Dataset Information

Calcium-dependent dynamics of cadherin interactions at cell-cell junctions.

ABSTRACT: Cadherins play a key role in the dynamics of cell-cell contact formation and remodeling of junctions and tissues. Cadherin-cadherin interactions are gated by extracellular Ca(2+), which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Direct measurements of transjunctional cadherin interactions revealed a sudden, but partial, loss of homophilic interactions (? = 1.17 ± 0.06 s(-1)) upon chelation of extracellular Ca(2+). A cadherin mutant with reduced adhesive activity (W2A) exhibited a faster, more substantial loss of homophilic interactions (? = 0.86 ± 0.02 s(-1)), suggesting two types of native cadherin interactions--one that is rapidly modulated by changes in extracellular Ca(2+) and another with relatively stable adhesive activity that is Ca(2+) independent. The Ca(2+)-sensitive dynamics of cadherin interactions were transmitted to the cell interior where ?-catenin translocated to N-cadherin at the junction in both cells. These data indicate that cadherins can rapidly convey dynamic information about the extracellular environment to both cells that comprise a junction.

SUBMITTER: Kim SA

PROVIDER: S-EPMC3116393 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Calcium-dependent dynamics of cadherin interactions at cell-cell junctions.

Kim Sally A SA Tai Chin-Yin CY Mok Lee-Peng LP Mosser Eric A EA Schuman Erin M EM

Proceedings of the National Academy of Sciences of the United States of America 20110525 24

Cadherins play a key role in the dynamics of cell-cell contact formation and remodeling of junctions and tissues. Cadherin-cadherin interactions are gated by extracellular Ca(2+), which serves to rigidify the cadherin extracellular domains and promote trans junctional interactions. Here we describe the direct visualization and quantification of spatiotemporal dynamics of N-cadherin interactions across intercellular junctions in living cells using a genetically encodable FRET reporter system. Dir ...[more]

PMID: 21613566

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Project description:E-cadherin-mediated cell-cell adhesion, which is essential for the maintenance of the architecture and integrity of epithelial tissues, is often lost during carcinoma progression. To better understand the nature of alterations of cell-cell interactions at the early stages of neoplastic evolution of epithelial cells, we examined the line of nontransformed IAR-2 epithelial cells and their descendants, lines of IAR-6-1 epithelial cells transformed with dimethylnitrosamine and IAR1170 cells transformed with N-RasG12D. IAR-6-1 and IAR1170 cells retained E-cadherin, displayed discoid or polygonal morphology, and formed monolayers similar to IAR-2 monolayer. Fluorescence staining, however, showed that in IAR1170 and IAR-6-1 cells the marginal actin bundle, which is typical of nontransformed IAR-2 cells, disappeared, and the continuous adhesion belt (tangential adherens junctions (AJs)) was replaced by radially oriented E-cadherin-based AJs. Time-lapse imaging of IAR-6-1 cells stably transfected with GFP-E-cadherin revealed that AJs in transformed cells are very dynamic and unstable. The regulation of AJ assembly by Rho family small GTPases was different in nontransformed and in transformed IAR epithelial cells. As our experiments with the ROCK inhibitor Y-27632 and the myosin II inhibitor blebbistatin have shown, the formation and maintenance of radial AJs critically depend on myosin II-mediated contractility. Using the RNAi technique for the depletion of mDia1 and loading cells with N17Rac, we established that mDia1 and Rac are involved in the assembly of tangential AJs in nontransformed epithelial cells but not in radial AJs in transformed cells. Neoplastic transformation changed cell-cell interactions, preventing contact paralysis after the establishment of cell-cell contact and promoting dynamic cell-cell adhesion and motile behavior of cells. It is suggested that the disappearance of the marginal actin bundle and rearrangements of AJs may change the adhesive function of E-cadherin and play an active role in migratory activity of carcinoma cells.

Project description:To test how cell-cell contacts regulate microtubule (MT) and actin cytoskeletal dynamics, we examined dynamics in cells that were contacted on all sides with neighboring cells in an epithelial cell sheet that was undergoing migration as a wound-healing response. Dynamics were recorded using time-lapse digital fluorescence microscopy of microinjected, labeled tubulin and actin. In fully contacted cells, most MT plus ends were quiescent; exhibiting only brief excursions of growth and shortening and spending 87.4% of their time in pause. This contrasts MTs in the lamella of migrating cells at the noncontacted leading edge of the sheet in which MTs exhibit dynamic instability. In the contacted rear and side edges of these migrating cells, a majority of MTs were also quiescent, indicating that cell-cell contacts may locally regulate MT dynamics. Using photoactivation of fluorescence techniques to mark MTs, we found that MTs in fully contacted cells did not undergo retrograde flow toward the cell center, such as occurs at the leading edge of motile cells. Time-lapse fluorescent speckle microscopy of fluorescently labeled actin in fully contacted cells revealed that actin did not flow rearward as occurs in the leading edge lamella of migrating cells. To determine if MTs were required for the maintenance of cell-cell contacts, cells were treated with nocodazole to inhibit MTs. After 1-2 h in either 10 microM or 100 nM nocodazole, breakage of cell-cell contacts occurred, indicating that MT growth is required for maintenance of cell-cell contacts. Analysis of fixed cells indicated that during nocodazole treatment, actin became reduced in adherens junctions, and junction proteins alpha- and beta-catenin were lost from adherens junctions as cell-cell contacts were broken. These results indicate that a MT plus end capping protein is regulated by cell-cell contact, and in turn, that MT growth regulates the maintenance of adherens junctions contacts in epithelia.

Dataset Information

Calcium-dependent dynamics of cadherin interactions at cell-cell junctions.

Publications

Calcium-dependent dynamics of cadherin interactions at cell-cell junctions.

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