UC Berkeley

Micron-scale spatial reorganization of cell membrane receptor protein clusters is emerging as an important early modulator of inter- and intracellular signaling input. Detailed within is an in-vitro investigative technique utilizing cultured cells, nanolithograpically-patterned supports for fluid biomolecule-functionalized phospholipid bilayer membranes and inverted optical microscopy. This method allows the user to visualize and, if desired, restrict the formation and transport of these transient macroclusters as they coalesce on the membrane of a stimulated cell. Multi-color fluorescence imaging allows the simultaneous tracking of multiple molecules, permitting real-time analysis of coupling between surface receptors and intracellular signaling and structural proteins. The two systems examined are the T cell immunological synapse and ephrinA1-EphA2 interactions in highly invasive breast cancer cells. In both cases, it is demonstrated that induced restrictions of receptor-mediated cytoskeletal reorganization is sufficient to alter downstream signaling outcomes, and the molecular mechanisms of feedback coupling are investigated.