Measurement of receptor distributions on cell surfaces is one important aspect of understanding the mechanism whereby receptors function. In recent years, scanning fluorescence correlation spectroscopy has emerged as an excellent tool for making quantitative measurements of cluster sizes and densities. However, the measurements are slow and usually require fixed preparations. Moreover, while the precision is good, the accuracy is limited by the relatively small amount of information in each measurement, such that many are required. Here we present a novel extension of the scanning correlation spectroscopy that solves a number of the present problems. The new technique, which we call image correlation spectroscopy, is based on quantitative analysis of confocal scanning laser microscopy images. Since these can be generated in a matter of a second or so, the measurements become more rapid. The image is collected over a large cell area so that more sampling is done, improving the accuracy. The sacrifice is a lower resolution in the sampling, which leads to a lower precision. This compromise of precision in favor of speed and accuracy still provides an enormous advantage for image correlation spectroscopy over scanning correlation spectroscopy. The present work demonstrates the underlying theory, showing how the principles can be applied to measurements on standard fluorescent beads and changes in distribution of receptors for platelet-derived growth factor on human foreskin fibroblasts.
Receptor aggregation is believed to be an important, early step when growth factors such as PDGF stimulate proliferation and differentiation of cell populations. To investigate receptor aggregation, we utilized a novel biophysical technique, image correlation spectroscopy, to study the distribution and aggregation state of PDGF-ß receptors on the surface of human dermal fibroblasts under various experimental conditions. It was found that the cell surface receptors were pre-clustered at 4°C and receptor aggregation increased for samples measured at 37°C. Treatment with PDGF-BB had no measurable effect on the receptor aggregation state. The results also indícate that additions of 10% serum or an inhibitor of tyrosine kinase activity, may disperse the receptors. The results of this study are consistent with organization of PDGF-ß receptors in pre-existing membrane domains.
When the receptors for platelet-derived growth factor (PDGF) are activated they aggregate, become tyrosine-phosphorylated and elicit a cascade of down-stream signals, including mobilization of Ca2+ from intra- and extracellular stores. Receptor mobility in the plane of the membrane is a prerequisite for receptor aggregation and further signalling. Using human foreskin fibroblasts (AG 1523) and fluorescence recovery after photobleaching (FRAP), we therefore assessed the lateral mobility characteristics of PDGF-beta2 receptors by their diffusion coefficient (D), and fraction of mobile receptors (R). This was done on cells stimulated with either normal human serum (NHS) or PDGF under different Ca2+-conditions. The results suggest that both intra- and extracellular free Ca2+ influence the mobility characteristics of the PDGF-beta2 receptor. Interestingly, the extracellular Ca2+ seems to impose general restrictions on the mobility of receptors, since R increased when extracellular Ca2+ was quenched with EGTA, whereas intracellular clamping of Ca2+ transients with MABTAM (BAPT/AM) primarily affected D. When both intra- and extracellular Ca2+ were quenced, D remained low and R high, further supporting the proposition that they achieve distinct effects. Inhibition of tyrosine phosphorylation with Erbstatin, partly inhibited the NHS effects and released PDGF-induced receptor immobilization. Ratio imaging with Fura-2 displayed that both NHS and PDGF induced changes in intracellular free [Ca2+]. In view of the present data it might have important effects on the state of the receptor in the membrane, for instance by regulating its lateral mobility, communication with other receptors and signalling functions in the membrane.
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