Analysis of membrane protein cluster densities and sizes in situ by image correlation spectroscopy

No. Petersen et al., Analysis of membrane protein cluster densities and sizes in situ by image correlation spectroscopy, FARADAY DIS, (111), 1998, pp. 289-305
Citations number
Categorie Soggetti
Physical Chemistry/Chemical Physics
Journal title
Faraday discussions
ISSN journal
1359-6640 → ACNP
Year of publication
289 - 305
SICI code
Communication between cells invariably involves interactions of a signallin g molecule with a receptor at the surface of the cell. Typically, the recep tor is imbedded in the membrane and it is hypothesized that the binding of the signalling molecule causes a change in the state of aggregation of the receptor which, in turn, initiates a biochemical signal within the cell. Su bsequently, many of the occupied receptors bind to membrane-associated stru ctures, called coated pits, which invaginate and pinch off to form coated v esicles, thereby removing the receptors from the cell surface. The state of aggregation of membrane receptors is obviously in constant flux. Any usefu l approach to measuring the state of aggregation must, therefore, allow for dynamic measurements in living cells. It is possible to use fluorescently labelled signalling molecules or antibodies directed at the receptor of int erest to visualize the receptor on the cell surface with a fluorescence mic roscope. By employing a laser confocal microscope, high resolution images c an be produced in which the fluorescence intensity is quantitatively imaged as a function of position across the surface of the cell. Calculations of autocorrelation functions of these images provide direct and accurate measu res of the density of fluorescent particles on the surface. Combined with t he average intensity in the image, which reflects the total average number of molecules, it is possible to estimate the degree of aggregation of the r eceptor molecules. We refer to this analysis as image correlation spectrosc opy (ICS). We show how ICS can be used to measure the density of several re ceptors on a variety of cells and how it can be used to measure the density of coated pits and the number of molecules per coated pit. We also show ho w the technique can be used to monitor fusion of virus particles to cell me mbranes. Further, we illustrate that, by calculating cross-correlation func tions between pairs of images, we can extend the analysis to measurements o f the distributions as a function of time, on the second timescale, as well as to measurements of the movement of the receptor aggregates on the surfa ce. Finally, we illustrate that, by this approach, we can measure the exten t of interaction between two different receptors as a function of time. Thi s represents the most quantitative measurement of the extent of co-localiza tion of receptors available and is independent of the spatial resolution of the confocal microscope. The theory of ICS and image cross-correlation spe ctroscopy (ICCS), focussing on the interpretation of the data in terms of t he biological phenomenon being probed, is discussed.