Supported lipid bilayers composed of a binary phospholipid mixtures have been widely used to investigate membrane heterogeneity 
. At constant temperature, surface heterogeneity and long range membrane diffusivity in the gel-fluid co-existence region vary according to the lipid composition, so that mobility within binary mixtures is governed by percolation theory 
. In this study, we have used supported planar membranes composed of DPPC and DMPC. Using FRAP measurements of D
to quantitatively describe long-range lateral movement of ligands in the presence of immobile domains, we identified lipid ratios that result in fluid, fluid-connected, fluid-confined, and low mobility membranes, and bound T cell stimulatory ligands to each of these membranes with similar efficiency and similar orientation. Using this experimental configuration, we have systematically explored the effect of varying ligand mobility on T cell activation.
Our results show that aggregation of the TCR complex within the cSMAC and centripetal movement of MCs containing key T cell signaling molecules are highly dependent on ligand mobility. Comparison of cSMAC formation in T cells interacting with fluid membranes and fluid confined membranes shows that CD3ζ centralization is delayed with fluid-connected membranes, but the final accumulation after 60 min is similar on these two surfaces. In contrast, CD3ζ accumulation plateaus at a lower level in T cells responding to fluid confined membranes. These results are consistent with a model in which the rate of TCR lateral transport is influenced by D while the overall level of TCR accumulated at the cSMAC depends on the connectivity of fluid regions.
The movement of MCs containing the tyrosine kinase ZAP70 toward the cSMAC also depends strongly on D
. This likely reflects the direct association of ZAP70 with phosphorylated ITAM motifs within the TCR complex. Several factors such as cluster size 
, differential coupling to actin flow 
, and integrin engagement 
have been proposed to affect the movement of signaling MCs at the IS. Our data suggest that membrane fluidity should also be considered. In comparison with ZAP70 MCs, MCs containing the adaptor molecule SLP76 show relatively little dependence on D
. This finding is consistent with previous work showing robust centripetal movement of SLP76 MCs in T cells responding to ligand-coated coverslips 
and indicates that the mechanisms that propel or constrain these two molecules at the IS are distinct. ZAP70 MCs are likely to be associated with the plasma membrane, while there is evidence that SLP76 MCs are associated with intracellular vesicles 
, and this could explain their differential sensitivity to ligand mobility. Alternatively, these two sets of MCs may interact differently with cytoskeletal components, e.g. actin and microtubules. Indeed, recent studies have demonstrated that dynein motors essentially co-distribute with signaling complexes and TCR MCs move along microtubules within the central region of the IS 
. In this context, it is interesting to note that retrograde flow of the actin cytoskeleton was not grossly affected by ligand mobility. Thus, ligand-dependent changes in mobility of TCR or ZAP70 cannot be attributed to slowing of the actin network.
In addition to promoting enhanced dynamics of T cell signaling proteins, we find that increased ligand mobility is associated with increased T cell activation as measured by tyrosine phosphorylation and elevated intracellular calcium. This result contrasts with previous studies, where augmented MC phosphorylation and elevated cytoplasmic calcium flux was associated with diminished
MC movement in cells costimulated with TCR and β1 integrin ligands 
or in cells plated on bilayer surfaces with metal grids that constrain the lateral movement of membrane components 
. In those experimental systems, augmented signaling was proposed to result from prolonged co-distribution between TCR and active kinases at peripheral signaling sites. The mechanism by which cell activation is related to ligand mobility may be different. In T cells responding to mobile ligands, we observed an overall increase in the accumulation of CD3ζ at the IS. The large reservoir of TCR agonist could continue to generate new TCR MCs, and to recruit downstream protein tyrosine kinases, raising the pool of phosphorylated molecules and sustaining elevated intracellular calcium levels 
. The positive correlation between ligand mobility and T cell activation cannot simply be an artifactual consequence of stimulation with anti-CD3 antibody, because similar results were obtained using peptide-loaded MHC Class II molecules and antigen-specific primary T cells. Nonetheless, the studies presented here used super-physiological concentrations of TCR agonist. In future work, it will be interesting to explore the effects of altered ligand mobility at limiting agonist dose, and to ask if the results differ depending on TCR-pMHC binding kinetics.
The reductionist system used here highlights the importance of ligand mobility and membrane heterogeneity as modulators of T cell activation. Clearly, ligand mobility in APC membranes lies somewhere between the extremes represented by the highly mobile and low mobility planar lipid bilayers. Indeed, live-cell FRAP experiments have revealed that many membrane-associated proteins exhibit relatively low long-range diffusional mobility and/or large immobile pools 
. Moreover, cell surface molecules exhibit distinct mobility properties depending on differential cytoskeletal association, aggregation state, or lipid microdomain association. Given the strong dependence of T cell activation on ligand mobility demonstrated here, it will be important to explore the parameter of ligand mobility on the surface of APCs. Future studies should address the differences in ligand mobility among different types of APCs, and the possibility that regulated changes in APC membrane fluidity are used to modulate T cell activation in vivo.