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 characteristics of MHC (major histocompatibility complex) class I molecules at the surface of HeLa cells at 22 degrees C and on different time scales. MHC class I was labeled using the Fab fragment of a monoclonal antibody (W6/32), covalently bound to either R-phycoerythrin or fluorescent microspheres, and the particles were tracked using high-sensitivity fluorescence imaging. Analysis of the data for a fixed time interval suggests a reasonable fit to a random diffusion model. The best fit values of the diffusion coefficient D decreased markedly, however, with increasing time interval, demonstrating the existence of anomalous diffusion. Further analysis of the data shows that the diffusion is anomalous over the complete time range investigated, 4-300 s. Fitting the results obtained with the R-phycoerythrin probe to D = D0talpha-1, where Do is a constant and t is the time, gave D0 = (6.7 +/- 4.5) x 10(-11) cm2 s-1 and alpha = 0.49 +/- 0.16. Experiments with fluorescent microspheres were less reproducible and gave slower anomalous diffusion. The R-phycoerythrin probe is considered more reliable for fluorescent SPT because it is small (11 x 8 nm) and monovalent. The type of motion exhibited by the class I molecules will greatly affect their ability to migrate in the plane of the membrane. Anomalous diffusion, in particular, greatly reduces the distance a class I molecule can travel on the time scale of minutes. The present data are discussed in relation to the possible role of diffusion and clustering in T-cell activation.
Smith
P R
PR
Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, England.
Morrison
I E
IE
Wilson
K M
KM
Fernández
N
N
Cherry
R J
RJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000911:Antibodies, Monoclonal; D055592:Biophysical Phenomena; D001703:Biophysics; D002462:Cell Membrane / Q000276:immunology; D004058:Diffusion; D005456:Fluorescent Dyes; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008954:Models, Biological
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D000255:Adenosine Triphosphate