Relative ligand binding to small or large aggregates measured by scanning correlation spectroscopy

Abstract

Cell surface receptors transduce signals, required to produce cellular activity, that may be mediated by ligand-induced receptor aggregation. Several receptor systems exhibit both low and high ligand affinities and some models of receptor activation associate receptor clusters with high or low ligand binding affinity. In the present work succinyl concanavalin A, which binds with both high and low affinity to receptors, was studied on 3T3 Swiss mouse fibroblasts, where preaggregation of receptors has been postulated. Scanning fluorescence correlation spectroscopy measurements were used to determine the relationship between the degree of ligand binding and the state of receptor aggregation. Correlation analysis of fluorescence fluctuations across the cell surface reveal that the variance of the fluctuations (quantitated by g[0]) increased when the ligand concentration was varied from 0.33 to 67 mg/L. The g(0) values reached a plateau at concentrations greater than approximately 10 mg/L. These data are incompatible with homogeneous receptor distributions or equal affinity receptor binding but are compatible with a partly aggregated receptor system with high affinity binding to small aggregates, and low affinity binding to large aggregates. Computer simulated scanning fluorescence correlation spectroscopy experiments confirm that background fluorescence from the cell does not account for the experimentally observed effects.

Applications of Fluorescence Correlation Spectroscopy: Polydispersity Measurements

“…[8]. The process of iteration is stopped when the relative difference in two consecutive R(c) values is less than a preset value.…”

“…As a result, the sensitivity has been substantially increased and it has become possible to perform single-molecule detection measurements (3,4). Until now the FCS method has been applied mainly in biochemical and biophysical investigations such as measurement of lateral transport on cell surfaces (5), nucleic acid hybridization (6,7), and receptor/ligand interaction (8,9). The method, however, can also be very helpful in environmental studies, both for analytical determination and interaction studies of small particles and trace components in natural water and for investigation of the particle dynamics of model systems.…”

Scanning Fluorescence Correlation Spectroscopy

“…The linescanning SFCS was applied to measure the mean number of aggregates of Sindbis virus and vesicular stromatitis virus glycoproteins on the membrane of fibroblasts. The same SFCS implementation was used by St-Pierre and Petersen (1990) to study ligand binding to aggregated receptors, or more precisely, to find the relationship between the degree of ligand binding and the state of receptor aggregation. Another application involved the measurement of epidermal growth factor receptor distribution on A431 cells (St-Pierre and Petersen 1992).…”

Applications of fluorescence correlation spectroscopy: Measurement of size-mass relationship of native and denatured schizophyllan

“…46 -53 One of the most commonly used is Broersma's relationship, which is derived for rigid rod [Eqs. (7) and (8) in Table I], 47 and we used it here to calculate the size of the nondenatured schizophyllan.…”

“…(7) and (8)] to calculate the length of nondenatured schizo- phyllan. Several values of the rod diameter d that are needed in Eqs.…”