Academic Appointments


Current Research and Scholarly Interests


Our research objectives are to understand the cellular mechanisms involved in the development and maintenance of epithelial cell polarity. Polarized epithelial cells play fundamental roles in the ontogeny and function of a variety of tissues and organs. Recent studies indicate that the development of epithelial cell polarity is a multistage process requiring instructive extracellular cues (eg. cell-cell and cell-substratum contact) and the reorganization of proteins in the cytoplasm and on the plasma membrane. Once established, polarity is maintained by targeting and retention of proteins to functionally distinct apical and basal-lateral plasma membrane domains.

We have developed three strategies to address molecular and biochemical aspects of the mechanisms involved in these processes:

(i). Analysis of membrane and cytoplasmic protein sorting, targeting and distribution in established cell lines of polarized renal epithelia in tissue culture (eg. MDCK cells). Biochemical and molecular analysis of protein sorting into vesicles, transport and docking of vesicles with specialized membrane domains.

(ii). Structural and functional analysis of cell adhesion in epithelial cells. Molecular genetic approaches to disrupt cadherin and associated protein (catenins) functions. Role of cell adhesion in establishing
docking machinery for transport vesicles.

(iii). In vitro studies of protein organization and interactions in purified and reconstituted cell systems.

(iv). In vivo studies of embryonic development of polarized renal epithelia in the normal mouse and the cpk mouse mutant, a murine strain with autosomal recessive polycystic kidney disease in which cell polarity is partially reversed.

2013-14 Courses


Journal Articles


  • ß-catenin at the centrosome: Discrete pools of ß-catenin communicate during mitosis and may co-ordinate centrosome functions and cell cycle progression. BioEssays Mbom, B. C., Nelson, W. J., Barth, A. 2013; 35 (9): 804-809

    Abstract

    Beta-catenin is a multifunctional protein with critical roles in cell-cell adhesion, Wnt-signaling and the centrosome cycle. Whereas the roles of β-catenin in cell-cell adhesion and Wnt-signaling have been studied extensively, the mechanism(s) involving β-catenin in centrosome functions are poorly understood. β-Catenin localizes to centrosomes and promotes mitotic progression. NIMA-related protein kinase 2 (Nek2), which stimulates centrosome separation, binds to and phosphorylates β-catenin. β-Catenin interacting proteins involved in Wnt signaling such as adenomatous polyposis coli, Axin, and GSK3β, are also localized at centrosomes and play roles in promoting mitotic progression. Additionally, proteins associated with cell-cell adhesion sites, such as dynein, regulate mitotic spindle positioning. These roles of proteins at the cell cortex and Wnt signaling that involve β-catenin indicate a cross-talk between different sub-cellular sites in the cell at mitosis, and that different pools of β-catenin may co-ordinate centrosome functions and cell cycle progression.

    View details for DOI 10.1002/bies.201300045

    View details for PubMedID 23804296

  • Microactuator device for integrated measurement of epithelium mechanics BIOMEDICAL MICRODEVICES Mukundan, V., Nelson, W. J., Pruitt, B. L. 2013; 15 (1): 117-123

    Abstract

    Mechanical forces are among important factors that drive cellular function and organization. We present a microfabricated device with on-chip actuation for mechanical testing of single cells. An integrated immersible electrostatic actuator system is demonstrated that applies calibrated forces to cells. We conduct stretching experiments by directly applying forces to epithelial cells adhered to device surfaces functionalized with collagen. We measure mechanical properties including stiffness, hysteresis and visco-elasticity of adherent cells.

    View details for DOI 10.1007/s10544-012-9693-0

    View details for Web of Science ID 000313517800012

    View details for PubMedID 22927158

  • Danio rerio ?E-catenin is a Monomeric F-actin Binding Protein With Distinct Properties from Mus musculus ?E-catenin. The Journal of biological chemistry Miller, P. W., Pokutta, S., Ghosh, A., Almo, S. C., Weis, W. I., Nelson, W. J., Kwiatkowski, A. V. 2013

    Abstract

    It is unknown whether homologs of the cadherin/catenin complex have conserved structures and functions across the Metazoa. Mammalian ?E-catenin is an allosterically regulated actin-binding protein that binds the cadherin/?-catenin complex as a monomer and whose dimerization potentiates F-actin association. We tested whether these functional properties are conserved in another vertebrate, the zebrafish Danio rerio. Here we show, despite 90% sequence identity, that D. rerio and M. musculus ?E-catenin have striking functional differences. We demonstrate that D. rerio ?E-catenin is monomeric using size exclusion chromatography, native-PAGE, and small angle X-ray scattering. D. rerio ?E-catenin binds F-actin in cosedimentation assays as a monomer and as an ?/?-catenin heterodimer complex. D. rerio ?E-catenin also bundles F-actin as shown by negative stained transmission electron microscopy, and does not inhibit Arp2/3 complex-mediated actin nucleation in bulk polymerization assays. Thus, core properties of ?-catenin function - F-actin and ?-catenin binding - are conserved between mouse and zebrafish. We speculate that unique regulatory properties have evolved to match specific developmental requirements.

    View details for PubMedID 23788645

  • An epithelial tissue in Dictyostelium challenges the traditional origin of metazoan multicellularity BIOESSAYS Dickinson, D. J., Nelson, W. J., Weis, W. I. 2012; 34 (10): 833-840

    Abstract

    We hypothesize that aspects of animal multicellularity originated before the divergence of metazoans from fungi and social amoebae. Polarized epithelial tissues are a defining feature of metazoans and contribute to the diversity of animal body plans. The recent finding of a polarized epithelium in the non-metazoan social amoeba Dictyostelium discoideum demonstrates that epithelial tissue is not a unique feature of metazoans, and challenges the traditional paradigm that multicellularity evolved independently in social amoebae and metazoans. An alternative view, presented here, is that the common ancestor of social amoebae, fungi, and animals spent a portion of its life cycle in a multicellular state and possessed molecular machinery necessary for forming an epithelial tissue. Some descendants of this ancestor retained multicellularity, while others reverted to unicellularity. This hypothesis makes testable predictions regarding tissue organization in close relatives of metazoans and provides a novel conceptual framework for studies of early animal evolution.

    View details for DOI 10.1002/bies.201100187

    View details for Web of Science ID 000308712600008

    View details for PubMedID 22930590

  • alpha-Catenin and IQGAP Regulate Myosin Localization to Control Epithelial Tube Morphogenesis in Dictyostelium DEVELOPMENTAL CELL Dickinson, D. J., Robinson, D. N., Nelson, W. J., Weis, W. I. 2012; 23 (3): 533-546

    Abstract

    Apical actomyosin activity in animal epithelial cells influences tissue morphology and drives morphogenetic movements during development. The molecular mechanisms leading to myosin II accumulation at the apical membrane and its exclusion from other membranes are poorly understood. We show that in the nonmetazoan Dictyostelium discoideum, myosin II localizes apically in tip epithelial cells that surround the stalk, and constriction of this epithelial tube is required for proper morphogenesis. IQGAP1 and its binding partner cortexillin I function downstream of ?- and ?-catenin to exclude myosin II from the basolateral cortex and promote apical accumulation of myosin II. Deletion of IQGAP1 or cortexillin compromises epithelial morphogenesis without affecting cell polarity. These results reveal that apical localization of myosin II is a conserved morphogenetic mechanism from nonmetazoans to vertebrates and identify a hierarchy of proteins that regulate the polarity and organization of an epithelial tube in a simple model organism.

    View details for DOI 10.1016/j.devcel.2012.06.008

    View details for Web of Science ID 000308776400011

    View details for PubMedID 22902739

  • E-cadherin is under constitutive actomyosin-generated tension that is increased at cell-cell contacts upon externally applied stretch PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Borghi, N., Sorokina, M., Shcherbakova, O. G., Weis, W. I., Pruitt, B. L., Nelson, W. J., Dunn, A. R. 2012; 109 (31): 12568-12573

    Abstract

    Classical cadherins are transmembrane proteins at the core of intercellular adhesion complexes in cohesive metazoan tissues. The extracellular domain of classical cadherins forms intercellular bonds with cadherins on neighboring cells, whereas the cytoplasmic domain recruits catenins, which in turn associate with additional cytoskeleton binding and regulatory proteins. Cadherin/catenin complexes are hypothesized to play a role in the transduction of mechanical forces that shape cells and tissues during development, regeneration, and disease. Whether mechanical forces are transduced directly through cadherins is unknown. To address this question, we used a Förster resonance energy transfer (FRET)-based molecular tension sensor to test the origin and magnitude of tensile forces transmitted through the cytoplasmic domain of E-cadherin in epithelial cells. We show that the actomyosin cytoskeleton exerts pN-tensile force on E-cadherin, and that this tension requires the catenin-binding domain of E-cadherin and ?E-catenin. Surprisingly, the actomyosin cytoskeleton constitutively exerts tension on E-cadherin at the plasma membrane regardless of whether or not E-cadherin is recruited to cell-cell contacts, although tension is further increased at cell-cell contacts when adhering cells are stretched. Our findings thus point to a constitutive role of E-cadherin in transducing mechanical forces between the actomyosin cytoskeleton and the plasma membrane, not only at cell-cell junctions but throughout the cell surface.

    View details for DOI 10.1073/pnas.1204390109

    View details for Web of Science ID 000307538200062

    View details for PubMedID 22802638

  • Competitive Regulation of E-Cadherin JuxtaMembrane Domain Degradation by p120-Catenin Binding and Hakai-Mediated Ubiquitination PLOS ONE Hartsock, A., Nelson, W. J. 2012; 7 (5)

    Abstract

    p120-Catenin binding to, and Hakai-mediated ubiquitination of the E-cadherin juxtamembrane domain (JMD) are thought to be involved in regulating E-cadherin internalization and degradation. However, the relationship between these two pathways is not understood. We targeted the E-cadherin JMD to mitochondria (WT-JMD) to isolate this domain from the plasma membrane and internalization, and to examine protein modifications and degradation. WT-JMD localized to mitochondria, but did not accumulate there except when proteasome activity was inhibited. We found WT-JMD was ubiquitinated, and arginine substitution of lysines at position 5 (K5R) and 83 (K83R) resulted in the stable accumulation of mutant JMD at mitochondria. p120-Catenin did not localize, or bind to WT-JMD even upon proteasome inhibition, whereas the K5,83R-JMD mutant bound and localized p120-catenin to mitochondria. Mutation of the p120-catenin binding site in combination with these lysine mutations inhibited p120-catenin binding, but did not decrease JMD stability or its accumulation at mitochondria. Thus, increased stability of JMD lysine mutants was due to inhibition of ubiquitination and not to p120-catenin binding. Finally, mutation of these critical lysines in full length E-cadherin had similar effects on protein stability as WT-JMD. Our results indicate that ubiquitination of the JMD inhibits p120-catenin binding, and targets E-cadherin for degradation.

    View details for DOI 10.1371/journal.pone.0037476

    View details for Web of Science ID 000305338500038

    View details for PubMedID 22693575

  • Adherens junction function and regulation during zebrafish gastrulation CELL ADHESION & MIGRATION Schepis, A., Nelson, W. J. 2012; 6 (3): 173-178

    Abstract

    The adherens junction (AJ) comprises multi-protein complexes required for cell-cell adhesion in embryonic development and adult tissue homeostasis. Mutations in key proteins and mis-regulation of AJ adhesive properties can lead to pathologies such as cancer. In recent years, the zebrafish has become an excellent model organism to integrate cell biology in the context of a multicellular organization. The combination of classical genetic approaches with new tools for live imaging and biophysical approaches has revealed new aspects of AJ biology, particularly during zebrafish gastrulation. These studies have resulted in progress in understanding the relationship between cell-cell adhesion, cell migration and plasma membrane blebbing.

    View details for DOI 10.4161/cam.20583

    View details for Web of Science ID 000307868700005

    View details for PubMedID 22568981

  • alpha E-catenin regulates cell-cell adhesion and membrane blebbing during zebrafish epiboly DEVELOPMENT Schepis, A., Sepich, D., Nelson, W. J. 2012; 139 (3): 537-546

    Abstract

    ?E-catenin is an actin-binding protein associated with the E-cadherin-based adherens junction that regulates cell-cell adhesion. Recent studies identified additional E-cadherin-independent roles of ?E-catenin in regulating plasma membrane dynamics and cell migration. However, little is known about the roles of ?E-catenin in these different cellular processes in vivo during early vertebrate development. Here, we examined the functions of ?E-catenin in cell-cell adhesion, cell migration and plasma membrane dynamics during morphogenetic processes that drive epiboly in early Danio rerio (zebrafish) development. We show that depletion of ?E-catenin caused a defect in radial intercalation that was associated with decreased cell-cell adhesion, in a similar manner to E-cadherin depletion. Depletion of ?E-catenin also caused deep cells to have protracted plasma membrane blebbing, and a defect in plasma membrane recruitment of ERM proteins that are involved in controlling membrane-to-cortex attachment and membrane blebbing. Significantly, depletion of both E-cadherin and ?E-catenin suppressed plasma membrane blebbing. We suggest that during radial intercalation the activities of E-cadherin and ?E-catenin in the maintenance of membrane-to-cortex attachment are balanced, resulting in stabilization of cell-cell adhesion and suppression of membrane blebbing, thereby enabling proper radial intercalation.

    View details for DOI 10.1242/dev.073932

    View details for Web of Science ID 000299168700011

    View details for PubMedID 22190637

  • Protein Evolution in Cell and Tissue Development: Going Beyond Sequence and Transcriptional Analysis DEVELOPMENTAL CELL Dickinson, D. J., Weis, W. I., Nelson, W. J. 2011; 21 (1): 32-34

    Abstract

    Studies of animal evolution often focus on sequence and transcriptional analysis, based on an assumption that the evolution of development is driven by changes in gene expression. We argue that biochemical and cell biological approaches are also required, because sequence-conserved proteins can have different biochemical, cellular, and developmental properties.

    View details for DOI 10.1016/j.devcel.2011.06.004

    View details for Web of Science ID 000293310200011

    View details for PubMedID 21763606

  • Ciliary Diffusion Barrier: The Gatekeeper for the Primary Cilium Compartment CYTOSKELETON Hu, Q., Nelson, W. J. 2011; 68 (6): 313-324

    Abstract

    The primary cilium is a cellular antenna that detects and transmits chemical and mechanical cues in the environment through receptors and downstream signal proteins enriched along the ciliary membrane. While it is known that ciliary membrane proteins enter the cilium by way of vesicular and intraflagellar transport, less is known about how ciliary membrane proteins are retained in, and how apical membrane proteins are excluded from the cilium. Here, we review evidence for a membrane diffusion barrier at the base of the primary cilium, and highlight the recent finding of a septin cytoskeleton diffusion barrier. We also discuss candidate ciliopathy genes that may be involved in formation of the barrier, and the role of a diffusion barrier as a common mechanism for compartmentalizing membranes and lipid domains.

    View details for DOI 10.1002/cm.20514

    View details for Web of Science ID 000292942600001

    View details for PubMedID 21634025

  • A Polarized Epithelium Organized by beta- and alpha-Catenin Predates Cadherin and Metazoan Origins SCIENCE Dickinson, D. J., Nelson, W. J., Weis, W. I. 2011; 331 (6022): 1336-1339

    Abstract

    A fundamental characteristic of metazoans is the formation of a simple, polarized epithelium. In higher animals, the structural integrity and functional polarization of simple epithelia require a cell-cell adhesion complex that contains a classical cadherin, the Wnt-signaling protein ?-catenin and the actin-binding protein ?-catenin. We show that the non-metazoan Dictyostelium discoideum forms a polarized epithelium that is essential for multicellular development. Although D. discoideum lacks a cadherin homolog, we identify an ?-catenin ortholog that binds a ?-catenin-related protein. Both proteins are essential for formation of the epithelium, polarized protein secretion, and proper multicellular morphogenesis. Thus, the organizational principles of metazoan multicellularity may be more ancient than previously recognized, and the role of the catenins in cell polarity predates the evolution of Wnt signaling and classical cadherins.

    View details for DOI 10.1126/science.1199633

    View details for Web of Science ID 000288215200050

    View details for PubMedID 21393547

  • Rapid Suppression of Activated Rac1 by Cadherins and Nectins during De Novo Cell-Cell Adhesion PLOS ONE Kitt, K. N., Nelson, W. J. 2011; 6 (3)

    Abstract

    Cell-cell adhesion in simple epithelia involves the engagement of E-cadherin and nectins, and the reorganization of the actin cytoskeleton and membrane dynamics by Rho GTPases, particularly Rac1. However, it remains unclear whether E-cadherin and nectins up-regulate, maintain or suppress Rac1 activity during cell-cell adhesion. Roles for Rho GTPases are complicated by cell spreading and integrin-based adhesions to the extracellular matrix that occur concurrently with cell-cell adhesion, and which also require Rho GTPases. Here, we designed a simple approach to examine Rac1 activity upon cell-cell adhesion by MDCK epithelial cells, without cell spreading or integrin-based adhesion. Upon initiation of cell-cell contact in 3-D cell aggregates, we observed an initial peak of Rac1 activity that rapidly decreased by ?66% within 5 minutes, and further decreased to a low baseline level after 30 minutes. Inhibition of E-cadherin engagement with DECMA-1 Fab fragments or competitive binding of soluble E-cadherin, or nectin2alpha extracellular domain completely inhibited Rac1 activity. These results indicate that cadherins and nectins cooperate to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion, which may be important in inhibiting the migratory cell phenotype and allowing the establishment of initially weak cell-cell adhesions.

    View details for DOI 10.1371/journal.pone.0017841

    View details for Web of Science ID 000288247800034

    View details for PubMedID 21412440

  • CellBio-X: celebrating the interface between Cell Biology and other disciplines TRENDS IN CELL BIOLOGY Nelson, W. J., Hosking, C. R., Alvania, R. 2010; 20 (12): 689-690

    View details for DOI 10.1016/j.tcb.2010.09.014

    View details for Web of Science ID 000285443500001

    View details for PubMedID 21095561

  • VE-cadherin: at the front, center, and sides of endothelial cell organization and function CURRENT OPINION IN CELL BIOLOGY Harris, E. S., Nelson, W. J. 2010; 22 (5): 651-658

    Abstract

    Endothelial cells form cell-cell adhesive structures, called adherens and tight junctions, which maintain tissue integrity, but must be dynamic for leukocyte transmigration during the inflammatory response and cellular remodeling during angiogenesis. This review will focus on Vascular Endothelial (VE)-cadherin, an endothelial-specific cell-cell adhesion protein of the adherens junction complex. VE-cadherin plays a key role in endothelial barrier function and angiogenesis, and consequently VE-cadherin availability and function are tightly regulated. VE-cadherin also participates directly and indirectly in intracellular signaling pathways that control cell dynamics and cell cycle progression. Here we highlight recent work that has advanced our understanding of multiple regulatory and signaling mechanisms that converge on VE-cadherin and have consequences for endothelial barrier function and angiogenic remodeling.

    View details for DOI 10.1016/j.ceb.2010.07.006

    View details for Web of Science ID 000283805700014

    View details for PubMedID 20708398

  • Formation of extra centrosomal structures is dependent on beta-catenin JOURNAL OF CELL SCIENCE Bahmanyar, S., Guiney, E. L., Hatch, E. M., Nelson, W. J., Barth, A. I. 2010; 123 (18): 3125-3135

    Abstract

    beta-Catenin has important roles in cell-cell adhesion and in the regulation of gene transcription. Mutations that stabilize beta-catenin are common in cancer, but it remains unclear how these mutations contribute to cancer progression. beta-Catenin is also a centrosomal component involved in centrosome separation. Centrosomes nucleate interphase microtubules and the bipolar mitotic spindle in normal cells, but their organization and function in human cancers are abnormal. Here, we show that expression of stabilized mutant beta-catenin, which mimics mutations found in cancer, results in extra non-microtubule nucleating structures that contain a subset of centrosome proteins including gamma-tubulin and centrin, but not polo-like kinase 4 (Plk4), SAS-6 or pericentrin. A transcriptionally inactive form of beta-catenin also gives rise to abnormal structures of centrosome proteins. HCT116 human colon cancer cell lines, from which the mutant beta-catenin allele has been deleted, have reduced numbers of cells with abnormal centrosome structures and S-phase-arrested, amplified centrosomes. RNAi-mediated depletion of beta-catenin from centrosomes inhibits S-phase-arrested amplification of centrosomes. These results indicate that beta-catenin is required for centrosome amplification, and mutations in beta-catenin might contribute to the formation of abnormal centrosomes observed in cancers.

    View details for DOI 10.1242/jcs.064782

    View details for Web of Science ID 000281575100011

    View details for PubMedID 20736306

  • In vitro and in vivo reconstitution of the cadherin-catenin-actin complex from Caenorhabditis elegans PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kwiatkowski, A. V., Maiden, S. L., Pokutta, S., Choi, H., Benjamin, J. M., Lynch, A. M., Nelson, W. J., Weis, W. I., Hardin, J. 2010; 107 (33): 14591-14596

    Abstract

    The ternary complex of cadherin, beta-catenin, and alpha-catenin regulates actin-dependent cell-cell adhesion. alpha-Catenin can bind beta-catenin and F-actin, but in mammals alpha-catenin either binds beta-catenin as a monomer or F-actin as a homodimer. It is not known if this conformational regulation of alpha-catenin is evolutionarily conserved. The Caenorhabditis elegans alpha-catenin homolog HMP-1 is essential for actin-dependent epidermal enclosure and embryo elongation. Here we show that HMP-1 is a monomer with a functional C-terminal F-actin binding domain. However, neither full-length HMP-1 nor a ternary complex of HMP-1-HMP-2(beta-catenin)-HMR-1(cadherin) bind F-actin in vitro, suggesting that HMP-1 is auto-inhibited. Truncation of either the F-actin or HMP-2 binding domain of HMP-1 disrupts C. elegans development, indicating that HMP-1 must be able to bind F-actin and HMP-2 to function in vivo. Our study defines evolutionarily conserved properties of alpha-catenin and suggests that multiple mechanisms regulate alpha-catenin binding to F-actin.

