1F). To analyze the interaction of LPL and calmodulin in more detail, we first analyzed the subcellular localization of calmodulin in T cells. In unstimulated cells that did not form a contact with APC, calmodulin and LPL were both equally distributed throughout the cytoplasm (Fig. 3A). Upon T-cell stimulation via superantigen-loaded APC for 45 min, in 48.09±0.16% of the T-cell/APC couples calmodulin translocated to the contact zone between T cells and APC where it colocalized with LPL. We reinforced this quantification by calculating the area corrected calmodulin
content within the contact zone of T cells and APC and subtracted an area corrected protein content within T-cell/T-cell and APC/APC contact zones 26. This analysis confirmed Carfilzomib mw that calmodulin and LPL accumulated in the T-cell/APC contact zone (Supporting
Information Fig. 2). The interaction of calmodulin and LPL was shown by calmodulin pull-down experiments (Fig. 3B). A binding of LPL to calmodulin could only be seen in the presence of EGTA. Note that the calcium/calmodulin dependent PD-1 inhibitor kinase type IV (CamKIV) was efficiently precipitated with calmodulin in the presence of calcium, whereas EGTA inhibited this interaction (Fig. 3C). These experiments explain at the same time the interaction of LPL and calmodulin in unstimulated cells, in which no calcium signal was induced (Fig. 3B). Although binding of LPL to calmodulin in the absence of calcium was Org 27569 unexpected, such interactions to calcium-free calmodulin (Apocalmodulin/ApoCam) were described for several proteins (reviewed in 27). We next analyzed whether inhibition
of calmodulin through the calmodulin antagonist W7 would lead to reduced LPL accumulation in the IS. MIFC analysis demonstrates that LPL recruitment was indeed diminished in the presence of W7 (Fig. 4A and B). The degree of inhibition is reminiscence of that observed for ΔCBD-LPL. Importantly, W7 also inhibited recruitment of the pSMAC-marker LFA-1, but not of the cSMAC-marker CD3 in the contact zone. The selective effects of W7 on the accumulation of pSMAC-markers in the IS was independently confirmed using LSM and EGFP-tagged LPL, F-actin or PKCΘ and staining of endogenous LFA-1 (Supporting Information Fig. 3). Also in these experiments the enrichment of LPL and the pSMAC-markers actin and LFA–1 were inhibited by W7, whereas it had no effect on the accumulation of the cSMAC-marker PKCθ in the IS. The reduced accumulation of ΔCBD-LPL (Fig. 1F), or of wt-LPL in the presence of calmodulin antagonists (Supporting Information Fig. 3) may be explained either by a diminished initial relocalization or a reduced maintenance of LPL in the contact zone. To discriminate between the two possibilities, we analyzed the relocalization kinetics and mean duration of wt-LPL and ΔCBD-LPL in the contact zone using time-lapse video-microscopy (TLV).