Cell-adhesion assays: fabrication of an E-cadherin substratum and isolation of lateral and Basal membrane patches.
Cell adhesion between cells and with the extracellular matrix (ECM) results in dramatic changes in cell organization and, in particular, the cytoskeleton and plasma membrane domains involved in adhesion. However, current methods to analyze these changes are limited because of the small areas of membrane involved in adhesion, compared to the areas of membrane not adhering (a signal to noise problem), and the difficulty in accessing native protein complexes directly for imaging or reconstitution with purified proteins. The methods described here overcome these problems. Using a mammalian expression system, a chimeric protein comprising the extracellular domain of E-cadherin fused at its C-terminus to the Fc domain of human IgG1 (E-cadherin:Fc) is expressed and purified. A chemical bridge of biotin-NeutrAvidin-biotinylated Protein G bound to a silanized glass cover slip is fabricated to which the E-cadherin:Fc chimera binds in the correct orientation for adhesion by cells. After cell attachment, the basal membrane (a contact formed between cellular E-cadherin and the E-cadherin:Fc substratum) is isolated by sonication; a similar method is described to isolate basal membranes of cells attached to ECM. These membrane patches provide direct access to protein complexes formed on the membrane following cell-cell or cell-ECM adhesion.
Drees
Frauke
F
Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, CA, USA.
Reilein
Amy
A
Nelson
W James
WJ
D000818:Animals; D015820:Cadherins / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D002465:Cell Movement / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology; D004285:Dogs; D005109:Extracellular Matrix / Q000502:physiology; D006801:Humans; D007141:Immunoglobulin Fc Fragments; D007668:Kidney; D051381:Rats
D015820:Cadherins; D007141:Immunoglobulin Fc Fragments