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1.  Type Iγ phosphatidylinositol phosphate kinase modulates adherens junction and E-cadherin trafficking via a direct interaction with μ1B adaptin 
The Journal of Cell Biology  2007;176(3):343-353.
Assembly of E-cadherin–based adherens junctions (AJ) is obligatory for establishment of polarized epithelia and plays a key role in repressing the invasiveness of many carcinomas. Here we show that type Iγ phosphatidylinositol phosphate kinase (PIPKIγ) directly binds to E-cadherin and modulates E-cadherin trafficking. PIPKIγ also interacts with the μ subunits of clathrin adaptor protein (AP) complexes and acts as a signalling scaffold that links AP complexes to E-cadherin. Depletion of PIPKIγ or disruption of PIPKIγ binding to either E-cadherin or AP complexes results in defects in E-cadherin transport and blocks AJ assembly. An E-cadherin germline mutation that loses PIPKIγ binding and shows disrupted basolateral membrane targeting no longer forms AJs and leads to hereditary gastric cancers. These combined results reveal a novel mechanism where PIPKIγ serves as both a scaffold, which links E-cadherin to AP complexes and the trafficking machinery, and a regulator of trafficking events via the spatial generation of phosphatidylinositol-4,5-bisphosphate.
doi:10.1083/jcb.200606023
PMCID: PMC2063960  PMID: 17261850
2.  Phosphatidylinositol 4,5-Bisphosphate Mediates the Targeting of the Exocyst to the Plasma Membrane for Exocytosis in Mammalian Cells 
Molecular Biology of the Cell  2007;18(11):4483-4492.
The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles at the plasma membrane for exocytosis. To elucidate the mechanism of vesicle tethering, it is important to understand how the exocyst physically associates with the plasma membrane (PM). In this study, we report that the mammalian exocyst subunit Exo70 associates with the PM through its direct interaction with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Furthermore, we have identified key conserved residues at the C-terminus of Exo70 that are crucial for the interaction of Exo70 with PI(4,5)P2. Disrupting Exo70-PI(4,5)P2 interaction abolished the membrane association of Exo70. We have also found that wild-type Exo70 but not the PI(4,5)P2-binding–deficient Exo70 mutant is capable of recruiting other exocyst components to the PM. Using the ts045 vesicular stomatitis virus glycoprotein trafficking assay, we demonstrate that Exo70-PI(4,5)P2 interaction is critical for the docking and fusion of post-Golgi secretory vesicles, but not for their transport to the PM.
doi:10.1091/mbc.E07-05-0461
PMCID: PMC2043555  PMID: 17761530
3.  Sec3-containing Exocyst Complex Is Required for Desmosome Assembly in Mammalian Epithelial Cells 
Molecular Biology of the Cell  2010;21(1):152-164.
In epithelial cells, Sec3 associates with Exocyst complexes enriched at desmosomes and centrosomes, distinct from Sec6/8 complexes at the apical junctional complex. RNAi-mediated suppression of Sec3 alters trafficking of desmosomal cadherins and impairs desmosome morphology and function, without noticeable effect on adherens junctions.
The Exocyst is a conserved multisubunit complex involved in the docking of post-Golgi transport vesicles to sites of membrane remodeling during cellular processes such as polarization, migration, and division. In mammalian epithelial cells, Exocyst complexes are recruited to nascent sites of cell–cell contact in response to E-cadherin–mediated adhesive interactions, and this event is an important early step in the assembly of intercellular junctions. Sec3 has been hypothesized to function as a spatial landmark for the development of polarity in budding yeast, but its role in epithelial cells has not been investigated. Here, we provide evidence in support of a function for a Sec3-containing Exocyst complex in the assembly or maintenance of desmosomes, adhesive junctions that link intermediate filament networks to sites of strong intercellular adhesion. We show that Sec3 associates with a subset of Exocyst complexes that are enriched at desmosomes. Moreover, we found that membrane recruitment of Sec3 is dependent on cadherin-mediated adhesion but occurs later than that of the known Exocyst components Sec6 and Sec8 that are recruited to adherens junctions. RNA interference-mediated suppression of Sec3 expression led to specific impairment of both the morphology and function of desmosomes, without noticeable effect on adherens junctions. These results suggest that two different exocyst complexes may function in basal–lateral membrane trafficking and will enable us to better understand how exocytosis is spatially organized during development of epithelial plasma membrane domains.
