MHC-I dynamics vs. T-cell recognition

Progress Report 1. Development of simFRAP method We have continued to develop the simulation-assisted method to analyze Fluorescence Recovery After Photobleaching (FRAP) data, that we have reported in the previous report.

The development was very successful and culminated in a publication in the prestigious “Scientific Reports” journal, from the Nature Publishing Group: Daniel Blumenthal, Leo Godstien, Michael Edidin and Levi A. Gheber. “Universal Approach to FRAP Analysis of Arbitrary Bleaching Patterns”, Scientific. Reports. 5, 11655 (2015).

Briefly, closed-solutions describing fluorescence recovery after photobleaching are very limited by boundary and initial conditions assumed. To overcome this severe limitation, the recovery of fluorescence following photobleaching is simulated and compared with the real (measured) recovery. From the comparison, we are able to extract absolute values of diffusion coefficient of the fluorescence species. This enabled us to assess and separate two components of the fluorescence recovery in our GFP tagged MHC-I cells: latera diffusion and delivery of newly synthesized proteins.

2. Assessment of MHC-I delivery rates Using Dynasore, a drug that inhibits the ability of Clathrin-coated vesicles to form, we partially blocked delivery of MHC-I vesicles to the membrane. The arrival rate of MHC-I was shown to decrease with increasing concentration of Dynasore, in a dose-dependent manner, as shown in the following figure.

3. Apparent size of MHC-I clusters.

Moreover, the apparent size of the MHC-I clusters was shown to increase proportionally, with increased concentration of Dynasore (and decrease in MHC-I delivery rate), as predicted by our model. This is shown in the following figure.

4. Sites of delivery of MHC-I vesicles Using our ability to photo-bleach a large portion of a cells membrane, we characterized the correlation between MHC-I clusters before (at steady-state) and following photo-bleaching (newly-formed clusters). We show that that approximately 85% of the newly formed clusters appear at the same location where there existed a cluster before photo-bleaching (following figure). This result strongly indicates that MHC-I delivery sites are non-random, and that the delivery may be targeted to preferred sites. All these results are being submitted these days for publication in Journal of Cell Science.