    View details for DOI 10.1073/pnas.1007349107

    View details for Web of Science ID 000281287600016

    View details for PubMedID 20689042

  • Adenomatous Polyposis Coli Regulates Endothelial Cell Migration Independent of Roles in beta-Catenin Signaling and Cell-Cell Adhesion MOLECULAR BIOLOGY OF THE CELL Harris, E. S., Nelson, W. J. 2010; 21 (15): 2611-2623

    Abstract

    Adenomatous polyposis coli (APC), a tumor suppressor commonly mutated in cancer, is a cytoskeletal organizer for cell migration and a scaffold for GSK3 beta/CKI-mediated phosphorylation and degradation of the Wnt effector beta-catenin. It remains unclear whether these different APC functions are coupled, or independently regulated and localized. In primary endothelial cells, we show that GSK3 beta/CKI-phosphorylated APC localizes to microtubule-dependent clusters at the tips of membrane extensions. Loss of GSK3 beta/CKI-phosphorylated APC from these clusters correlates with a decrease in cell migration. GSK3 beta/CKI-phosphorylated APC and beta-catenin at clusters is degraded rapidly by the proteasome, but inhibition of GSK3 beta/CKI does not increase beta-catenin-mediated transcription. GSK3 beta/CKI-phosphorylated and -nonphosphorylated APC also localize along adherens junctions, which requires actin and cell-cell adhesion. Significantly, inhibition of cell-cell adhesion results in loss of lateral membrane APC and a concomitant increase in GSK3 beta/CKI-phosphorylated APC in clusters. These results uncouple different APC functions and show that GSK3 beta/CKI phosphorylation regulates APC clusters and cell migration independently of cell-cell adhesion and beta-catenin transcriptional activity.

    View details for DOI 10.1091/mbc.E10-03-0235

    View details for Web of Science ID 000280531400007

    View details for PubMedID 20519433

  • Regulation of cell motile behavior by crosstalk between cadherin- and integrin-mediated adhesions PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Borghi, N., Lowndes, M., Maruthamuthu, V., Gardel, M. L., Nelson, W. J. 2010; 107 (30): 13324-13329

    Abstract

    During normal development and in disease, cohesive tissues undergo rearrangements that require integration of signals from cell adhesions to neighboring cells and to the extracellular matrix (ECM). How a range of cell behaviors is coordinated by these different adhesion complexes is unknown. To analyze epithelial cell motile behavior in response to combinations of cell-ECM and cell-cell adhesion cues, we took a reductionist approach at the single-cell scale by using unique, functionalized micropatterned surfaces comprising alternating stripes of ECM (collagenIV) and adjustable amounts of E-cadherin-Fc (EcadFc). On these surfaces, individual cells spatially segregated integrin- and cadherin-based complexes between collagenIV and EcadFc surfaces, respectively. Cell migration required collagenIV and did not occur on surfaces functionalized with only EcadFc. However, E-cadherin adhesion dampened lamellipodia activity on both collagenIV and EcadFc surfaces and biased the direction of cell migration without affecting the migration rate, all in an EcadFc concentration-dependent manner. Traction force microscopy showed that spatial confinement of integrin-based adhesions to collagenIV stripes induced anisotropic cell traction on collagenIV and migration directional bias. Selective depletion of different pools of alphaE-catenin, an E-cadherin and actin binding protein, identified a membrane-associated pool required for E-cadherin-mediated adhesion and down-regulation of lamellipodia activity and a cytosolic pool that down-regulated the migration rate in an E-cadherin adhesion-independent manner. These results demonstrate that there is crosstalk between E-cadherin- and integrin-based adhesion complexes and that E-cadherin regulates lamellipodia activity and cell migration directionality, but not cell migration rate.

    View details for DOI 10.1073/pnas.1002662107

    View details for Web of Science ID 000280602800027

    View details for PubMedID 20566866

  • A Septin Diffusion Barrier at the Base of the Primary Cilium Maintains Ciliary Membrane Protein Distribution SCIENCE Hu, Q., Milenkovic, L., Jin, H., Scott, M. P., Nachury, M. V., Spiliotis, E. T., Nelson, W. J. 2010; 329 (5990): 436-439

    Abstract

    In animal cells, the primary cilium transduces extracellular signals through signaling receptors localized in the ciliary membrane, but how these ciliary membrane proteins are retained in the cilium is unknown. We found that ciliary membrane proteins were highly mobile, but their diffusion was impeded at the base of the cilium by a diffusion barrier. Septin 2 (SEPT2), a member of the septin family of guanosine triphosphatases that form a diffusion barrier in budding yeast, localized at the base of the ciliary membrane. SEPT2 depletion resulted in loss of ciliary membrane protein localization and Sonic hedgehog signal transduction, and inhibited ciliogenesis. Thus, SEPT2 is part of a diffusion barrier at the base of the ciliary membrane and is essential for retaining receptor-signaling pathways in the primary cilium.

    View details for DOI 10.1126/science.1191054

    View details for Web of Science ID 000280196500036

    View details for PubMedID 20558667

  • alpha E-catenin regulates actin dynamics independently of cadherin-mediated cell-cell adhesion JOURNAL OF CELL BIOLOGY Benjamin, J. M., Kwiatkowski, A. V., Yang, C., Korobova, F., Pokutta, S., Svitkina, T., Weis, W. I., Nelson, W. J. 2010; 189 (2): 339-352

    Abstract

    alphaE-catenin binds the cell-cell adhesion complex of E-cadherin and beta-catenin (beta-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of alphaE-catenin to the E-cadherin-beta-cat complex lowers alphaE-catenin affinity for F-actin, and alphaE-catenin alone can bind F-actin and inhibit Arp2/3 complex-mediated actin polymerization. In cells, to test whether alphaE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic alphaE-catenin pool was sequestered to mitochondria without affecting overall levels of alphaE-catenin or the cadherin-catenin complex. Sequestering cytosolic alphaE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell-cell adhesion. In contrast, sequestration of cytosolic alphaE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of alphaE-catenin regulates actin dynamics independently of cell-cell adhesion.

    View details for DOI 10.1083/jcb.200910041

    View details for Web of Science ID 000276825200016

    View details for PubMedID 20404114

  • Remodeling Epithelial Cell Organization: Transitions Between Front-Rear and Apical-Basal Polarity COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY Nelson, W. J. 2009; 1 (1)

    Abstract

    Polarized epithelial cells have a distinctive apical-basal axis of polarity for vectorial transport of ions and solutes across the epithelium. In contrast, migratory mesenchymal cells have a front-rear axis of polarity. During development, mesenchymal cells convert to epithelia by coalescing into aggregates that undergo epithelial differentiation. Signaling networks and protein complexes comprising Rho family GTPases, polarity complexes (Crumbs, PAR, and Scribble), and their downstream effectors, including the cytoskeleton and the endocytic and exocytic vesicle trafficking pathways, together regulate the distributions of plasma membrane and cytoskeletal proteins between front-rear and apical-basal polarity. The challenge is to understand how these regulators and effectors are adapted to regulate symmetry breaking processes that generate cell polarities that are specialized for different cellular activities and functions.

    View details for DOI 10.1101/cshperspect.a000513

    View details for Web of Science ID 000279845000003

    View details for PubMedID 20066074

  • Cell autonomous defects in cortical development revealed by two-color chimera analysis MOLECULAR AND CELLULAR NEUROSCIENCE Kwiatkowski, A. V., Garner, C. C., Nelson, W. J., Gertler, F. B. 2009; 41 (1): 44-50

    Abstract

    A complex program of cell intrinsic and extrinsic signals guide cortical development. Although genetic studies in mice have uncovered roles for numerous genes and gene families in multiple aspects of corticogenesis, determining their cell autonomous functions is often complicated by pleiotropic defects. Here we describe a novel lentiviral-based method to analyze cell autonomy by generating two-color chimeric mouse embryos. Ena/VASP-deficient mutant and control embryonic stem (ES) cells were labeled with different fluorescent chimeric proteins (EGFP and mCherry) that were modified to bind to the plasma membrane. These labeled ES cells were used to generate two-color chimeric embryos possessing two genetically distinct populations of cortical cells, permitting multiple aspects of neuronal morphogenesis to be analyzed and compared between the two cell populations. We observed little difference between the ability of control and Ena/VASP-deficient cells to contribute to cortical organization during development. In contrast, we observed axon fiber tracts originating from control neurons but not Ena/VASP-deficient neurons, indicating that loss of Ena/VASP causes a cell autonomous defect in cortical axon formation. This technique could be applied to determine other cell autonomous functions in different stages of cortical development.

    View details for DOI 10.1016/j.mcn.2009.01.008

    View details for Web of Science ID 000265715700005

    View details for PubMedID 19386231

  • Resolving cadherin interactions and binding cooperativity at the single-molecule level PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhang, Y., Sivasankar, S., Nelson, W. J., Chu, S. 2009; 106 (1): 109-114

    Abstract

    The cadherin family of Ca(2+)-dependent cell adhesion proteins are critical for the morphogenesis and functional organization of tissues in multicellular organisms, but the molecular interactions between cadherins that are at the core of cell-cell adhesion are a matter of considerable debate. A widely-accepted model is that cadherins adhere in 3 stages. First, the functional unit of cadherin adhesion is a cis dimer formed by the binding of the extracellular regions of 2 cadherins on the same cell surface. Second, formation of low-affinity trans interactions between cadherin cis dimers on opposing cell surfaces initiates cell-cell adhesion. Third, lateral clustering of cadherins cooperatively strengthens intercellular adhesion. Evidence of these cadherin binding states during adhesion is, however, contradictory, and evidence for cooperativity is lacking. We used single-molecule structural (fluorescence resonance energy transfer) and functional (atomic force microscopy) assays to demonstrate directly that cadherin monomers interact via their N-terminal EC1 domain to form trans adhesive complexes. We could not detect the formation of cadherin cis dimers, but found that increasing the density of cadherin monomers cooperatively increased the probability of trans adhesive binding.

    View details for DOI 10.1073/pnas.0811350106

    View details for Web of Science ID 000262263900023

    View details for PubMedID 19114658

  • Role of APC and Its Binding Partners in Regulating Microtubules in Mitosis APC PROTEINS Bahmanyar, S., Nelson, W. J., Barth, A. I. 2009; 656: 65-74

    Abstract

    Adenomatous polyposis coli (APC) is a multifunctional protein commonly mutated in colon cancer. APC contains binding sites for multiple proteins with diverse roles in signaling and the structural and functional organization of cells. Recent evidence suggests roles for APC and some of its binding partners in regulating microtubules in mitosis. APC localizes to three key locations in mitosis: kinetochores, the cortex and centrosomes. Here, we discuss possible mechanisms for APC function at these sites and suggest new pathways by which APC mutations promote tumorigenesis.

    View details for Web of Science ID 000270230600006

    View details for PubMedID 19928353

  • Epithelial cell surface polarity: the early steps FRONTIERS IN BIOSCIENCE-LANDMARK Nejsum, L. N., Nelson, W. J. 2009; 14: 1088-1098

    Abstract

    Establishment and maintenance of epithelial cell surface polarity is of vital importance for the correct function of transporting epithelia. To maintain normal cell function, the distribution of apical and basal-lateral proteins is highly regulated and defects in expression levels or plasma membrane targeting can have severe consequences. It has been shown recently that initiation of cell-surface polarity occurs immediately upon cell-cell contact, and requires components of the lateral targeting patch, the Exocyst and the lateral SNARE complex to specify delivery of basolateral proteins to the site of cell-cell adhesion. The Exocyst and SNARE complex are present in the cytoplasm in single epithelial cells before adhesion. Upon initial cell-cell adhesion, E-cadherin accumulates at the forming contact between cells. Shortly hereafter, components of the lateral targeting patch, the Exocyst and the lateral SNARE complex, co-localize with E-cadherin at the forming contact, where they function in specifying the delivery of basal-lateral.

    View details for DOI 10.2741/3295

    View details for Web of Science ID 000262352400068

    View details for PubMedID 19273117

  • Forchlorfenuron Alters Mammalian Septin Assembly, Organization, and Dynamics JOURNAL OF BIOLOGICAL CHEMISTRY Hu, Q., Nelson, W. J., Spiliotis, E. T. 2008; 283 (43): 29563-29571

    Abstract

    Septins are filamentous GTPases that associate with cell membranes and the cytoskeleton and play essential roles in cell division and cellular morphogenesis. Septins are implicated in many human diseases including cancer and neuropathies. Small molecules that reversibly perturb septin organization and function would be valuable tools for dissecting septin functions and could be used for therapeutic treatment of septin-related diseases. Forchlorfenuron (FCF) is a plant cytokinin previously shown to disrupt septin localization in budding yeast. However, it is unknown whether FCF directly targets septins and whether it affects septin organization and functions in mammalian cells. Here, we show that FCF alters septin assembly in vitro without affecting either actin or tubulin polymerization. In live mammalian cells, FCF dampens septin dynamics and induces the assembly of abnormally large septin structures. FCF has a low level of cytotoxicity, and these effects are reversed upon FCF washout. Significantly, FCF treatment induces mitotic and cell migration defects that phenocopy the effects of septin depletion by small interfering RNA. We conclude that FCF is a promising tool to study mammalian septin organization and functions.

    View details for DOI 10.1074/jbc.M804962200

    View details for Web of Science ID 000260179900082

    View details for PubMedID 18713753

  • Role of adenomatous polyposis coli (APC) and microtubules in directional cell migration and neuronal polarization SEMINARS IN CELL & DEVELOPMENTAL BIOLOGY Barth, A. I., Caro-Gonzalez, H. Y., Nelson, W. J. 2008; 19 (3): 245-251

    Abstract

    In response to extracellular signals during embryonic development, cells undergo directional movements to specific sites and establish proper connections to other cells to form organs and tissues. Cell extension and migration in the direction of extracellular cues is mediated by the actin and microtubule cytoskeletons, and recent results have shed new light on how these pathways are activated by neurotrophins, Wnt or extracellular matrix. These signals lead to modifications of microtubule-associated proteins (MAPs) and point to glycogen synthase kinase (GSK) 3beta as a key regulator of microtubule function during directional migration. This review will summarize these results and then focus on the role of microtubule-binding protein adenomatous polyposis coli (APC) in neuronal polarization and directed migration, and on its regulation by GSK3beta.

    View details for DOI 10.1016/j.semcdb.2008.02.003

    View details for Web of Science ID 000255730600005

    View details for PubMedID 18387324

  • Biochemical and structural analysis of alpha-catenin in cell-cell contacts BIOCHEMICAL SOCIETY TRANSACTIONS Pokutta, S., Drees, F., Yamada, S., Nelson, W. J., Weis, W. I. 2008; 36: 141-147

    Abstract

    Cadherins are transmembrane adhesion molecules that mediate homotypic cell-cell contact. In adherens junctions, the cytoplasmic domain of cadherins is functionally linked to the actin cytoskeleton through a series of proteins known as catenins. E-cadherin binds to beta-catenin, which in turn binds to alpha-catenin to form a ternary complex. alpha-Catenin also binds to actin, and it was assumed previously that alpha-catenin links the cadherin-catenin complex to actin. However, biochemical, structural and live-cell imaging studies of the cadherin-catenin complex and its interaction with actin show that binding of beta-catenin to alpha-catenin prevents the latter from binding to actin. Biochemical and structural data indicate that alpha-catenin acts as an allosteric protein whose conformation and activity changes depending on whether or not it is bound to beta-catenin. Initial contacts between cells occur on dynamic lamellipodia formed by polymerization of branched actin networks, a process controlled by the Arp2/3 (actin-related protein 2/3) complex. alpha-Catenin can suppress the activity of Arp2/3 by competing for actin filaments. These findings lead to a model for adherens junction formation in which clustering of the cadherin-beta-catenin complex recruits high levels of alpha-catenin that can suppress the Arp2/3 complex, leading to cessation of lamellipodial movement and formation of a stable contact. Thus alpha-catenin appears to play a central role in cell-cell contact formation.

    View details for DOI 10.1042/BST0360141

    View details for Web of Science ID 000255371000001

    View details for PubMedID 18363554

  • Regulation of cell-cell adhesion by the cadherin-catenin complex BIOCHEMICAL SOCIETY TRANSACTIONS Nelson, W. J. 2008; 36: 149-155

    Abstract

    Ca(2+)-dependent cell-cell adhesion is regulated by the cadherin family of cell adhesion proteins. Cadherins form trans-interactions on opposing cell surfaces which result in weak cell-cell adhesion. Stronger cell-cell adhesion occurs by clustering of cadherins and through changes in the organization of the actin cytoskeleton. Although cadherins were thought to bind directly to the actin cytoskeleton through cytoplasmic proteins, termed alpha- and beta-catenin, recent studies with purified proteins indicate that the interaction is not direct, and instead an allosteric switch in alpha-catenin may mediate actin cytoskeleton reorganization. Organization and function of the cadherin-catenin complex are additionally regulated by phosphorylation and endocytosis. Direct studies of cell-cell adhesion has revealed that the cadherin-catenin complex and the underlying actin cytoskeleton undergo a series of reorganizations that are controlled by the Rho GTPases, Rac1 and RhoA, that result in the expansion and completion of cell-cell adhesion. In the present article, in vitro protein assembly studies and live-cell studies of de novo cell-cell adhesion are discussed in the context of how the cadherin-catenin complex and the actin cytoskeleton regulate cell-cell adhesion.

    View details for DOI 10.1042/BST0360149

    View details for Web of Science ID 000255371000002

    View details for PubMedID 18363555

  • Adherens and tight junctions: Structure, function and connections to the actin cytoskeleton BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES Hartsock, A., Nelson, W. J. 2008; 1778 (3): 660-669

    Abstract

    Adherens junctions and Tight junctions comprise two modes of cell-cell adhesion that provide different functions. Both junctional complexes are proposed to associate with the actin cytoskeleton, and formation and maturation of cell-cell contacts involves reorganization of the actin cytoskeleton. Adherens junctions initiate cell-cell contacts, and mediate the maturation and maintenance of the contact. Adherens junctions consist of the transmembrane protein E-cadherin, and intracellular components, p120-catenin, beta-catenin and alpha-catenin. Tight junctions regulate the paracellular pathway for the movement of ions and solutes in-between cells. Tight junctions consist of the transmembrane proteins occludin and claudin, and the cytoplasmic scaffolding proteins ZO-1, -2, and -3. This review discusses the binding interactions of the most studied proteins that occur within each of these two junctional complexes and possible modes of regulation of these interactions, and the different mechanisms that connect and regulate interactions with the actin cytoskeleton.

    View details for DOI 10.1016/j.bbamem.2007.07.012

    View details for Web of Science ID 000254691500009

    View details for PubMedID 17854762

  • Immediate-early signaling induced by E-cadherin engagement and adhesion JOURNAL OF BIOLOGICAL CHEMISTRY Perez, T. D., Tamada, M., Sheetz, M. P., Nelson, W. J. 2008; 283 (8): 5014-5022

    Abstract

    Epithelial cell-cell interactions require localized adhesive interactions between E-cadherin on opposing membranes and the activation of downstream signaling pathways that affect membrane and actin dynamics. However, it is not known whether E-cadherin engagement and activation of these signaling pathways are locally coordinated or whether signaling is sustained or locally down-regulated like other receptor-mediated pathways. To obtain high spatiotemporal resolution of immediate-early signaling events upon E-cadherin engagement, we used laser tweezers to place beads coated with functional E-cadherin extracellular domain on cells. We show that cellular E-cadherin accumulated rapidly around beads, reaching a sustained plateau level in 1-3 min. Phosphoinositides and Rac1 co-accumulated with E-cadherin, reached peak levels with E-cadherin, but then rapidly dispersed. Both E-cadherin and Rac1 accumulated independently of Rac1 GTP binding/hydrolysis, but these activities were required for Rac1 dispersal. E-cadherin accumulation was dependent on membrane dynamics and actin polymerization, but actin did not stably co-accumulate with E-cadherin; mathematical modeling showed that diffusion-mediated trapping could account for the initial E-cadherin accumulation. We propose that initial E-cadherin accumulation requires active membrane dynamics and involves diffusion-mediated trapping at contact sites; to propagate further contacts, phosphatidylinositol 3-kinase and Rac1 are transiently activated by E-cadherin engagement and initiate a new round of membrane dynamics, but they are subsequently suppressed at that site to allow maintenance of weak E-cadherin mediated adhesion.

    View details for DOI 10.1074/jbc.M705209200

    View details for Web of Science ID 000253426500059

    View details for PubMedID 18089563

  • Bench to bedside and back again: Molecular mechanisms of alpha-catenin function and roles in tumorigenesis SEMINARS IN CANCER BIOLOGY Benjamin, J. M., Nelson, W. J. 2008; 18 (1): 53-64

    Abstract

    The cadherin/catenin complex, comprised of E-cadherin, beta-catenin and alpha-catenin, is essential for initiating cell-cell adhesion, establishing cellular polarity and maintaining tissue organization. Disruption or loss of the cadherin/catenin complex is common in cancer. As the primary cell-cell adhesion protein in epithelial cells, E-cadherin has long been studied in cancer progression. Similarly, additional roles for beta-catenin in the Wnt signaling pathway has led to many studies of the role of beta-catenin in cancer. Alpha-catenin, in contrast, has received less attention. However, recent data demonstrate novel functions for alpha-catenin in regulating the actin cytoskeleton and cell-cell adhesion, which when perturbed could contribute to cancer progression. In this review, we use cancer data to evaluate molecular models of alpha-catenin function, from the canonical role of alpha-catenin in cell-cell adhesion to non-canonical roles identified following conditional alpha-catenin deletion. This analysis identifies alpha-catenin as a prognostic factor in cancer progression.

    View details for DOI 10.1016/j.semcancer.2007.08.003

    View details for Web of Science ID 000253064200007

    View details for PubMedID 17945508

  • Epithelial polarity requires septin coupling of vesicle transport to polyglutamylated microtubules JOURNAL OF CELL BIOLOGY Spiliotis, E. T., Hunt, S. J., Hu, Q., Kinoshita, M., Nelson, W. J. 2008; 180 (2): 295-303

    Abstract

    In epithelial cells, polarized growth and maintenance of apical and basolateral plasma membrane domains depend on protein sorting from the trans-Golgi network (TGN) and vesicle delivery to the plasma membrane. Septins are filamentous GTPases required for polarized membrane growth in budding yeast, but whether they function in epithelial polarity is unknown. Here, we show that in epithelial cells septin 2 (SEPT2) fibers colocalize with a subset of microtubule tracks composed of polyglutamylated (polyGlu) tubulin, and that vesicles containing apical or basolateral proteins exit the TGN along these SEPT2/polyGlu microtubule tracks. Tubulin-associated SEPT2 facilitates vesicle transport by maintaining polyGlu microtubule tracks and impeding tubulin binding of microtubule-associated protein 4 (MAP4). Significantly, this regulatory step is required for polarized, columnar-shaped epithelia biogenesis; upon SEPT2 depletion, cells become short and fibroblast-shaped due to intracellular accumulation of apical and basolateral membrane proteins, and loss of vertically oriented polyGlu microtubules. We suggest that septin coupling of the microtubule cytoskeleton to post-Golgi vesicle transport is required for the morphogenesis of polarized epithelia.

    View details for Web of Science ID 000252746900008

    View details for PubMedID 18209106

  • beta-Catenin is a Nek2 substrate involved in centrosome separation GENES & DEVELOPMENT Bahmanyar, S., Kaplan, D. D., DeLuca, J. G., Giddings, T. H., O'Toole, E. T., Winey, M., Salmon, E. D., Casey, P. J., Nelson, W. J., Barth, A. I. 2008; 22 (1): 91-105

    Abstract

    beta-Catenin plays important roles in cell adhesion and gene transcription, and has been shown recently to be essential for the establishment of a bipolar mitotic spindle. Here we show that beta-catenin is a component of interphase centrosomes and that stabilization of beta-catenin, mimicking mutations found in cancers, induces centrosome splitting. Centrosomes are held together by a dynamic linker regulated by Nek2 kinase and its substrates C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin. We show that beta-catenin binds to and is phosphorylated by Nek2, and is in a complex with Rootletin. In interphase, beta-catenin colocalizes with Rootletin between C-Nap1 puncta at the proximal end of centrioles, and this localization is dependent on C-Nap1 and Rootletin. In mitosis, when Nek2 activity increases, beta-catenin localizes to centrosomes at spindle poles independent of Rootletin. Increased Nek2 activity disrupts the interaction of Rootletin with centrosomes and results in binding of beta-catenin to Rootletin-independent sites on centrosomes, an event that is required for centrosome separation. These results identify beta-catenin as a component of the intercentrosomal linker and define a new function for beta-catenin as a key regulator of mitotic centrosome separation.

    View details for DOI 10.1101/gad.1596308

    View details for Web of Science ID 000252095700010

    View details for PubMedID 18086858

  • Parallels between global transcriptional programs of polarizing caco-2 intestinal epithelial cells in vitro and gene expression programs in normal colon and colon cancer MOLECULAR BIOLOGY OF THE CELL Saeaef, A. M., Halbleib, J. M., Chen, X., Yuen, S. T., Leung, S. Y., Nelson, W. J., Brown, P. O. 2007; 18 (11): 4245-4260

    Abstract

    Posttranslational mechanisms are implicated in the development of epithelial cell polarity, but little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized temporal patterns of gene expression during cell-cell adhesion-initiated polarization of cultured human Caco-2 cells, which develop structural and functional polarity resembling enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts. Comparison to gene expression patterns in normal human colon and colon tumors revealed that the pattern in proliferating, nonpolarized Caco-2 cells paralleled patterns seen in human colon cancer in vivo, including expression of genes involved in cell proliferation. The pattern switched in polarized Caco-2 cells to one more closely resembling that in normal colon tissue, indicating that regulation of transcription underlying Caco-2 cell polarization is similar to that during enterocyte differentiation in vivo. Surprisingly, the temporal program of gene expression in polarizing Caco-2 cells involved changes in signaling pathways (e.g., Wnt, Hh, BMP, FGF) in patterns similar to those during migration and differentiation of intestinal epithelial cells in vivo, despite the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. The full data set is available at http://microarray-pubs.stanford.edu/CACO2.

    View details for DOI 10.1091/mbc.E07-04-0309

    View details for Web of Science ID 000250740000005

    View details for PubMedID 17699589

  • Transcriptional modulation of genes encoding structural characteristics of differentiating Enterocytes during development of a polarized epithelium in vitro MOLECULAR BIOLOGY OF THE CELL Halbleib, J. M., Saeaef, A. M., Brown, P. O., Nelson, W. J. 2007; 18 (11): 4261-4278

    Abstract

    Although there is considerable evidence implicating posttranslational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of genes involved in cell proliferation was down-regulated concomitant with induction of genes necessary for functional specialization of polarized epithelial cells. Transcriptional up-regulation of these latter genes correlated with formation of important structural and functional features in enterocyte differentiation and establishment of structural and functional cell polarity; components of the apical microvilli were induced as the brush border formed during polarization; as barrier function was established, expression of tight junction transmembrane proteins peaked; transcripts encoding components of the apical, but not the basal-lateral trafficking machinery were increased during polarization. Coordinated expression of genes encoding components of functional cell structures were often observed indicating temporal control of expression and assembly of multiprotein complexes.

    View details for DOI 10.1091/mbc.E07-04-0308

    View details for Web of Science ID 000250740000006

    View details for PubMedID 17699590

  • Catenins: playing both sides of the synapse CURRENT OPINION IN CELL BIOLOGY Kwiatkowski, A. V., Weis, W. I., Nelson, W. J. 2007; 19 (5): 551-556

    Abstract

    Synapses of the central nervous system (CNS) are specialized cell-cell junctions that mediate intercellular signal transmission from one neuron to another. The directional nature of signal relay requires synaptic contacts to be morphologically asymmetric with distinct protein components, while changes in synaptic communication during neural network formation require synapses to be plastic. Synapse morphology and plasticity require a dynamic actin cytoskeleton. Classical cadherins, which are junctional proteins associated with the actin cytoskeleton, localize to synapses and regulate synaptic adhesion, stability and remodeling. The major intracellular components of cadherin junctions are the catenin proteins, and increasing evidence suggests that cadherin-catenin complexes modulate an array of synaptic processes. Here we review the role of catenins in regulating the development of pre- and postsynaptic compartments and function in synaptic plasticity, with particular focus on their role in regulating the actin cytoskeleton.

    View details for DOI 10.1016/j.ceb.2007.08.005

    View details for Web of Science ID 000251164200009

    View details for PubMedID 17936606

  • Fabrication of a dual substrate display to test roles of cell adhesion proteins in vesicle targeting to plasma membrane domains FEBS LETTERS Hunt, S. J., Nelson, W. J. 2007; 581 (23): 4539-4543

    Abstract

    While much is known of the molecular machinery involved in protein sorting during exocytosis, less is known about the spatial regulation of exocytosis at the plasma membrane (PM). This study outlines a novel method, dual substrate display, used to formally test the hypothesis that E-cadherin-mediated adhesion directs basolateral vesicle exocytosis to specific sites at the PM. We show that vesicles containing the basolateral marker protein VSV-G preferentially target to sites of adhesion to E-cadherin rather than collagen VI or a control peptide. These results support the hypothesis that E-cadherin adhesion initiates signaling at the PM resulting in targeted sites for exocytosis.

    View details for DOI 10.1016/j.febslet.2007.08.037

    View details for Web of Science ID 000249864200029

    View details for PubMedID 17803993

  • Localized zones of Rho and Rac activities drive initiation and expansion of epithelial cell-cell adhesion JOURNAL OF CELL BIOLOGY Yamada, S., Nelson, W. J. 2007; 178 (3): 517-527

    Abstract

    Spatiotemporal coordination of cell-cell adhesion involving lamellipodial interactions, cadherin engagement, and the lateral expansion of the contact is poorly understood. Using high-resolution live-cell imaging, biosensors, and small molecule inhibitors, we investigate how Rac1 and RhoA regulate actin dynamics during de novo contact formation between pairs of epithelial cells. Active Rac1, the Arp2/3 complex, and lamellipodia are initially localized to de novo contacts but rapidly diminish as E-cadherin accumulates; further rounds of activation and down-regulation of Rac1 and Arp2/3 occur at the contacting membrane periphery, and this cycle repeats as a restricted membrane zone that moves outward with the expanding contact. The cortical bundle of actin filaments dissolves beneath the expanding contacts, leaving actin bundles at the contact edges. RhoA and actomyosin contractility are activated at the contact edges and are required to drive expansion and completion of cell-cell adhesion. We show that zones of Rac1 and lamellipodia activity and of RhoA and actomyosin contractility are restricted to the periphery of contacting membranes and together drive initiation, expansion, and completion of cell-cell adhesion.

    View details for DOI 10.1083/jcb.200701058

    View details for Web of Science ID 000248544500018

    View details for PubMedID 17646397

  • A molecular mechanism directly linking E-cadherin adhesion to initiation of epithelial cell surface polarity JOURNAL OF CELL BIOLOGY Nejsum, L. N., Nelson, W. J. 2007; 178 (2): 323-335

    Abstract

    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.

    View details for DOI 10.1083/jcb.200705094

    View details for Web of Science ID 000248151500013

    View details for PubMedID 17635938

  • Characterization of mammalian Par 6 as a dual-location protein MOLECULAR AND CELLULAR BIOLOGY Cline, E. G., Nelson, W. J. 2007; 27 (12): 4431-4443

    Abstract

    Par 6 acts as a scaffold protein to facilitate atypical protein kinase C-mediated phosphorylation of cytoplasmic protein complexes, leading to epithelial and neuronal cell polarization. In addition to its location in the cytoplasm, Par 6 is localized to the nucleus. However, its organization and potential functions in the nucleus have not been examined. Using an affinity-purified Par 6 antibody and a chimera of Par 6 and green fluorescent protein, we show that Par 6 localizes precisely to nuclear speckles, but not to other nuclear structures, and displays characteristics of speckle proteins. We show that Par 6 colocalizes in the nucleus with Tax, a transcriptional activator of the human T-cell leukemia virus type 1 long terminal repeat, but multiple lines of evidence show that Par 6 is not directly involved in known functions of speckle proteins, including general transcription, splicing, or mRNA transport. Significantly, however, the structure of nuclear speckles is lost when Par 6 levels are reduced by Par 6-specific small interfering RNA. Therefore, we hypothesize that Par 6 in the nucleus acts as a scaffolding protein in nuclear speckle complexes, similar to its role in the cytoplasm.

    View details for DOI 10.1128/MCB.02235-06

    View details for Web of Science ID 000247150000021

    View details for PubMedID 17420281

  • Separation of cell-cell adhesion complexes by differential centrifugation. Methods in molecular biology (Clifton, N.J.) Vogelmann, R., Nelson, W. J. 2007; 370: 11-22

    Abstract

    The number of proteins found associated with cell-cell adhesion substructures is growing rapidly. Based on potential protein-protein interactions, complex protein networks at cell-cell contacts can be modeled. Traditional studies to examine protein-protein interactions include co-immunoprecipitation or pull-down experiments of tagged proteins. These studies provide valuable information that proteins can associate directly or indirectly through other proteins in a complex. However, they do not clarify if a given protein is part of other protein complexes or inform about the specificity of those interactions in the context of adhesion substructures. Thus, it is not clear if models compiled from these types of studies reflect the combination of protein interactions in the adhesion complex in vivo for a specific cell type. Therefore, we present here a method to separate cell-cell contact membrane substructures with their associated protein complexes based on their buoyant behavior in iodixanol density gradients. Analysis of 16 proteins of the apical junctional complex (AJC) in epithelial Madin-Darby canine kidney cells revealed a more simple organization of the AJC adhesion complex than that predicted from the combination of all possible protein-protein interactions defined from co-immunoprecipitation and pull-down experiments.

    View details for PubMedID 17416984

  • Cadherins in development: cell adhesion, sorting, and tissue morphogenesis GENES & DEVELOPMENT Halbleib, J. M., Nelson, W. J. 2006; 20 (23): 3199-3214

    Abstract

    Tissue morphogenesis during development is dependent on activities of the cadherin family of cell-cell adhesion proteins that includes classical cadherins, protocadherins, and atypical cadherins (Fat, Dachsous, and Flamingo). The extracellular domain of cadherins contains characteristic repeats that regulate homophilic and heterophilic interactions during adhesion and cell sorting. Although cadherins may have originated to facilitate mechanical cell-cell adhesion, they have evolved to function in many other aspects of morphogenesis. These additional roles rely on cadherin interactions with a wide range of binding partners that modify their expression and adhesion activity by local regulation of the actin cytoskeleton and diverse signaling pathways. Here we examine how different members of the cadherin family act in different developmental contexts, and discuss the mechanisms involved.

    View details for DOI 10.1101/gad.1486806

    View details for Web of Science ID 000242527100001

    View details for PubMedID 17158740

  • Re-solving the cadherin-catenin-actin conundrum JOURNAL OF BIOLOGICAL CHEMISTRY Weis, W. I., Nelson, W. J. 2006; 281 (47): 35593-35597

    View details for DOI 10.1074/jbc.R600027200

    View details for Web of Science ID 000242100500002

    View details for PubMedID 17005550

  • Wingless signaling modulates cadherin-mediated cell adhesion in Drosophila imaginal disc cells JOURNAL OF CELL SCIENCE Wodarz, A., Stewart, D. B., Nelson, W. J., Nusse, R. 2006; 119 (12): 2425-2434

    Abstract

    Armadillo, the Drosophila homolog of beta-catenin, plays a crucial role in both the Wingless signal transduction pathway and cadherin-mediated cell-cell adhesion, raising the possibility that Wg signaling affects cell adhesion. Here, we use a tissue culture system that allows conditional activation of the Wingless signaling pathway and modulation of E-cadherin expression levels. We show that activation of the Wingless signaling pathway leads to the accumulation of hypophosphorylated Armadillo in the cytoplasm and in cellular processes, and to a concomitant reduction of membrane-associated Armadillo. Activation of the Wingless pathway causes a loss of E-cadherin from the cell surface, reduced cell adhesion and increased spreading of the cells on the substratum. After the initial loss of E-cadherin from the cell surface, E-cadherin gene expression is increased by Wingless. We suggest that Wingless signaling causes changes in Armadillo levels and subcellular localization that result in a transient reduction of cadherin-mediated cell adhesion, thus facilitating cell shape changes, division and movement of cells in epithelial tissues.

    View details for DOI 10.1242/jcs.02973

    View details for Web of Science ID 000238413500004

    View details for PubMedID 16720643

  • Here come the septins: novel polymers that coordinate intracellular functions and organization JOURNAL OF CELL SCIENCE Spiliotis, E. T., NELSON, W. J. 2006; 119 (1): 4-10

    Abstract

    Septins are conserved GTP-binding proteins that associate with cellular membranes and the actin and microtubule cytoskeletons. They polymerize to form filamentous structures that act as diffusion barriers between different membrane domains and as molecular scaffolds for membrane- and cytoskeleton-binding proteins. In yeast, septins are central to the spatio-temporal coordination of membrane polarity and cell division, but the roles of their mammalian counterparts have remained poorly understood. However, recent findings have shed light on the dynamics and regulation of mammalian septin assembly and our understanding of septin functions in cytoskeleton and membrane organization. The mammalian septins appear to form a novel network of hetero-polymers that are multi-functional, inter-changeable and respond dynamically to signals that coordinate events at the interface between cytoskeleton and membrane biology. Hence, studies of these molecules might provide new insights not only into how cells coordinate their functions, but also into the pathogenesis of cancer and other diseases in which septins are abnormally expressed.

    View details for DOI 10.1242/jcs.02746

    View details for Web of Science ID 000234901200003

    View details for PubMedID 16371649

  • Neurite outgrowth involves adenomatous polyposis coil protein and beta-catenin JOURNAL OF CELL SCIENCE Votin, V., NELSON, W. J., Barth, A. I. 2005; 118 (24): 5699-5708

    Abstract

    Neuronal morphogenesis involves the initial formation of neurites and then differentiation of neurites into axons and dendrites. The mechanisms underlying neurite formation are poorly understood. A candidate protein for controlling neurite extension is the adenomatous polyposis coli (APC) protein, which regulates membrane extensions, microtubules and beta-catenin-mediated transcription downstream of Wnt signaling. APC is enriched at the tip of several neurites of unpolarized hippocampal neurons and the tip of only the long axon in polarized hippocampal neurons. Significantly, APC localized to the tip of only one neurite, marked by dephospho-tau as the future axon, before that neurite had grown considerably longer than other neurites. To determine whether neurite outgrowth was affected by beta-catenin accumulation and signaling, a stabilized beta-catenin mutant was expressed in PC12 cells, and neurite formation was measured. Stabilized beta-catenin mutants accumulated in APC clusters and inhibited neurite formation and growth. Importantly, these effects were also observed was independently of the gene transcriptional activity of beta-catenin. These results indicate that APC is involved in both early neurite outgrowth and increased growth of the future axon, and that beta-catenin has a structural role in inhibiting APC function in neurite growth.

    View details for DOI 10.1242/jcs.02679

    View details for Web of Science ID 000234811600006

    View details for PubMedID 16303851

  • E-cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion LANGMUIR Perez, T. D., NELSON, W. J., Boxer, S. G., Kam, L. 2005; 21 (25): 11963-11968

    Abstract

    Cell-cell adhesion is a dynamic process requiring recruitment, binding, and reorganization of signaling proteins in the plane of the plasma membrane. Here, we describe a new system for investigating how this lateral mobility influences cadherin-based cell signaling. This model is based on tethering of a GPI-modified E-cadherin protein (hEFG) to a supported lipid bilayer. In this report, membrane microfluidics and micropatterning techniques are used to adopt this tethered protein system for studies with the anchorage-dependent cells. As directly formed from proteoliposomes, hEFG exhibits a diffusion coefficient of 0.6 +/- 0.3 microm(2)/s and mobile fraction of 30-60%. Lateral structuring of the supported lipid bilayer is used to isolate mobile proteins from this mixed mobile/immobile population, and should be widely applicable to other proteins. MCF-7 cells seeded onto hEFG-containing bilayers recognize and cluster this protein, but do not exhibit cell spreading required for survival. By micropatterning small anchors into the supported lipid bilayer, we have achieved cell spreading across the bilayer surface and concurrent interaction with mobile hEFG protein. Together, these techniques will allow more detailed analysis of the cellular dynamics involved in cadherin-dependent adhesion events.

    View details for DOI 10.1021/la052264a

    View details for Web of Science ID 000233730200063

    View details for PubMedID 16316139

  • Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells JOURNAL OF CELL BIOLOGY Reilein, A., Yamada, S., Nelson, W. J. 2005; 171 (5): 845-855

    Abstract

    Mechanisms underlying the organization of centrosome-derived microtubule arrays are well understood, but less is known about how acentrosomal microtubule networks are formed. The basal cortex of polarized epithelial cells contains a microtubule network of mixed polarity. We examined how this network is organized by imaging microtubule dynamics in acentrosomal basal cytoplasts derived from these cells. We show that the steady-state microtubule network appears to form by a combination of microtubule-microtubule and microtubule-cortex interactions, both of which increase microtubule stability. We used computational modeling to determine whether these microtubule parameters are sufficient to generate a steady-state acentrosomal microtubule network. Microtubules undergoing dynamic instability without any stabilization points continuously remodel their organization without reaching a steady-state network. However, the addition of increased microtubule stabilization at microtubule-microtubule and microtubule-cortex interactions results in the rapid assembly of a steady-state microtubule network in silico that is remarkably similar to networks formed in situ. These results define minimal parameters for the self-organization of an acentrosomal microtubule network.

    View details for DOI 10.1083/jcb.200505071

    View details for Web of Science ID 000233753700011

    View details for PubMedID 16314429

  • alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly CELL Drees, F., Pokutta, S., Yamada, S., Nelson, W. J., Weis, W. I. 2005; 123 (5): 903-915

    Abstract

    Epithelial cell-cell junctions, organized by adhesion proteins and the underlying actin cytoskeleton, are considered to be stable structures maintaining the structural integrity of tissues. Contrary to the idea that alpha-catenin links the adhesion protein E-cadherin through beta-catenin to the actin cytoskeleton, in the accompanying paper we report that alpha-catenin does not bind simultaneously to both E-cadherin-beta-catenin and actin filaments. Here we demonstrate that alpha-catenin exists as a monomer or a homodimer with different binding properties. Monomeric alpha-catenin binds more strongly to E-cadherin-beta-catenin, whereas the dimer preferentially binds actin filaments. Different molecular conformations are associated with these different binding states, indicating that alpha-catenin is an allosteric protein. Significantly, alpha-catenin directly regulates actin-filament organization by suppressing Arp2/3-mediated actin polymerization, likely by competing with the Arp2/3 complex for binding to actin filaments. These results indicate a new role for alpha-catenin in local regulation of actin assembly and organization at sites of cadherin-mediated cell-cell adhesion.

    View details for DOI 10.1016/j.cell.2005.09.021

    View details for Web of Science ID 000233814100022

    View details for PubMedID 16325583

  • Deconstructing the cadherin-catenin-actin complex CELL Yamada, S., Pokutta, S., Drees, F., Weis, W. I., Nelson, W. J. 2005; 123 (5): 889-901

    Abstract

    Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.

    View details for DOI 10.1016/j.cell.2005.09.020

    View details for Web of Science ID 000233814100021

    View details for PubMedID 16325582

  • APC is a component of an organizing template for cortical microtubule networks NATURE CELL BIOLOGY Reilein, A., Nelson, W. J. 2005; 7 (5): 463-U20

    Abstract

    A microtubule network on the basal cortex of polarized epithelial cells consists of non-centrosomal microtubules of mixed polarity. Here, we investigate the proteins that are involved in organizing this network, and we show that end-binding protein 1 (EB1), adenomatous polyposis coli protein (APC) and p150Glued - although considered to be microtubule plus-end-binding proteins - are localized along the entire length of microtubules within the network, and at T-junctions between microtubules. The network shows microtubule behaviours that arise from physical interactions between microtubules, including microtubule plus-end stabilization on the sides of other microtubules, and sliding of microtubule ends along other microtubules. APC also localizes to the basal cortex. Microtubules grew over and paused at APC puncta; an in vitro reconstituted microtubule network overlaid APC puncta; and microtubule network reconstitution was inhibited by function-blocking APC antibodies. Thus, APC is a component of a cortical template that guides microtubule network formation.

    View details for DOI 10.1038/ncb1248

    View details for Web of Science ID 000229123500013

    View details for PubMedID 15892196

  • A mitotic septin scaffold required for mammalian chromosome congression and segregation SCIENCE Spiliotis, E. T., Kinoshita, M., Nelson, W. J. 2005; 307 (5716): 1781-1785

    Abstract

    Coordination of cytokinesis with chromosome congression and segregation is critical for proper cell division, but the mechanism is unknown. Here, septins, a conserved family of polymerizing guanosine triphosphate-binding proteins, localized to the metaphase plate during mitosis. Septin depletion resulted in chromosome loss from the metaphase plate, lack of chromosome segregation and spindle elongation, and incomplete cytokinesis upon delayed mitotic exit. These defects correlated with loss of the mitotic motor and the checkpoint regulator centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes. Mammalian septins may thus form a mitotic scaffold for CENP-E and other effectors to coordinate cytokinesis with chromosome congression and segregation.