doi:10.1091/mbc.E09-06-0459
PMCID: PMC2801709  PMID: 19889837
4.  A βPIX-PAK2 complex confers protection against Scrib-dependent and cadherin-mediated apoptosis 
Current biology : CB  2012;22(19):1747-1754.
SUMMARY
Background
During epithelial morphogenesis, a complex comprised of the βPIX (PAK-interacting exchange factor β) and class I PAKs (p21-activated kinases) is recruited to adherens junctions. Scrib, the mammalian orthologue of the Drosophila polarity determinant and tumor suppressor Scribble, binds βPIX directly. Scrib is also targeted to adherens junctions by E-cadherin, where Scrib strengthens cadherin-mediated cell-cell adhesion. While a role for the Scrib-βPIX-PAK signaling complex in promoting membrane protrusion at wound edges has been elucidated, a function for this complex at adherens junctions remains unknown.
Results
Here, we establish that Scrib targets βPIX and PAK2 to adherens junctions where a βPIX-PAK2 complex counterbalances apoptotic stimuli transduced by Scrib and elicited by cadherin-mediated cell-cell adhesion. Moreover, we show that this signaling pathway regulates cell survival in response to osmotic stress. Finally, we determine that in suspension cultures, the Scrib-βPIX-PAK2 complex functions to regulate anoikis elicited by cadherin engagement, with Scrib promoting and the βPIX-PAK2 complex suppressing anoikis, respectively.
Conclusions
Our findings demonstrate that the Scrib-βPIX-PAK2 signaling complex functions as an essential modulator of cell survival when localized to adherens junctions of polarized epithelia. The activity of this complex at adherens junctions is thereby essential for normal epithelial morphogenesis and tolerance of physiological stress. Furthermore, when localized to adherens junctions, the Scrib-βPIX-PAK2 signaling complex serves as a key determinant of anoikis sensitivity, a pivotal mechanism in tumor suppression. Thus, this work also reveals the need to expand the definition of anoikis to include a central role for adherens junctions.
doi:10.1016/j.cub.2012.07.011
PMCID: PMC3470768  PMID: 22863318
5.  ERK1/2 Regulate Exocytosis through Direct Phosphorylation of the Exocyst Component Exo70 
Developmental Cell  2012;22(5):967-978.
The exocyst is a multi-protein complex essential for exocytosis and plasma membrane remodeling. The assembly of the exocyst complex mediates the tethering of post-Golgi secretory vesicles to the plasma membrane prior to fusion. Elucidating the mechanisms regulating exocyst assembly is important for the understanding of exocytosis. Here we show that the exocyst component Exo70 is a direct substrate of the Extracellular signal-Regulated Kinases 1/2 (ERK1/2). ERK1/2 phosphorylation enhances the binding of Exo70 to other exocyst components and promotes the assembly the exocyst complex in response to epidermal growth factor (EGF) signaling. We further demonstrate that ERK1/2 regulates exocytosis as blocking ERK1/2 signaling by a chemical inhibitor or the expression of an Exo70 mutant defective in ERK1/2 phosphorylation inhibited exocytosis. In tumor cells, blocking Exo70 phosphorylation inhibits matrix metalloproteinase secretion and invadopodia formation. ERK1/2 phosphorylation of Exo70 may thus coordinate exocytosis with other cellular events in response to growth factor signaling.
