α-Catenin-independent Recruitment of ZO-1 to Nectin-based Cell-Cell Adhesion Sites through Afadin

ZO-1 is an actin filament (F-actin)–binding protein that localizes to tight junctions and connects claudin to the actin cytoskeleton in epithelial cells. In nonepithelial cells that have no tight junctions, ZO-1 localizes to adherens junctions (AJs) and may connect cadherin to the actin cytoskeleton indirectly through β- and α-catenins as one of many F-actin–binding proteins. Nectin is an immunoglobulin-like adhesion molecule that localizes to AJs and is associated with the actin cytoskeleton through afadin, an F-actin–binding protein. Ponsin is an afadin- and vinculin-binding protein that also localizes to AJs. The nectin-afadin complex has a potency to recruit the E-cadherin–β-catenin complex through α-catenin in a manner independent of ponsin. By the use of cadherin-deficient L cell lines stably expressing various components of the cadherin-catenin and nectin-afadin systems, and α-catenin–deficient F9 cell lines, we examined here whether nectin recruits ZO-1 to nectin-based cell-cell adhesion sites. Nectin showed a potency to recruit not only α-catenin but also ZO-1 to nectin-based cell-cell adhesion sites. This recruitment of ZO-1 was dependent on afadin but independent of α-catenin and ponsin. These results indicate that ZO-1 localizes to cadherin-based AJs through interactions not only with α-catenin but also with the nectin-afadin system.

Contact interactions between epitheliocytes and fibroblasts: Formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization

Contact interactions between different cell types play a number of important roles in development, for example in cell sorting, tissue organization, and ordered migration of cells. The nature of such heterocellular interactions, in contrast to interactions between cells of the same type, remains largely unknown. In this report, we present experimental data examining the dynamics of heterocellular interactions between epitheliocytes and fibroblasts, which express different cadherin cell adhesion molecules and possess different actin cytoskeletal organizations. Our analysis revealed two striking features of heterocellular contact. First, the active free edge of an epitheliocyte reorganizes its actin cytoskeleton after making contact with a fibroblast. Upon contact with the leading edge of a fibroblast, epitheliocytes disassemble their marginal bundle of actin filaments and reassemble actin filaments into a geometric organization more typical of a fibroblast lamella. Second, epitheliocytes and fibroblasts form cell–cell adhesion structures that have an irregular organization and are associated with components of cell adhesion complexes. The structural organization of these adhesions is more closely related to the type of contacts formed between fibroblasts rather than to those between epitheliocytes. Heterotypic epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts, are transient and do not lead to formation of stable contact interactions. We suggest that heterocellular contact interactions in culture may be regarded as models of how tissue systems consisting of epithelia and mesenchyme interact and become organized in vivo.

The human formin-binding protein 17 (FBP17) interacts with sorting nexin, SNX2, and is an MLL-fusion partner in acute myelogeneous leukemia

We have cloned a fusion partner of the MLL gene at 11q23 and identified it as the gene encoding the human formin-binding protein 17, FBP17. It maps to chromosome 9q34 centromeric to ABL. The gene fusion results from a complex chromosome rearrangement that was resolved by fluorescence in situ hybridization with various probes on chromosomes 9 and 11 as an ins(11;9)(q23;q34)inv(11)(q13q23). The rearrangement resulted in a 5′-MLL/FBP17-3′ fusion mRNA. We retrovirally transduced murine-myeloid progenitor cells with MLL/FBP17 to test its transforming ability. In contrast to MLL/ENL, MLL/ELL and other MLL-fusion genes, MLL/FBP17 did not give a positive readout in a serial replating assay. Therefore, we assume that additional cooperating genetic abnormalities might be needed to establish a full malignant phenotype. FBP17 consists of a C-terminal Src homology 3 domain and an N-terminal region that is homologous to the cell division cycle protein, cdc15, a regulator of the actin cytoskeleton in Schizosaccharomyces pombe. Both domains are separated by a consensus Rho-binding motif that has been identified in different Rho-interaction partners such as Rhotekin and Rhophilin. We evaluated whether FBP17 and members of the Rho family interact in vivo with a yeast two-hybrid assay. None of the various Rho proteins tested, however, interacted with FBP17. We screened a human kidney library and identified a sorting nexin, SNX2, as a protein interaction partner of FBP17. These data provide a link between the epidermal growth factor receptor pathway and an MLL fusion protein.

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with the most severe pathology in the T lymphocytes and platelets. The disease arises from mutations in the gene encoding the WAS protein. T lymphocytes of affected males with WAS exhibit a severe disturbance of the actin cytoskeleton, suggesting that the WAS protein could regulate its organization. We show here that WAS protein interacts with a member of the Rho family of GTPases, Cdc42. This interaction, which is guanosine 5'-triphosphate (GTP)-dependent, was detected in cell lysates, in transient transfections and with purified recombinant proteins. A weaker interaction was also detected with Rac1 using WAS protein from cell lysates. It was also found that different mutant WAS proteins from three affected males retained their ability to interact with Cdc42 and that the level of expression of the WAS protein in these mutants was only 2-5% of normal. Taken together these data suggest that the WAS protein might function as a signal transduction adaptor downstream of Cdc42, and in affected males, the cytoskeletal abnormalities may result from a defect in Cdc42 signaling.