    View details for DOI 10.1126/science.1106823

    View details for Web of Science ID 000227883900047

    View details for PubMedID 15774761

  • Fractionation of the epithelial apical junctional complex: Reassessment of protein distributions in different substructures MOLECULAR BIOLOGY OF THE CELL Vogelmann, R., Nelson, W. J. 2005; 16 (2): 701-716

    Abstract

    The epithelial apical junctional complex (AJC) is an important regulator of cell structure and function. The AJC is compartmentalized into substructures comprising the tight and adherens junctions, and other membrane complexes containing the membrane proteins nectin, junctional adhesion molecule, and crumbs. In addition, many peripheral membrane proteins localize to the AJC. Studies of isolated proteins indicate a complex map of potential binding partners in which there is extensive overlap in the interactions between proteins in different AJC substructures. As an alternative to a direct search for specific protein-protein interactions, we sought to separate membrane substructures of the AJC in iodixanol density gradients and define their protein constituents. Results show that the AJC can be fractured into membrane substructures that contain specific membrane and peripheral membrane proteins. The composition of each substructure reveals a more limited overlap in common proteins than predicted from the inventory of potential interactions; some of the overlapping proteins may be involved in stepwise recruitment and assembly of AJC substructures.

    View details for DOI 10.1091/mbc.E04-09-0827

    View details for Web of Science ID 000226563600022

    View details for PubMedID 15548593

  • Interaction of cadherin with the actin cytoskeleton. Novartis Foundation symposium Nelson, W. J., Drees, F., Yamada, S. 2005; 269: 159-168

    Abstract

    Cadherins regulate cell-cell adhesion throughout embryonic development and in the adult organism, and defects in cadherin expression and function are characteristic of many disease states including cancer. Although extracellular binding between cadherins specifies adhesion between cells, the strength of the interaction is thought to be regulated by cadherin clustering through reorganization of the actin cytoskeleton. Protein-protein interactions have been described that could link cadherins either directly or indirectly to the actin cytoskeleton. Here, we describe these protein interactions, and examine critically the evidence that they link cadherins to the actin cytoskeleton.

    View details for PubMedID 16358407

  • Cell-adhesion assays: fabrication of an E-cadherin substratum and isolation of lateral and Basal membrane patches. Methods in molecular biology (Clifton, N.J.) Drees, F., Reilein, A., Nelson, W. J. 2005; 294: 303-320

    Abstract

    Cell adhesion between cells and with the extracellular matrix (ECM) results in dramatic changes in cell organization and, in particular, the cytoskeleton and plasma membrane domains involved in adhesion. However, current methods to analyze these changes are limited because of the small areas of membrane involved in adhesion, compared to the areas of membrane not adhering (a signal to noise problem), and the difficulty in accessing native protein complexes directly for imaging or reconstitution with purified proteins. The methods described here overcome these problems. Using a mammalian expression system, a chimeric protein comprising the extracellular domain of E-cadherin fused at its C-terminus to the Fc domain of human IgG1 (E-cadherin:Fc) is expressed and purified. A chemical bridge of biotin-NeutrAvidin-biotinylated Protein G bound to a silanized glass cover slip is fabricated to which the E-cadherin:Fc chimera binds in the correct orientation for adhesion by cells. After cell attachment, the basal membrane (a contact formed between cellular E-cadherin and the E-cadherin:Fc substratum) is isolated by sonication; a similar method is described to isolate basal membranes of cells attached to ECM. These membrane patches provide direct access to protein complexes formed on the membrane following cell-cell or cell-ECM adhesion.

    View details for PubMedID 15576920

  • Convergence of Wnt, beta-catenin, and cadherin pathways SCIENCE Nelson, W. J., Nusse, R. 2004; 303 (5663): 1483-1487

    Abstract

    The specification and proper arrangements of new cell types during tissue differentiation require the coordinated regulation of gene expression and precise interactions between neighboring cells. Of the many growth factors involved in these events, Wnts are particularly interesting regulators, because a key component of their signaling pathway, beta-catenin, also functions as a component of the cadherin complex, which controls cell-cell adhesion and influences cell migration. Here, we assemble evidence of possible interrelations between Wnt and other growth factor signaling, beta-catenin functions, and cadherin-mediated adhesion.

    View details for Web of Science ID 000220000100031

    View details for PubMedID 15001769

  • Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes JOURNAL OF CELL SCIENCE Louie, R. K., Bahmanyar, S., Siemers, K. A., Votin, V., Chang, P., Stearns, T., NELSON, W. J., Barth, A. I. 2004; 117 (7): 1117-1128

    Abstract

    Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by epsilon-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, our data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole.

    View details for DOI 10.1242/jcs.00939

    View details for Web of Science ID 000220762900015

    View details for PubMedID 14970257

  • Breaking into the epithelial apical-junctional complex - news from pathogen hackers CURRENT OPINION IN CELL BIOLOGY Vogelmann, R., Amieva, M. R., FALKOW, S., Nelson, W. J. 2004; 16 (1): 86-93

    Abstract

    The epithelial apical-junctional complex is a key regulator of cellular functions. In addition, it is an important target for microbial pathogens that manipulate the cell to survive, proliferate and sometimes persist within a host. Out of a myriad of potential molecular targets, some bacterial and viral pathogens have selected a subset of protein targets at the apical-junctional complex of epithelial cells. Studying how microbes use these targets also teaches us about the inherent physiological properties of host molecules in the context of normal junctional structure and function. Thus, we have learned that three recently uncovered components of the apical-junctional complex of the Ig superfamily--junctional adhesion molecule, Nectin and the coxsackievirus and adenovirus receptor--are important regulators of junction structure and function and represent critical targets of microbial virulence gene products.

    View details for DOI 10.1016/j.ceb.2003.12.002

    View details for Web of Science ID 000188769900013

    View details for PubMedID 15037310

  • Mechanism of recruiting Sec6/8 (exocyst) complex to the apical junctional complex during polarization of epithelial cells JOURNAL OF CELL SCIENCE Yeaman, C., Grindstaff, K. K., NELSON, W. J. 2004; 117 (4): 559-570

    Abstract

    Sec6/8 (exocyst) complex regulates vesicle delivery and polarized membrane growth in a variety of cells, but mechanisms regulating Sec6/8 localization are unknown. In epithelial cells, Sec6/8 complex is recruited to cell-cell contacts with a mixture of junctional proteins, but then sorts out to the apex of the lateral membrane with components of tight junction and nectin complexes. Sec6/8 complex fractionates in a high molecular mass complex with tight junction proteins and a portion of E-cadherin, and co-immunoprecipitates with cell surface-labeled E-cadherin and nectin-2alpha. Recruitment of Sec6/8 complex to cell-cell contacts can be achieved in fibroblasts when E-cadherin and nectin-2alpha are co-expressed. These results support a model in which localized recruitment of Sec6/8 complex to the plasma membrane by specific cell-cell adhesion complexes defines a site for vesicle delivery and polarized membrane growth during development of epithelial cell polarity.

    View details for DOI 10.1242/jcs.00893

    View details for Web of Science ID 000189080100007

    View details for PubMedID 14709721

  • Cadherin adhesion: mechanisms and molecular interactions. Handbook of experimental pharmacology Perez, T. D., NELSON, W. J. 2004: 3-21

    Abstract

    Cadherins constitute a superfamily of cell-cell adhesion molecules expressed in many different cell types that are required for proper cellular function and maintenance of tissue architecture. Classical cadherins are the best understood class of cadherins. They are single membrane spanning proteins with a divergent extracellular domain of five repeats and a conserved cytoplasmic domain. Binding between cadherin extracellular domains is weak, but strong cell-cell adhesion develops during lateral clustering of cadherins by proteins that link the cadherin cytoplasmic domain to the actin cytoskeleton. Understanding how different regions of cadherins regulate cell-cell adhesion has been a major focus of study. Here, we examine evidence of the structure and function of the extracellular domain of classical cadherins in regard to the control of recognition and adhesive contacts between cadherins on opposing cell surfaces. Early experiments that focused on understanding the homotypic, Ca(++)-dependent characteristics of cadherin adhesion are discussed, and data supporting the widely accepted cis- and trans-dimer models of cadherins are analyzed.

    View details for DOI 10.1007/978-3-540-68170-0_1

    View details for PubMedID 20455088

  • Tube morphogenesis: closure, but many openings remain TRENDS IN CELL BIOLOGY NELSON, W. J. 2003; 13 (12): 615-621

    Abstract

    Epithelial and endothelial tubes form the basic structure of many organs and tissues in the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, zebrafish and mammals. Comparison of how tubes form during development defines several pathways that generate a single unbranched tube or dichotomously branching tubular networks. The formation of tubes can be induced directly by intrinsic signals within epithelial primordia or by inductive signaling between adjacent epithelia and the mesenchyme. Both processes are hierarchically controlled by master transcriptional regulators, growth factors and their receptors, directed cell migration and cellular reorganization, which is controlled by changes in the cytoskeleton and protein trafficking. This review provides a summary of these pathways based upon articles published in the Tube Morphogenesis Series in Trends in Cell Biology.

    View details for DOI 10.1016/j.tcb.2003.10.005

    View details for Web of Science ID 000186909700004

    View details for PubMedID 14624839

  • Mum, this bud's for you: where do you want it? Roles for Cdc42 in controlling bud site selection in Saccharomyces cerevisiae BIOESSAYS NELSON, W. J. 2003; 25 (9): 833-836

    Abstract

    The generation of asymmetric cell shapes is a recurring theme in biology. In budding yeast, one form of cell asymmetry occurs for division and is generated by anisotropic growth of the mother cell to form a daughter cell bud. Previous genetic studies uncovered key roles for the small GTPase Cdc42 in organizing the actin cytoskeleton and vesicle delivery to the site of bud growth, but a recent paper has also raised questions about how control of Cdc42 activity is integrated into a proposed hierarchical regulatory pathway that specifies a unique site of bud formation.

    View details for DOI 10.1002/bies.10335

    View details for Web of Science ID 000185779600003

    View details for PubMedID 12938172

  • Epithelial cell polarity from the outside looking in NEWS IN PHYSIOLOGICAL SCIENCES NELSON, W. J. 2003; 18: 143-146

    Abstract

    Epithelial cell polarity may be regulated by protein sorting in the Golgi and delivery to different membrane domains, a view from the inside looking out. But from the outside looking in, cell adhesion may be required first to establish sites for delivery, retention, and separation of membrane proteins, and delivery of presorted proteins from the Golgi subsequently reinforces and maintains different membrane domains.

    View details for DOI 10.1152/nips.01435.2002

    View details for Web of Science ID 000184429700002

    View details for PubMedID 12869613

  • Spatial control of exocytosis CURRENT OPINION IN CELL BIOLOGY Spiliotis, E. T., Nelson, W. J. 2003; 15 (4): 430-437

    Abstract

    During many key biological processes, exocytosis is confined to distinct regions of the plasma membrane. Spatial control of exocytosis correlates with altered membrane skeleton dynamics and assembly of local membrane microdomains. These domains act as local stages for the assembly and the regulation of molecular complexes (targeting patches) that mediate vesicle-membrane fusion. Furthermore, local activation of signaling pathways reinforces formation of these patches and might effect global repositioning of the secretory pathway toward sites of localized exocytosis.

    View details for DOI 10.1016/S0955-0674(03)00074-7

    View details for Web of Science ID 000184641200009

    View details for PubMedID 12892783

  • Urinary excretion of viable podocytes in health and renal disease AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY Vogelmann, S. U., NELSON, W. J., Myers, B. D., Lemley, K. V. 2003; 285 (1): F40-F48

    Abstract

    The loss of glomerular visceral epithelial cells (podocytes) has been associated with the development of glomerular sclerosis and loss of renal function. Viability of podocytes recovered from urine of subjects with glomerular disease and of healthy controls was investigated by propidium iodide exclusion and TUNEL staining. Podocyte loss was quantified by cytospin. The growth behavior in culture of urinary cells and their expression of specific markers were examined. The majority of urinary podocytes are viable, although apoptosis occurs in about one-half of the cells. Patients with active glomerular disease excreted up to 388 podocytes/mg creatinine, whereas healthy controls and patients with quiescent disease generally excreted <0.5 podocytes/mg creatinine. The identity of cultured cells was confirmed by their morphology, growth behavior, and expression of podocyte-specific markers. The difference in growth behavior between healthy controls and subjects with active glomerular disease suggests that in active disease viable podocytes detach from the glomerular tuft due to local environmental factors rather than defects in the podocytes per se, whereas in healthy individuals mostly senescent podocytes are shed.

    View details for DOI 10.1152/ajprenal.00404.2002

    View details for Web of Science ID 000183314500005

    View details for PubMedID 12631553

  • Disruption of the epithelial apical-junctional complex by Helicobacter pylori CagA SCIENCE Amieva, M. R., Vogelmann, R., Covacci, A., Tompkins, L. S., NELSON, W. J., FALKOW, S. 2003; 300 (5624): 1430-1434

    Abstract

    Helicobacter pylori translocates the protein CagA into gastric epithelial cells and has been linked to peptic ulcer disease and gastric carcinoma. We show that injected CagA associates with the epithelial tight-junction scaffolding protein ZO-1 and the transmembrane protein junctional adhesion molecule, causing an ectopic assembly of tight-junction components at sites of bacterial attachment, and altering the composition and function of the apical-junctional complex. Long-term CagA delivery to polarized epithelia caused a disruption of the epithelial barrier function and dysplastic alterations in epithelial cell morphology. CagA appears to target H. pylori to host cell intercellular junctions and to disrupt junction-mediated functions.

    View details for Web of Science ID 000183181800045

    View details for PubMedID 12775840

  • Adaptation of core mechanisms to generate cell polarity NATURE NELSON, W. J. 2003; 422 (6933): 766-774

    Abstract

    Cell polarity is defined as asymmetry in cell shape, protein distributions and cell functions. It is characteristic of single-cell organisms, including yeast and bacteria, and cells in tissues of multi-cell organisms such as epithelia in worms, flies and mammals. This diversity raises several questions: do different cell types use different mechanisms to generate polarity, how is polarity signalled, how do cells react to that signal, and how is structural polarity translated into specialized functions? Analysis of evolutionarily diverse cell types reveals that cell-surface landmarks adapt core pathways for cytoskeleton assembly and protein transport to generate cell polarity.

    View details for DOI 10.1038/nature01602

    View details for Web of Science ID 000182272300051

    View details for PubMedID 12700771

  • Molecular mechanism for orienting membrane and actin dynamics to nascent cell-cell contacts in epithelial cells JOURNAL OF BIOLOGICAL CHEMISTRY Hansen, M. D., Ehrlich, J. S., Nelson, W. J. 2002; 277 (47): 45371-45376

    Abstract

    The small GTPase Rac1 has been implicated in regulation of cell migration and cell-cell adhesion in epithelial cells. Little is known, however, about the spatial and temporal coordination of Rac1 activity required to balance these competing processes. We fractionated endogenous Rac1-containing protein complexes from membranes of Madin-Darby canine kidney cells and identified three major complexes comprising a Rac1.PAK (p21-activated kinase) complex, and 11 S and 16 S Rac1 complexes. Significantly, Rac1 shifts from the 11 S to a 16 S particle during initiation of cell-cell adhesion. This shift may reflect a diffusion trapping mechanism by which these Rac1 complexes are localized to cadherin-mediated cell-cell contacts through an interaction with annexin II.

    View details for DOI 10.1074/jbc.M207747200

    View details for Web of Science ID 000179404800100

    View details for PubMedID 12244058

  • What can humans learn from flies about adenomatous polyposis coli BIOESSAYS Barth, A. I., NELSON, W. J. 2002; 24 (9): 771-774

    Abstract

    Somatic or inherited mutations in the adenomatous polyposis coli (APC) gene are a frequent cause of colorectal cancer in humans. APC protein has an important tumor suppression function to reduce cellular levels of the signaling protein beta-catenin and, thereby, inhibit beta-catenin and T-cell-factor-mediated gene expression. In addition, APC protein binds to microtubules in vertebrate cells and localizes to actin-rich adherens junctions in epithelial cells of the fruit fly Drosophila (Fig. 1). Very little is known, however, about the function of these cytoskeletal associations. Recently, Hamada and Bienz have described a potential role for Drosophila E-APC in cellular adhesion, which offers new clues to APC function in embryonic development, and potentially colorectal adenoma formation and tumor progression in humans.

    View details for DOI 10.1002/bies.10152

    View details for Web of Science ID 000177747100001

    View details for PubMedID 12210511

  • Spatio-temporal regulation of Rac1 localization and lamellipodia dynamics during epithelial cell-cell adhesion DEVELOPMENTAL CELL Ehrlich, J. S., Hansen, M. D., Nelson, W. J. 2002; 3 (2): 259-270

    Abstract

    Cadherin-dependent epithelial cell-cell adhesion is thought to be regulated by Rho family small GTPases and PI 3-kinase, but the mechanisms involved are poorly understood. Using time-lapse microscopy and quantitative image analysis, we show that cell-cell contact in MDCK epithelial cells coincides with a spatio-temporal reorganization of plasma membrane Rac1 and lamellipodia from noncontacting to contacting surfaces. Within contacts, Rac1 and lamellipodia transiently concentrate at newest sites, but decrease at older, stabilized sites. Significantly, Rac1 mutants alter kinetics of cell-cell adhesion and strengthening, but not the eventual generation of cell-cell contacts. Products of PI 3-kinase activity also accumulate dynamically at contacts, but are not essential for either initiation or development of cell-cell adhesion. These results define a role for Rac1 in regulating the rates of initiation and strengthening of cell-cell adhesion.

    View details for Web of Science ID 000177325700015

    View details for PubMedID 12194856

  • Biochemical and structural definition of the 1-afadin- and actin-binding sites of alpha-catenin JOURNAL OF BIOLOGICAL CHEMISTRY Pokutta, S., Drees, F., Takai, Y., NELSON, W. J., Weis, W. I. 2002; 277 (21): 18868-18874

    Abstract

    alpha-Catenin is an integral component of adherens junctions, where it links cadherins to the actin cytoskeleton. alpha-Catenin is also required for the colocalization of the nectin/afadin/ponsin adhesion system to adherens junctions, and it specifically associates with the nectin-binding protein afadin. A proteolytic fragment of alpha-catenin, residues 385-651, contains the afadin-binding site. The three-dimensional structure of this fragment comprises two side-by-side four-helix bundles, both of which are required for afadin binding. The alpha-catenin fragment 385-651 binds afadin more strongly than the full-length protein, suggesting that the full-length protein harbors a cryptic binding site for afadin. Comparison of the alpha-catenin 385-651 structure with the recently solved structure of the alpha-catenin M-fragment (Yang, J., Dokurno, P., Tonks, N. K., and Barford, D. (2001) EMBO J. 20, 3645-3656) reveals a surprising flexibility in the orientation of the two four-helix bundles. alpha-Catenin and the actin-binding protein vinculin share sequence and most likely structural similarity within their actin-binding domains. Despite this homology, actin binding requires additional sequences adjacent to this region.

    View details for DOI 10.1074/jbc.M201463200

    View details for Web of Science ID 000175975800081

    View details for PubMedID 11907041

  • Dissecting interactions between EB1, microtubules and APG in cortical clusters at the plasma membrane JOURNAL OF CELL SCIENCE Barth, A. I., Siemers, K. A., NELSON, W. J. 2002; 115 (8): 1583-1590

    Abstract

    End-binding protein (EB) 1 binds to the C-terminus of adenomatous polyposis coli (APC) protein and to the plus ends of microtubules (MT) and has been implicated in the regulation of APC accumulation in cortical clusters at the tip of extending membranes. We investigated which APC domains are involved in cluster localization and whether binding to EB1 or MTs is essential for APC cluster localization. Armadillo repeats of APC that lack EB1- and MT-binding domains are necessary and sufficient for APC localization in cortical clusters; an APC fragment lacking the armadillo repeats, but containing MT- and EB1-binding domains, does not localize to the cortical clusters but instead co-aligns with MTs throughout the cell. Significantly, analysis of endogenous proteins reveals that EB1 does not accumulate in the APC clusters. However, overexpressed EB1 does accumulate in APC clusters; the APC-binding domain in EB1 is located in the C-terminal region of EB1 between amino acids 134 and 268. Overexpressed APC- or MT-binding domains of EB1 localize to APC cortical clusters and MT, respectively, without affecting APC cluster formation itself. These results show that localization of APC in cortical clusters is different from that of EB1 at MT plus ends and appears to be independent of EB1.

    View details for Web of Science ID 000175479800004

    View details for PubMedID 11950877

  • Protein trafficking in the exocytic pathway of polarized epithelial cells TRENDS IN CELL BIOLOGY Nelson, W. J., Yeaman, C. 2001; 11 (12): 483-486

    Abstract

    Ten years ago, we knew much about the function of polarized epithelia from the work of physiologists, but, as cell biologists, our understanding of how these cells were constructed was poor. We knew proteins were sorted and targeted to different plasma membrane domains and that, in some cells, the Golgi was the site of sorting, but we did not know the mechanisms involved. Between 1991 and the present, significant advances were made in defining sorting motifs for apical and basal-lateral proteins, describing the sorting machinery in the trans-Golgi network (TGN) and plasma membrane, and in understanding how cells specify delivery of transport vesicles to different membrane domains. The challenge now is to extend this knowledge to defining molecular mechanisms in detail in vitro and comprehending the development of complex epithelial structures in vivo.

    View details for Web of Science ID 000172530100010

    View details for PubMedID 11719053

  • Plasma selenium level before diagnosis and the risk of prostate cancer development JOURNAL OF UROLOGY Brooks, J. D., Metter, E. J., Chan, D. W., Sokoll, L. J., Landis, P., Nelson, W. G., Muller, D., Andres, R., Carter, H. B. 2001; 166 (6): 2034-2038

    Abstract

    Epidemiological studies and a randomized intervention trial suggest that the risk of prostate cancer may be reduced by selenium intake. We investigated whether plasma selenium level before diagnosis correlated with the risk of later developing prostate cancer.A case control study was performed on men from the Baltimore Longitudinal Study of Aging registry, including 52 with known prostate cancer and 96 age matched controls with no detectable prostatic disease. Plasma selenium was measured at an average time plus or minus standard deviation of 3.83 +/- 1.85 years before the diagnosis of prostate cancer by graphite furnace atomic absorption spectrophotometry. Adjusted odds ratio and 95% confidence interval were computed with logistic regression.After correcting for years before diagnosis, body mass index, and smoking and alcohol use history, higher selenium was associated with a lower risk of prostate cancer. Compared with the lowest quartile of selenium (range 8.2 to 10.7 microg./dl.), the odds ratios of the second (10.8 to 11.8), third (11.9 to 13.2) and fourth (13.3 to 18.2) quartiles were 0.15 (95% confidence interval 0.05 to 0.50), 0.21 (0.07 to 0.68) and 0.24 (0.08 to 0.77, respectively, p =0.01). Furthermore, plasma selenium decreased significantly with patient age (p <0.001).Low plasma selenium is associated with a 4 to 5-fold increased risk of prostate cancer. These results support the hypothesis that supplemental selenium may reduce the risk of prostate cancer. Because plasma selenium decreases with patient age, supplementation may be particularly beneficial to older men.

