Time-resolved fluorescence anisotropy and fluctuation correlation analysis of major histocompatibility complex class I proteins in fibroblast cells

Abstract

Major histocompatibility complex class I proteins, MHC(I), are expressed in almost all nucleated cells and synthesized in the endoplasmic reticulum (ER). The orientation and mobility of these complexes are crucial in their biological function in the immune system, i.e., the cytosolic pathogen peptides loading and their presentation to T-cell receptors at the plasma membrane, where cell destruction is triggered. Here, we investigate the structural flexibility and associations of GFP-encoded MHC(I) alleles (H2Ld), namely H2LdGFPin and H2LdGFPout, in cultured mouse fibroblast cells. Time-resolved fluorescence anisotropy of H2LdGFPin in the ER indicates a dominant overall tumbling motion of 56±7ns (ER), with a fast conformational flexibility, as compared with a restricted rotation of H2LdGFPout. At the single-molecule level, the diffusion coefficient of H2LdGFPin and H2LdGFPout in the ER is (1.8±0.5)×10−9 and (2.1±0.6)×10−9cm2/s, respectively, as revealed by fluorescence correlation spectroscopy. A complementary immunoblotting of H2LdGFP constructs, isolated from mouse fibroblast cells, reveals band at 75kDa as compared with 29kDa of the free EGFP. These real-time dynamics provide new insights into the structural flexibility and intracellular associations of GFP-labeled MHC(I) alleles (H2Ld) in living cells.