Dynamics of molecules involved in antigen presentation: effects of fixation

Abstract

Antigen presentation by MHC class II molecules can be enhanced by paraformaldehyde fixation of antigen-presenting cells prior to assay. This treatment might be expected to aggregate membrane proteins and thus stabilize and strengthen transient protein-protein interactions involved in intercellular cooperation. Lateral and rotational dynamics of the MHC class II antigen I-A d on A20 cells fixed with various concentrations of paraformaldehyde were examined by fluorescence photobleaching recovery and time-resolved phosphorescence anisotropy, respectively. Probes were tetramethylrhodamine and erythrosin conjugates of MKD6 Fab fragments. Increasing concentrations of paraformaldehyde led to a progressive increase in the limiting anisotropy of I-A d at 4°C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 μs unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from ∼2×10 −10 cm 2 s −1, while the fractional recovery remained unchanged at about 40–50%. Taken together, these results suggest that fixation crosslinks class II molecules to each other or to other membrane proteins into structures large enough (>500,000 kDa) to diffuse translationally with perceptibly size-dependent rates. The fixation effects on both class II rotation and lateral diffusion were half-maximal at paraformaldehyde concentrations of ∼0.2%. Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed.