Anomalous diffusion of major histocompatibility complex class I molecules on HeLa cells determined by single particle tracking

Single-particle tracking (SPT) was used to determine the mobility character istics of MHC (major histocompatibility complex) class I molecules at the s urface of HeLa cells at 22 degrees C and on different time scales. MHC clas s I was labeled using the Fab fragment of a monoclonal antibody (W6/32), co valently bound to either R-phycoerythrin or fluorescent microspheres, and t he particles were tracked using high-sensitivity fluorescence imaging. Anal ysis of the data for a fixed time interval suggests a reasonable fit to a r andom diffusion model. The best fit values of the diffusion coefficient D d ecreased markedly, however, with increasing time interval, demonstrating th e existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-30 0 s. Fitting the results obtained with the R-phycoerythrin probe to D = D(o )t(alpha-1), where D-o is a constant and t is the time, gave D-o = (6.7 +/- 4.5) x 10(-11) cm(2) s(-1) and alpha = 0.49 +/- 0.16. Experiments with flu orescent microspheres were less reproducible and gave slower anomalous diff usion. The R-phycoerythrin probe is considered more reliable for fluorescen t SPT because it is small (11 x 8 nm) and monovalent. The type of motion ex hibited by the class I molecules will greatly affect their ability to migra te in the plane of the membrane. Anomalous diffusion, in particular, greatl y reduces the distance a class I molecule can travel on the time scale of m inutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.