Schedule Info

Correlating Fc\epsilon RI signaling with dynamics, topography and microdomains via single quantum dot imaging

Nicholas Andrews



Abstract: The high affinity IgE receptor (Fc\epsilon RI) is present on human mast cells and basophils and is responsible for allergen-induced release of histamine and other mediators of allergic inflammation. Although biochemical techniques have provided tremendous insight into the cascade of events that occurs after allergen binding, the precise mechanism by which binding initiates this cascade remains to be elucidated. Single particle tracking (SPT) using bright and photostable quantum dots (QDs) provides information about protein dynamics on living cells with high spatial (~10 nm) and temporal (>30 Hz) resolution. We used SPT to explore the roles of receptor dynamics and the actin cytoskeleton in the initiation of Fc\epsilon RI signal transduction. Simultaneous observations of Fc\epsilon RI motion and GFP-tagged actin dynamics provide direct evidence that membrane-proximal actin filament bundles restrict receptor motion. Dynamic reorganization of actin structures occurs over seconds, thus making the location and dimensions of actin-defined domains time dependent. We also show that receptors become immobilized within seconds of crosslinking and that disruption of the actin cytoskeleton results in delayed immobilization kinetics and increased diffusion of crosslinked clusters. These results implicate actin in membrane partitioning that not only restricts diffusion of membrane proteins, but also dynamically influences their long-range mobility, sequestration and response to ligand binding.