doi:10.1016/j.devcel.2012.03.005
PMCID: PMC3356571  PMID: 22595671
ERK1/2; exocyst; Exo70; vesicle tethering; exocytosis; EGF; invadopodia
6.  Type I gamma phosphatidylinositol phosphate kinase modulates invasion and proliferation and its expression correlates with poor prognosis in breast cancer 
Introduction
The loss of E-cadherin based cell-cell contacts and tumor cell migration to the vasculature and lymphatic system are hallmarks of metastasis of epithelial cancers. Type I gamma phosphatidylinositol phosphate kinase (PIPKIγ), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PI4,5P2) a lipid messenger and precursor to many additional second messengers, was found to regulate E-cadherin cell-cell contacts and growth factor-stimulated directional cell migration, indicating that PIPKIγ regulates key steps in metastasis. Here, we assess the expression of PIPKIγ in breast cancers and have shown that expression correlated with disease progression and outcome.
Methods
Using a tissue microarray, we analyzed 438 breast carcinomas for the levels of PIPKIγ and investigated the correlation of PIPKIγ expression with patient survival via Kaplan-Meier survival analysis. Moreover, via knockdown of the expression of PIPKIγ in cultured breast cancer cells with siRNA, the roles of PIPKIγ in breast cancer migration, invasion, and proliferation were examined.
Results
Tissue microarray data shows that ~18% of the cohort immunostained showed high expression of PIPKIγ. The Kaplan-Meier survival analysis revealed a significant inverse correlation between strong PIPKIγ expression and overall patient survival. Expression of PIPKIγ correlated positively with epidermal growth factor receptor (EGFR) expression, which regulates breast cancer progression and metastasis. In cultured breast cancer cells, PIPKIγ is required for growth factor stimulated migration, invasion, and proliferation of cells.
Conclusions
The results reveal a significant correlation between PIPKIγ expression and the progression of breast cancer. This is consistent with PIPKIγ 's role in breast cancer cell migration, invasion, and proliferation.
doi:10.1186/bcr2471
PMCID: PMC2880426  PMID: 20074374
7.  Lethal giant larvae proteins interact with the exocyst complex and are involved in polarized exocytosis 
The Journal of Cell Biology  2005;170(2):273-283.
The tumor suppressor lethal giant larvae (Lgl) plays a critical role in epithelial cell polarization. However, the molecular mechanism by which Lgl carries out its functions is unclear. In this study, we report that the yeast Lgl proteins Sro7p and Sro77p directly interact with Exo84p, which is a component of the exocyst complex that is essential for targeting vesicles to specific sites of the plasma membrane for exocytosis, and that this interaction is important for post-Golgi secretion. Genetic analyses demonstrate a molecular pathway from Rab and Rho GTPases through the exocyst and Lgl to SNAREs, which mediate membrane fusion. We also found that overexpression of Lgl and t-SNARE proteins not only improves exocytosis but also rescues polarity defects in exocyst mutants. We propose that, although Lgl is broadly distributed in the cells, its localized interaction with the exocyst and kinetic activation are important for the establishment and reenforcement of cell polarity.
doi:10.1083/jcb.200502055
PMCID: PMC2171422  PMID: 16027223
8.  par-1, Atypical pkc, and PP2A/B55 sur-6 Are Implicated in the Regulation of Exocyst-Mediated Membrane Trafficking in Caenorhabditis elegans 
G3: Genes|Genomes|Genetics  2013;4(1):173-183.