    View details for Web of Science ID 000172133900004

    View details for PubMedID 11696701

  • Sec6/8 complexes on trans-Golgi network and plasma membrane regulate late stages of exocytosis in mammalian cells JOURNAL OF CELL BIOLOGY Yeaman, C., Grindstaff, K. K., Wright, J. R., NELSON, W. J. 2001; 155 (4): 593-604

    Abstract

    Sec6/8 complex regulates delivery of exocytic vesicles to plasma membrane docking sites, but how it is recruited to specific sites in the exocytic pathway is poorly understood. We identified an Sec6/8 complex on trans-Golgi network (TGN) and plasma membrane in normal rat kidney (NRK) cells that formed either fibroblast- (NRK-49F) or epithelial-like (NRK-52E) intercellular junctions. At both TGN and plasma membrane, Sec6/8 complex colocalizes with exocytic cargo protein, vesicular stomatitis virus G protein (VSVG)-tsO45. Newly synthesized Sec6/8 complex is simultaneously recruited from the cytosol to both sites. However, brefeldin A treatment inhibits recruitment to the plasma membrane and other treatments that block exocytosis (e.g., expression of kinase-inactive protein kinase D and low temperature incubation) cause accumulation of Sec6/8 on the TGN, indicating that steady-state distribution of Sec6/8 complex depends on continuous exocytic vesicle trafficking. Addition of antibodies specific for TGN- or plasma membrane-bound Sec6/8 complexes to semiintact NRK cells results in cargo accumulation in a perinuclear region or near the plasma membrane, respectively. These results indicate that Sec6/8 complex is required for several steps in exocytic transport of vesicles between TGN and plasma membrane.

    View details for Web of Science ID 000172291400011

    View details for PubMedID 11696560

  • The cadherin cytoplasmic domain is unstructured in the absence of beta-catenin - A possible-mechanism for regulating cadherin turnover JOURNAL OF BIOLOGICAL CHEMISTRY Huber, A. H., STEWART, D. B., Laurents, D. V., NELSON, W. J., Weis, W. I. 2001; 276 (15): 12301-12309

    Abstract

    Cadherins are single pass transmembrane proteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion by linking the cytoskeletons of adjacent cells. In adherens junctions, the cytoplasmic domain of cadherins bind to beta-catenin, which in turn binds to the actin-associated protein alpha-catenin. The physical properties of the E-cadherin cytoplasmic domain and its interactions with beta-catenin have been investigated. Proteolytic sensitivity, tryptophan fluorescence, circular dichroism, and (1)H NMR measurements indicate that murine E-cadherin cytoplasmic domain is unstructured. Upon binding to beta-catenin, the domain becomes resistant to proteolysis, suggesting that it structures upon binding. Cadherin-beta-catenin complex stability is modestly dependent on ionic strength, indicating that, contrary to previous proposals, the interaction is not dominated by electrostatics. Comparison of 18 cadherin sequences indicates that their cytoplasmic domains are unlikely to be structured in isolation. This analysis also reveals the presence of PEST sequences, motifs associated with ubiquitin/proteosome degradation, that overlap the previously identified beta-catenin-binding site. It is proposed that binding of cadherins to beta-catenin prevents recognition of degradation signals that are exposed in the unstructured cadherin cytoplasmic domain, favoring a cell surface population of catenin-bound cadherins capable of participating in cell adhesion.

    View details for Web of Science ID 000168081800115

    View details for PubMedID 11121423

  • Serum-activated assembly and membrane translocation of an endogenous Rac1 : effector complex CURRENT BIOLOGY Hansen, M. D., NELSON, W. J. 2001; 11 (5): 356-360

    Abstract

    Rho family GTPases (Cdc42, Rac1, and RhoA) function downstream of Ras [1], and in a variety of cellular processes [2]. Studies to examine these functions have not directly linked endogenous protein interactions with specific in vivo functions of Rho GTPases. Here, we show that endogenous Rac1 and two known binding partners, Rho GDP dissociation inhibitor (RhoGDI) and p21-activated kinase (PAK), fractionate as distinct cytosolic complexes. A Rac1:PAK complex is translocated from the cytosol to ruffling membranes upon cell activation by serum. Overexpression of dominant-negative (T17N) Rac1 does not affect the assembly or distribution of this Rac1:PAK complex. This is the first direct evidence of how a specific function of Rac1 is selected by the assembly and membrane translocation of a distinct Rac1:effector complex.

    View details for Web of Science ID 000169076500021

    View details for PubMedID 11267873

  • Mutant cadherin affects epithelial morphogenesis and invasion, but not transformation JOURNAL OF CELL SCIENCE Troxell, M. L., Loftus, D. J., NELSON, W. J., MARRS, J. A. 2001; 114 (6): 1237-1246

    Abstract

    MDCK cells were engineered to reversibly express mutant E-cadherin protein with a large extracellular deletion. Mutant cadherin overexpression reduced the expression of endogenous E- and K-cadherins in MDCK cells to negligible levels, resulting in decreased cell adhesion. Despite severe impairment of the cadherin adhesion system, cells overexpressing mutant E-cadherin formed fluid-filled cysts in collagen gel cultures and responded to hepatocyte growth factor/scatter factor (HGF/SF) that induced cellular extension formation with a frequency similar to that of control cysts. However, cells were shed from cyst walls into the lumen and into the collagen matrix prior to and during HGF/SF induced tubule extension. Despite the propensity for cell dissociation, MDCK cells lacking cadherin adhesion molecules were not capable of anchorage-independent growth in soft agar and cell proliferation rate was not affected. Thus, cadherin loss does not induce transformation, despite inducing an invasive phenotype, a later stage of tumor progression. These experiments are especially relevant to tumor progression in cells with altered E-cadherin expression, particularly tumor samples with identified E-cadherin extracellular domain genomic mutations.

    View details for Web of Science ID 000167952200022

    View details for PubMedID 11228167

  • Differential regulation of endogenous cadherin expression in Madin-Darby canine kidney cells by cell-cell adhesion and activation of beta-catenin signaling JOURNAL OF BIOLOGICAL CHEMISTRY STEWART, D. B., Barth, A. I., Nelson, W. J. 2000; 275 (27): 20707-20716

    Abstract

    Cadherins mediate cell-cell adhesion, but little is known about how their expression is regulated. In Madin-Darby canine kidney (MDCK) cells, the cadherin-associated cytoplasmic proteins alpha- and beta-catenin form high molecular weight protein complexes with two glycoproteins (Stewart, D. B., and Nelson, W. J. (1997) J. Biol. Chem. 272, 29652-29662), one of which is E-cadherin and the other we show here is the type II cadherin, cadherin-6 (K-cadherin). In low density, motile MDCK cells, the steady-state level of cadherin-6 is low, but protein is synthesized. However, following cell-cell adhesion, cadherin-6 becomes stabilized and accumulates by >50-fold at cell-cell contacts while the E-cadherin level increases only 5-fold during the same period. To investigate a role of beta-catenin in regulation of cadherin expression in MDCK cells, we examined the effects of expressing signaling-active beta-catenin mutants (DeltaGSK, DeltaN90, and DeltaN131). In these cells, while levels of E-cadherin, alpha- and beta-catenin are similar to those in control cells, levels of cadherin-6 are significantly reduced due to rapid degradation of newly synthesized protein. Additionally, these cells appeared more motile and less cohesive, as expression of DeltaGSK-beta-catenin delayed the establishment of tight confluent cell monolayers compared with control cells. These results indicate that the level of cadherin-6, but not that of E-cadherin, is strictly regulated post-translationally in response to Wnt signaling, and that E-cadherin and cadherin-6 may contribute different properties to cell-cell adhesion and the epithelial phenotype.

    View details for Web of Science ID 000088084500069

    View details for PubMedID 10747916

  • Colocalization and redistribution of dishevelled and actin during Wnt-induced mesenchymal morphogenesis JOURNAL OF CELL BIOLOGY Torres, M. A., Nelson, W. J. 2000; 149 (7): 1433-1442

    Abstract

    Activation of the Wnt signaling pathway is important for induction of gene expression and cell morphogenesis throughout embryonic development. We examined the subcellular localization of dishevelled, the immediate downstream component from the Wnt receptor, in the embryonic mouse kidney. Using immunofluorescence staining, confocal microscopy, and coimmunoprecipitation experiments, we show that dishevelled associates with actin fibers and focal adhesion plaques in metanephric mesenchymal cells. Stimulation of Wnt signaling leads to profound changes in metanephric mesenchymal cell morphology, including disruption of the actin cytoskeleton, increased cell spreading, and increased karyokinesis. Upon activation of Wnt signaling, dishevelled also accumulates in and around the nucleus. Casein kinase Iepsilon colocalizes with dishevelled along actin fibers and in the perinuclear region, whereas axin and GSK-3 are only present around the nucleus. These data indicate a branched Wnt signaling pathway comprising a canonical signal that targets the nucleus and gene expression, and another signal that targets the cytoskeleton and regulates cell morphogenesis.

    View details for Web of Science ID 000087946600009

    View details for PubMedID 10871283

  • W(h)ither the Golgi during mitosis? JOURNAL OF CELL BIOLOGY Nelson, W. J. 2000; 149 (2): 243-248

    View details for Web of Science ID 000086598600001

    View details for PubMedID 10769017

  • Inhibiting cadherin function by dominant mutant E-cadherin expression increases the extent of tight junction assembly JOURNAL OF CELL SCIENCE Troxell, M. L., Gopalakrishnan, S., McCormack, J., Poteat, B. A., Pennington, J., Garringer, S. M., Schneeberger, E. E., NELSON, W. J., Marrs, J. A. 2000; 113 (6): 985-996

    Abstract

    Previous studies have shown that induction of cadherin-mediated cell-cell adhesion leads to tight junction formation, and that blocking cadherin-mediated cell-cell adhesion inhibits tight junction assembly. Here we report analysis of tight junction assembly in MDCK cells overexpressing a mutant E-cadherin protein that lacks an adhesive extracellular domain (T151 cells). Mutant E-cadherin overexpression caused a dramatic reduction in endogenous cadherin levels. Despite this, tight junction assembly was extensive. The number of tight junction strands observed by freeze-fracture electron microscopy significantly increased in T151 cells compared to that in control cells. Our data indicate that the hierarchical regulation of junctional complex assembly is not absolute, and that inhibition of cadherin function has both positive and negative effects on tight junction assembly.

    View details for Web of Science ID 000086312400010

    View details for PubMedID 10683147

  • Selective alterations in biosynthetic and endocytic protein traffic in Madin-Darby canine kidney epithelial cells expressing mutants of the small GTPase Rac1 MOLECULAR BIOLOGY OF THE CELL Jou, T. S., Leung, S. M., Fung, L. M., Ruiz, W. G., NELSON, W. J., Apodaca, G. 2000; 11 (1): 287-304

    Abstract

    Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.

    View details for Web of Science ID 000085016300025

    View details for PubMedID 10637309

  • Listeria monocytogenes exploits normal host cell processes to spread from cell to cell JOURNAL OF CELL BIOLOGY Robbins, J. R., Barth, A. I., Marquis, H., de Hostos, E. L., NELSON, W. J., Theriot, J. A. 1999; 146 (6): 1333-1349

    Abstract

    The bacterial pathogen, Listeria monocytogenes, grows in the cytoplasm of host cells and spreads intercellularly using a form of actin-based motility mediated by the bacterial protein ActA. Tightly adherent monolayers of MDCK cells that constitutively express GFP-actin were infected with L. monocytogenes, and intercellular spread of bacteria was observed by video microscopy. The probability of formation of membrane-bound protrusions containing bacteria decreased with host cell monolayer age and the establishment of extensive cell-cell contacts. After their extension into a recipient cell, intercellular membrane-bound protrusions underwent a period of bacterium-dependent fitful movement, followed by their collapse into a vacuole and rapid vacuolar lysis. Actin filaments in protrusions exhibited decreased turnover rates compared with bacterially associated cytoplasmic actin comet tails. Recovery of motility in the recipient cell required 1-2 bacterial generations. This delay may be explained by acid-dependent cleavage of ActA by the bacterial metalloprotease, Mpl. Importantly, we have observed that low levels of endocytosis of neighboring MDCK cell surface fragments occurs in the absence of bacteria, implying that intercellular spread of bacteria may exploit an endogenous process of paracytophagy.

    View details for Web of Science ID 000082765400011

    View details for PubMedID 10491395

  • Cell polarity: Versatile scaffolds keep things in place CURRENT BIOLOGY Yeaman, C., Grindstaff, K. K., Hansen, M. D., NELSON, W. J. 1999; 9 (14): R515-R517

    Abstract

    Protein scaffolds organize transmembrane and cytoplasmic proteins and serve to integrate both structure and signaling at the apical junctional complex of polarized epithelial cells. These scaffolds are important in coordinating local and global changes in cell organization.

    View details for Web of Science ID 000081516100008

    View details for PubMedID 10421564

  • T cell factor-activated transcription is not sufficient to induce anchorage-independent growth of epithelial cells expressing mutant beta-catenin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Barth, A. I., STEWART, D. B., NELSON, W. J. 1999; 96 (9): 4947-4952

    Abstract

    N-terminal mutations in beta-catenin that inhibit beta-catenin degradation are found in primary tumors and cancer cell lines, and increased beta-catenin/T cell factor (TCF)-activated transcription in these cells has been correlated with cancer formation. However, the role of mutant beta-catenin in cell transformation is poorly understood. Here, we compare the ability of different N-terminal mutations of beta-catenin (DeltaN131, DeltaN90, DeltaGSK) to induce TCF-activated transcription and anchorage-independent growth in Madin-Darby canine kidney epithelial cells. Expression of DeltaN90 or DeltaGSK beta-catenin increased TCF-activated transcription but did not induce significant anchorage-independent cell growth. In contrast, deletion of the alpha-catenin-binding site in DeltaN131 beta-catenin reduced TCF-activated transcription, compared with that induced by DeltaN90 or DeltaGSK beta-catenin, but significantly enhanced anchorage-independent cell growth.

    View details for Web of Science ID 000080130200041

    View details for PubMedID 10220399

  • Sodium reabsorption and distribution of Na+/K+-ATPase during postischemic injury to the renal allograft KIDNEY INTERNATIONAL Kwon, O., Corrigan, G., Myers, B. D., Sibley, R., Scandling, J. D., Dafoe, D., Alfrey, E., NELSON, W. J. 1999; 55 (3): 963-975

    Abstract

    A loss of proximal tubule cell polarity is thought to activate tubuloglomerular feedback, thereby contributing to glomerular filtration rate depression in postischemic acute renal failure (ARF).We used immunomicroscopy to evaluate the segmental distribution of Na+/K+-ATPase in tubules of recipients of cadaveric renal allografts. Fractional excretion (FE) of sodium and lithium was determined simultaneously. Observations were made on two occasions: one to three hours after graft reperfusion (day 0) and again on post-transplant day 7. An inulin clearance below or above 25 ml/min on day 7 was used to divide subjects into groups with sustained (N = 15) or recovering (N = 16) ARF, respectively.In sustained ARF, the fractional excretion of sodium (FENa) was 40 +/- 6% and 11 +/- 5%, and the fractional excretion of lithium (FELi) was 76 +/- 5% and 70 +/- 2% on days 0 and 7, respectively. Corresponding findings in recovering ARF were 28 +/- 2% and 6 +/- 2% for the FENa and 77 +/- 4% and 55 +/- 3% (P < 0.05 vs. sustained) for FELi. Na+/K+-ATPase distribution in both groups was mainly basolateral in distal straight and convoluted tubule segments and collecting ducts. However, Na+/K+-ATPase was poorly retained in the basolateral membrane of proximal convoluted and straight tubule segments in sustained and recovering ARF on both days 0 and 7.We conclude that loss of proximal tubule cell polarity for Na+/K+-ATPase distribution is associated with enhanced delivery of filtered Na+ to the macula densa for seven days after allograft reperfusion. Whether an ensuing activation of tubuloglomerular feedback is an important cause of glomerular filtration rate depression in this form of ARF remains to be determined.

    View details for Web of Science ID 000078682200019

    View details for PubMedID 10027933

  • Coupling assembly of the E-cadherin/beta-catenin complex to efficient endoplasmic reticulum exit and basal-lateral membrane targeting of E-cadherin in polarized MDCK cells JOURNAL OF CELL BIOLOGY Chen, Y. T., STEWART, D. B., NELSON, W. J. 1999; 144 (4): 687-699

    Abstract

    The E-cadherin/catenin complex regulates Ca++-dependent cell-cell adhesion and is localized to the basal-lateral membrane of polarized epithelial cells. Little is known about mechanisms of complex assembly or intracellular trafficking, or how these processes might ultimately regulate adhesion functions of the complex at the cell surface. The cytoplasmic domain of E-cadherin contains two putative basal-lateral sorting motifs, which are homologous to sorting signals in the low density lipoprotein receptor, but an alanine scan across tyrosine residues in these motifs did not affect the fidelity of newly synthesized E-cadherin delivery to the basal-lateral membrane of MDCK cells. Nevertheless, sorting signals are located in the cytoplasmic domain since a chimeric protein (GP2CAD1), comprising the extracellular domain of GP2 (an apical membrane protein) and the transmembrane and cytoplasmic domains of E-cadherin, was efficiently and specifically delivered to the basal-lateral membrane. Systematic deletion and recombination of specific regions of the cytoplasmic domain of GP2CAD1 resulted in delivery of <10% of these newly synthesized proteins to both apical and basal-lateral membrane domains. Significantly, >90% of each mutant protein was retained in the ER. None of these mutants formed a strong interaction with beta-catenin, which normally occurs shortly after E-cadherin synthesis. In addition, a simple deletion mutation of E-cadherin that lacks beta-catenin binding is also localized intracellularly. Thus, beta-catenin binding to the whole cytoplasmic domain of E-cadherin correlates with efficient and targeted delivery of E-cadherin to the lateral plasma membrane. In this capacity, we suggest that beta-catenin acts as a chauffeur, to facilitate transport of E-cadherin out of the ER and the plasma membrane.

    View details for Web of Science ID 000082932600008

    View details for PubMedID 10037790

  • Cadherin function in junctional complex rearrangement and posttranslational control of cadherin expression AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Troxell, M. L., Chen, Y. T., Cobb, N., NELSON, W. J., MARRS, J. A. 1999; 276 (2): C404-C418

    Abstract

    The role of E-cadherin, a calcium-dependent adhesion protein, in organizing and maintaining epithelial junctions was examined in detail by expressing a fusion protein (GP2-Cad1) composed of the extracellular domain of a nonadherent glycoprotein (GP2) and the transmembrane and cytoplasmic domains of E-cadherin. All studies shown were also replicated using an analogous cell line that expresses a mutant cadherin construct (T151) under the control of tet repressor. Mutant cadherin was expressed at approximately 10% of the endogenous E-cadherin level and had no apparent effect on tight junction function or on distributions of adherens junction, tight junction, or desmosomal marker proteins in established Madin-Darby canine kidney cell monolayers. However, GP2-Cad1 accelerated the disassembly of epithelial junctional complexes and delayed their reassembly in calcium switch experiments. Inducing expression of GP2-Cad1 to levels approximately threefold greater than endogenous E-cadherin expression levels in control cells resulted in a decrease in endogenous E-cadherin levels. This was due in part to increased protein turnover, indicating a cellular mechanism for sensing and controlling E-cadherin levels. Cadherin association with catenins is necessary for strong cadherin-mediated cell-cell adhesion. In cells expressing low levels of GP2-Cad1, protein levels and stoichiometry of the endogenous cadherin-catenin complex were unaffected. Thus effects of GP2-Cad1 on epithelial junctional complex assembly and stability were not due to competition with endogenous E-cadherin for catenin binding. Rather, we suggest that GP2-Cad1 interferes with the packing of endogenous cadherin-catenin complexes into higher-order structures in junctional complexes that results in junction destabilization.

    View details for Web of Science ID 000078595500015

    View details for PubMedID 9950768

  • New perspectives on mechanisms involved in generating epithelial cell polarity PHYSIOLOGICAL REVIEWS Yeaman, C., Grindstaff, K. K., NELSON, W. J. 1999; 79 (1): 73-98

    Abstract

    Polarized epithelial cells form barriers that separate biological compartments and regulate homeostasis by controlling ion and solute transport between those compartments. Receptors, ion transporters and channels, signal transduction proteins, and cytoskeletal proteins are organized into functionally and structurally distinct domains of the cell surface, termed apical and basolateral, that face these different compartments. This review is about mechanisms involved in the establishment and maintenance of cell polarity. Previous reports and reviews have adopted a Golgi-centric view of how epithelial cell polarity is established, in which the sorting of apical and basolateral membrane proteins in the Golgi complex is a specialized process in polarized cells, and the generation of cell surface polarity is a direct consequence of this process. Here, we argue that events at the cell surface are fundamental to the generation of cell polarity. We propose that the establishment of structural asymmetry in the plasma membrane is the first, critical event, and subsequently, this asymmetry is reinforced and maintained by delivery of proteins that were constitutively sorted in the Golgi. We propose a hierarchy of stages for establishing cell polarity.

    View details for Web of Science ID 000080163000002

    View details for PubMedID 9922368

  • Distribution of cell membrane-associated proteins along the human nephron JOURNAL OF HISTOCHEMISTRY & CYTOCHEMISTRY Kwon, O., Myers, B. D., Sibley, R., Dafoe, D., Alfrey, E., NELSON, W. J. 1998; 46 (12): 1423-1434

    Abstract

    Cytoskeletal proteins associate with specific cell adhesion complexes and membrane proteins and influence the structural and functional organization of polarized epithelial cells in the kidney. Among such proteins that have been studied in cultured cell lines and in animals are the tight junction complex (ZO-1 and occludin), the adherens cell-cell adhesion complex (alpha-, beta-catenin and plakoglobin), and Na+,K+-ATPase, with its associated membrane skeleton proteins ankyrin and fodrin. Although abnormal distribution of these proteins has been implicated in the pathogenesis of various renal diseases, the relevance of these findings to corresponding disease of the human kidney remains to be established. As a first step towards elucidating a role for such proteins in human kidney disease, we undertook a histochemical analysis of the distribution of these proteins in biopsy specimens of human kidney taken from healthy kidney transplant donors. We found each protein to have a characteristic subcellular localization and an intensity of staining that varied among different segments of the nephron in a manner that is consistent with discrete, segmental nephron function.

