Monitoring Distribution of N-Cadherin and E-Cadherin with HT Imaging
Introduction: N-cadherin and E-cadherin are widely distributed cell adhesion molecules that feature large extracellular domains which bind to each other on neighboring cells in a zipper-like fashion (Figure 1). Intracellular domains of cadherins bind to catenins, which, in turn, bind actin fibers in the cytoskeleton. The cadherin-catenin system provides a mechanical linkage between cells essential for proper differentiation, embryogenesis, and cell migration. In addition to providing mechanical support, cadherins also participate in cell signaling, helping to couple mechanical stresses to regulatory molecules such as the fibroblast growth factor receptor, Rho-GTPase, and tyrosine kinases (Derycke and Bracke 2004; Walker et al., in press).
Furthermore, cadherins participate in the regulation of cell division. Downregulation of E-cadherin leads to release of β-catenin from cellular junctions; β-catenin then interacts with the Wnt signaling pathway, and becomes localized to the nucleus where it helps to activate the transcription of proto-oncogenes, stimulating mitosis (Daniels et al., 2001). Tumor cells often feature lost or reduced junctional E-cadherin and prominent nuclear β-catenin. Loss of cadherin expression may also generally promote the invasiveness of tumor cells by reducing the interaction of the cells with neighboring tissue. While E-cadherin is an endogenous tumor suppressor, the situation with N-cadherin is less clear. Emerging data suggest that N-cadherin upregulation can increase cell invasiveness and that tumor progression is often accompanied by a “cadherin switch” in which E-cadherin is replaced by N-cadherin (Derycke and Bracke 2004; Cavallaro 2004). Thus, there is considerable interest in identifying novel pharmaceuticals that regulate cadherin expression and localization, as such agents might be candidates for anti-neoplastic therapies.
Assay of N-cadherin and E-cadherin utilizing high throughput (HT) techniques: To meet the need for cadherin-targeted screening assays, Vala Sciences Inc announces cell-image-based kits designed for the detection and quantification of cadherins in cultured cells in HT screening applications (Cat #K002 for N-cadherin, Cat #K003 for E-cadherin). Each kit includes reagents sufficient for visualizing the cadherins for cells cultured in five 96-well dishes via fluorescence photomicroscopy. Each kit also features Vala’s CyteSeer® imaging analysis software platform, which will automatically analyze digital photomicrographs of cells visualized for the cadherins. To demonstrate the sensitivity and specificity of our cadherin kits, 96-well microtiter plates were prepared with wells containing either HeLa or A431 cells. For one experiment, PMA (an activator of conventional Protein Kinase C isoforms) was added at either 12 or 100 nM concentrations for a 3 hr incubation period. As shown in Figure 2, the Vala kit reagents enabled quantification of N-cadherin at the cell to cell junctions in HeLa cells, and junctional E-cadherin in A431 cells.
Figure 1.
Cadherin-mediated cell to cell adhesion (see Goodwin and Yap 2004 for review).
Figure 2. Quantification of N-cadherin and E-cadherin via Vala Sciences kits.
Cells cultured in a 96-well dish were visualized for N-cadherin (upper left) or E-cadherin (upper right); Vala CyteSeer® software was used to automatically identify junctional regions (masks) and quantify protein distribution (lower graphs). For N-cadherin, 12 nM PMA (3 hr) induced a 2-fold increase in junctional protein in HeLa cells but was virtually undetectable in A431 cells. For E-cadherin, PMA induced a dose-dependent increase in A431 cells, but was virtually undetectable in HeLa cells. Each bar is the mean ± SD (n=14 wells).
Conclusions
Vala’s membrane structure and function algorithm correctly identified (masks) and quantified (graphs) the cadherin-specific junctional fluorescence associated with each experimental condition. As expected, N-cadherin was virtually undetectable in A431 cells, whereas E-cadherin was not found in HeLa cells. Interestingly, PMA strongly upregulated junctional N-cadherin in HeLa cells (approximately 2-fold), and upregulated junctional E-cadherin in A431 cells in a dose-dependent manner (15% at 12 nM, 30% at 100 nM, p < 0.01 for each dose vs all other conditions). The results suggest that PKC activity may alter the distribution and expression of these cadherins. The increase in N-cadherin in HeLa is consistent with the ability of PMA to promote tumors. Notably, the Z’ score (Zhang et al., 1999) for the N-cadherin assay was 0.45 for the comparison of HeLa cells + PMA with the A431. The Z’ score for the E-cadherin assay was 0.86 for A431 cells + 100 nM PMA vs. HeLa. Assays with Z’ scores of 0.2-0.6 are considered useful for HT screening applications (Zhang et al., 1999). The excellent Z’ scores for the Vala Sciences N-cadherin and E-cadherin assay kits suggest that these kits will enable HT screening experiments for these key regulatory molecules. The cadherin kits will also be of use to investigators wishing to conduct fundamental biological research on cadherin expression and distribution, providing rapid, quantitative analysis of experimental results.
References
Cavallaro, U. 2004. N-cadherin as an invasion promoter: a novel target for antitumor therapy? Current Opinion in Investigational Drugs 5:1274-1278.
Daniels, D. L., Spink, K. E., Weis, W. I. 2001. β-Catenin: molecular plasticity and drug design. Trends in Biochemical Sciences 26:672-678.
Derycke, L. D. M., and Bracke, M. E. 2004. N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling. Int. J. Dev. Biol. 48:463-476.
Goodwin, M., Yap, A. S. 2004. Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton. J. Molecular Histology 35:839-844.
Walker, J. L., Fournier, A. K., Assoian, R. K. Regulation of growth factor signaling and cell cycle progression by cell adhesion and adhesion-dependent changes in celluar tension. In press, Cytokine & Growth Factor Reviews.
Zhang et al., 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomolecular Scr. 4:67-73
Vala Sciences Contact Information
Vala Sciences is a cell biology company offering software, kits, services and custom development for analyzing a wide variety of cell types and conditions from adipocytes & stem cells to primary & well established cell lines.
For further information on products and ordering information please call or email:
Vala Sciences Inc:
Sales and technical information:
858 481-6862
info@valasciences.com
http://valasciences.com
CyteSeer is a registered trademark of Vala Sciences Inc. All rights reserved 2005-2009.
Related Reagents
The following reagents are available to order online from our reagents store:
