Rotational dynamics of type I Fc epsilon receptors on individually-selected rat mast cells studied by polarized fluorescence depletion.

We report the first application of polarized fluorescence depletion (PFD), a technique which combines the sensitivity of fluorescence detection with the long lifetimes of triplet probes, to the measurement of membrane protein rotational diffusion on individually selected, intact mammalian cells. We have examined the rotation of type I Fc epsilon receptors (Fc epsilon RI) on rat mucosal mast cells of the RBL-2H3 line in their resting monomeric and differently oligomerized states using as probes IgE and three monoclonal antibodies (mAbs; H10, J17, and F4) specific for the Fc epsilon RI. PFD experiments using eosin (EITC)-IgE show that individual Fc epsilon RI on cells have a rotational correlation time (RCT) at 4 degrees C of 79 +/- 4 microseconds. Similarly, Fc epsilon RI-bound EITC-Fab fragments of the J17 Fc epsilon RI-specific mAb exhibit an RCT of 76 +/- 6 microseconds. These values agree with previous measurements of Fc epsilon RI-bound IgE rotation by time-resolved phosphorescence anisotropy methods. Receptor-bound EITC-conjugated divalent J17 antibody exhibits an increased RCT of 140 +/- 6 microseconds. This is consistent with the ability of this mAb to form substantial amounts of Fc epsilon RI dimers on these cell surfaces. The ratio of limiting to initial anisotropy in these experiments remains constant at about 0.5 from 5 degrees C through 25 degrees C for IgE, Fab, and intact mAb receptor ligands. Extensive cross-linking by second antibody of cell-bound IgE, of intact Fc epsilon RI-specific mAbs or of their Fab fragments, however, produced large fixed anisotropies demonstrating, under these conditions, receptor immobilization in large aggregates. PFD using the mAbs H10 and F4 as receptor probes yielded values for triplet lifetimes, RCT values, and anisotropy parameters essentially indistinguishable from those obtained with the mAb J17 clone. Possible explanations for these observations are discussed.