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Biomedical subjects

W James Nelson

Publications and source records attributed to W James Nelson.

At least 19 recordsLinked to original sources
PubMed2007

Two distinct modes of myosin assembly and dynamics during epithelial wound closure.

Actomyosin contraction powers the sealing of epithelial sheets during embryogenesis and wound closure; however, the mechanisms are poorly understood. After laser ablation wounding of Madin-Darby canine kidney cell monolayers, we observed distinct steps in wound closure from time-lapse images of myosin distribution during resealing. Immediately upon wounding, actin and myosin II regulatory light chain accumulated at two locations: (1) in a ring adjacent to the tight junction that circumscribed the wound and (2) in fibers at the base of the cell in membranes extending over the wound site. Rho-kinase activity was required for assembly of the myosin ring, and myosin II activity was required for contraction but not for basal membrane extension. As it contracted, the myosin ring moved toward the basal membrane with ZO-1 and Rho-kinase. Thus, we suggest that tight junctions serve as attachment points for the actomyosin ring during wound closure and that Rho-kinase is required for localization and activation of the contractile ring.

Actomyosin↗
PubMed2006

Cadherins in development: cell adhesion, sorting, and tissue morphogenesis.

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.

Animals↗
PubMed2006

Wingless signaling modulates cadherin-mediated cell adhesion in Drosophila imaginal disc cells.

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.

Animals↗
PubMed2006

Here come the septins: novel polymers that coordinate intracellular functions and organization.

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.

Animals↗
PubMed2005

E-cadherin tethered to micropatterned supported lipid bilayers as a model for cell adhesion.

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.

Cadherins↗
PubMed2005

Deconstructing the cadherin-catenin-actin complex.

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.

Actins↗
PubMed2005

Alpha-catenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actin-filament assembly.

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.

Actin-Related Protein 2-3 Complex↗
PubMed2005

Self-organization of an acentrosomal microtubule network at the basal cortex of polarized epithelial cells.

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.

Animals↗
PubMed2005

Neurite outgrowth involves adenomatous polyposis coli protein and beta-catenin.

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.

Adenomatous Polyposis Coli Protein↗
PubMed2005

A mitotic septin scaffold required for Mammalian chromosome congression and segregation.

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.

Animals↗
PubMed2005

APC is a component of an organizing template for cortical microtubule networks.

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.

Adenomatous Polyposis Coli Protein↗
PubMed2005

Cell-adhesion assays: fabrication of an E-cadherin substratum and isolation of lateral and Basal membrane patches.

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.

Animals↗
PubMed2005

Interaction of cadherin with the actin cytoskeleton.

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.

Actins↗
PubMed2004

Fractionation of the epithelial apical junctional complex: reassessment of protein distributions in different substructures.

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.

Animals↗
PubMed2004

Convergence of Wnt, beta-catenin, and cadherin pathways.

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.

Animals↗
PubMed2004

Adenomatous polyposis coli and EB1 localize in close proximity of the mother centriole and EB1 is a functional component of centrosomes.

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.

Adenomatous Polyposis Coli Protein↗