1). A 3D reconstruction of the contact zone confirmed that LPL localized outside the cSMAC and thus in the p/dSMAC of the BYL719 research buy IS (Fig. 1C). A quantification of the contact zone of 150 cell couples from three experiments

revealed that LPL was found in 62% in and around (i.e. distal) the LFA-1 staining (p/dSMAC) (Fig. 1D). The mechanism by which LPL is targeted to the IS was so far unknown. To scrutinize the mechanisms that promoted the relocalization and stabilization of LPL in the contact zone, we created LPL cDNA constructs lacking the potential calmodulin-binding domain (ΔCBD-LPL), actin-binding domain (ΔABD-LPL) or EF-hand calcium-binding domains (ΔEF1/2-LPL). In addition, we mutated the phosphorylation site at Ser5 to alanine (5A-LPL) 17. The expression of theses mutants in T cells was at a similar level as wt-LPL (Fig. 1E). After incubation with superantigen-loaded APC, the localization of these

mutants in the IS was analyzed and judged as enriched if at least 50% of the protein was found in the cell interface (Fig. 1F). Compared to wt-LPL the number of cells that contained ΔCBD-LPL in the contact zone was reduced by about 50% as analyzed by LSM. Hardly any cells displayed ΔABD-LPL in the contact zone. In contrast, mutation of the phosphorylation site (5A-LPL) or deletion of the calcium-binding sites (ΔEF1/2-LPL) had no effect on the relocalization of LPL. Thus, the binding sites of LPL for calmodulin and F-actin GW-572016 ic50 are important for the redistribution of LPL to the T-cell/APC contact zone. MIFC allows quantification of the F-actin content of T cells that form a contact with APC (Supporting Information Fig. 1). These analyses revealed

that T cells had an elevated total F-actin content if they formed a contact Buspirone HCl with their APC compared to solitaire T cells within the same sample (Supporting Information Fig. 1D). Actin polymerization could account for the enrichment of LPL at the T-cell APC contact zone since plastins bind to F-actin during actin polymerization 14, 15 and LPL mutants lacking the actin-binding domain did not localize to the IS (See Fig. 1D). Vice versa, LPL is an actin-bundling protein that stabilizes F-actin. Therefore, one would assume that lack of LPL affects the F-actin content of T cells. We knocked down LPL expression with small interference RNA (siRNA) to test this hypothesis. T cells that were transfected with LPL-specific siRNA (LP) displayed a reduction of LPL-expression by at least 90% compared to cells that were transfected with a non-targeting control siRNA (con) (Fig. 2A). The mean F-actin content in unstimulated LPL knock-down T cells (T-cell singlets) was only slightly lower compared to unstimulated control siRNA-treated cells (Fig. 2B). However, the rise in the F-actin content observed upon APC encounter was significantly lower than in control siRNA-treated T cells (Fig. 2B).