Flow cytometric detection of the association between cell surface receptors and the cytoskeleton

The cytoskeleton can serve as a structure at which receptors and signaling molecules can be immobilized to react with each other and induce signal transduction, which, consequently, leads to functional responses of the cell. Furthermore, transduction of mechanical forces into the cell can be realized by a physical linkage between receptor and cytoskeleton. We present a flow cytometric approach to analyze integrin receptors that are physically linked to the cytoskeleton. Epithelial cells were suspended and extracted with Triton X-100 containing lysis buffer to obtain the detergent-insoluble cytoskeletal fraction. To detect immobilized receptors, the fractions were incubated with antibodies against the receptors. We were able to measure these cytoskeletons as single particles in flow cytometry. The extracted fractions revealed distinct lower forward and side light scatter intensities compared with normal cells. Our results demonstrated that integrin receptor cross linking induced their association to the cytoskeleton. Incubation of cells with a receptor antibody alone had no effect. We conclude that flow cytometry enables the evaluation of the receptor-cytoskeleton linkage on the basis of objective fluorescence data and on a single cell level.