Minimal mutation of the cytoplasmic tail inhibits the ability of E-cadherin to activate Rac but not phosphatidylinositol 3-kinase - Direct evidence of a role for cadherin-activated Rac signaling in adhesion and contact formation

Classic cadherins are adhesion-activated cell signaling receptors. In particular, homophilic cadherin ligation can directly activate Rho family GTPases and phosphatidylinositol 3-kinase (PI3-kinase), signaling molecules with the capacity to support the morphogenetic effects of these adhesion molecules during development and disease. However, the molecular basis for cadherin signaling has not been elucidated, nor is its precise contribution to cadherin function yet understood. One attractive hypothesis is that cadherin-activated signaling participates in stabilizing adhesive contacts ( Yap, A. S., and Kovacs, E. M. ( 2003) J. Cell Biol. 160, 11-16). We now report that minimal mutation of the cadherin cytoplasmic tail to uncouple binding of p120-ctn ablated the ability of E-cadherin to activate Rac. This was accompanied by profound defects in the capacity of cells to establish stable adhesive contacts, defects that were rescued by sustained Rac signaling. These data provide direct evidence for a role of cadherin-activated Rac signaling in contact formation and adhesive stabilization. In contrast, cadherin-activated PI3-kinase signaling was not affected by loss of p120-ctn binding. The molecular requirements for E-cadherin to activate Rac signaling thus appear distinct from those that stimulate PI3-kinase, and we postulate that p120-ctn may play a central role in the E-cadherin-Rac signaling pathway.

Direct cadherin-activated cell signaling: a view from the plasma membrane

Classical cadherin adhesion molecules are key determinants of cell recognition and tissue morphogenesis, with diverse effects on cell behavior. Recent developments indicate that classical cadherins are adhesion-activated signaling receptors. In particular, early-immediate Rac signaling is emerging as a mechanism to coordinate cadherin-actin integration at the plasma membrane.

Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.

Isolation and characterization of a Cotesia rubecula bracovirus gene expressed in the lepidopteran Pieris rapae

Polydnaviruses are endogenous particles that are crucial for the survival of endoparasitoid wasps, providing active suppression of the immune function of the lepidopteran host in which wasp larvae develop. The Cotesia rubecula bracovirus (CrBV) is unique in that only four gene products are detected in larval host (Pieris rapae) tissues and expression of CrBV genes is transient, occurring between 4 and 12 h post-parasitization. Two of the four genes, CrV1 and CrV3, have been characterized. CrV1 is a secreted glycoprotein that has been implicated in depolymerization of the actin cytoskeleton of host haemocytes, leading to haemocyte inactivation; CrV3 is a multimeric C-type lectin that shares homology with insect immune lectins. Here, a third CrBV-specific gene is described, CrV2, which is expressed in larval P. rapae tissues. CrV2, which is transcribed in haemocytes and fat body cells, has an ORF of 963 bp that produces a glycoprotein of approximately 40 kDa. CrV2 is secreted into haemolymph and appears to be internalized by host haemocytes. CrV2 has a coiled-coil region predicted at its C-terminus, which may be involved in the formation of putative CrV2 trimers that are detected in haemolymph of parasitized host larvae.

Lectin-induced haemocyte inactivation in insects

Most multimeric lectins are adhesion molecules, promoting attachment and spreading on surface glycodeterminants. In addition, some lectins have counter-adhesion properties, detaching already spread cells which then acquire round or spindle-formed cell shapes. Since lectin-mediated adhesion and detachment is observed in haemocyte-like Drosophila cells, which have haemomucin as the major lectin-binding glycoprotein, the two opposite cell behaviours may be the result of lectin-mediated receptor rearrangements on the cell surface. To investigate oligomeric lectins as a possible extracellular driving force affecting cell shape changes, we examined lectin-mediated reactions in lepidopteran haemocytes after cytochalasin D-treatment and observed that while cell-spreading was dependent on F-actin, lectin-uptake was less dependent on F-actin. We propose a model of cell shape changes involving a dynamic balance between adhesion and uptake reactions. (C) 2004 Elsevier Ltd. All rights reserved.

Evolution of polydnaviruses as insect immune suppressors

Polydnaviruses (PDVs) are endogenous particles that are used by some endoparasitic hymenoptera to disrupt host immunity and development. Recent analyses of encapsidated PDV genes have increased the number of known PDV gene families, which are often closely related to insect genes. Several PDV proteins inactivate host haemocytes by damaging their actin cytoskeleton. These proteins share no significant sequence homology and occur in polyphyletic PDV genera, possibly indicating that convergent evolution has produced functionally similar immune-suppressive molecules causing a haemocyte phenotype characterised by damaged cytoskeleton and inactivation. These phenomena provide further insights into the immune-suppressive activity of PDVs and raise interesting questions about PDV evolution, a topic that has puzzled researchers ever since the discovery of PDVs.

Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

The challenges of abundance: epithelial junctions and small GTPase signalling

Small GTPases of the Ras superfamily play critical roles in epithelial biogenesis. Many key morphogenetic functions occur when small GTPases act at epithelial junctions, where they mediate an increasingly complex interplay between cell-cell adhesion molecules and fundamental cellular processes, such as cytoskeletal activity, polarity and trafficking. Important recent advances in this field include the role of additional members of the Ras superfamily in cell-cell contact stability and the capacity for polarity determinants to regulate small GTPase signalling. Interestingly, small GTPases may participate in the cross-talk between different adhesive receptors: in tissues classical cadherins can selectively regulate other junctions through cell signalling rather than through a global influence on cell-cell cohesion.

Rho and vascular disease

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling. (c) 2005 Elsevier Ireland Ltd. All rights reserved.