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1.  Mitogen-Activated Protein Kinase (MAPK) Pathway Regulates Branching by Remodeling Epithelial Cell Adhesion 
PLoS Genetics  2014;10(3):e1004193.
Although the growth factor (GF) signaling guiding renal branching is well characterized, the intracellular cascades mediating GF functions are poorly understood. We studied mitogen-activated protein kinase (MAPK) pathway specifically in the branching epithelia of developing kidney by genetically abrogating the pathway activity in mice lacking simultaneously dual-specificity protein kinases Mek1 and Mek2. Our data show that MAPK pathway is heterogeneously activated in the subset of G1- and S-phase epithelial cells, and its tissue-specific deletion results in severe renal hypodysplasia. Consequently to the deletion of Mek1/2, the activation of ERK1/2 in the epithelium is lost and normal branching pattern in mutant kidneys is substituted with elongation-only phenotype, in which the epithelium is largely unable to form novel branches and complex three-dimensional patterns, but able to grow without primary defects in mitosis. Cellular characterization of double mutant epithelium showed increased E-cadherin at the cell surfaces with its particular accumulation at baso-lateral locations. This indicates changes in cellular adhesion, which were revealed by electron microscopic analysis demonstrating intercellular gaps and increased extracellular space in double mutant epithelium. When challenged to form monolayer cultures, the mutant epithelial cells were impaired in spreading and displayed strong focal adhesions in addition to spiky E-cadherin. Inhibition of MAPK activity reduced paxillin phosphorylation on serine 83 while remnants of phospho-paxillin, together with another focal adhesion (FA) protein vinculin, were augmented at cell surface contacts. We show that MAPK activity is required for branching morphogenesis, and propose that it promotes cell cycle progression and higher cellular motility through remodeling of cellular adhesions.
Author Summary
Development of the ureter and collecting ducts of the kidney requires extensive growth and branching of an epithelial tube, the ureteric bud. While many genes that control this process are known, the intracellular signaling pathways that underlie renal morphogenesis remain poorly understood. The cellular changes that contribute to ureteric bud morphogenesis, such as adhesion and movements, are guided by intracellular signaling triggered by stimuli at the cell surface. Mitogen-activated protein kinase (MAPK) pathway is known to regulate proliferation in general, but its precise functions during different cell cycle phases are debatable. Moreover, the role of MAPK activity in control of cell adhesion has been demonstrated in cultured cells, but such regulation in vivo remains to be elucidated. Here, we examine the importance of the MAPK activity in ureteric bud branching, and find that simultaneous lack of Mek1 and Mek2 genes allows elongation of the bud but specifically arrests new branch formation. We show that lack of MAPK activity leads to changes in focal adhesion molecules and E-cadherin mediated cell adhesion and delay in cell cycle progression. Our findings may help to understand the origins of certain congenital malformations in humans.
PMCID: PMC3945187  PMID: 24603431
2.  Src-mediated caveolin-1 phosphorylation affects the targeting of active Src to specific membrane sites 
Molecular Biology of the Cell  2013;24(24):3881-3895.
Biophysical and biochemical studies show that caveolin-1 phosphorylation by Src at Tyr-14, followed by binding of the SH2 domain of activated Src to phospho–Tyr-14, enhances Src–plasma membrane interaction. This targets activated Src preferentially to focal adhesions, providing a mechanism that potentially regulates focal adhesion function.
Src interactions with the plasma membrane are an important determinant of its activity. In turn, Src activity modulates its association with the membrane through binding of activated Src to phosphotyrosylated proteins. Caveolin-1 (Cav-1), a major component of caveolae, is a known Src phosphorylation target, and both were reported to regulate cell transformation. However, the nature of Src-Cav-1 interactions, a potential mechanism of their coregulation, remained unclear. Here we used fluorescence recovery after photobleaching beam-size analysis, coimmunoprecipitation, quantitative imaging, and far-Western studies with cells expressing wild type, as well as structural and activity mutants of Src–green fluorescent protein and Cav-1–monomeric red fluorescent protein, to measure their interactions with the membrane and with each other. We show dynamic Src–plasma membrane interactions, which are augmented and stabilized by Cav-1. The mechanism involves phosphorylation of Cav-1 at Tyr-14 by Src and subsequent binding of the Src SH2 domain to phospho–Cav-1, leading to accumulation of activated Src in focal adhesions. This novel Cav-1 function potentially modulates focal adhesion dynamics.
PMCID: PMC3861084  PMID: 24131997
3.  JAM-A associates with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and regulate epithelial barrier function 
Molecular Biology of the Cell  2013;24(18):2849-2860.
Intestinal barrier function is regulated by epithelial tight junctions, structures that control paracellular permeability. JAM-A regulates epithelial permeability through association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.
Intestinal barrier function is regulated by epithelial tight junctions (TJs), structures that control paracellular permeability. Junctional adhesion molecule-A (JAM-A) is a TJ-associated protein that regulates barrier; however, mechanisms linking JAM-A to epithelial permeability are poorly understood. Here we report that JAM-A associates directly with ZO-2 and indirectly with afadin, and this complex, along with PDZ-GEF1, activates the small GTPase Rap2c. Supporting a functional link, small interfering RNA–mediated down-regulation of the foregoing regulatory proteins results in enhanced permeability similar to that observed after JAM-A loss. JAM-A–deficient mice and cultured epithelial cells demonstrate enhanced paracellular permeability to large molecules, revealing a potential role of JAM-A in controlling perijunctional actin cytoskeleton in addition to its previously reported role in regulating claudin proteins and small-molecule permeability. Further experiments suggest that JAM-A does not regulate actin turnover but modulates activity of RhoA and phosphorylation of nonmuscle myosin, both implicated in actomyosin contraction. These results suggest that JAM-A regulates epithelial permeability via association with ZO-2, afadin, and PDZ-GEF1 to activate Rap2c and control contraction of the apical cytoskeleton.
PMCID: PMC3771947  PMID: 23885123
4.  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
5.  Chemical-genetic disruption of clathrin function spares adaptor complex 3–dependent endosome vesicle biogenesis 
Molecular Biology of the Cell  2013;24(15):2378-2388.
Clathrin–AP-3 association is dispensable for AP-3 vesicle budding from endosomes, which suggests that AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis.
A role for clathrin in AP-3–dependent vesicle biogenesis has been inferred from biochemical interactions and colocalization between this adaptor and clathrin. The functionality of these molecular associations, however, is controversial. We comprehensively explore the role of clathrin in AP-3–dependent vesicle budding, using rapid chemical-genetic perturbation of clathrin function with a clathrin light chain–FKBP chimera oligomerizable by the drug AP20187. We find that AP-3 interacts and colocalizes with endogenous and recombinant FKBP chimeric clathrin polypeptides in PC12-cell endosomes. AP-3 displays, however, a divergent behavior from AP-1, AP-2, and clathrin chains. AP-3 cofractionates with clathrin-coated vesicle fractions isolated from PC12 cells even after clathrin function is acutely inhibited by AP20187. We predicted that AP20187 would inhibit AP-3 vesicle formation from endosomes after a brefeldin A block. AP-3 vesicle formation continued, however, after brefeldin A wash-out despite impairment of clathrin function by AP20187. These findings indicate that AP-3–clathrin association is dispensable for endosomal AP-3 vesicle budding and suggest that endosomal AP-3–clathrin interactions differ from those by which AP-1 and AP-2 adaptors productively engage clathrin in vesicle biogenesis.
PMCID: PMC3727930  PMID: 23761069
6.  The WASH complex, an endosomal Arp2/3 activator, interacts with the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo phosphatidylinositol-4-kinase type IIα 
Molecular Biology of the Cell  2013;24(14):2269-2284.
The WASH complex, an endosomal activator of the Arp2/3 complex involved in branched actin polymerization, is identified as a new factor in vesicle traffic mediated by the Hermansky–Pudlak syndrome complex BLOC-1.
Vesicle biogenesis machinery components such as coat proteins can interact with the actin cytoskeleton for cargo sorting into multiple pathways. It is unknown, however, whether these interactions are a general requirement for the diverse endosome traffic routes. In this study, we identify actin cytoskeleton regulators as previously unrecognized interactors of complexes associated with the Hermansky–Pudlak syndrome. Two complexes mutated in the Hermansky–Pudlak syndrome, adaptor protein complex-3 and biogenesis of lysosome-related organelles complex-1 (BLOC-1), interact with and are regulated by the lipid kinase phosphatidylinositol-4-kinase type IIα (PI4KIIα). We therefore hypothesized that PI4KIIα interacts with novel regulators of these complexes. To test this hypothesis, we immunoaffinity purified PI4KIIα from isotope-labeled cell lysates to quantitatively identify interactors. Strikingly, PI4KIIα isolation preferentially coenriched proteins that regulate the actin cytoskeleton, including guanine exchange factors for Rho family GTPases such as RhoGEF1 and several subunits of the WASH complex. We biochemically confirmed several of these PI4KIIα interactions. Of importance, BLOC-1 complex, WASH complex, RhoGEF1, or PI4KIIα depletions altered the content and/or subcellular distribution of the BLOC-1–sensitive cargoes PI4KIIα, ATP7A, and VAMP7. We conclude that the Hermansky–Pudlak syndrome complex BLOC-1 and its cargo PI4KIIα interact with regulators of the actin cytoskeleton.
PMCID: PMC3708732  PMID: 23676666
7.  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
8.  Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization 
Molecular Biology of the Cell  2013;24(12):1996-2007.
The mechanisms that regulate the apical sorting of proteins are unclear, but clustering may play an important role. A role for dimerization and higher-order oligomerization in the biosynthetic transport of the model O-glycosylated protein p75 has been identified. This study also suggests that the O-glycans of p75 have a structural role in apical sorting.
The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75–green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery.
PMCID: PMC3681702  PMID: 23637462
9.  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.
PMCID: PMC3140688  PMID: 21576256
10.  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
11.  Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin–Darby canine kidney cells 
Molecular Biology of the Cell  2013;24(8):1196-1207.
Four-dimensional live-cell imaging is combined with single-particle tracking to identify key steps in polarized epithelium cell entry by the prototype enteric virus reovirus.
Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin–Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.
PMCID: PMC3623640  PMID: 23427267
12.  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
13.  A Rab11a-Rab8a-Myo5B network promotes stretch-regulated exocytosis in bladder umbrella cells 
Molecular Biology of the Cell  2013;24(7):1007-1019.
Rab11a and Rab8 work in conjunction with myosin5B to promote discoidal/fusiform vesicle exocytosis at the apical surface of umbrella cells. It is predicted that similar Rab cascades will be common to other regulated secretory pathways.
Multiple Rabs are associated with secretory granules/vesicles, but how these GTPases are coordinated to promote regulated exocytosis is not well understood. In bladder umbrella cells a subapical pool of discoidal/fusiform-shaped vesicles (DFVs) undergoes Rab11a-dependent regulated exocytosis in response to bladder filling. We show that Rab11a-associated vesicles are enmeshed in an apical cytokeratin meshwork and that Rab11a likely acts upstream of Rab8a to promote exocytosis. Surprisingly, expression of Rabin8, a previously described Rab11a effector and guanine nucleotide exchange factor for Rab8, stimulates stretch-induced exocytosis in a manner that is independent of its catalytic activity. Additional studies demonstrate that the unconventional motor protein myosin5B motor (Myo5B) works in association with the Rab8a–Rab11a module to promote exocytosis, possibly by ensuring transit of DFVs through a subapical, cortical actin cytoskeleton before fusion. Our results indicate that Rab11a, Rab8a, and Myo5B function as part of a network to promote stretch-induced exocytosis, and we predict that similarly organized Rab networks will be common to other regulated secretory pathways.
PMCID: PMC3608489  PMID: 23389633
14.  Small heat shock proteins target mutant cystic fibrosis transmembrane conductance regulator for degradation via a small ubiquitin-like modifier–dependent pathway 
Molecular Biology of the Cell  2013;24(2):74-84.
Selective degradation of the mutant protein responsible for most cystic fibrosis, F508del cystic fibrosis transmembrane conductance regulator (CFTR), is initiated by Hsp27, which associates with the small ubiquitin-like modifier (SUMO) E2, Ubc9. They modify F508del with SUMO-2/3, directing F508del to a SUMO-targeted ubiquitin ligase, RNF4. This work implicates SUMO and RNF4 in quality control of a cytosolic transmembrane protein.
Small heat shock proteins (sHsps) bind destabilized proteins during cell stress and disease, but their physiological functions are less clear. We evaluated the impact of Hsp27, an sHsp expressed in airway epithelial cells, on the common protein misfolding mutant that is responsible for most cystic fibrosis. F508del cystic fibrosis transmembrane conductance regulator (CFTR), a well-studied protein that is subject to cytosolic quality control, selectively associated with Hsp27, whose overexpression preferentially targeted mutant CFTR to proteasomal degradation. Hsp27 interacted physically with Ubc9, the small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, implying that F508del SUMOylation leads to its sHsp-mediated degradation. Enhancing or disabling the SUMO pathway increased or blocked Hsp27’s ability to degrade mutant CFTR. Hsp27 promoted selective SUMOylation of F508del NBD1 in vitro and of full-length F508del CFTR in vivo, which preferred endogenous SUMO-2/3 paralogues that form poly-chains. The SUMO-targeted ubiquitin ligase (STUbL) RNF4 recognizes poly-SUMO chains to facilitate nuclear protein degradation. RNF4 overexpression elicited F508del degradation, whereas Hsp27 knockdown blocked RNF4’s impact on mutant CFTR. Similarly, the ability of Hsp27 to degrade F508del CFTR was lost during overexpression of dominant-negative RNF4. These findings link sHsp-mediated F508del CFTR degradation to its SUMOylation and to STUbL-mediated targeting to the ubiquitin–proteasome system and thereby implicate this pathway in the disposal of an integral membrane protein.
PMCID: PMC3541966  PMID: 23155000
15.  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
16.  Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia 
Molecular Biology of the Cell  2012;23(21):4188-4202.
Functional expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuates expression and secretion of the proinflammatory cytokines IL-6, IL-8, and CXCL1/2 in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport may contribute to lung inflammation in cystic fibrosis.
Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air–liquid but not liquid–liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr∆F508/∆F508 immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor–mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.
PMCID: PMC3484098  PMID: 22973054
17.  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
18.  Sialylation of N-linked glycans mediates apical delivery of endolyn in MDCK cells via a galectin-9–dependent mechanism 
Molecular Biology of the Cell  2012;23(18):3636-3646.
The sialomucin endolyn is implicated in adhesion, migration, and differentiation of various cell types. Apical delivery of endolyn requires recognition of sialic acids on its N-glycans possibly (or likely) mediated by galectin-9.
The sialomucin endolyn is implicated in adhesion, migration, and differentiation of various cell types. Along rat kidney tubules, endolyn is variously localized to the apical surface and endosomal/lysosomal compartments. Apical delivery of newly synthesized rat endolyn predominates over direct lysosomal delivery in polarized Madin–Darby canine kidney cells. Apical sorting depends on terminal processing of a subset of lumenal N-glycans. Here we dissect the requirements of N-glycan processing for apical targeting and investigate the underlying mechanism. Modulation of glycan branching and subsequent polylactosamine elongation by knockdown of N-acetylglucosaminyltransferase III or V had no effect on apical delivery of endolyn. In contrast, combined but not individual knockdown of sialyltransferases ST3Gal-III, ST3Gal-IV, and ST6Gal-I, which together are responsible for addition of α2,3- and α2,6-linked sialic acids on N-glycans, dramatically decreased endolyn surface polarity. Endolyn synthesized in the presence of kifunensine, which blocks terminal N-glycan processing, reduced its interaction with several recombinant canine galectins, and knockdown of galectin-9 (but not galectin-3, -4, or -8) selectively disrupted endolyn polarity. Our data suggest that sialylation enables recognition of endolyn by galectin-9 to mediate efficient apical sorting. They raise the intriguing possibility that changes in glycosyltransferase expression patterns and/or galectin-9 distribution may acutely modulate endolyn trafficking in the kidney.
