A flow cytometry-based assay for the measurement of protein regulation of E-cadherin-mediated adhesion.
Epithelial (E)-cadherin is a transmembrane protein that mediates calcium-dependent cell adhesion. E-cadherin has significant roles in tissue development, adhesion between keratinocytes and retention of Langerhans cells in the epidermis, and its loss on tumour cells is frequently associated with metastasis. Here we describe a simple, flow cytometric adhesion assay to measure the effects of potential regulators of cell surface E-cadherin expression on E-cadherin-mediated adhesion between cells. In this assay, cells that have been transiently transfected to express the protein of interest are enzymatically treated to remove cell surface E-cadherin. Cells are then incubated in low attachment plates, during which time the E-cadherin is re-expressed and E-cadherin-mediated aggregation occurs. The effect of the protein of interest on the percentage of E-cadherin-mediated aggregates that form during incubation is measured flow cytometrically, following staining with an E-cadherin specific antibody. A major advantage of this assay is that a potential regulatory protein of interest can be tested in a transient expression system by co-expression with green fluorescent protein and analysis of adhesion conducted on green fluorescent cells only. We have applied this assay to measure the regulatory effects of E6 protein from human papillomavirus type 16 on E-cadherin-mediated adhesion but this assay potentially has broad applicability for testing the effects of other proteins on E-cadherin-mediated adhesion in an accurate and highly reproducible manner.
Virus Research Unit, Department of Microbiology and Immunology, University of Otago, P.O. Box 56, Dunedin, New Zealand.
Cheng-Mee
CM
Leong
Merilyn H
MH
Hibma
D015820:Cadherins / Q000502:physiology*; D002448:Cell Adhesion / Q000235:genetics; D002449:Cell Aggregation / Q000235:genetics; D002461:Cell Line, Transformed; D005434:Flow Cytometry / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D015603:Keratinocytes / Q000502:physiology; D009856:Oncogene Proteins, Viral / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000502:physiology; D014162:Transfection
D015820:Cadherins; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins