CPM Seminar

Mapping Protein Diffusion, Velocity Vectors, and Interactions in Living Cells by Spatio-Temporal Image Correlation Spectroscopy

Paul Wiseman

McGill University

Transport Map of actinin dynamics at CHO cell basal membrane. Circles rms diffusion distance 10 min. Arrows mean flow distance 10 min. Bars fraction of proteins diffusing (green), flowing (yellow), immobile (cyan). Scale bar 10 μm.

The seminar will cover recent advances in image correlation methods and their application for measurements in living cells. The talk will focus on the development of image correlation spectroscopy (ICS) as an imaging extension of fluorescence correlation spectroscopy (FCS). The ICS technique is ideally suited to measure transport and clustering of fluorescently tagged proteins in cellular membranes where transport is slow and static proteins abound. The image correlation methods are based on the measurement of fluorescence intensity fluctuations as a function of space and time in cells collected as image time series using a laser scanning microscope (either confocal or two-photon). Spatial and temporal variants of the basic ICS method will be introduced and the power of these approaches to measure both aggregation and transport of cell surface proteins will be explained with the aid of computer simulations to demonstrate the measurement detection limits. The use of two-photon microscopy to perform image cross-correlation spectroscopy (ICCS) studies will also be discussed. ICCS allows direct measurement of the interactions of two co-localized proteins labeled with fluorophores having different emission wavelengths even in a high density environment. The transport properties of the co-localized proteins are also measured simultaneously by ICCS. Recent applications of the ICS and ICCS methods for characterizing the transport and clustering of GFP labeled integrin adhesion proteins in living fibroblasts will be presented. Image correlation studies which demonstrate simultaneous measurement of diffusing and flowing populations of integrin clusters, and correlated transport between the alpha-5 integrin and intracellular alpha-actinin in CHO fibroblasts at 37�C will be shown. We will highlight our recent development of spatio-temportal ICS and present measurements of full vector maps of directed protein transport in living cells.