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Paul Wiseman"Probing protein transport and interactions in cells and neurons using image correlation microscopy"

Abstract :


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 recently 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 STICS and its two color extension, spatio-temporal image cross-correlation spectroscopy (STICCS), for measuring transport maps of adhesion related macromolecules such as integrin, alpha-actinin, paxillin, talin, etc. within, or associated with the basal membrane in living fibroblast and CHO cells. These measurements have allowed us to propose a model for the molecular clutch that regulates connections between the extracellular matrix, integrins in the membrane and the cytoskeleton during cell protrusion and migration. We will also highlight recent advances we have made with a new form of reciprocal 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. We will describe kICS measurements of the transport properties of quantum dot labeled CD-73 GPI anchored proteins in the membranes of living IMR-90 fibroblast cells.


Velocity map of retrograde transport of alpha-actinin/EGFP in an MEF cell. Measured by TIRF microscopy and STICS analysis.


 

Selected publications

D. L. Kolin, P. W. Wiseman. Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently-labeled macromolecules in cells. Cell Biochemistry and Biophysics 49 (3):141-164 (2007).
N. Durisic, A. I. Bachir, D. L. Kolin, B. Hebert, B. C. Lagerholm, P. Grutter, P. W. Wiseman. Detection and correction of blinking bias in image correlation transport measurements of quantum dot tagged macromolecules. Biophysical Journal 93: 1338-1346 (2007).
C. M. Brown, B. Hebert, D. L. Kolin, J. Zareno, L. Whitmore, A. R. Horwitz, P. W. Wiseman. Probing the integrin-actin linkage using high resolution protein velocity mapping. Journal of Cell Science 119:5204-5214 (2006).

Daniel Choquet