    View details for Web of Science ID 000077379000011

    View details for PubMedID 9815284

  • Biogenesis of polarized epithelial cells during kidney development in situ: Roles of E-cadherin-mediated cell-cell adhesion and membrane cytoskeleton organization MOLECULAR BIOLOGY OF THE CELL Piepenhagen, P. A., Nelson, W. J. 1998; 9 (11): 3161-3177

    Abstract

    Organization of proteins into structurally and functionally distinct plasma membrane domains is an essential characteristic of polarized epithelial cells. Based on studies with cultured kidney cells, we have hypothesized that a mechanism for restricting Na/K-ATPase to the basal-lateral membrane involves E-cadherin-mediated cell-cell adhesion and integration of Na/K-ATPase into the Triton X-100-insoluble ankyrin- and spectrin-based membrane cytoskeleton. In this study, we examined the relevance of these in vitro observations to the generation of epithelial cell polarity in vivo during mouse kidney development. Using differential detergent extraction, immunoblotting, and immunofluorescence histochemistry, we demonstrate the following. First, expression of the 220-kDa splice variant of ankyrin-3 correlates with the development of resistance to Triton X-100 extraction for Na/K-ATPase, E-cadherin, and catenins and precedes maximal accumulation of Na/K-ATPase. Second, expression of the 190-kDa slice variant of ankyrin-3 correlates with maximal accumulation of Na/K-ATPase. Third, Na/K-ATPase, ankyrin-3, and fodrin specifically colocalize at the basal-lateral plasma membrane of all epithelial cells in which they are expressed and during all stages of nephrogenesis. Fourth, the relative immunofluorescence staining intensities of Na/K-ATPase, ankyrin-3, and fodrin become more similar during development until they are essentially identical in adult kidney. Thus, renal epithelial cells in vivo regulate the accumulation of E-cadherin-mediated adherens junctions, the membrane cytoskeleton, and Na/K-ATPase through sequential protein expression and assembly on the basal-lateral membrane. These results are consistent with a mechanism in which generation and maintenance of polarized distributions of these proteins in vivo and in vitro involve cell-cell adhesion, assembly of the membrane cytoskeleton complex, and concomitant integration and retention of Na/K-ATPase in this complex.

    View details for Web of Science ID 000076888800014

    View details for PubMedID 9802904

  • Mechanisms of epithelial cell-cell adhesion and cell compaction revealed by high-resolution tracking of E-cadherin-green fluorescent protein JOURNAL OF CELL BIOLOGY Adams, C. L., Chen, Y. T., Smith, S. J., NELSON, W. J. 1998; 142 (4): 1105-1119

    Abstract

    Cadherin-mediated adhesion initiates cell reorganization into tissues, but the mechanisms and dynamics of such adhesion are poorly understood. Using time-lapse imaging and photobleach recovery analyses of a fully functional E-cadherin/GFP fusion protein, we define three sequential stages in cell-cell adhesion and provide evidence for mechanisms involving E-cadherin and the actin cytoskeleton in transitions between these stages. In the first stage, membrane contacts between two cells initiate coalescence of a highly mobile, diffuse pool of cell surface E-cadherin into immobile punctate aggregates along contacting membranes. These E-cadherin aggregates are spatially coincident with membrane attachment sites for actin filaments branching off from circumferential actin cables that circumscribe each cell. In the second stage, circumferential actin cables near cell-cell contact sites separate, and the resulting two ends of the cable swing outwards to the perimeter of the contact. Concomitantly, subsets of E-cadherin puncta are also swept to the margins of the contact where they coalesce into large E-cadherin plaques. This reorganization results in the formation of a circumferential actin cable that circumscribes both cells, and is embedded into each E-cadherin plaque at the contact margin. At this stage, the two cells achieve maximum contact, a process referred to as compaction. These changes in E-cadherin and actin distributions are repeated when additional single cells adhere to large groups of cells. The third stage of adhesion occurs as additional cells are added to groups of >3 cells; circumferential actin cables linked to E-cadherin plaques on adjacent cells appear to constrict in a purse-string action, resulting in the further coalescence of individual plaques into the vertices of multicell contacts. The reorganization of E-cadherin and actin results in the condensation of cells into colonies. We propose a model to explain how, through strengthening and compaction, E-cadherin and actin cables coordinate to remodel initial cell-cell contacts to the final condensation of cells into colonies.

    View details for Web of Science ID 000075619400019

    View details for PubMedID 9722621

  • Effects of regulated expression of mutant RhoA and Rac1 small GTPases on the development of epithelial (MDCK) cell polarity JOURNAL OF CELL BIOLOGY Jou, T. S., NELSON, W. J. 1998; 142 (1): 85-100

    Abstract

    MDCK cells expressing RhoA or Rac1 mutants under control of the tetracycline repressible transactivator were used to examine short-term effects of known amounts of each mutant before, during, or after development of cell polarity. At low cell density, Rac1V12 cells had a flattened morphology and intact cell-cell contacts, whereas Rac1N17 cells were tightly compacted. Abnormal intracellular aggregates formed between Rac1N17, F-actin, and E-cadherin in these nonpolarized cells. At all subsequent stages of polarity development, Rac1N17 and Rac1V12 colocalized with E-cadherin and F-actin in an unusual beaded pattern at lateral membranes. In polarized cells, intracellular aggregates formed with Rac1V12, F-actin, and an apical membrane protein (GP135). At low cell density, RhoAV14 and RhoAN19 were localized in the cytoplasm, and cells were generally flattened and more fibroblastic than epithelial in morphology. In polarized RhoAV14 cells, F-actin was diffuse at lateral membranes and prominent in stress fibers on the basal membrane. GP135 was abnormally localized to the lateral membrane and in intracellular aggregates, but E-cadherin distribution appeared normal. In RhoAN19 cells, F-actin, E-cadherin, and GP135 distributions were similar to those in controls. Expression of either RhoAV14 or RhoAN19 in Rac1V12 cells disrupted Rac1V12 distribution and caused cells to adopt the more fibroblastic, RhoA mutant phenotype. We suggest that Rac1 and RhoA are involved in the transition of epithelial cells from a fibroblastic to a polarized structure and function by direct and indirect regulation of actin and actin-associated membrane protein organizations.

    View details for Web of Science ID 000074945000008

    View details for PubMedID 9660865

  • Structural and functional regulation of tight junctions by RhoA and Rac1 small GTPases JOURNAL OF CELL BIOLOGY Jou, T. S., Schneeberger, E. E., NELSON, W. J. 1998; 142 (1): 101-115

    Abstract

    Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.

    View details for Web of Science ID 000074945000009

    View details for PubMedID 9660866

  • Sec6/8 complex is recruited to cell-cell contacts and specifies transport vesicle delivery to the basal-lateral membrane in epithelial cells CELL Grindstaff, K. K., Yeaman, C., Anandasabapathy, N., Hsu, S. C., Rodriguez-Boulan, E., Scheller, R. H., NELSON, W. J. 1998; 93 (5): 731-740

    Abstract

    In budding yeast, the Sec6/8p complex is essential for generating cell polarity by specifying vesicle delivery to the bud tip. We show that Sec6/8 homologs are components of a cytosolic, approximately 17S complex in nonpolarized MDCK epithelial cells. Upon initiation of calcium-dependent cell-cell adhesion, approximately 70% of Sec6/8 is rapidly (t(1/2) approximately 3-6 hr) recruited to sites of cell-cell contact. In streptolysin-O-permeabilized MDCK cells, Sec8 antibodies inhibit delivery of LDL receptor to the basal-lateral membrane, but not p75NTR to the apical membrane. These results indicate that lateral membrane recruitment of the Sec6/8 complex is a consequence of cell-cell adhesion and is essential for the biogenesis of epithelial cell surface polarity.

    View details for Web of Science ID 000073956700010

    View details for PubMedID 9630218

  • Backleak, light junctions, and cell-cell adhesion in postischemic injury to the renal allograft JOURNAL OF CLINICAL INVESTIGATION Kwon, O., NELSON, W. J., Sibley, R., Huie, P., Scandling, J. D., Dafoe, D., Alfrey, E., Myers, B. D. 1998; 101 (10): 2054-2064

    Abstract

    Postischemic injury in recipients of 3-7-d-old renal allografts was classified into sustained (n = 19) or recovering (n = 20) acute renal failure (ARF) according to the prevailing inulin clearance. Recipients of optimally functioning, long-standing allografts and living donors undergoing nephrectomy served as functional (n = 14) and structural controls (n = 10), respectively. Marked elevation above control of fractional clearance of dextrans of graded size was consistent with transtubular backleak of 57% of filtrate (inulin) in sustained ARF. No backleak was detected in recovering ARF. To explore a structural basis for backleak, allograft biopsies were taken intraoperatively, 1 h after reperfusion in all recipients, and again on day 7 after transplant in a subset (n = 10). Electron microscopy revealed disruption of both apical and basolateral membranes of proximal tubule cells in both sustained and recovering ARF, but cell exfoliation and tubule basement membrane denudation were negligible. Histochemical analysis of membrane-associated adhesion complexes confirmed an abnormality of proximal but not distal tubule cells, marked in sustained ARF but not in recovering ARF. Staining for the zonula occludens complex (ZO-1) and adherens complex (alpha, beta, and gamma catenins) revealed diminished intensity and redistribution of each cytoskeletal protein from the apico-lateral membrane boundary. We conclude that impaired integrity of tight junctions and cell-cell adhesion in the proximal tubule provides a paracellular pathway through which filtrate leaks back in sustained allograft ARF.

    View details for Web of Science ID 000073808800004

    View details for PubMedID 9593761

  • Apiconuclear organization of microtubules does not specify protein delivery from the trans-Golgi network to different membrane domains in polarized epithelial cells MOLECULAR BIOLOGY OF THE CELL Grindstaff, K. K., Bacallao, R. L., NELSON, W. J. 1998; 9 (3): 685-699

    Abstract

    In nonpolarized epithelial cells, microtubules originate from a broad perinuclear region coincident with the distribution of the Golgi complex and extend outward to the cell periphery (perinuclear [PN] organization). During development of epithelial cell polarity, microtubules reorganize to form long cortical filaments parallel to the lateral membrane, a meshwork of randomly oriented short filaments beneath the apical membrane, and short filaments at the base of the cell; the Golgi becomes localized above the nucleus in the subapical membrane cytoplasm (apiconuclear [AN] organization). The AN-type organization of microtubules is thought to be specialized in polarized epithelial cells to facilitate vesicle trafficking between the trans-Golgi Network (TGN) and the plasma membrane. We describe two clones of MDCK cells, which have different microtubule distributions: clone II/G cells, which gradually reorganize a PN-type distribution of microtubules and the Golgi complex to an AN-type during development of polarity, and clone II/J cells which maintain a PN-type organization. Both cell clones, however, exhibit identical steady-state polarity of apical and basolateral proteins. During development of cell surface polarity, both clones rapidly establish direct targeting pathways for newly synthesized gp80 and gp135/170, and E-cadherin between the TGN and apical and basolateral membrane, respectively; this occurs before development of the AN-type microtubule/Golgi organization in clone II/G cells. Exposure of both clone II/G and II/J cells to low temperature and nocodazole disrupts >99% of microtubules, resulting in: 1) 25-50% decrease in delivery of newly synthesized gp135/170 and E-cadherin to the apical and basolateral membrane, respectively, in both clone II/G and II/J cells, but with little or no missorting to the opposite membrane domain during all stages of polarity development; 2) approximately 40% decrease in delivery of newly synthesized gp80 to the apical membrane with significant missorting to the basolateral membrane in newly established cultures of clone II/G and II/J cells; and 3) variable and nonspecific delivery of newly synthesized gp80 to both membrane domains in fully polarized cultures. These results define several classes of proteins that differ in their dependence on intact microtubules for efficient and specific targeting between the Golgi and plasma membrane domains.

    View details for Web of Science ID 000072545800011

    View details for PubMedID 9487135

  • Identification of four distinct pools of catenins in mammalian cells and transformation-dependent changes in catenin distributions among these pools JOURNAL OF BIOLOGICAL CHEMISTRY STEWART, D. B., NELSON, W. J. 1997; 272 (47): 29652-29662

    Abstract

    Catenins are cytoplasmic proteins that were initially identified in a complex with cadherins, a superfamily of transmembrane glycoproteins important for cell adhesion in normal and disease states. We have used gel filtration to identify four complexes of catenins in extracts from normal and transformed epithelial cells. In normal Madin-Darby canine kidney epithelial cells, a significant fraction of alpha- and beta-catenin and plakoglobin co-elute with cadherin in a high molecular weight complex (complex I). A portion of alpha-catenin and the remainder of beta-catenin and plakoglobin co-elute in a high molecular weight complex that does not contain cadherin (complex II). The remainder of alpha-catenin elutes in a low molecular weight fraction (complex III). In extracts from two colon carcinoma cell lines, HCT116 and SW480, beta-catenin elutes in an additional low molecular weight pool (complex IV) not present in Madin-Darby canine kidney cell extracts. In two subclones derived from SW480 cells, SW-E8 and SW-R2, beta-catenin is distributed evenly between high and low molecular weight pools in SW-E8 cells, whereas it elutes primarily in the low molecular weight pool (complex IV) in SW-R2 cells. These changes in beta-catenin elution profiles correlate with an increase in transformed phenotype and decreased cell-cell adhesion in the SW-R2 cells.

    View details for Web of Science ID A1997YG64700042

    View details for PubMedID 9368032

  • Cadherins, catenins and APC protein: interplay between cytoskeletal complexes and signaling pathways CURRENT OPINION IN CELL BIOLOGY Barth, A. I., Nathke, I. S., NELSON, W. J. 1997; 9 (5): 683-690

    Abstract

    Cadherins play important roles in cell-cell adhesion during tissue differentiation. Cadherins are linked to the actin cytoskeleton by catenins (beta-catenin/armadillo, plakoglobin, and alpha-catenin). Recent results show that beta-catenin also binds to another cytoskeletal complex containing the adenomatous polyposis coli protein and microtubules, and interacts with several signaling pathways that include tyrosine kinases and phosphatases and Wnt/Wingless. Interplay between these cytoskeletal complexes and signaling pathways may regulate morphogenesis.

    View details for Web of Science ID A1997XX92200012

    View details for PubMedID 9330872

  • Three-dimensional structure of the armadillo repeat region of beta-catenin CELL Huber, A. H., NELSON, W. J., Weis, W. I. 1997; 90 (5): 871-882

    Abstract

    Beta-catenin is essential for cadherin-based cell adhesion and Wnt/Wingless growth factor signaling. In these roles, it binds to cadherins, Tcf-family transcription factors, and the tumor suppressor gene product Adenomatous Polyposis Coli (APC). A core region of beta-catenin, composed of 12 copies of a 42 amino acid sequence motif known as an armadillo repeat, mediates these interactions. The three-dimensional structure of a protease-resistant fragment of beta-catenin containing the armadillo repeat region has been determined. The 12 repeats form a superhelix of helices that features a long, positively charged groove. Although unrelated in sequence, the beta-catenin binding regions of cadherins, Tcfs, and APC are acidic and are proposed to interact with this groove.

    View details for Web of Science ID A1997XV56300008

    View details for PubMedID 9298899

  • Cell polarity: Par for the polar course CURRENT BIOLOGY NELSON, W. J., Grindstaff, K. K. 1997; 7 (9): R562-R564

    Abstract

    The nematode PAR-1 gene is required for asymmetric cell divisions during development. Recently identified mammalian Par-1 homologues are kinases that phosphorylate microtubule-associated proteins; their overexpression disrupts the microtubule cytoskeleton, and alters cellular structure and organization.

    View details for Web of Science ID A1997XW88900017

    View details for PubMedID 9285696

  • Golgi membrane skeleton: Identification, localization and oligomerization of a 195 kDa ankyrin isoform associated with the Golgi complex JOURNAL OF CELL SCIENCE Beck, K. A., Buchanan, J. A., NELSON, W. J. 1997; 110: 1239-1249

    Abstract

    To extend our finding of a Golgi-localized form of the membrane skeleton protein spectrin, we have identified an isoform of ankyrin that associates at steady state with the Golgi complex. Immuno-light and -electron microscopy show that this ankyrin isoform localizes to the perinuclear cytoplasm on tubular vesicular structures that co-stain with Golgi marker proteins. An antiserum raised against erythrocyte ankyrin, which was used to identify the Golgi ankyrin, recognized three prominent polypeptides of 220, 213 and 195 kDa in MDCK cells. Affinity purification of this antiserum against each of these MDCK cell ankyrins revealed that only an antibody specific for the 195 kDa form retained the ability to stain the Golgi complex; affinity purified antibody preparations specific for both the 220 and 213 kDa forms stained punctate and reticular cytoplasmic structures distinct from the Golgi complex. Antibody specific for the 195 kDa ankyrin did not recognize a recently identified 119 kDa ankyrin that is also localized to the Golgi. The 195 kDa Golgi ankyrin binds purified erythrocyte spectrin, and rapidly co-sediments with Golgi beta-spectrin during brief, low speed centrifugation of Triton X-100 extracts of MDCK cells. Golgi ankyrin and beta-spectrin are retained on tubular vesicular 'Golgi ghosts' following extraction of cultured cells with Triton X-100. Significantly, Golgi ghost tubules containing ankyrin/spectrin are co-linear with individual microtubules, suggesting a role for both Golgi membrane skeleton and microtubules in spatial localization of the Golgi. Golgi ankyrin dissociates from Golgi membranes during mitosis and in cells treated with brefeldin A, indicating that Golgi ankyrin has a dynamic assembly state similar to that of Golgi spectrin and other Golgi membrane coat proteins.

    View details for Web of Science ID A1997XE50500010

    View details for PubMedID 9191047

  • NH2-terminal deletion of beta-catenin results in stable colocalization of mutant beta-catenin with adenomatous polyposis coli protein and altered MDCK cell adhesion JOURNAL OF CELL BIOLOGY Barth, A. I., Pollack, A. L., Altschuler, Y., MOSTOV, K. E., NELSON, W. J. 1997; 136 (3): 693-706

    Abstract

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

    View details for Web of Science ID A1997WH75300017

    View details for PubMedID 9024698

  • NH2-terminal deletion of b-catenin results in stable co-localization of mutant b-catenin with APC protein and altered MDCK cell adhesion J. Cell Biol Nelson,W. J., Barth, A. I. M., Pollack, A. L., Altschuler, Y., Mostov, K. E. 1997
  • Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium BIOESSAYS Sjaastad, M. D., NELSON, W. J. 1997; 19 (1): 47-55

    Abstract

    Integrins are ubiquitous trans-membrane adhesion molecules that mediate the interaction of cells with the extracellular matrix (ECM). Integrins link cells to the ECM by interacting with the cell cytoskeleton. In cases such as leukocyte binding, integrins mediate cell-cell interactions and cell-ECM interactions. Recent research indicates that integrins also function as signal transduction receptors, triggering a number of intracellular signaling pathways that regulate cell behavior and development. A number of integrins are known to stimulate changes in intracellular calcium levels, resulting in integrin activation. Although changes in intracellular calcium regulate a vast number of cellular functions, this review will discuss the stimulation of calcium signaling by integrins and the role of intracellular calcium in the regulation of integrin-mediated adhesion.

    View details for Web of Science ID A1997WC70000008

    View details for PubMedID 9008416

  • Dynamics of b-catenin interactions with APC protein regulate epithelial tubulogenesis J. Cell Biol Nelson, W. J., Pollack, A. L., Barth, A. I. M., Altschuler, Y., Mostov, K. E. 1997
  • Quantitative analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion JOURNAL OF CELL BIOLOGY Adams, C. L., NELSON, W. J., Smith, S. J. 1996; 135 (6): 1899-1911

    Abstract

    Epithelial cell-cell adhesion requires interactions between opposing extracellular domains of E-cadherin, and among the cytoplasmic domain of E-cadherin, catenins, and actin cytoskeleton. Little is known about how the cadherin-catenin-actin complex is assembled upon cell-cell contact, or how these complexes initiate and strengthen adhesion. We have used time-lapse differential interference contrast (DIC) imaging to observe the development of cell-cell contacts, and quantitative retrospective immunocytochemistry to measure recruitment of proteins to those contacts. We show that E-cadherin, alpha-catenin, and beta-catenin, but not plakoglobin, coassemble into Triton X-100 insoluble (TX-insoluble) structures at cell-cell contacts with kinetics similar to those for strengthening of E-cadherin-mediated cell adhesion (Angres, B., A. Barth, and W.J. Nelson. 1996. J. Cell Biol. 134:549-557). TX-insoluble E-cadherin, alpha-catenin, and beta-catenin colocalize along cell-cell contacts in spatially discrete micro-domains which we designate "puncta," and the relative amounts of each protein in each punctum increase proportionally. As the length of the contact increases, the number of puncta increases proportionally along the contact and each punctum is associated with a bundle of actin filaments. These results indicate that localized clustering of E-cadherin/catenin complexes into puncta and their association with actin is involved in initiating cell contacts. Subsequently, the spatial ordering of additional puncta along the contact may be involved in zippering membranes together, resulting in rapid strengthening of adhesion.

    View details for Web of Science ID A1996WA87900019

    View details for PubMedID 8991100

  • Continuous production of soluble extracellular domain of a type-I transmembrane protein in mammalian cells using an Epstein-Barr virus Ori-P-based expression vector ANALYTICAL BIOCHEMISTRY Chen, Y. T., NELSON, W. J. 1996; 242 (2): 276-278

    View details for Web of Science ID A1996VV31800018

    View details for PubMedID 8937575

  • The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration JOURNAL OF CELL BIOLOGY Nathke, I. S., Adams, C. L., Polakis, P., Sellin, J. H., NELSON, W. J. 1996; 134 (1): 165-179

    Abstract

    Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. We show that APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration.

