Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand mechanism underlying cadherin regulation, it is essential to elucidate homophilic binding that underlies all functions. Therefore, we have investigated structural and functional properties extracellular segment Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported CHO cells expressing C-cadherin. The was also capable mediating aggregation microspheres. Chemical...

We have obtained evidence that a known intracellular component of the cadherin cell-cell adhesion machinery, beta-catenin, contributes to development body axis in frog Xenopus laevis. Vertebrate beta-catenin is homologous Drosophila segment polarity gene product armadillo, and vertebrate plakoglobin (McCrea, P. D., C. W. Turck, B. Gumbiner. 1991. Science (Wash. DC). 254: 1359-1361.). Beta-Catenin was found present all embryonic stages examined, associated with C-cadherin, major early...

Turnover of actin filaments in cells requires rapid disassembly a cytoplasmic environment that thermodynamically favors assembly because high concentrations polymerizable monomers. We here image the single by cofilin, coronin, and actin-interacting protein 1, purified system reconstitutes rapid, monomer-insensitive (Brieher, W.M., H.Y. Kueh, B.A. Ballif, T.J. Mitchison. 2006. J. Cell Biol. 175:315–324). In this three-component system, disassemble abrupt bursts initiate preferentially, but...

We have developed an in vitro assay to study actin assembly at cadherin-enriched cell junctions. Using this assay, we demonstrate that junctions can polymerize new filaments but cannot capture preexisting filaments, suggesting a mechanism involving de novo synthesis. In agreement with hypothesis, inhibition of Arp2/3-dependent nucleation abolished cell-cell Reconstitution biochemistry using the identified α-actinin-4/focal segmental glomerulosclerosis 1 (FSGS1) as essential factor....

Direct-force measurements of the interactions between recombinant C-cadherin from Xenopus demonstrated that ectodomain cadherin exhibits multiple adhesive contacts involve successive domains along extracellular region protein. Contacts fully interdigitated antiparallel proteins form strongest interaction. A second weaker minimum was measured when were separated by a distance equal to length one domain (EC) fragment and corresponding alignment through four (EC1 EC4). The rupture these...

Treatment of Xenopus animal pole tissue with activin results in the induction mesodermal cell types and a dramatic elongation tissue. The morphogenetic movements involved appear similar to those normal gastrulation, which is driven by rearrangement intercalations. We have used this system explore potential regulation cell-cell adhesion cadherin function during morphogenesis. Quantitative blastomere aggregation assays revealed that reduced calcium-dependent between blastomeres....

Actin filaments in cells depolymerize rapidly despite the presence of high concentrations polymerizable G actin. Cofilin is recognized as a key regulator that promotes actin depolymerization. In this study, we show although pure cofilin can disassemble Listeria monocytogenes comet tails, it cannot efficiently tails Thymus extracts also and reaction more efficient than when normalized to concentration. By biochemical fractionation, identify Aip1 coronin two proteins present thymus extract...

By combining in vitro reconstitution biochemistry with a cross-linking approach, we have identified focal segmental glomerulosclerosis 3/CD2-associated protein (FSGS3/CD2AP) as novel actin barbed-end capping responsible for stability at the adherens junction. FSGS3/CD2AP colocalizes E-cadherin and α-actinin-4 apical junction polarized Madin-Darby canine kidney (MDCK) cells. Knockdown of compromised decreased accumulation Using apparatus to apply mechanical stress cell–cell junctions, showed...

Cadherin-mediated cell-cell adhesion is actin-dependent, but the precise role of actin in maintaining not fully understood. Actin polymerization-dependent protrusive activity required to push distally separated cells close enough initiate contact. Whether maintain confluent sheets epithelial known. By electron microscopy as well live cell imaging, we have identified a population protruding microspikes that operate continuously near apical junctions polarized Madin-Darby canine kidney (MDCK)...

The regulation of cadherin-mediated adhesion at the cell surface underlies several morphogenetic processes. To investigate role cadherin in morphogenesis and to begin analyze molecular mechanisms regulation, we have screened for monoclonal antibodies (mAbs) that allow us manipulate adhesive state molecule. Xenopus C-cadherin is regulated during convergent extension movements gastrulation. Treatment animal pole tissue explants (animal caps) with mesoderm-inducing factor activin induces...

Although small molecule actin modulators have been widely used as research tools, only one cell-permeable inhibitor of depolymerization (jasplakinolide) is commercially available. We report that the natural product cucurbitacin E inhibits and show its mechanism action different from jasplakinolide. In assays using pure fluorescently labeled actin, specifically affects without affecting polymerization. It at substoichiometric concentrations up to 1:6 E:actin. Cucurbitacin binds filamentous...

The actin cytoskeleton is constantly assembling and disassembling. Cells harness the energy of these turnover dynamics to drive cell motility organize cytoplasm. Although much known about how cells control polymerization, we do not understand filaments depolymerize inside cells. I briefly describe combination imaging filament in using vitro biochemistry progressively altered our views depolymerization. why think that prevailing model turnover--cofilin-mediated severing--can account for...

Actin-dependent propulsion of Listeria monocytogenes is thought to require frequent nucleation actin polymerization by the Arp2/3 complex. We demonstrate that L. motility can be separated into an Arp2/3-dependent phase and Arp2/3-independent elongation phase. Elongation-based requires a unique set biochemical factors in addition those required for motility. isolated fascin from brain extracts as only soluble factor during this type movement. The reaction assembles comet tail branched...

We report here that actin filaments in vitro exist two populations with significantly different shrinkage rates. Newly polymerized shrink rapidly, primarily from barbed ends, at 1.8/s, but as they age switch to a stable state shrinks slowly, pointed approximately 0.1/s. This dynamic filament stabilization runs opposite the classical prediction become more unstable hydrolyze their bound ATP and release phosphate. Upon cofilin treatment, aged revert shows accelerated both ends combined rate of...

Significance A major question in tissue biology is how cells manage to maintain stable cell–cell adhesion using the intrinsically weak homophilic bonds of cadherin molecules. One suggestion that cluster cadherins into large puncta promote strong adhesion. However, we now show are not clusters but rather interdigitated protruding microspikes. Actin polymerization–dependent protrusive activity generates fingers help hold together by increasing membrane surface area available for...