PMCID: PMC3442411  PMID: 22855528
19.  Actin polymerization controls the activation of multidrug efflux at fertilization by translocation and fine-scale positioning of ABCB1 on microvilli 
Molecular Biology of the Cell  2012;23(18):3663-3672.
Multidrug efflux is activated at fertilization in sea urchin eggs, but it is unclear how cortical reorganization initiates transport. Using structured illumination microscopy, we found that the multidrug transporter ABCB1a translocates along polymerizing actin filaments to the microvillar tips. This short-range (micrometer scale) translocation is necessary for up-regulation of efflux activity.
Fertilization changes the structure and function of the cell surface. In sea urchins, these changes include polymerization of cortical actin and a coincident, switch-like increase in the activity of the multidrug efflux transporter ABCB1a. However, it is not clear how cortical reorganization leads to changes in membrane transport physiology. In this study, we used three-dimensional superresolution fluorescence microscopy to resolve the fine-scale movements of the transporter along polymerizing actin filaments, and we show that efflux activity is established after ABCB1a translocates to the tips of the microvilli. Inhibition of actin poly­merization or bundle formation prevents tip localization, resulting in the patching of ABCB1a at the cell surface and decreased efflux activity. In contrast, enhanced actin polymerization promotes tip localization. Finally, interference with Rab11, a regulator of apical recycling, inhibits activation of efflux activity in embryos. Together our results show that actin-mediated, short-range traffic and positioning of transporters at the cell surface regulates multidrug efflux activity and highlight the multifaceted roles of microvilli in the spatial distribution of membrane proteins.
PMCID: PMC3442413  PMID: 22855533
20.  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
21.  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
22.  Electron tomography of late stages of FcRn-mediated antibody transcytosis in neonatal rat small intestine 
Molecular Biology of the Cell  2012;23(13):2537-2545.
Structural aspects of later stages of FcRn transcytosis in neonatal rat intestinal epithelia are studied. FcRn pools are found in dilated regions of the lateral intercellular space, representing possible exit sites from the epithelium. Clathrin and multivesicular bodies play a role in the neonatal transcytotic pathway.
The neonatal Fc receptor (FcRn) transports maternal immunoglobulin (IgG) across epithelia to confer passive immunity to mammalian young. In newborn rodents, FcRn transcytoses IgG from ingested milk across the intestinal epithelium for release into the bloodstream. We used electron tomography to examine FcRn transport of Nanogold-labeled Fc (Au-Fc) in neonatal rat jejunum, focusing on later aspects of transport by chasing Au-Fc before fixation. We observed pools of Au-Fc in dilated regions of the lateral intercellular space (LIS), likely representing exit sites where Au-Fc accumulates en route to the blood. Before weaning, the jejunum functions primarily in IgG transport and exhibits unusual properties: clathrin-rich regions near/at the basolateral LIS and multivesicular bodies (MVBs) expressing early endosomal markers. To address whether these features are related to IgG transport, we examined LIS and endocytic/transcytotic structures from neonatal and weaned animals. Weaned samples showed less LIS-associated clathrin. MVBs labeled with late endosomal/lysosomal markers were smaller than their neonatal counterparts but contained 10 times more internal compartments. These results are consistent with hypotheses that clathrin-rich basolateral regions in neonatal jejunum are involved in IgG exocytosis and that MVBs function in IgG transport while FcRn is expressed but switch to degradative functions after weaning, when the jejunum does not express FcRn or transport IgG.
PMCID: PMC3386217  PMID: 22573886
23.  Postlipolytic insulin-dependent remodeling of micro lipid droplets in adipocytes 
Molecular Biology of the Cell  2012;23(10):1826-1837.
