Dynamics of molecules involved in antigen presentation: effects of fixation.
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-Ad 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-Ad at 4 degrees C from the value of 0.042 for untreated cells, indicative of large aggregate formation, while leaving the rotational correlation time of 29 micros unchanged, a measure of the unperturbed molecule. On the other hand, the translational diffusion constants decreased from approximately 2x10(-10) cm2 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 approximately 0.2%. Possible relations between the biological effector functions of class II and the physical sizes of fixation-induced aggregates are discussed.
Barisas
B G
BG
Department of Chemistry, Colorado State University, Fort Collins 80523, USA. barisas@lamar.colostate.edu
Wade
W F
WF
Jovin
T M
TM
Arndt-Jovin
D
D
Roess
D A
DA
D000818:Animals; D016880:Anisotropy; D017951:Antigen Presentation; D000938:Antigen-Presenting Cells / Q000276:immunology; D002460:Cell Line; D004058:Diffusion; D005404:Fixatives; D005557:Formaldehyde; D000949:Histocompatibility Antigens Class II / Q000737:chemistry / Q000378:metabolism*; D008163:Luminescent Measurements; D051379:Mice; D011108:Polymers
D005404:Fixatives; D000949:Histocompatibility Antigens Class II; C111389:I-Ad antigen; D011108:Polymers; D005557:Formaldehyde; C003043:paraform