The exocyst is a conserved protein complex that is involved in tethering secretory vesicles to the plasma membrane and regulating cell polarity. Despite a large body of work, little is known how exocyst function is controlled. To identify regulators for exocyst function, we performed a targeted RNA interference (RNAi) screen in Caenorhabditis elegans to uncover kinases and phosphatases that genetically interact with the exocyst. We identified seven kinase and seven phosphatase genes that display enhanced phenotypes when combined with hypomorphic alleles of exoc-7 (exo70), exoc-8 (exo84), or an exoc-7;exoc-8 double mutant. We show that in line with its reported role in exocytotic membrane trafficking, a defective exoc-8 caused accumulation of exocytotic soluble NSF attachment protein receptor (SNARE) proteins in both intestinal and neuronal cells in C. elegans. Down-regulation of the phosphatase protein phosphatase 2A (PP2A) phosphatase regulatory subunit sur-6/B55 gene resulted in accumulation of exocytic SNARE proteins SNB-1 and SNAP-29 in wild-type and in exoc-8 mutant animals. In contrast, RNAi of the kinase par-1 caused reduced intracellular green fluorescent protein signal for the same proteins. Double RNAi experiments for par-1, pkc-3, and sur-6/B55 in C. elegans suggest a possible cooperation and involvement in postembryo lethality, developmental timing, as well as SNARE protein trafficking. Functional analysis of the homologous kinases and phosphatases in Drosophila median neurosecretory cells showed that atypical protein kinase C kinase and phosphatase PP2A regulate exocyst-dependent, insulin-like peptide secretion. Collectively, these results characterize kinases and phosphatases implicated in the regulation of exocyst function, and suggest the possibility for interplay between the par-1 and pkc-3 kinases and the PP2A phosphatase regulatory subunit sur-6 in this process.
doi:10.1534/g3.113.006718
PMCID: PMC3887533  PMID: 24192838
Caenorhabditis elegans; exocyst; PP2A; par-1; pkc-3
9.  Exo70 Stimulates the Arp2/3 Complex-mediated Actin Branching for Lamellipodia Formation and Cell Migration 
Current biology : CB  2012;22(16):1510-1515.
Summary
Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling [1,2]. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration [3-9]. Using in vitro actin polymerization assay and time-lapse TIRF microscopy, we found Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors (NPFs). At the cellular level, the stimulatory effect of Exo70 on Arp2/3 is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.
doi:10.1016/j.cub.2012.05.055
PMCID: PMC3427469  PMID: 22748316
10.  In Candida albicans, phosphorylation of Exo84 by Cdk1-Hgc1 is necessary for efficient hyphal extension 
Molecular Biology of the Cell  2014;25(7):1097-1110.
Candida albicans hyphae continue to grow throughout mitosis, and phosphorylation of Exo84 by Cdk is necessary for efficient hyphal growth. In contrast, phosphorylation of Exo84 by Cdk halts cell growth in Saccharomyces cerevisiae. The location of Cdk1 target sites in Exo84 explains how phosphoregulation mediates these different patterns of growth.
The exocyst, a conserved multiprotein complex, tethers secretory vesicles before fusion with the plasma membrane; thus it is essential for cell surface expansion. In both Saccharomyces cerevisiae and mammalian cells, cell surface expansion is halted during mitosis. In S. cerevisiae, phosphorylation of the exocyst component Exo84 by Cdk1-Clb2 during mitosis causes the exocyst to disassemble. Here we show that the hyphae of the human fungal pathogen Candida albicans continue to extend throughout the whole of mitosis. We show that CaExo84 is phosphorylated by Cdk1, which is necessary for efficient hyphal extension. This action of Cdk1 depends on the hyphal-specific cyclin Hgc1, the homologue of G1 cyclins in budding yeast. Phosphorylation of CaExo84 does not alter its localization but does alter its affinity for phosphatidylserine, allowing it to recycle at the plasma membrane. The different action of Cdk1 on CaExo84 and ScExo84 is consistent with the different locations of the Cdk1 target sites in the two proteins. Thus this conserved component of polarized growth has evolved so that its phosphoregulation mediates the dramatically different patterns of growth shown by these two organisms.
doi:10.1091/mbc.E13-11-0688
PMCID: PMC3967973  PMID: 24501427
11.  The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth 
Molecular Biology of the Cell  2014;25(8):1312-1326.
The Neurospora crassa exocyst presents two distinct localization patterns. EXO-70 and -84 colocalize with a region of the Spitzenkörper occupied by secretory macrovesicles. In contrast, SEC-3, -5, -6, -8, and -15 localize distinctively at the apical plasma membrane.
Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.
doi:10.1091/mbc.E13-06-0299
PMCID: PMC3982996  PMID: 24523289
12.  Determinants of phosphatidylinositol-4-phosphate 5-kinase type Iγ90 uropod location in T-lymphocytes and its role in uropod formation 
PeerJ  2013;1:e131.
