Repeated FRAP of the actin-binding protein CapG in the cell nucleus - a functional assay for EGF signaling in the single live breast cancer cell

Abstract

AbstractCompartmentalization and differential distribution of proteins within a cell maintain cellular function and viability. CapG is the only gelsolin-related actin-binding protein that distributes in steady state diffusively throughout cytoplasm and cell nucleus. To detect changes in CapG’s nuclear shuttling in response to external stimuli on the single cell level, we established repeated FRAP experiments of one and the same breast cancer cell. With this experimental set up, we found that ATP-depletion reversibly decreased CapG’s shuttling into the cell nucleus. The addition of epidermal growth factor (EGF) increased CapG’s nuclear shuttling within minutes. Serum-starvation doubled the number of breast cancer cells from 40% to 80% displaying increased CapG shuttling in response to EGF. Testing five different potential CapG phosphorylation sites, we found that serine 70 mediates the increase in CapG’s nuclear shuttling triggered by EGF. Thus, repeated FRAP of CapG in the cell nucleus can be used as functional readout of signaling cascades in the same single live breast cancer cell.