LFA-1 and kindlin-3 enable the collaborative transport of SLP-76 microclusters by myosin and dynein motors.

Integrin engagement within the immune synapse enhances T cell activation, but our understanding of this process is incomplete. In response to T cell receptor (TCR) ligation, SLP-76 (LCP2), ADAP (FYB1) and SKAP55 (SKAP1) are recruited into microclusters and activate integrins via the effectors talin-1 and kindlin-3 (FERMT3). We postulated that integrins influence the centripetal transport and signaling of SLP-76 microclusters via these linkages. We show that contractile myosin filaments surround and are co-transported with SLP-76 microclusters, and that TCR ligand density governs the centripetal movement of both structures. Centripetal transport requires formin activity, actomyosin contraction, microtubule integrity and dynein motor function. Although immobilized VLA-4 (α4ß1 integrin) and LFA-1 (αLß2 integrin) ligands arrest the centripetal movement of SLP-76 microclusters and myosin filaments, VLA-4 acts distally, while LFA-1 acts in the lamellum. Integrin ß2, kindlin-3 and zyxin are required for complete centripetal transport, while integrin ß1 and talin-1 are not. CD69 upregulation is similarly dependent on integrin ß2, kindlin-3 and zyxin, but not talin-1. These findings highlight the integration of cytoskeletal systems within the immune synapse and reveal extracellular ligand-independent roles for LFA-1 and kindlin-3. This article has an associated First Person interview with the first author of the paper.

ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters.

Antigen recognition by the T cell receptor (TCR) directs the assembly of essential signaling complexes known as SLP-76 (also known as LCP2) microclusters. Here, we show that the interaction of the adhesion and degranulation-promoting adaptor protein (ADAP; also known as FYB1) with SLP-76 enables the formation of persistent microclusters and the stabilization of T cell contacts, promotes integrin-independent adhesion and enables the upregulation of CD69. By analyzing point mutants and using a novel phospho-specific antibody, we show that Y595 is essential for normal ADAP function, that virtually all tyrosine phosphorylation of ADAP is restricted to a Y595-phosphorylated (pY595) pool, and that multivalent interactions between the SLP-76 SH2 domain and its binding sites in ADAP are required to sustain ADAP phosphorylation. Although pY595 ADAP enters SLP-76 microclusters, non-phosphorylated ADAP is enriched in protrusive actin-rich structures. The pre-positioning of ADAP at the contact sites generated by these structures favors the retention of nascent SLP-76 oligomers and their assembly into persistent microclusters. Although ADAP is frequently depicted as an effector of SLP-76, our findings reveal that ADAP acts upstream of SLP-76 to convert labile, Ca2+-competent microclusters into stable adhesive junctions with enhanced signaling potential.

The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms.

The T cell receptor (TCR) triggers the assembly of "SLP-76 microclusters," which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase-associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A "tandem dimer" containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP-interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and "inside-out" signaling to ß1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and ß1 integrins.

Motion-based angiogenesis analysis: a simple method to quantify blood vessel growth.

Existing methods to quantify angiogenesis range from image analysis of photographs to fluorescent microscopy. These methods are often time consuming and costly; they also may not detect capillaries if they are indistinct from the background of the image. We have developed a simple method based on the motion of blood to create an image that reveals the entire angiogenic vasculature. Two image analysis software programs were used separately to demonstrate the method. Using either ImageJ or Environment for Visualizing Images, we analyzed a video clip of regenerated tissue from the partially amputated caudal fin of a zebrafish (Danio rerio). The deviations among the frames in the video stack were calculated to reveal pixels where motion has occurred. The resulting image highlighted all vessels through which blood flowed and allowed for automatic quantification of the newly developed vasculature. Using this method, we quantified the angiogenic action of basic fibroblast growth factor and vascular endothelial growth factor, as well as suppression of angiogenesis by an inhibitor. In a preliminary study, we also found that it could be used to trace the developing vasculature in zebrafish embryos. Thus, motion-based angiogenesis analysis may provide an easy and accurate quantification of angiogenesis.