PIP2 Controls T Cell Rigidity and Organization Figure 5. PIP5K overexpression interferes with ERM dephosphorylation. A, B. 5C.C7 T cells were activated with a-CD3 and a-CD28 antibodies for 1 or 2 min upon manipulation of PIP5K expression as indicated. T cell extracts were blotted for phospho-Ezrin T567/Radixin T564/ Moesin T558. Representative blots are given in (A). Phospho-ERM levels after two minutes of T cell activation are given with standard errors relative to those of T cell extracts from non-stimulated, non-transduced T cells in (B). Modest differences in ERM phosphorylation prior to T cell stimulation were not significant. Data from 3–10 independent experiments are given. doi:10.1371/journal.pone.0027227.g005 spatiotemporal organization of T cell signaling [6], is dependent of cytoskeletal dynamics [37,38], and is thus likely to be influenced by ERM-regulated cytoskeleton plasma membrane interactions. Upon tight cell coupling, the TCR is recruited to the T cell/ APC interface with a preference for the interface center. In the activation of 5C.C7 T cells by APCs such central clustering is related to the efficiency of T cell signaling [6,7]. Moreover, TCR clustering has a strong distal component as in about half of the cell couples the TCR is recruited transiently to the T cell distal pole [6](Figs. 6A, S6A). Rapid release of the TCR from the distal pole is associated with increasing interface accumulation. Overexpression of PIP5K b and c90 interfered with TCR accumulation at the T cell/APC interface within the first minute of tight cell coupling (Figs. 6B, C, S6B, C). For example upon overexpression of PIP5K c90, interface accumulation of the TCR in any pattern and in a central pattern at 1 min was reduced from 7966% and 4968% of control T cell/APC couples with such accumulation to 4268% and 1766%, respectively (p,0.005) (Fig. 6B). Delayed interface accumulation was accompanied by delayed TCR release from the distal pole. For example at 2 min no distal pole accumulation was observed in control T cell/APC couples any more, while in cell couples with T cell overexpression of PIP5K c90 or b 3368% and 865% of cell couples, respectively, still displayed distal accumulation of the TCR (p#0.01)(Fig. 6B, C). The phenotype upon PIP5K b overexpres- sion was most complex, as both the inducible recruitment of the TCR to the distal pole and the subsequent release were defective (Fig. 6C). Intriguingly, the severity of the defects in the early spatiotemporal organization of T cell signaling upon overexpres- sion of the different PIP5K isoforms matched the degree of impairment of IL-2 secretion, in that distal PIP5K isoforms were most effective. Effects of overexpression of PIP5K c87 (Fig. S6D, E), PIP5K c knockdown (Fig. S6F, G), and PIP2 blockade (Fig. S6H, I) were less severe to not significant, again consistent with data on IL-2 secretion. The TCR localization experiments tie our data together into a consistent scenario of how cytoskeletal roles of PIP2 control T cell activation: Increased rigidity of the T cell upon PIP5K overexpression (Figs. 4, S4) could trap the TCR, in particular at the distal pole (Fig. 6), thus delaying and impairing the formation of a TCR-anchored signaling complex at the center of the T cell/ APC interface (Fig. 6). A smaller interface would also limit receptor engagement. Less central TCR clustering and receptor engagement is linked to less efficient proximal signaling in 5C.C7 T cell/APC couples [6,7], as also seen here (Fig. 3C, D), and thus to less IL-2 secretion (Fig. 3A, B). PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27227