Mapping protein transport waves in podosome clusters in live immune dendritic cells with image correlation spectroscopy

Image correlation methods provide a new window of analysis for measurement of protein-protein interactions and macromolecular transport properties from fluorescence images of living cells. These approaches are based on space and time correlation analysis of fluctuations in fluorescence intensity within images recorded as a time series on a laser scanning or TIRF microscope. We previously introduced spatio-temporal image correlation spectroscopy (STICS) which measures vectors of protein flux in cells based on the calculation of a spatial correlation function as a function of time from an image time series. Here we will describe the application of time window STICS and its two color extension, spatio-temporal image cross-correlation spectroscopy (STICCS), for measuring transport waves of adhesion related macromolecules talin and vinculin as well as cytoskeletal actin between assembling and disassembling podosomes in dendritic immune cells. Podosomes are cylindrical membrane complexes with an integrin adhesive ring and an actin rich core that are associated with cellular migration and invasion in specific cell types. Finally, if there is time, we will also highlight recent advances we have made with a new form of reciprocal (k-) space ICS, (kICS) that allows us to measure unbiased transport coefficients of fluorescently labeled membrane proteins even if there is complex photophysics (such as emission blinking) of the probe. We will describe an application of kICS to map membrane transport and confinement in lipid domains of the