    View details for Web of Science ID A1996UW67600013

    View details for PubMedID 8698812

  • Mechanisms of integrin-mediated calcium signaling in MDCK cells: Regulation of adhesion by IP3 and store-independent calcium influx MOLECULAR BIOLOGY OF THE CELL Sjaastad, M. D., Lewis, R. S., NELSON, W. J. 1996; 7 (7): 1025-1041

    Abstract

    Peptides containing Arg-Gly-Asp (RGD) immobilized on beads bind to integrins and trigger biphasic, transient increases in intracellular free Ca2+ ([Ca2+]i) in Madin-Darby canine kidney epithelial cells. The [Ca2+]i increase participates in feedback regulation of integrin-mediated adhesion in these cells. We examined influx pathways and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ store release as possible sources of the [Ca2+]i rise. The RGD-induced [Ca2+]i response requires external Ca2+ (threshold approximately 150 microM), and its magnitude is proportional to extracellular calcium. RGD-induced transients were attenuated by Ca2+ channel inhibitors (Ni2+ and carboxy-amidotriazole) or by plasma membrane depolarization, indicating that Ca2+ influx contributes to the response. Loading cells with heparin reduced the size of RGD-induced [Ca2+]i transients, indicating that IP3-mediated release of Ca2+ from stores may also contribute to the RGD response. Depletion of Ca2+ stores with thapsigargin activated Ni(2+)-sensitive Ca2+ influx that might also be expected to occur after IP3-mediated depletion of stored Ca2-. However, RGD elicited a Ni(2+)-sensitive Ca2+ influx even after pretreatment with thapsigargin, indicating that Ca2+ influx is controlled by a mechanism independent of IP3-mediated store depletion. We conclude that RGD-induced [Ca2+]i transients in Madin-Darby canine kidney cells result primarily from the combination of two distinct mechanisms: 1) IP3-mediated release of intracellular stores, and 2) activation of a Ca2+ influx pathway regulated independently of IP3 and Ca2+ store release. Because Ni2+ and carboxy-amidotriazole inhibited adhesion, whereas store depletion with thapsigargin had little effect, we suggest that the Ca2+ influx mechanism is most important for feedback regulation of integrin-mediated adhesion by increased [Ca2+]i.

    View details for Web of Science ID A1996UX91800003

    View details for PubMedID 8862518

  • Mechanism for transition from initial to stable cell-cell adhesion: Kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay JOURNAL OF CELL BIOLOGY Angres, B., Barth, A., NELSON, W. J. 1996; 134 (2): 549-557

    Abstract

    A centrifugal force-based adhesion assay has been used to quantitatively examine the kinetics of formation of cell-cell contacts mediated specifically by expression of E-cadherin under the control of a glucocorticoid-inducible promoter in mouse fibroblasts. Analysis of cells expressing maximal or minimal levels of E-cadherin showed that the strength of E-cadherin-mediated adhesion developed in a single exponential step over a short time (half-maximal adhesion, 13-17 min). At 37 degrees C, adhesion strength increased rapidly in the first 20 min without an apparent lag phase. After 90 min, adhesion strength reached a plateau. Differences in final strengths of adhesion were commensurate with the level of E-cadherin expression. Strengthening of adhesion was temperature dependent. At 19 degrees C, strengthening of adhesion was delayed and subsequently developed with a slower rate compared to adhesion at 37 degrees C. At 4 degrees C, adhesion was completely inhibited. Strengthening of adhesion was absolutely dependent on a functional actin cytoskeleton since adhesion did not develop when cells were treated with cytochalasin D. Together, our current and previous (McNeill et al., 1993.J. Cell Biol. 120:1217-1226) studies indicate that the rate of initial strengthening of E-cadherin-mediated adhesion is neither dependent on the amount of E-cadherin expressed nor on long-range protein diffusion in the membrane to the adhesion site. However, initial strengthening of adhesion is dependent on temperature-sensitive cellular activities that may locally couple clusters of E-cadherin to the actin cytoskeleton.

    View details for Web of Science ID A1996UX94600024

    View details for PubMedID 8707837

  • The spectrin-based membrane skeleton as a membrane protein-sorting machine AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Beck, K. A., NELSON, W. J. 1996; 270 (5): C1263-C1270

    Abstract

    Normal cell function is dependent on the existence of membrane compartments that have unique populations of membrane proteins. Sorting of membrane proteins forms the basis for the biogenesis of distinct membrane compartments. There are many examples of membrane protein-sorting events in cells, but the molecular machinery involved is poorly understood. We discuss characteristics of a putative membrane protein-sorting machine and show that the spectrin-based membrane skeleton conforms to these characteristics. The spectrin-based membrane skeleton is a submembranous, spatially limited, two-dimensional lattice that binds a subset of membrane proteins. These properties allow the membrane skeleton to facilitate the formation of distinct membrane domains and thus reveal its potential as a membrane protein-sorting machine.

    View details for Web of Science ID A1996UJ81400001

    View details for PubMedID 8967424

  • Origins of cell polarity CELL Drubin, D. G., NELSON, W. J. 1996; 84 (3): 335-344

    View details for Web of Science ID A1996TV70800002

    View details for PubMedID 8608587

  • Quantitative, single-cell analysis of cadherin-catenin-actin reorganization during development of cell-cell adhesion J. Cell Biol Nelson, W. J.,, Adams, C. A., Smith, S J. 1996
  • Differential expression of cell-cell and cell-substratum adhesion proteins along the kidney nephron AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Piepenhagen, P. A., NELSON, W. J. 1995; 269 (6): C1433-C1449

    Abstract

    Structural and functional differences among epithelial cells of kidney nephrons may be regulated by variations in cell-to-cell (cell-cell) and cell-to-substratum (cell-substratum) junctions. Using immunofluorescence microscopy, we demonstrate that the cadherin-associated proteins alpha- and beta-catenin are localized to basolateral membranes of cells in all nephron segments, whereas plakoglobin, a protein associated with both classical and desmosomal cadherins, is localized to noninterdigitated lateral membranes in the distal half of the nephron where it colocalizes with desmoplakin and cytokeratin K8. Plakoglobin is also present in capillary endothelial cells where staining for the other catenins and desmosomal proteins is not observed. Immunofluorescence for laminin A and alpha 6-integrin, proteins that mediate cell-substratum contacts, reveal no correlations with the other staining patterns observed. These data indicate that plakoglobin and beta-catenin subserve distinct functions in cell-cell adhesion and suggest that E-cadherin-mediated contacts generate a basal level of cell-cell adhesion, whereas desmosomal junctions provide additional strength to cell-cell contacts in the distal nephron.

    View details for Web of Science ID A1995TL69500012

    View details for PubMedID 8572172

  • Differential expression of Na+-K+-ATPase, ankyrin, fodrin, and E-cadherin along the kidney nephron AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY Piepenhagen, P. A., Peters, L. L., Lux, S. E., NELSON, W. J. 1995; 269 (6): C1417-C1432

    Abstract

    Ionic homeostasis in vertebrates is maintained by epithelial cells that line kidney nephrons. Transport of ions and solutes is coupled to Na+ reabsorption from the ultrafiltrate and requires specific subcellular distribution and activity of Na(+)-K(+)-ATPase along the nephron. Studies using cell culture models of renal epithelia indicate that the subcellular distribution of Na(+)-K(+)-ATPase is regulated by interactions with the submembrane cytoskeleton and E-cadherin-mediated adherens junctions. We have now examined the relevance of these in vitro observations to the subcellular organization of these proteins in different nephron segments of the adult mouse kidney using immunofluorescence microscopy. Our results demonstrate that segmental and subcellular distributions of Na(+)-K(+)-ATPase and the membrane-cytoskeletal proteins, ankyrin and fodrin, vary in parallel along the nephron and do not parallel variations in expression of the tight junction protein ZO-1 or E-cadherin. These data indicate that a mechanism for restricting Na(+)-K(+)-ATPase subcellular distributions through interactions with the membrane cytoskeleton is likely to be relevant in vivo.

    View details for Web of Science ID A1995TL69500011

    View details for PubMedID 8572171

  • HIERARCHY OF MECHANISMS INVOLVED IN GENERATING NA/K-ATPASE POLARITY IN MDCK EPITHELIAL-CELLS JOURNAL OF CELL BIOLOGY Mays, R. W., Siemers, K. A., Fritz, B. A., Lowe, A. W., VANMEER, G., NELSON, W. J. 1995; 130 (5): 1105-1115

    Abstract

    We have studied mechanisms involved in generating a polarized distribution of Na/K-ATPase in the basal-lateral membrane of two clones of MDCK II cells. Both clones exhibit polarized distributions of marker proteins of the apical and basal-lateral membranes, including Na/K-ATPase, at steady state. Newly synthesized Na/K-ATPase, however, is delivered from the Golgi complex to both apical and basal-lateral membranes of one clone (II/J), and to the basal-lateral membrane of the other clone (II/G); Na/K-ATPase is selectively retained in the basal-lateral membrane resulting in the generation of complete cell surface polarity in both clones. Another basal-lateral membrane protein, E-cadherin, is sorted to the basal-lateral membrane in both MDCK clones, demonstrating that there is not a general sorting defect for basal-lateral membrane proteins in clone II/J cells. A glycosyl-phosphatidylinositol (GPI)-anchored protein (GP-2) and a glycosphingolipid (glucosylceramide, GlcCer) are preferentially transported to the apical membrane in clone II/G cells, but, in clone II/J cells, GP-2 and GlcCer are delivered equally to both apical and basal-lateral membranes, similar to Na/K-ATPase. To examine this apparent inter-relationship between sorting of GlcCer, GP-2 and Na/K-ATPase, sphingolipid synthesis was inhibited in clone II/G cells with the fungal metabolite, Fumonisin B1 (FB1). In the presence of FB1, GP-2 and Na/K-ATPase are delivered to both apical and basal-lateral membranes, similar to clone II/J cells; FB1 had no effect on sorting of E-cadherin to the basal-lateral membrane of II/G cells. Addition of exogenous ceramide, to circumvent the FB1 block, restored GP-2 and Na/K-ATPase sorting to the apical and basal-lateral membranes, respectively. These results show that the generation of complete cell surface polarity of Na/K-ATPase involves a hierarchy of sorting mechanisms in the Golgi complex and plasma membrane, and that Na/K-ATPase sorting in the Golgi complex of MDCK cells may be regulated by exclusion from an apical pathway(s). These results also provide new insights into sorting pathways for other apical and basal-lateral membrane proteins.

    View details for Web of Science ID A1995RR84100009

    View details for PubMedID 7657695

  • GENETIC AND BIOCHEMICAL DISSECTION OF PROTEIN LINKAGES IN THE CADHERIN-CATENIN COMPLEX PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jou, T. S., STEWART, D. B., STAPPERT, J., NELSON, W. J., MARRS, J. A. 1995; 92 (11): 5067-5071

    Abstract

    The cadherin-catenin complex is important for mediating homotypic, calcium-dependent cell-cell interactions in diverse tissue types. Although proteins of this complex have been identified, little is known about their interactions. Using a genetic assay in yeast and an in vitro protein-binding assay, we demonstrate that beta-catenin is the linker protein between E-cadherin and alpha-catenin and that E-cadherin does not bind directly to alpha-catenin. We show that a 25-amino acid sequence in the cytoplasmic domain of E-cadherin and the amino-terminal domain of alpha-catenin are independent binding sites for beta-catenin. In addition to beta-catenin and plakoglobin, another member of the armadillo family, p120 binds to E-cadherin. However, unlike beta-catenin, p120 does not bind alpha-catenin in vitro, although a complex of p120 and endogenous alpha-catenin could be immunoprecipitated from cell extracts. In vitro protein-binding assays using recombinant E-cadherin cytoplasmic domain and alpha-catenin revealed two catenin pools in cell lysates: an approximately 1000- to approximately 2000-kDa complex bound to E-cadherin and an approximately 220-kDa pool that did not contain E-cadherin. Only beta-catenin in the approximately 220-kDa pool bound exogenous E-cadherin. Delineation of these molecular linkages and the demonstration of separate pools of catenins in different cell lines provide a foundation for examining regulatory mechanisms involved in the assembly and function of the cadherin-catenin complex.

    View details for Web of Science ID A1995RA15000072

    View details for PubMedID 7761449

  • PLASTICITY IN EPITHELIAL-CELL PHENOTYPE - MODULATION BY EXPRESSION OF DIFFERENT CADHERIN CELL-ADHESION MOLECULES JOURNAL OF CELL BIOLOGY MARRS, J. A., ANDERSSONFISONE, C., Jeong, M. C., COHENGOULD, L., ZURZOLO, C., Nabi, I. R., RODRIGUEZBOULAN, E., NELSON, W. J. 1995; 129 (2): 507-519

    Abstract

    A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.

    View details for Web of Science ID A1995QU09100017

    View details for PubMedID 7536748

  • BETA-CATENIN - A COMMON TARGET FOR THE REGULATION OF CELL-ADHESION BY WNT-1 AND SRC SIGNALING PATHWAYS TRENDS IN BIOCHEMICAL SCIENCES Hinck, L., Nathke, I. S., Papkoff, J., NELSON, W. J. 1994; 19 (12): 538-542

    Abstract

    Beta-catenin is a cytosolic protein originally identified through its association with the cadherin class of cell-adhesion proteins. However, recent studies have demonstrated that there are cadherin-independent pools of beta-catenin and that beta-catenin binds at least one other protein, the product of the tumor-suppressor gene APC. Furthermore, beta-catenin is the target of two signal transduction pathways mediated by the proto-oncogenes src and wnt-1. This raises the possibility that beta-catenin plays a pivotal role in balancing cellular responses to both adhesive and proliferative signals.

    View details for Web of Science ID A1994PX38500010

    View details for PubMedID 7846766

  • GOLGI SPECTRIN - IDENTIFICATION OF AN ERYTHROID BETA-SPECTRIN HOMOLOG ASSOCIATED WITH THE GOLGI-COMPLEX JOURNAL OF CELL BIOLOGY Beck, K. A., Buchanan, J. A., Malhotra, V., NELSON, W. J. 1994; 127 (3): 707-723

    Abstract

    Spectrin is a major component of a membrane-associated cytoskeleton involved in the maintenance of membrane structural integrity and the generation of functionally distinct membrane protein domains. Here, we show that a homolog of erythrocyte beta-spectrin (beta I sigma*) co-localizes with markers of the Golgi complex in a variety of cell types, and that microinjected beta-spectrin codistributes with elements of the Golgi complex. Significantly, we show a dynamic relationship between beta-spectrin and the structural and functional organization of the Golgi complex. Disruption of both Golgi structure and function, either in mitotic cells or following addition of brefeldin A, is accompanied by loss of beta-spectrin from Golgi membranes and dispersal in the cytoplasm. In contrast, perturbation of Golgi structure without a loss of function, by the addition of nocodazole, results in retention of beta-spectrin with the dispersed Golgi elements. These results indicate that the association of beta-spectrin with Golgi membranes is coupled to Golgi organization and function.

    View details for Web of Science ID A1994PP69400010

    View details for PubMedID 7962054

  • FEEDBACK-REGULATION OF CELL-SUBSTRATUM ADHESION BY INTEGRIN-MEDIATED INTRACELLULAR CA2+ SIGNALING PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sjaastad, M. D., Angres, B., Lewis, R. S., NELSON, W. J. 1994; 91 (17): 8214-8218

    Abstract

    Integrin binding to extracellular matrix (ECM) regulates cell migration and gene expression in embryogenesis, metastasis, would healing, and the inflammatory response. In many cases, binding of integrins to ECM triggers intracellular signaling pathways. The regulatory roles of intracellular signaling mechanisms in these events are poorly understood. Using single-cell analysis, we demonstrate that beads coated with peptide containing Arg-Gly-Asp (RGD), an integrin recognition motif found in many ECM proteins, elicit a rapid transient increase in intracellular calcium in Madin-Darby canine kidney (MDCK) epithelial cells. Also, significantly more beads bind to responding cells than to nonresponders. Several independent methods that inhibit RGD-induced Ca2+ signaling decrease both the number of beads bound and the strength of adhesion to an RGD-coated substratum. These results indicate that intracellular Ca2+ signaling participates in a positive feedback loop that enhances integrin-mediated cell adhesion.

    View details for Web of Science ID A1994PC24200080

    View details for PubMedID 8058782

  • DETERMINANTS OF APICAL MEMBRANE FORMATION AND DISTRIBUTION IN MULTICELLULAR EPITHELIAL MDCK CYSTS AMERICAN JOURNAL OF PHYSIOLOGY Wang, A. Z., Wang, J. C., Ojakian, G. K., NELSON, W. J. 1994; 267 (2): C473-C481

    Abstract

    Madin-Darby canine kidney epithelial cells form three-dimensional cysts in spinner culture with a defined cell surface polarity. Transfer of cysts from spinner culture to a collagen gel matrix results in rapid loss of apical membrane proteins from the outside surface of the cyst, degradation of extracellular matrix (ECM) from the cyst lumen, and de novo formation of the apical membrane at the luminal surface. Degradation of endogenous ECM was inhibited with 1,10-phenanthroline, an inhibitor of metalloproteinases, resulting in cysts in which cells are surrounded by either cell-cell or cell-substratum contacts. The consequence of the lack of a free cell surface on the formation of a new apical membrane domain in these cysts was analyzed. Changes in cell surface polarity were followed with antibodies to marker proteins of the apical or basolateral membranes. In the absence of a free cell surface, the apical membrane formed de novo by accumulation and fusion of presorted vesicles containing apical membrane proteins; the coalescence of these vesicles results in the formation of a central lumen. These results provide novel insights into the generation of membrane domains and formation of a lumen in complex, three-dimensional epithelial structures in development.

    View details for Web of Science ID A1994PC43900017

    View details for PubMedID 8074182

  • DYNAMICS OF CADHERIN/CATENIN COMPLEX-FORMATION - NOVEL PROTEIN INTERACTIONS AND PATHWAYS OF COMPLEX ASSEMBLY JOURNAL OF CELL BIOLOGY Hinck, L., Nathke, I. S., Papkoff, J., NELSON, W. J. 1994; 125 (6): 1327-1340

    Abstract

    Calcium-dependent cell-cell adhesion is mediated by the cadherin family of cell adhesion proteins. Transduction of cadherin adhesion into cellular reorganization is regulated by cytosolic proteins, termed alpha-, beta-, and gamma-catenin (plakoglobin), that bind to the cytoplasmic domain of cadherins and link them to the cytoskeleton. Previous studies of cadherin/catenin complex assembly and organization relied on the coimmunoprecipitation of the complex with cadherin antibodies, and were limited to the analysis of the Triton X-100 (TX-100)-soluble fraction of these proteins. These studies concluded that only one complex exists which contains cadherin and all of the catenins. We raised antibodies specific for each catenin to analyze each protein independent of its association with E-cadherin. Extracts of Madin-Darby canine kidney epithelial cells were sequentially immunoprecipitated and immunoblotted with each antibody, and the results showed that there were complexes of E-cadherin/alpha-catenin, and either beta-catenin or plakoglobin in the TX-100-soluble fraction. We analyzed the assembly of cadherin/catenin complexes in the TX-100-soluble fraction by [35S]methionine pulse-chase labeling, followed by sucrose density gradient fractionation of proteins. Immediately after synthesis, E-cadherin, beta-catenin, and plakoglobin cosedimented as complexes. alpha-Catenin was not associated with these complexes after synthesis, but a subpopulation of alpha-catenin joined the complex at a time coincident with the arrival of E-cadherin at the plasma membrane. The arrival of E-cadherin at the plasma membrane coincided with an increase in its insolubility in TX-100, but extraction of this insoluble pool with 1% SDS disrupted the cadherin/catenin complex. Therefore, to examine protein complex assembly in both the TX-100-soluble and -insoluble fractions, we used [35S]methionine labeling followed by chemical cross-linking before cell extraction. Analysis of cross-linked complexes from cells labeled to steady state indicates that, in addition to cadherin/catenin complexes, there were cadherin-independent pools of catenins present in both the TX-100-soluble and -insoluble fractions. Metabolic labeling followed by chase showed that immediately after synthesis, cadherin/beta-catenin, and cadherin/plakoglobin complexes were present in the TX-100-soluble fraction. Approximately 50% of complexes were titrated into the TX-100-insoluble fraction coincident with the arrival of the complexes at the plasma membrane and the assembly of alpha-catenin. Subsequently, > 90% of labeled cadherin, but no additional labeled catenin complexes, entered the TX-100-insoluble fraction.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1994NT42000012

    View details for PubMedID 8207061

  • DEFINING INTERACTIONS AND DISTRIBUTIONS OF CADHERIN AND CATENIN COMPLEXES IN POLARIZED EPITHELIAL-CELLS JOURNAL OF CELL BIOLOGY Nathke, I. S., Hinck, L., Swedlow, J. R., Papkoff, J., NELSON, W. J. 1994; 125 (6): 1341-1352

    Abstract

    The cadherin/catenin complex plays important roles in cell adhesion, signal transduction, as well as the initiation and maintenance of structural and functional organization of cells and tissues. In the preceding study, we showed that the assembly of the cadherin/catenin complex is temporally regulated, and that novel combinations of catenin and cadherin complexes are formed in both Triton X-100-soluble and -insoluble fractions; we proposed a model in which pools of catenins are important in regulating assembly of E-cadherin/catenin and catenin complexes. Here, we sought to determine the spatial distributions of E-cadherin, alpha-catenin, beta-catenin, and plakoglobin, and whether different complexes of these proteins accumulate at steady state in polarized Madin-Darby canine kidney cells. Protein distributions were visualized by wide field, optical sectioning, and double immunofluorescence microscopy, followed by reconstruction of three-dimensional images. In cells that were extracted with Triton X-100 and then fixed (Triton X-100-insoluble fraction), more E-cadherin was concentrated at the apical junction relative to other areas of the lateral membrane. alpha-Catenin and beta-catenin colocalize with E-cadherin at the apical junctional complex. There is some overlap in the distribution of these proteins in the lateral membrane, but there are also areas where the distributions are distinct. Plakoglobin is excluded from the apical junctional complex, and its distribution in the lateral membrane is different from that of E-cadherin. Cells were also fixed and then permeabilized to reveal the total cellular pool of each protein (Triton X-100-soluble and -insoluble fractions). This analysis showed lateral membrane localization of alpha-catenin, beta-catenin, and plakoglobin, and it also revealed that they are distributed throughout the cell. Chemical cross-linking of proteins and analysis with specific antibodies confirmed the presence at steady state of E-cadherin/catenin complexes containing either beta-catenin or plakoglobin, and catenin complexes devoid of E-cadherin. Complexes containing E-cadherin/beta-catenin and E-cadherin/alpha-catenin are present in both the Triton X-100-soluble and -insoluble fractions, but E-cadherin/plakoglobin complexes are not detected in the Triton X-100-insoluble fraction. Taken together, these results show that different complexes of cadherin and catenins accumulate in fully polarized epithelial cells, and that they distribute to different sites. We suggest that cadherin/catenin and catenin complexes at different sites have specialized roles in establishing and maintaining the structural and functional organization of polarized epithelial cells.

