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Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)
Published on: May 27, 2012
Anomalous diffusion of major histocompatibility complex class I molecules on HeLa cells determined by single particle
P R Smith1, I E Morrison, K M Wilson
1Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, England.
Insights
Single-particle tracking reveals major histocompatibility complex (MHC) class I molecules exhibit anomalous diffusion on HeLa cell surfaces. This restricted movement impacts their ability to migrate and potentially influences T-cell activation.
Area of Science:
- Cellular and Molecular Biology
- Biophysics
- Immunology
Background:
- Major histocompatibility complex (MHC) class I molecules are crucial for immune responses.
- Understanding their surface mobility is key to deciphering immune cell interactions.
- Previous studies suggested random diffusion, but detailed mobility characteristics remained unclear.
Purpose of the Study:
- To investigate the mobility characteristics of MHC class I molecules on HeLa cell surfaces.
- To determine if MHC class I diffusion is random or anomalous.
- To assess the impact of diffusion type on molecular migration and T-cell activation.
Main Methods:
- Single-particle tracking (SPT) using high-sensitivity fluorescence imaging.
- Labeling MHC class I with R-phycoerythrin or fluorescent microspheres via antibody fragments (W6/32).
- Analyzing diffusion coefficients over time intervals from 4 to 300 seconds.
Main Results:
- MHC class I molecules exhibit anomalous diffusion, not simple random diffusion.
- Diffusion coefficient (D) decreased significantly with increasing time intervals.
- R-phycoerythrin probe showed more reliable results than microspheres due to its small size and monovalency.
- The anomalous diffusion exponent (alpha) was found to be 0.49 ± 0.16.
Conclusions:
- MHC class I mobility on cell surfaces is anomalous, significantly limiting their travel distance over time.
- Anomalous diffusion may play a role in regulating molecular interactions relevant to T-cell activation.
- The choice of probe in SPT is critical for accurate characterization of molecular dynamics.
Abstract:
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.
