization on Transwell filters. Upregulation of N-glycosylation enhanced binding, whereas pharmacologic inhibition of N-glycosylation or infection of MDCK cells defective in N-glycosylation resulted in decreased binding. In contrast, at the BL surface, the HS moiety of HSPGs mediated P. aeruginosa binding, cytotoxicity, and invasion. In incompletely polarized epithelium, HSPG abundance was increased at the AP surface, explaining its increased susceptibility to P. aeruginosa colonization and damage. Using MDCK cells grown as three-dimensional cysts as a model for epithelial organs, we show that P. aeruginosa specifically colocalized with HS-rich areas at the BL membrane but with complex N-glycans at the AP surface. Finally, P. aeruginosa bound to HS chains and N-glycans coated on plastic surfaces, showing the highest binding affinity toward isolated HS chains. Together, these findings demonstrate that P. aeruginosa recognizes distinct receptors on the AP and BL surfaces of polarized epithelium. Changes in the composition of N-glycan chains and/or in the distribution of HSPGs may explain the enhanced susceptibility of damaged epithelium to P. aeruginosa.
doi:10.1128/IAI.01215-09
PMCID: PMC2825949  PMID: 20008530
20.  A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma 
Cancer cell  2006;9(5):341-349.
Summary
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.
doi:10.1016/j.ccr.2006.03.029
PMCID: PMC2925230  PMID: 16697955
21.  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.
doi:10.1038/nrm2523
PMCID: PMC2921794  PMID: 18946477
22.  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.
doi:10.1111/j.1600-0854.2009.00941.x
PMCID: PMC2920487  PMID: 19522755
Epithelial polarity; apical sorting; transcytosis; glycosylation; polymeric immunoglobulin receptor
23.  Polarity is Destiny 
Cell  2009;139(4):660-662.
Summary
Cdc42 specifies polarity in various biological processes. Kesavan et al. (2009) now demonstrate that Cdc42 also regulates epithelial cell polarity in the mouse pancreas, where it is required for tubule initiation and maintenance. Furthermore, the polarization of epithelial tissue influences the differentiation of pancreatic progenitor cells, linking cell polarity to cell specification.
doi:10.1016/j.cell.2009.10.040
PMCID: PMC2900917  PMID: 19914162
24.  A computational approach to resolve cell level contributions to early glandular epithelial cancer progression 
BMC Systems Biology  2009;3:122.
Background
Three-dimensional (3D) embedded cell cultures provide an appropriate physiological environment to reconstruct features of early glandular epithelial cancer. Although these are orders of magnitude simpler than tissues, they too are complex systems that have proven challenging to understand. We used agent-based, discrete event simulation modeling methods to build working hypotheses of mechanisms of epithelial 3D culture phenotype and early cancer progression. Starting with an earlier software analogue, we validated an improved in silico epithelial analogue (ISEA) for cardinal features of a normally developed MDCK cyst. A set of axiomatic operating principles defined simulated cell actions. We explored selective disruption of individual simulated cell actions. New framework features enabled recording detailed measures of ISEA cell activities and morphology.
Results
Enabled by a small set of cell operating principles, ISEA cells multiplied and self-organized into cyst-like structures that mimicked those of MDCK cells in a 3D embedded cell culture. Selective disruption of "anoikis" or directional cell division caused the ISEA to develop phenotypic features resembling those of in vitro tumor reconstruction models and cancerous tissues in vivo. Disrupting either process, or both, altered cell activity patterns that resulted in morphologically similar outcomes. Increased disruption led to a prolonged presence of intraluminal cells.
Conclusions
ISEA mechanisms, behaviors, and morphological properties may have biological counterparts. To the extent that in silico-to-in vitro mappings are valid, the results suggest plausible, additional mechanisms of in vitro cancer reconstruction or reversion, and raise potentially significant implications for early cancer diagnosis based on histology. Further ISEA development and use are expected to provide a viable platform to complement in vitro methods for unraveling the mechanistic basis of epithelial morphogenesis and cancer progression.
doi:10.1186/1752-0509-3-122
PMCID: PMC2814811  PMID: 20043854
25.  Focal adhesion components are essential for mammalian cell cytokinesis 
Cell cycle (Georgetown, Tex.)  2008;7(18):2868-2876.
The final stages in mammalian cytokinesis are poorly understood. Previously, we reported that the ADP-ribosyltransferase activity of Pseudomonas aeruginosa type III secreted toxin ExoT inhibits late stages of cytokinesis. Given that Crk adaptor proteins are the major substrates of ExoT ADP-ribosyltransferase activity, we tested the involvement of Crk in cytokinesis. We report that the focal adhesion-associated proteins, Crk and paxillin are essential for completion of cytokinesis. When their function is absent, the cytoplasmic bridge fails to resolve and the daughter cells fuse to form a binucleated cell. During cytokinesis, Crk is required for syntaxin-2 recruitment to the midbody, while paxillin is required for both Crk and syntaxin-2 localization to this compartment. Our data demonstrate that the subcellular localization and the activity of RhoA and citron K, which are essential for early stages of cytokinesis, are not dependent on paxillin, Crk or syntaxin-2. These studies reveal a novel role for Crk and paxillin in cytokinesis and suggest that focal adhesion complex, as a unit, may partake in this fundamental cellular process.
PMCID: PMC2745397  PMID: 18787414
cytokinesis; Crk; Src; paxillin; focal adhesion; midbody; abscission

Results 1-25 (53)