DYNAMICS OF MEMBRANE RAFTS, TALIN, AND ACTIN AT NASCENT AND MECHANICALLY PERTURBED FOCAL ADHESIONS

摘要

A single endothelial cell was deformed at the apical surface by binding a functionalized nanoelectrode probe to a predetermined location on the surface of the cell. After identifying the point of contact, as recognized by the electronic signature of the nanoelectrode, and allowing binding to the cell of the fibronectin-fiinctionalized tip, a focal adhesion site was induced at the probe site. The probe was displaced thereby applying a prescribed shear deformation to the surface of the cell. Locations of membrane rafts were identified by cholera toxin, and focal adhesion proteins were assessed using RFP-talin, and GFP-actin. Mechanical coupling and kinetics of assembly of these labeled proteins were measured using time-lapse fluorescent images taken under 60X with a multi-point confocal scanner. Raft marker GM1, Actin, and Talin were observed to sequentially accumulate at probe site with different kinetics not only upon probe contact but also upon deformation. Following deformation, later transient motion of rafts in the opposite direction of initial deformation was observed suggesting that rafts recoil. In conclusion, we report a novel nanoelectrode-based method for controlled manipulation of the cell surface and observed mechanical coupling of focal adhesions and cross-linked lipid rafts.