Abstract
T cell receptor (TCR) ligation induces rapid polarization of the actin cytoskeleton resulting in the formation and stabilization of the immunological synapse (IS), recruitment of signaling molecules, and initiation of signaling cascades of T cell activation. Actin remodeling is essential for these events and is mandatory for T cell activation. Specific recruitment, redistribution and organization of signaling molecules in the IS is facilitated by lipid raft microdomains, which provide a scafold for focal protein assembly. Fyn and ZAP-70 are the most proximal TCR signaling molecules that localized in the IS and are redistributed in the lipid rafts during T cell activation. Currently, it is poorly understood how signals originating from the activated TCR are linked to the cytoskeletal rearrangement and the focal redistribution of signaling proteins. Recently, we identified RIAM, a novel adaptor molecule that contains a RA (Ras Association) domain, a PH (Plekstrin Homology) domain and proline-rich motifs. RIAM interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Profilin associates with G-actin and promotes actin polymerization by adding actin monomers to the barbed ends of F-actin. Ena/VASP family proteins are cytoskeletal proteins and regulate actin dynamics. Via these interactions, RIAM functions as a regulator of the actin cytoskeleton. To examine whether RIAM plays a role in the local reorganization of the actin cytoskeleton during IS formation, stable GFP-transfected Jurkat T cells were incubated with Raji B cells as APC, in the presence or absence of SEE. T cell:APC conjugates and formation of IS were examined by confocal microscopy. Synapse formation between GFP-Jurkat cells and Raji B cells was detected only in the presence of SEE. Under these conditions, polymerized (F) actin was highly recruited at the IS, as determined by phalloidin staining. Staining with RIAM-specific antibody indicated that in the absence of antigen, RIAM was diffusely expressed in the cytoplasm and at the plasma membrane. Impressively, upon incubation with SEE loaded Raji cells, RIAM was redistributed at the IS where it co-localized with polymerized (F) actin. Staining with ZAP-70 specific antibody revealed that ZAP-70 was also recruited at the IS upon activation and co-localized with RIAM and polymerized (F) actin. Sucrose gradient centrifugation indicated that RIAM translocated to the lipid rafts and co-localized in the same raft fraction with fyn and ZAP-70. RIAM was also a substrate of fyn and ZAP-70, which induced its tyrosine phosphorylation. Co-expression studies, by transfection of RIAM with fyn or ZAP-70 cDNA in COS cells, indicated that RIAM was a specific substrate of fyn and ZAP-70 but not lck. In vivo association of endogenous RIAM with fyn and ZAP-70 was also observed in primary T cells and in RIAM-transfected Jurkat cells and this interaction was highly upregulated upon activation. In the same complex, RIAM associated with grb2, crkL and the cytoskeletal proteins VASP and Profilin, indicating that RIAM functions as a link between the actin cytoskeleton and TCR signaling. Taken together these observations indicate a unique role of RIAM as an adaptor protein integrating TCR-mediated events that result in focal redistribution of polymerized actin, recruitment of signaling molecules at the IS and initiation of downstream signaling pathways.
Actin Cytoskeleton Regulates Calcium Dynamics and NFAT Nuclear Duration