Receptor-regulated cellular signaling often is mediated by formation of transient, heterogeneous protein complexes of undefined structure. We used single and two-color photoactivated-localization microscopy (PALM) to study complexes downstream of the T cell antigen receptor (TCR) in single molecule detail at the plasma membrane of intact T cells. The kinase ZAP-70 distributed completely with the TCRζ chain and both partially mixed with the adapter LAT in activated cells thus showing localized activation of LAT by TCR-coupled ZAP-70. In resting and activated cells LAT primarily resided in nanoscale clusters as small as dimers whose formation depended on protein-protein and protein-lipid interactions. Surprisingly, the adapter SLP-76 localized to the periphery of LAT clusters. This nanoscale structure depended on polymerized actin and its disruption affected TCR-dependent cell function. These results extend our understanding of the mechanism of T cell activation and the formation and organization of TCR-mediated signaling complexes, findings also relevant to other receptor systems.

Speciation--the splitting of one species into two--occurs by the evolution of any of several forms of reproductive isolation between taxa, including the intrinsic sterility and inviability of hybrids. Abundant evidence shows that these hybrid fitness problems are caused by incompatible interactions between loci: new alleles that become established in one species are sometimes functionally incompatible with alleles at interacting loci from another species. However, almost nothing is known about the genes involved in such hybrid incompatibilities or the evolutionary forces that drive their divergence. Here we identify a gene that causes epistatic inviability in hybrids between two fruitfly species, Drosophila melanogaster and D. simulans. Our population genetic analysis reveals that this gene--which encodes a nuclear pore protein--evolved by positive natural selection in both species' lineages. These results show that a lethal hybrid incompatibility has evolved as a by-product of adaptive protein evolution.

The adapter molecule LAT is a nucleating site for multiprotein signaling complexes that are vital for the function and differentiation of T cells. Extensive investigation of LAT in multiple experimental systems has led to an integrated understanding of the formation, composition, regulation, dynamic movement, and function of LAT-nucleated signaling complexes. This review discusses interactions of signaling molecules that bind directly or indirectly to LAT and the role of cooperativity in stabilizing LAT-nucleated signaling complexes. In addition, it focuses on how imaging studies visualize signaling assemblies as signaling clusters and demonstrate their dynamic nature and cellular fate. Finally, this review explores the function of LAT based on the interpretation of mouse models using various LAT mutants.

Cytoskeletal forces are implicated in T-cell–receptor activation, but their determinants are not known. Traction force microscopy was used to measure forces generated during T-cell activation. Whereas actin dynamics were essential for force generation, myosin contractility played a limited role. T-cells were also found to be mechanosensitive.