We have previously identified phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90 as a T cell uropod component. However, the molecular determinants and functional consequences of its localization remain unknown. In this report, we seek to better understand the mechanisms involved in PIPKIγ90 uropod targeting and the role that PIPKIγ90 plays in T cell uropod formation. During T cell activation, PIPKIγ90 cocaps with the membrane microdomain-associated proteins flotillin-1 and -2 and accumulates in the uropod. We report that the C-terminal 26 amino acid extension of PIPKIγ90 is required for its localization to the uropod. We further use T cells from PIPKIγ90−/− mice and human T cells expressing a kinase-dead PIPKIγ90 mutant to examine the role of PIPKIγ90 in a T cell uropod formation. We find that PIPKIγ90 deficient T cells have elongated uropods on ICAM-1. Moreover, in human T cells overexpression of PIPKIγ87, a naturally occurring isoform lacking the last 26 amino acids, suppresses uropod formation and impairs capping of uropod proteins such as flotillins. Transfection of human T cells with a dominant-negative mutant of flotillin-2 in turn attenuates capping of PIPKIγ90. Our data contribute to the understanding of the molecular mechanisms that regulate T cell uropod formation.
doi:10.7717/peerj.131
PMCID: PMC3757496  PMID: 24010013
T cell; Phosphatidylinositol-4-phosphate 5-kinase type I (PIPKI)γ90; PIPKIγ87; Uropod; Flotillin
13.  Conserved Domains of the Nullo Protein Required for Cell-Surface Localization and Formation of Adherens Junctions 
Molecular Biology of the Cell  2002;13(1):146-157.
During cellularization, the Drosophila melanogaster embryo undergoes a transition from syncytial to cellular blastoderm with the de novo generation of a polarized epithelial sheet in the cortex of the embryo. This process couples cytokinesis with the establishment of apical, basal, and lateral membrane domains that are separated by two spatially distinct adherens-type junctions. In nullo mutant embryos, basal junctions fail to form at the onset of cellularization, leading to the failure of cleavage furrow invagination and the generation of multinucleate cells. Nullo is a novel protein that appears to stabilize the initial accumulation of cadherins and catenins as they form a mature basal junction. In this article we characterize a nullo homologue from D. virilis and identify conserved domains of Nullo that are required for basal junction formation. We also demonstrate that Nullo is a myristoylprotein and that the myristate group acts in conjunction with a cluster of basic amino acids to target Nullo to the plasma membrane. The membrane association of Nullo is required in vivo for its role in basal junction formation and for its ability to block apical junction formation when ectopically expressed during late cellularization.
doi:10.1091/mbc.01-08-0418
PMCID: PMC65079  PMID: 11809829
14.  The Type III Toxins of Pseudomonas aeruginosa Disrupt Epithelial Barrier Function▿  
Journal of Bacteriology  2007;190(8):2814-2821.
The type III secreted toxins of Pseudomonas aeruginosa are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to ΔSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance P. aeruginosa's invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.
doi:10.1128/JB.01567-07
PMCID: PMC2293221  PMID: 18165298
15.  Signaling function of α-catenin in microtubule regulation 
Cell cycle (Georgetown, Tex.)  2008;7(15):2377-2383.
Centrosomes control microtubule dynamics in many cell types, and their removal from the cytoplasm leads to a shift from dynamic instability to treadmilling behavior and to a dramatic decrease of microtubule mass (Rodionov et al., 1999; PNAS 96:115). In cadherin-expressing cells, these effects can be reversed: non-centrosomal cytoplasts that form cadherin-mediated adherens junctions display dense arrays of microtubules (Chausovsky et al., 2000; Nature Cell Biol 2:797). In adherens junctions, cadherin’s cytoplasmic domain binds p120 catenin and β-catenin, which in turn binds α-catenin. To elucidate the roles of the cadherin-associated proteins in regulating microtubule dynamics, we prepared GFP-tagged, plasma membrane targeted or untargeted p120 catenin, α-catenin and β-catenin and tested their ability to rescue the loss of microtubule mass caused by centrosomal removal in the poorly adhesive cell line CHO-K1. Only membrane targeting of α-catenin led to a significant increase in microtubule length and density in centrosome-free cytoplasts. Expression of non-membrane-targeted α-catenin produced only a slight effect, while both membrane-targeted and non-targeted p120 and β-catenin were ineffective in this assay. Together, these findings suggest that α-catenin is able to regulate microtubule dynamics in a centrosome-independent manner.
