Séminaire externe : Paul Wiseman
Séance du lundi 10 octobre 2016, 11:00 - 11:00,
Salle du Conseil de l'UFR de Physique (252, P5bis)
The image correlation spectroscopy toolkit and its application in live cells to measure protein transport and interactions
Paul Wiseman
Department of Physics and Chemistry, McGill University, Montreal
Image correlation methods are an extension of fluorescence fluctuation spectroscopy that can measure protein-protein interactions and macromolecular transport properties from input fluorescence microscopy 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 total internal reflection fluorescence (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 cellular 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. EM and super-resolution microscopy of cells shows radial actin filaments that connect neighboring podosomes so we applied radial and podosome pair correlation analysis to further characterize the transport waves within connected podosome clusters. These analyses combined with pharmacological perturbation experiments show that podosome turnover is coordinated within local clusters in cells. Finally we will also highlight recent advances we have made with a new form of reciprocal (k-) space ICS, called kICS, that allows us to measure unbiased transport coefficients of fluorescently labeled membrane proteins even if there is complex photophysics (such as nanoparticle emission blinking) of the probe.