Molecular-scale complexes of IL-2/15 receptors and MHC molecules persist but become more mobile, while microdomain-level aggregates are dispersed on actin-free membrane blebs of T cells

Abstract Earlier we found that interleukin-2 and -15 receptors (IL-2R, IL-15R) and MHC I and II, all proteins essential in T cell activation, form co-clusters at different hierarchic levels in lipid rafts of T cells. To investigate whether the membrane-associated actin cytoskeleton has a role in organizing these clusters, we used membrane blebs formed spontaneously on starved human T lymphoma cells for comparison, as they have a smooth surface without cytoskeletal connections. STED microscopy showed that the patchy cell surface distribution of IL-2Rα, IL-15Rα, MHC I and the lipid raft marker GM1 ganglioside observed on intact cell membranes became more homogenous on blebs. Spatial autocorrelation analysis revealed that cluster sizes diminished. Fluorescence correlation spectroscopy and single molecule tracking showed a two- to four-fold increase in the mobility of the investigated proteins. FRET efficiencies between homo- or hetero-associated proteins remained constant or decreased moderately and the associated fractions of protein pairs determined by fluorescence cross-correlation spectroscopy remained unchanged or increased. Our results show that the higher-order organization of the membrane becomes more homogeneous, clusters become smaller and the mobility of molecules increases on actin-free membrane blebs, but the investigated protein-protein interactions are preserved and do not depend on the actin cytoskeleton.