PMCID: PMC2668206  PMID: 18677116
alpha-catenin; microtubules; beta-catenin; p120ctn; adherens junction; centrosome; cadherins; cytoplasts
16.  Adherens junctions in C. elegans embryonic morphogenesis 
Sub-cellular biochemistry  2012;60:279-299.
C. elegans provides a simplified, in vivo model system in which to study adherens junctions and their role in morphogenesis. The core adherens junction components – HMR-1/E-cadherin, HMP-2/β-catenin and HMP-1/α-catenin – were initially identified through genetic screens for mutants with body axis elongation defects. In early embryos, adherens junction proteins are found at sites of contact between blastomeres, and in epithelial cells adherens junction proteins localize to the multifaceted apical junction (CeAJ) – a single structure that combines the adhesive and barrier functions of vertebrate adherens and tight junctions. The apically localized polarity proteins PAR-3 and PAR-6 mediate formation and maturation of junctions, while the basolaterally localized regulator LET-413/Scribble ensures that junctions remain apically positioned. Adherens junctions promote robust adhesion between epithelial cells and provide mechanical resistance for the physical strains of morphogenesis. However, in contrast to vertebrates, C. elegans adherens junction proteins are not essential for general cell adhesion or for epithelial cell polarization. A combination of conserved and novel proteins localizes to the CeAJ and works together with adherens junctions proteins to mediate adhesion.
doi:10.1007/978-94-007-4186-7_12
PMCID: PMC3633470  PMID: 22674076
17.  A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity 
The Journal of Cell Biology  2007;178(2):323-335.
Mechanisms involved in maintaining plasma membrane domains in fully polarized epithelial cells are known, but when and how directed protein sorting and trafficking occur to initiate cell surface polarity are not. We tested whether establishment of the basolateral membrane domain and E-cadherin–mediated epithelial cell–cell adhesion are mechanistically linked. We show that the basolateral membrane aquaporin (AQP)-3, but not the equivalent apical membrane AQP5, is delivered in post-Golgi structures directly to forming cell–cell contacts where it co-accumulates precisely with E-cadherin. Functional disruption of individual components of a putative lateral targeting patch (e.g., microtubules, the exocyst, and soluble N-ethylmaleimide–sensitive factor attachment protein receptors) did not inhibit cell–cell adhesion or colocalization of the other components with E-cadherin, but each blocked AQP3 delivery to forming cell–cell contacts. Thus, components of the lateral targeting patch localize independently of each other to cell–cell contacts but collectively function as a holocomplex to specify basolateral vesicle delivery to nascent cell–cell contacts and immediately initiate cell surface polarity.
doi:10.1083/jcb.200705094
PMCID: PMC2064450  PMID: 17635938
18.  The Ras Target AF-6 Interacts with ZO-1 and Serves as a Peripheral Component of Tight Junctions in Epithelial Cells  
The Journal of Cell Biology  1997;139(3):785-795.
The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.
PMCID: PMC2141704  PMID: 9348294
19.  Reggies/flotillins regulate E-cadherin–mediated cell contact formation by affecting EGFR trafficking 
Molecular Biology of the Cell  2012;23(10):1812-1825.
In epithelial cells, the reggie/flotillin proteins regulate—in association with PrP—the formation of E-cadherin adherens junctions (AJs) via the EGFR. Reggies control the EGF-mediated phosphorylation and internalization of EGFR. EGF signaling at the plasma membrane induces the macropinocytosis of E-cadherin and thus the formation of AJs.