Micro lipid droplets (mLDs) form during lipolysis in both primary and cultured adipocytes. Surprisingly, mLDs do not appear to bud from large LDs or require an intact cytoskeleton to form. Insulin and fatty acids trigger fusion and growth of mLDs to reform macroLDs, a process that is microtubule dependent.
Despite the lipolysis–lipogenesis cycle being a fundamental process in adipocyte biology, very little is known about the morphological changes that occur during this process. The remodeling of lipid droplets to form micro lipid droplets (mLDs) is a striking feature of lipolysis in adipocytes, but once lipolysis ceases, the cell must regain its basal morphology. We characterized mLD formation in cultured adipocytes, and in primary adipocytes isolated from mouse epididymal fat pads, in response to acute activation of lipolysis. Using real-time quantitative imaging and electron tomography, we show that formation of mLDs in cultured adipocytes occurs throughout the cell to increase total LD surface area by ∼30% but does not involve detectable fission from large LDs. Peripheral mLDs are monolayered structures with a neutral lipid core and are sites of active lipolysis. Electron tomography reveals preferential association of mLDs with the endoplasmic reticulum. Treatment with insulin and fatty acids results in the reformation of macroLDs and return to the basal state. Insulin-dependent reformation of large LDs involves two distinct processes: microtubule-dependent homotypic fusion of mLDs and expansion of individual mLDs. We identify a physiologically important role for LD fusion that is involved in a reversible lipolytic cycle in adipocytes.
PMCID: PMC3350548  PMID: 22456503
24.  A VE-cadherin–PAR3–α-catenin complex regulates the Golgi localization and activity of cytosolic phospholipase A2α in endothelial cells 
Molecular Biology of the Cell  2012;23(9):1783-1796.
The rapid regulation of phospholipase A2 activity is essential for vascular function. Evidence is found for a VE-cadherin–α-catenin–PAR3 complex regulating the reversible association of cPLA2α with the Golgi apparatus in confluent endothelial cells. This regulation is important for controlling both cPLA2α activity and angiogenesis.
Phospholipase A2 enzymes hydrolyze phospholipids to liberate arachidonic acid for the biosynthesis of prostaglandins and leukotrienes. In the vascular endothelium, group IV phospholipase A2α (cPLA2α) enzyme activity is regulated by reversible association with the Golgi apparatus. Here we provide evidence for a plasma membrane cell adhesion complex that regulates endothelial cell confluence and simultaneously controls cPLA2α localization and enzymatic activity. Confluent endothelial cells display pronounced accumulation of vascular endothelial cadherin (VE-cadherin) at cell–cell junctions, and mechanical wounding of the monolayer stimulates VE-cadherin complex disassembly and cPLA2α release from the Golgi apparatus. VE-cadherin depletion inhibits both recruitment of cPLA2α to the Golgi and formation of tubules by endothelial cells. Perturbing VE-cadherin and increasing the soluble cPLA2α fraction also stimulated arachidonic acid and prostaglandin production. Of importance, reverse genetics shows that α-catenin and δ-catenin, but not β-catenin, regulates cPLA2α Golgi localization linked to cell confluence. Furthermore, cPLA2α Golgi localization also required partitioning defective protein 3 (PAR3) and annexin A1. Disruption of F-actin internalizes VE-cadherin and releases cPLA2α from the adhesion complex and Golgi apparatus. Finally, depletion of either PAR3 or α-catenin promotes cPLA2α-dependent endothelial tubule formation. Thus a VE-cadherin–PAR3–α-catenin adhesion complex regulates cPLA2α recruitment to the Golgi apparatus, with functional consequences for vascular physiology.
PMCID: PMC3338442  PMID: 22398721
25.  Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis 
Molecular Biology of the Cell  2012;23(6):996-1009.
cAMP/PKA stimulation elicited posttranslational increases in CFTR expression and the interaction of specific 14-3-3 proteins with phosphorylated sites within the R region. This improved the efficiency of nascent CFTR biogenesis and reduced its interaction with the COPI retrograde retrieval mechanism, making more CFTR available for anion secretion.
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)–regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.
PMCID: PMC3302758  PMID: 22278744

Results 1-25 (192)