Abstract
T cell receptor (TCR) engagement induces clustering and recruitment to the plasma membrane of many signaling molecules, including the protein tyrosine kinase zeta-chain associated protein of 70 kDa (ZAP70) and the adaptor SH2 domain-containing leukocyte protein of 76 kDa (SLP76). This molecular rearrangement results in formation of the immunological synapse (IS), a dynamic protein array that modulates T cell activation. The current study investigates the effects of apparent long-range ligand mobility on T cell signaling activity and IS formation. We formed stimulatory lipid bilayers on glass surfaces from binary lipid mixtures with varied composition, and characterized these surfaces with respect to diffusion coefficient and fluid connectivity. Stimulatory ligands coupled to these surfaces with similar density and orientation showed differences in their ability to activate T cells. On less mobile membranes, central supramolecular activation cluster (cSMAC) formation was delayed and the overall accumulation of CD3ζ at the IS was reduced. Analysis of signaling microcluster (MC) dynamics showed that ZAP70 MCs exhibited faster track velocity and longer trajectories as a function of increased ligand mobility, whereas movement of SLP76 MCs was relatively insensitive to this parameter. Actin retrograde flow was observed on all surfaces, but cell spreading and subsequent cytoskeletal contraction were more pronounced on mobile membranes. Finally, increased tyrosine phosphorylation and persistent elevation of intracellular Ca2+ were observed in cells stimulated on fluid membranes. These results point to ligand mobility as an important parameter in modulating T cell responses.
Highlights
Cell membranes have unique physical properties including lateral heterogeneity, fluid nature, and diverse surface topology
Surface heterogeneity and long range membrane diffusivity in the gel-fluid coexistence region vary according to the lipid composition, so that mobility within binary mixtures is governed by percolation theory [31,36,37]
Using fluorescence recovery after photobleaching (FRAP) measurements of D and R 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, fluidconfined, and low mobility membranes, and bound T cell stimulatory ligands to each of these membranes with similar efficiency and similar orientation
Summary
Cell membranes have unique physical properties including lateral heterogeneity, fluid nature, and diverse surface topology. Signal transduction across the plasma membrane is commonly accompanied by the coordinated reorganization of membrane components. This is illustrated by the interaction between T cells and antigen presenting cells (APCs). After formation of IS, TCR-proximal MCs are continuously assembled near the cell periphery and translocate to the cSMAC region [5]. These newlygenerated TCR MCs are essential for the maintenance of IS structure [3] and for sustained signaling [5]. It is generally believed that MCs serve as primary signaling sites where early tyrosine phosphorylation events take place, leading to downstream signaling events such as calcium flux and transcriptional activation [5,6,7]
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