The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.
doi:10.1091/mbc.E11-12-1006
PMCID: PMC3350547  PMID: 22438585
20.  Sinorhizobium meliloti ExoR Is the Target of Periplasmic Proteolysis 
Journal of Bacteriology  2012;194(15):4029-4040.
Sinorhizobium meliloti ExoR regulates the production of succinoglycan and flagella through the ExoS/ChvI two-component regulatory system. ExoR has been proposed to inhibit the ExoS sensor through direct interaction in the periplasm. To understand how ExoR suppression of ExoS is relieved, which is required for the expression of ExoS/ChvI-regulated symbiosis genes, we characterized wild-type ExoR and ExoR95 mutant proteins. In addition to the previously identified precursor and mature forms of ExoR (designated ExoRp and ExoRm, respectively), we detected a 20-kDa form of ExoR (designated ExoRc20) derived from the wild-type ExoR protein, but not from the ExoR95 mutant protein. ExoRc20 was isolated directly from S. meliloti periplasm to identify its N-terminal amino acids and the site of the proteolysis, which is highly conserved among ExoR homologs. ExoRc20 retains the C terminus of the wild-type ExoR. When expressed directly, ExoRc20 did not complement the exoR95 mutation, suggesting that ExoRc20 does not function directly in the ExoR-ExoS/ChvI regulatory pathway and that ExoRm is the functional form of ExoR. A single-amino-acid change (ExoRL81A) at the site of ExoR periplasmic proteolysis resulted in the reduction of the amount of ExoRm and the loss of the regulatory function of the ExoR protein. These findings suggest that ExoRm is a target of periplasmic proteolysis and that the amount of ExoRm could be reduced through effective proteolysis to relieve its suppression of ExoS.
doi:10.1128/JB.00313-12
PMCID: PMC3416547  PMID: 22636773
21.  Membrane association and functional regulation of Sec3 by phospholipids and Cdc42 
The Journal of Cell Biology  2008;180(1):145-158.
The exocyst is an octameric protein complex implicated in tethering post-Golgi secretory vesicles at the plasma membrane in preparation for fusion. However, it is not clear how the exocyst is targeted to and physically associates with specific domains of the plasma membrane and how its functions are regulated at those regions. We demonstrate that the N terminus of the exocyst component Sec3 directly interacts with phosphatidylinositol 4,5-bisphosphate. In addition, we have identified key residues in Sec3 that are critical for its binding to the guanosine triphosphate–bound form of Cdc42. Genetic analyses indicate that the dual interactions of Sec3 with phospholipids and Cdc42 control its function in yeast cells. Disrupting these interactions not only blocks exocytosis and affects exocyst polarization but also leads to defects in cell morphogenesis. We propose that the interactions of Sec3 with phospholipids and Cdc42 play important roles in exocytosis and polarized cell growth.
doi:10.1083/jcb.200704128
PMCID: PMC2213614  PMID: 18195105
22.  Tyrosine phosphorylation of type Iγ phosphatidylinositol phosphate kinase by Src regulates an integrin–talin switch 
The Journal of Cell Biology  2003;163(6):1339-1349.
Engagement of integrin receptors with the extracellular matrix induces the formation of focal adhesions (FAs). Dynamic regulation of FAs is necessary for cells to polarize and migrate. Key interactions between FA scaffolding and signaling proteins are dependent on tyrosine phosphorylation. However, the precise role of tyrosine phosphorylation in FA development and maturation is poorly defined. Here, we show that phosphorylation of type Iγ phosphatidylinositol phosphate kinase (PIPKIγ661) on tyrosine 644 (Y644) is critical for its interaction with talin, and consequently, localization to FAs. PIPKIγ661 is specifically phosphorylated on Y644 by Src. Phosphorylation is regulated by focal adhesion kinase, which enhances the association between PIPKIγ661 and Src. The phosphorylation of Y644 results in an ∼15-fold increase in binding affinity to the talin head domain and blocks β-integrin binding to talin. This defines a novel phosphotyrosine-binding site on the talin F3 domain and a “molecular switch” for talin binding between PIPKIγ661 and β-integrin that may regulate dynamic FA turnover.
