Lateral dimerization is required for the homophilic binding activity of C-cadherin

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the hemophilic binding mechanism that underlies all cadherin-mediated functions. Therefore, we have investigated the structural and functional properties of the extracellular segment of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of microspheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Analysis of the functional activity of purified dimers and monomers demonstrated that dimers retained substantially greater homophilic binding activity than monomers. These results demonstrate that lateral dimerization is necessary for hemophilic binding between cadherin extracellular segments and suggest multiple potential mechanisms for the regulation of cadherin activity

Actin disassembly by cofilin, coronin, and Aip1 occurs in bursts and is inhibited by barbed-end cappers

Turnover of actin filaments in cells requires rapid actin disassembly in a cytoplasmic environment that thermodynamically favors assembly because of high concentrations of polymerizable monomers. We here image the disassembly of single actin filaments by cofilin, coronin, and actin-interacting protein 1, a purified protein system that reconstitutes rapid, monomer-insensitive disassembly (Brieher, W.M., H.Y. Kueh, B.A. Ballif, and T.J. Mitchison. 2006. J. Cell Biol. 175:315–324). In this three-component system, filaments disassemble in abrupt bursts that initiate preferentially, but not exclusively, from both filament ends. Bursting disassembly generates unstable reaction intermediates with lowered affinity for CapZ at barbed ends. CapZ and cytochalasin D (CytoD), a barbed-end capping drug, strongly inhibit bursting disassembly. CytoD also inhibits actin disassembly in mammalian cells, whereas latrunculin B, a monomer sequestering drug, does not. We propose that bursts of disassembly arise from cooperative separation of the two filament strands near an end. The differential effects of drugs in cells argue for physiological relevance of this new disassembly pathway and potentially explain discordant results previously found with these drugs

Rapid actin monomer–insensitive depolymerization of Listeria actin comet tails by cofilin, coronin, and Aip1

Actin filaments in cells depolymerize rapidly despite the presence of high concentrations of polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show that although pure cofilin can disassemble Listeria monocytogenes actin comet tails, it cannot efficiently disassemble comet tails in the presence of polymerizable actin. Thymus extracts also rapidly disassemble comet tails, and this reaction is more efficient than pure cofilin when normalized to cofilin concentration. By biochemical fractionation, we identify Aip1 and coronin as two proteins present in thymus extract that facilitate the cofilin-mediated disassembly of Listeria comet tails. Together, coronin and Aip1 lower the amount of cofilin required to disassemble the comet tail and permit even low concentrations of cofilin to depolymerize actin in the presence of polymerizable G actin. The cooperative activities of cofilin, coronin, and Aip1 should provide a biochemical basis for understanding how actin filaments can grow in some places in the cell while shrinking in others

The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p129(ctn)

Cadherin cell-cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308-315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94-amino acid region of the cytoplasmic tail, but not the beta-catenin-binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was Ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120(ctn). These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120(ctn) is involved in clustering and cell adhesion

New Proposed Mechanism of Actin-Polymerization-Driven Motility

We present the first numerical simulation of actin-driven propulsion by elastic filaments. Specifically, we use a Brownian dynamics formulation of the dendritic nucleation model of actin-driven propulsion. We show that the model leads to a self-assembled network that exerts forces on a disk and pushes it with an average speed. This simulation approach is the first to observe a speed that varies non-monotonically with the concentration of branching proteins (Arp2/3), capping protein and depolymerization rate (ADF), in accord with experimental observations. Our results suggest a new interpretation of the origin of motility that can be tested readily by experiment.Comment: 31 pages, 5 figure

Functional Analysis of VopF Activity Required for Colonization in Vibrio cholerae

Vibrio cholerae, a Gram-negative facultative pathogen, is the etiologic agent for the diarrheal disease cholera. We previously characterized a clinical isolate, AM-19226, that translocates a type III secretion system (T3SS) effector protein with actin-nucleating activity, VopF, into the host cells. From comparative genomic studies, we identified a divergent T3SS island in additional isolates which possess a VopF homolog, VopN. Unlike the VopF-mediated protrusion formation, VopN localizes to stress fiber in host cells similarly to VopL, which is present in the pandemic strain of Vibrio parahaemolyticus. Chimera and yeast two-hybrid studies indicated that the amino-terminal regions of VopF and VopN proteins interact with distinct host cell factors. We determined that AM-19226-infected cells are arrested at S phase of the cell cycle and that VopF/VopN are antiapoptotic factors. To understand how VopF may contribute to the pathogenesis of AM-19226, we examined the effect of VopF in an in vitro polarized-epithelial model and an in vivo adult rabbit diarrheal model. Within the T3SS pathogenicity island is VopE, a homolog of YopE from Yersinia, which has been shown to loosen tight junctions. In polarized intestinal epithelia, VopF and VopE compromised the integrity of tight junctions by inducing cortical actin depolymerization and aberrant localization of the tight-junction protein ZO-1. An assay for pathogenicity in the adult rabbit diarrhea model suggested that these effectors are involved in eliciting the diarrheal response in infected rabbits