Measurement of the lateral diffusion of human MHC class I molecules on HeLa cells by fluorescence recovery after photobleaching using a phycoerythrin probe.
The mobility of cell surface MHC class I molecules on HeLa cells was measured by fluorescence recovery after photobleaching (FRAP). The probe used for these studies was the phycobiliprotein R-phycoerythrin coupled to Fab fragments of a monoclonal antibody specific for human monomorphic MHC class I molecules. It was found that the recovery curves could be equally well fitted by either a random diffusion model with an immobile component or by an anomalous diffusion model. In the latter case, the anomalous diffusion exponent was consistent with that previously determined by single-particle tracking (SPT) experiments using the same probe (P. R. Smith, I. E. G. Morrison, K. M. Wilson, N. Fernandez, and R. J. Cherry. 1999. Biophys. J. 76:3331-3344). The FRAP experiments, however, yielded a considerably higher value of D(0), the diffusion coefficient for a time interval of 1 s. To determine whether the results were probe dependent, FRAP measurements were also performed with the same monoclonal antibody labeled with Oregon Green. These experiments gave similar results to those obtained with the phycoerythrin probe. FRAP experiments with the lipid probe 5-N-(octadecanoyl) aminofluoroscein (ODAF) bound to HeLa cells gave typical results for lipid diffusion. Overall, our observations and analysis are consistent with anomalous diffusion of MHC class I diffusion on HeLa cells, but quantitative differences between FRAP and SPT data remain to be explained.
Georgiou
George
G
Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdom.
Bahra
Sukhvinder S
SS
Mackie
Alan R
AR
Wolfe
Caroline A
CA
O'Shea
Paul
P
Ladha
Shab
S
Fernandez
Nelson
N
Cherry
Richard J
RJ
D000911:Antibodies, Monoclonal; D002851:Chromatography, High Pressure Liquid; D004058:Diffusion; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D005805:Genes, MHC Class I; D006367:HeLa Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G / Q000378:metabolism; D008027:Light; D008055:Lipids / Q000737:chemistry; D010799:Phycoerythrin / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors
C116138:5-(N-octadecanoyl)aminofluorescein; D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D008055:Lipids; C401939:oregon green 514; D010799:Phycoerythrin; D019793:Fluorescein