doi:10.1083/jcb.200310067
PMCID: PMC2173703  PMID: 14691141
PIPKIγ661; focal adhesion; phosphotyrosine-binding domain; FAK; β-integrin
23.  Adherens Junction: Molecular Architecture and Regulation 
The adherens junction (AJ) is an element of the cell–cell junction in which cadherin receptors bridge the neighboring plasma membranes via their homophilic interactions. Cadherins associate with cytoplasmic proteins, called catenins, which in turn bind to cytoskeletal components, such as actin filaments and microtubules. These molecular complexes further interact with other proteins, including signaling molecules, rendering the AJs into highly dynamic and regulatable structures. The AJs of such nature contribute to the physical linking of cells, as well as to the regulation of cell–cell contacts, which is essential for morphogenesis and remodeling of tissues and organs. Thus, elucidating the molecular architecture of the AJs and their regulatory mechanisms are crucial for understanding how the multicellular system is organized.
Adherens junctions preserve tissue integrity by linking cells and connecting to actin filaments, but they also interact with microtubules and polarity regulators.
doi:10.1101/cshperspect.a002899
PMCID: PMC2882120  PMID: 20457565
24.  Cell Polarity Development and Protein Trafficking in Hepatocytes Lacking E-Cadherin/β-Catenin–based Adherens Junctions 
Molecular Biology of the Cell  2007;18(6):2313-2321.
Using a mutant hepatocyte cell line in which E-cadherin and β-catenin are completely depleted from the cell surface, and, consequently, fail to form adherens junctions, we have investigated adherens junction requirement for apical–basolateral polarity development and polarized membrane trafficking. It is shown that these hepatocytes retain the capacity to form functional tight junctions, develop full apical–basolateral cell polarity, and assemble a subapical cortical F-actin network, although with a noted delay and a defect in subsequent apical lumen remodeling. Interestingly, whereas hepatocytes typically target the plasma membrane protein dipeptidyl peptidase IV first to the basolateral surface, followed by its transcytosis to the apical domain, hepatocytes lacking E-cadherin–based adherens junctions target dipeptidyl peptidase IV directly to the apical surface. Basolateral surface-directed transport of other proteins or lipids tested was not visibly affected in hepatocytes lacking E-cadherin–based adherens junctions. Together, our data show that E-cadherin/β-catenin–based adherens junctions are dispensable for tight junction formation and apical lumen biogenesis but not for apical lumen remodeling. In addition, we suggest a possible requirement for E-cadherin/β-catenin–based adherens junctions with regard to the indirect apical trafficking of specific proteins in hepatocytes.
doi:10.1091/mbc.E06-11-1040
PMCID: PMC1877101  PMID: 17429067
25.  Thinking outside the cell: how cadherins drive adhesion 
Trends in Cell Biology  2012;22(6):299-310.
Cadherins embody a superfamily of cell-surface glycoproteins whose ectodomains contain multiple repeats of β-sandwich EC (extracellular cadherin) domains that adopt a similar fold to immunoglobulin domains. The best characterized cadherins are the vertebrate “classical” cadherins, which mediate adhesion via trans homodimerization between their membrane-distal EC1 domains that extend from apposed cells, and assemble intercellular adherens junctions through cis clustering. To form mature trans adhesive dimers, cadherin domains from apposed cells dimerize in a “strand-swapped” conformation. This occurs in a two-step binding process involving a fast-binding intermediate called the “X-dimer”. Trans dimers are less flexible than cadherin monomers, a factor which drives junction assembly following cell-cell contact by reducing the entropic cost associated with the formation of lateral cis oligomers. Cadherins outside of the classical subfamily appear to have evolved distinct adhesive mechanisms which are just now beginning to be understood.
doi:10.1016/j.tcb.2012.03.004
PMCID: PMC3385655  PMID: 22555008

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