    View details for Web of Science ID A1994NT42000013

    View details for PubMedID 8207062

  • WNT-1 MODULATES CELL-CELL ADHESION IN MAMMALIAN-CELLS BY STABILIZING BETA-CATENIN BINDING TO THE CELL-ADHESION PROTEIN CADHERIN JOURNAL OF CELL BIOLOGY Hinck, L., NELSON, W. J., Papkoff, J. 1994; 124 (5): 729-741

    Abstract

    Wnt-1 homologs have been identified in invertebrates and vertebrates and play important roles in cellular differentiation and organization. In Drosophila, the products of the segment polarity genes wingless (the Wnt-1 homolog) and armadillo participate in a signal transduction pathway important for cellular boundary formation in embryonic development, but functional interactions between the proteins are unknown. We have examined Wnt-1 function in mammalian cells in which armadillo (beta-catenin and plakoglobin) is known to bind to and regulate cadherin cell adhesion proteins. We show that Wnt-1 expression results in the accumulation of beta-catenin and plakoglobin. In addition, binding of beta-catenin to the cell adhesion protein, cadherin, is stabilized, resulting in a concomitant increase in the strength of calcium-dependent cell-cell adhesion. Thus, a consequence of the functional interaction between Wnt-1 and armadillo family members is the strengthening of cell-cell adhesion, which may lead to the specification of cellular boundaries.

    View details for Web of Science ID A1994MY84600009

    View details for PubMedID 8120095

  • ORGANIZATION AND FUNCTION OF THE CYTOSKELETON IN POLARIZED EPITHELIAL-CELLS - A COMPONENT OF THE PROTEIN SORTING MACHINERY CURRENT OPINION IN CELL BIOLOGY Mays, R. W., Beck, K. A., NELSON, W. J. 1994; 6 (1): 16-24

    Abstract

    Development and maintenance of cell-surface polarity in epithelial cells requires specialized localization of proteins to functionally and structurally distinct plasma membrane domains. The organization of these domains is dependent upon targeted delivery of transport vesicles between different membrane compartments, and upon protein sorting in the membranes of the Golgi complex and cell surface. Increasing evidence has been gathered in recent years that cytoskeletal components facilitate these processes.

    View details for Web of Science ID A1994NF80000004

    View details for PubMedID 8167021

  • DISTINGUISHING ROLES OF THE MEMBRANE-CYTOSKELETON AND CADHERIN MEDIATED CELL-CELL ADHESION IN GENERATING DIFFERENT NA+,K+-ATPASE DISTRIBUTIONS IN POLARIZED EPITHELIA JOURNAL OF CELL BIOLOGY MARRS, J. A., Napolitano, E. W., MURPHYERDOSH, C., Mays, R. W., Reichardt, L. F., NELSON, W. J. 1993; 123 (1): 149-164

    Abstract

    In simple epithelia, the distribution of ion transporting proteins between the apical or basal-lateral domains of the plasma membrane is important for determining directions of vectorial ion transport across the epithelium. In the choroid plexus, Na+,K(+)-ATPase is localized to the apical plasma membrane domain where it regulates sodium secretion and production of cerebrospinal fluid; in contrast, Na+,K(+)-ATPase is localized to the basal-lateral membrane of cells in the kidney nephron where it regulates ion and solute reabsorption. The mechanisms involved in restricting Na+,K(+)-ATPase distribution to different membrane domains in these simple epithelia are poorly understood. Previous studies have indicated a role for E-cadherin mediated cell-cell adhesion and membrane-cytoskeleton (ankyrin and fodrin) assembly in regulating Na+,K(+)-ATPase distribution in absorptive kidney epithelial cells. Confocal immunofluorescence microscopy reveals that in chicken and rat choroid plexus epithelium, fodrin, and ankyrin colocalize with Na+,K(+)-ATPase at the apical plasma membrane, but fodrin, ankyrin, and adducin also localize at the lateral plasma membrane where Na+,K(+)-ATPase is absent. Biochemical analysis shows that fodrin, ankyrin, and Na+,K(+)-ATPase are relatively resistant to extraction from cells in buffers containing Triton X-100. The fractions of Na+,K(+)-ATPase, fodrin, and ankyrin that are extracted from cells cosediment in sucrose gradients at approximately 10.5 S. Further separation of the 10.5 S peak of proteins by electrophoresis in nondenaturing polyacrylamide gels revealed that fodrin, ankyrin, and Na+,K(+)-ATPase comigrate, indicating that these proteins are in a high molecular weight complex similar to that found previously in kidney epithelial cells. In contrast, the anion exchanger (AE2), a marker protein of the basal-lateral plasma membrane in the choroid plexus, did not cosediment in sucrose gradients or comigrate in nondenaturing polyacrylamide gels with the complex of Na+,K(+)-ATPase, ankyrin, and fodrin. Ca(++)-dependent cell adhesion molecules (cadherins) were detected at lateral membranes of the choroid plexus epithelium and colocalized with a distinct fraction of ankyrin, fodrin, and adducin. Cadherins did not colocalize with Na+,K(+)-ATPase and were absent from the apical membrane. The fraction of cadherins that was extracted with buffers containing Triton X-100 cosedimented with ankyrin and fodrin in sucrose gradients and comigrated in nondenaturing gels with ankyrin and fodrin in a high molecular weight complex. Since a previous study showed that E-cadherin is an instructive inducer of Na+,K(+)-ATPase distribution, we examined protein distributions in fibroblasts transfected with B-cadherin, a prominent cadherin expressed in the choroid plexus epithelium.(ABSTRACT TRUNCATED AT 400 WORDS)

    View details for Web of Science ID A1993LZ63100015

    View details for PubMedID 8408194

  • Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion. Seminars in cell biology Schmidt, J. W., Piepenhagen, P. A., NELSON, W. J. 1993; 4 (3): 161-173

    Abstract

    Cell-cell and cell-extracellular matrix (ECM) interactions control many developmental decisions of epithelial cell fate and morphogenesis. Protein tyrosine kinases are one class of regulatory molecules that have been implicated in the modulation of these processes. Several protein tyrosine kinases co-localize with cell-cell (cadherin) and cell-ECM (integrin) adhesion molecules at specific adhesion domains of epithelial cells. Protein tyrosine kinases may regulate epithelial development by modulating cell-cell and cell-ECM interactions and by relaying signals initiated by these interactions to other cellular components that determine cell structure and function.

    View details for PubMedID 8347833

  • DEFINING E-CADHERIN-ASSOCIATED PROTEIN COMPLEXES IN EPITHELIAL-CELLS - PLAKOGLOBIN, BETA-CATENIN AND GAMMA-CATENIN ARE DISTINCT COMPONENTS JOURNAL OF CELL SCIENCE Piepenhagen, P. A., NELSON, W. J. 1993; 104: 751-762

    Abstract

    Ca(2+)-dependent cell adhesion is mediated by a family of proteins termed cadherins, and is modulated by cytosolic proteins that include alpha-, beta-, and gamma-catenin and other cytoskeletal proteins that bind to the cytoplasmic domain of cadherins. Recent studies have suggested that either beta- or gamma-catenin may be identical to plakoglobin, a protein associated with adherens junctions. However, the relationship between these proteins, and their interaction with cadherins, are not well understood. In this study, we have further defined the relationship between plakoglobin and the catenins in complexes with E-cadherin in Madin-Darby canine kidney (MDCK) cells. Specific immunoprecipitations revealed that plakoglobin (86 kDa) and beta-catenin (92 kDa) have different detergent extractabilities and apparent molecular weights in these cells; however, plakoglobin has an apparent molecular weight similar to that of gamma-catenin (86 kDa). Immunoblotting of E-cadherin immunoprecipitates demonstrated that both plakoglobin and beta-catenin co-immunoprecipitate with E-cadherin. Laser-scanning confocal microscopy demonstrated temporally and spatially co-ordinate redistribution of plakoglobin and E-cadherin following induction of cell-cell contact in MDCK cells. Although plakoglobin comigrated with gamma-catenin on SDS-PAGE, quantitative analysis of E-cadherin and plakoglobin immunoprecipitates revealed that plakoglobin accounted for < 50% of the gamma-catenin signal. Two-dimensional gel electrophoresis resolved the gamma-catenin protein band into two proteins. One protein was identified as plakoglobin, based upon apparent molecular weight, immunoreactivity and isoelectric point (pI approximately 6.1). The other protein comigrated with gamma-catenin on SDS-PAGE, did not react with plakoglobin antibodies and had a pI of approximately 4.25; we refer to this protein as gamma-catenin to distinguish it from plakoglobin. Two-dimensional gel electrophoresis further revealed that plakoglobin comprised multiple isoelectric variants, but that, within the newly synthesized pool of plakoglobin, only the most basic of these variants co-immunoprecipitated with E-cadherin; phosphorylation did not account for the plakoglobin isoelectric variants seen by two-dimensional gel electrophoresis. These results demonstrate directly that plakoglobin associates and co-localizes with the E-cadherin in MDCK epithelial cells in a complex that contains alpha-, beta-, and gamma-catenin. Although plakoglobin shares sequence similarity with beta-catenin, and comigrates with gamma-catenin in SDS-PAGE, plakoglobin is distinct from the catenins. The association of plakoglobin with E-cadherin may be regulated by post-translational modifications of plakoglobin.

    View details for Web of Science ID A1993KY98300015

    View details for PubMedID 8267793

  • SPATIAL AND TEMPORAL DISSECTION OF IMMEDIATE AND EARLY EVENTS FOLLOWING CADHERIN-MEDIATED EPITHELIAL-CELL ADHESION JOURNAL OF CELL BIOLOGY McNeill, H., Ryan, T. A., Smith, S. J., NELSON, W. J. 1993; 120 (5): 1217-1226

    Abstract

    Cell-cell adhesion is at the top of a molecular cascade of protein interactions that leads to the remodeling of epithelial cell structure and function. The earliest events that initiate this cascade are poorly understood. Using high resolution differential interference contrast microscopy and retrospective immunohistochemistry, we observed that cell-cell contact in MDCK epithelial cells consists of distinct stages that correlate with specific changes in the interaction of E-cadherin with the cytoskeleton. We show that formation of a stable contact is preceded by numerous, transient contacts. During this time and immediately following formation of a stable contact, there are no detectable changes in the distribution, relative amount, or Triton X-100 insolubility of E-cadherin at the contact. After a lag period of approximately 10 min, there is a rapid acquisition of Triton X-100 insolubility of E-cadherin localized to the stable contact. Significantly, the total amount of E-cadherin at the contact remains unchanged during this time. The increase in the Triton X-100 insoluble pool of E-cadherin does not correlate with changes in the distribution of actin or fodrin, suggesting that the acquisition of the Triton X-100 insolubility is due to changes in E-cadherin itself, or closely associated proteins such as the catenins. The 10 minute lag period, and subsequent prompt and localized nature of E-cadherin reorganization indicate a form of signaling is occurring.

    View details for Web of Science ID A1993KN66900015

    View details for PubMedID 8436592

  • EPITHELIAL-CELL ADHESION AND DEVELOPMENT OF CELL-SURFACE POLARITY - POSSIBLE MECHANISMS FOR MODULATION OF CADHERIN FUNCTION, ORGANIZATION AND DISTRIBUTION JOURNAL OF CELL SCIENCE Nathke, I. S., HINCK, L. E., NELSON, W. J. 1993: 139-145

    Abstract

    Epithelial cell adhesion is principally regulated by calcium-dependent cell adhesion proteins, termed cadherins. Recent studies indicate that cadherin function is modulated by a class of proteins, termed catenins, that bind to the cytoplasmic domain of cadherin. Here we review the evidence that catenins regulate cadherin function in cell-cell adhesion, and discuss their role in initiating cell surface polarity in epithelial cells.

    View details for Web of Science ID A1993MW45500021

    View details for PubMedID 8144690

  • REGULATION OF CELL-SURFACE POLARITY FROM BACTERIA TO MAMMALS SCIENCE NELSON, W. J. 1992; 258 (5084): 948-955

    Abstract

    The generation of unique domains on the cell, cell surface polarity, is critical for differentiation into the diversity of cell structures and functions found in a wide variety of organisms and cells, including the bacterium Caulobacter crescentus, the budding yeast Saccharomyces cerevisiae, and mammalian polarized epithelial cells. Comparison of the mechanisms for establishing polarity in these cells indicates that restricted membrane protein distributions are generated by selective protein targeting to, and selective protein retention at, the cell surface. Initiation of these mechanisms involves reorientation of components of the cytoskeleton and protein transport pathways toward restricted sites at the cell surface and formation of a targeting patch at those sites for selective recruitment and retention of proteins.

    View details for Web of Science ID A1992JW79600024

    View details for PubMedID 1439806

  • Renal epithelial cell polarity. Current opinion in nephrology and hypertension NELSON, W. J. 1992; 1 (1): 59-67

    Abstract

    Recent cell biologic studies of protein trafficking, sorting, and distribution in polarized renal epithelial cells have begun to provide important new insights into the mechanisms involved in generating and maintaining cell surface polarity. Advances in this field have been rapid in the last year, due in part to the development of new approaches to analyzing protein delivery and distribution in polarized renal cells grown in vitro. Sorting signals within apical and basal-lateral membrane proteins have been described that may be involved in the segregation of proteins into different populations of transport vesicles in the trans-Golgi network; the nature of these signals has provided insight into the mechanisms involved. Elements of the cytoskeleton appear to be involved in the delivery of these transport vesicles to the appropriate membrane domain (microtubules) and in the retention of specific proteins in the correct membrane domain (membrane skeleton). Finally, detailed analysis of two prominent renal diseases, ischemia and polycystic kidney disease, indicates that abnormalities in the regulation of membrane protein distribution may be a contributing factor in generating the disease state.

    View details for PubMedID 1365832

  • ESTABLISHING AND MAINTAINING EPITHELIAL-CELL POLARITY - ROLES OF PROTEIN SORTING, DELIVERY AND RETENTION JOURNAL OF CELL SCIENCE WOLLNER, D. A., NELSON, W. J. 1992; 102: 185-190

    View details for Web of Science ID A1992JB71100001

    View details for PubMedID 1400627

  • Mechanisms for regulating the cell surface distribution of Na/K-ATPase in polarized epithelial cells. Chest Mays, R. W., NELSON, W. J. 1992; 101 (3): 50S-52S

    View details for PubMedID 1311669

  • REMODELING THE CELL-SURFACE DISTRIBUTION OF MEMBRANE-PROTEINS DURING THE DEVELOPMENT OF EPITHELIAL-CELL POLARITY JOURNAL OF CELL BIOLOGY WOLLNER, D. A., KRZEMINSKI, K. A., NELSON, W. J. 1992; 116 (4): 889-899

    Abstract

    The development of polarized epithelial cells from unpolarized precursor cells follows induction of cell-cell contacts and requires resorting of proteins into different membrane domains. We show that in MDCK cells the distributions of two membrane proteins, Dg-1 and E-cadherin, become restricted to the basal-lateral membrane domain within 8 h of cell-cell contact. During this time, however, 60-80% of newly synthesized Dg-1 and E-cadherin is delivered directly to the forming apical membrane and then rapidly removed, while the remainder is delivered to the basal-lateral membrane and has a longer residence time. Direct delivery of greater than 95% of these proteins from the Golgi complex to the basal-lateral membrane occurs greater than 48 h later. In contrast, we show that two apical proteins are efficiently delivered and restricted to the apical cell surface within 2 h after cell-cell contact. These results provide insight into mechanisms involved in the development of epithelial cell surface polarity, and the establishment of protein sorting pathways in polarized cells.

    View details for Web of Science ID A1992HD55000005

    View details for PubMedID 1734022

  • REGULATION OF EPITHELIAL-CELL POLARITY - A VIEW FROM THE CELL-SURFACE COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY NELSON, W. J., Wilson, R., Wollner, D., Mays, R., McNeill, H., Siemers, K. 1992; 57: 621-630

    View details for Web of Science ID A1992LV41600066

    View details for PubMedID 1339699

  • Cytoskeleton functions in membrane traffic in polarized epithelial cells. Seminars in cell biology NELSON, W. J. 1991; 2 (6): 375-385

    Abstract

    The complexity of membrane traffic in polarized epithelial cells between the Golgi complex and either the apical or basal-lateral membrane domain, and between different membrane domains (transcytosis) requires that vesicles leaving one membrane compartment efficiently and rapidly reach their (correct) destination. There is increasing evidence that microtubules, actin microfilaments and the membrane-cytoskeleton are involved in several aspects of vesicle transport and in the regulation of protein distributions in polarized epithelial cells. These possible functions are discussed in the context of the development and maintenance of cell polarity.

    View details for PubMedID 1813027

  • MECHANISM FOR REGULATING CELL-SURFACE DISTRIBUTION OF NA+, K+-ATPASE IN POLARIZED EPITHELIAL-CELLS SCIENCE HAMMERTON, R. W., KRZEMINSKI, K. A., Mays, R. W., Ryan, T. A., WOLLNER, D. A., NELSON, W. J. 1991; 254 (5033): 847-850

    Abstract

    Restriction of sodium, potassium adenosine triphosphatase (Na+,K(+)-ATPase) to either the apical or basal-lateral membrane domain of polarized epithelial cells is fundamental to vectorial ion and solute transport in many tissues and organs. A restricted membrane distribution of Na+,K(+)-ATPase in Madin-Darby canine kidney (MDCK) epithelial cells was found experimentally to be generated by preferential retention of active enzyme in the basal-lateral membrane domain and selective inactivation and loss from the apical membrane domain, rather than by vectorial targeting of newly synthesized protein from the Golgi complex to the basal-lateral membrane domain. These results show how different distributions of the same subunits of Na+,K(+)-ATPase may be generated in normal polarized epithelial and in disease states.

    View details for Web of Science ID A1991GN83300042

    View details for PubMedID 1658934

Conference Proceedings


  • Roles of the membrane cytoskeleton in protein sorting NELSON, W. J., Beck, K. A., Piepenhagen, P. A. ROCKEFELLER UNIV PRESS. 1997: 47-54

    View details for Web of Science ID A1997BJ08L00004

    View details for PubMedID 9210219

  • Generation of epithelial cell polarity: Roles for protein trafficking, membrane-cytoskeleton, and E-cadherin-mediated cell adhesion Mays, R. W., NELSON, W. J., MARRS, J. A. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1995: 763-773

    View details for Web of Science ID A1995VA12500081

    View details for PubMedID 8824451

  • REGULATION OF CELL-SURFACE POLARITY IN RENAL EPITHELIA NELSON, W. J. SPRINGER VERLAG. 1993: 599-604

    Abstract

    In the kidney, polarized epithelial cells play critical roles in ion, fluid and solute reabsorption from the ultrafiltrate to the blood supply. Detailed analysis of protein distributions has revealed that ion channels, transporters and pumps are restricted to distinct domains of the plasma membrane that face either the ultrafiltrate (apical membrane) or the blood supply (basal-lateral membrane). The importance of the development and maintenance of the polarized distributions of these proteins in renal epithelia for normal cell function is demonstrated by the fact that several disease states are characterized by abnormal distributions of proteins; for example in polycystic kidney disease, Na+/K(+)-ATPase has been detected in the apical and lateral membranes, compared with normal cells where Na+/K(+)-ATPase is localized in the basal-lateral membrane domain. Recent studies indicate that the development of restricted distributions of proteins at the cell surface of Madin Darby canine kidney epithelial cells is determined by direct sorting of proteins in the trans Golgi network into vesicles that are delivered vectorially to either the apical or basal-lateral membrane. Upon arrival at the plasma membrane, some proteins, such as Na+/K(+)-ATPase, may be selectively retained by binding to the membrane cytoskeleton.

    View details for Web of Science ID A1993LY41100024

    View details for PubMedID 8251331

  • GENERATION OF PLASMA-MEMBRANE DOMAINS IN POLARIZED EPITHELIAL-CELLS - ROLE OF CELL CELL CONTACTS AND ASSEMBLY OF THE MEMBRANE CYTOSKELETON NELSON, W. J. PORTLAND PRESS LTD. 1991: 1055-1059

    View details for Web of Science ID A1991GV01900060

    View details for PubMedID 1665428

  • ROLE OF THE MEMBRANE-CYTOSKELETON IN THE SPATIAL-ORGANIZATION OF THE NA,K-ATPASE IN POLARIZED EPITHELIAL-CELLS NELSON, W. J., HAMMERTON, R. W., McNeill, H. ROCKEFELLER UNIV PRESS. 1991: 77-87

    Abstract

    Vectorial function of polarized transporting epithelia requires the establishment and maintenance of a nonrandom distribution of Na,K-ATPase on the cell surface. In many epithelia, the Na,K-ATPase is located at the basal-lateral domain of the plasma membrane. The mechanisms involved in the spatial organization of the Na,K-ATPase in these cells are poorly understood. We have been investigating the roles of regulated cell-cell contacts and assembly of the membrane-cytoskeleton in the development of the cell surface polarity of Na,K-ATPase. We have shown that the Na,K-ATPase colocalizes with distinct components of the membrane-cytoskeleton in polarized Madin-Darby canine kidney (MDCK) epithelial cells. Significantly, we showed directly that Na,K-ATPase is a high affinity binding site for the membrane-cytoskeletal proteins ankyrin and fodrin, and that all three proteins exist in a high molecular weight protein complex that also contains the cell adhesion molecule (CAM) uvomorulin. We have proposed that these interactions are important in the assembly at sites of cell-cell contact of the membrane-cytoskeleton, which in turn initiates the development of the nonrandom distribution of the Na,K-ATPase. To directly investigate the functional significance of these protein-protein interactions in the spatial organization of the Na,K-ATPase, we analyzed the distribution of the Na,K-ATPase in fibroblasts transfected with a cDNA encoding the epithelial CAM, uvomorulin. Our results showed that expression of uvomorulin is sufficient to induce a redistribution of Na,K-ATPase from an unrestricted distribution over the entire cell surface in nontransfected cells to a restricted distribution at sites of uvomorulin-mediated cell-cell contacts in the transfected cells; this distribution is similar to that in polarized epithelial cells. This restricted distribution of the Na,K-ATPase occurred in the absence of tight junctions, but coincided with the reorganization of the membrane-cytoskeleton. These results support a model in which the epithelial CAM uvomorulin functions as an inducer of cell surface polarity of Na,K-ATPase through cytoplasmic linkage to the membrane-cytoskeleton.

    View details for Web of Science ID A1991BT71X00006

    View details for PubMedID 1653995