We used fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT) techniques to compare diffusion of class I major histocompatibility complex molecules (MHC) on normal and α-spectrin-deficient murine erythroleukemia (MEL) cells. Because the cytoskeleton mesh acts as a barrier to lateral mobility of membrane proteins, we expected that diffusion of membrane proteins in α-spectrin-deficient MEL cells would differ greatly from that in normal MEL cells. In the event, diffusion coefficients derived from either FRAP or SPT analysis were similar for α-spectrin-deficient and normal MEL cells, differing by a factor of ∼2, on three different timescales: tens of seconds, 1-10 s, and 100 ms. SPT analysis showed that the diffusion of most class I MHC molecules was confined on both cell types. On the normal MEL cells, the mean diagonal length of the confined area was 330 nm with a mean residency time of 40s. On the α-spectrin-deficient MEL cells, the mean diagonal length was 650 nm with a mean residency time of 45s. Thus there are fewer barriers to lateral diffusion on cytoskeleton mutant MEL cells than on normal MEL cells, but this difference does not strongly affect lateral diffusion on the scales measured here.
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