Place: Kleiner Physik-Hörsaal
Date: 06.12.2013, Time: 16:30h
Exploring diffusion and confinement of GPI receptors and CFTR in cell membranes using k-space image correlation spectroscopy
Prof. Paul Wiseman
The modern model of the cell membrane posits that it is spatially heterogeneous with domains that restrict molecular transport. Lipid rafts, or microdomains, are one such membrane domain that are enriched in sphingolipids, and function to sequester proteins in transient complexes that are believed to be a few 10s to 100s of nanometers in size. Membrane proteins may also be sequestered by the membrane proximal actin cytoskeleton. Here we show that image correlation based techniques, applied to standard laser scanning or TIRF fluorescence microscopy image series, can be used to discriminate between the different mechanisms of confined diffusion. Using k-space image correlation spectroscopy (kICS) to analyze images series, we can then fit the correlation functions for two transport components: free and confined. The correlation function output can be averaged to obtain the Mean Square Displacement (MSD) from the time dependent correlation function radii. Furthermore, we show how one can extract from the correlation function data the characteristic parameters of the system such as domain size, density, diffusion coefficients and partition rates. To verify the validity of this tool, we performed simulations of confined diffusion in meshwork and microdomains where we varied the domain size (10-1000 nm radius), density (up to 10 % area coverage), confinement probability and diffusion coefficients (0.002-0.1 µm2/s). We used the simulations to establish the limits due to the spatio-temporal sampling and noise. We applied this analysis to the study of dynamics of membrane proteins known to be raft associated: GPI-GFP and Cholera Toxin Subunit B, and verified that our tools can detect changes in the confinement parameters following the application of drugs that disrupt rafts. Finally we will show how an extension of kICS was used to measure the transport properties of Cystic fibrosis transmembrane conductance regulator (CFTR) and detect transiently confined and more freely diffusing populations of this ion channel in the cell membrane.