PIP2 Controls T Cell Rigidity and Organization Figure 4. PIP5K overexpression rigidifies T cells. A. 5C.C7 T cells were activated with CH27 APCs and 10 mM MCC agonist peptide. The diameter of the T cell/APC interface at the time of tight cell coupling is given with standard errors relative to that of the T cell body upon manipulation of PIP5K expression as indicated. An asterisk indicates statistical significance with p,0.01 relative to control. 28–54 cell couples were analyzed per condition. B. 5C.C7 T cells were transduced to express TCRf-GFP and were activated with CH27 APCs and 10 mM MCC agonist peptide. For all cell couples with persistent interface accumulation of TCRf-GFP it was determined whether preceding cell coupling occurred with (e.g. Figs. S1C, S2E, and S6A) or without (Fig. S4A, Movie S7) the formation of a visible lamellum. Of all T cell/APC couples with persistent TCRf-GFP interface accumulation, the percentage of cell couples without visible lamellum upon cell coupling is given with standard errors upon manipulation of PIP5K expression, as indicated. An asterisk indicates statistical significance with p,0.001 relative to control. 25–56 cell couples were analyzed per condition. C. 5C.C7 T cells were activated with CH27 APCs and 10 mM MCC agonist peptide. The percentage of cell couples with a visible uropod is given with standard errors relative to tight cell coupling upon manipulation of PIP5K expression and PIP2 blockade, as indicated. 17–68 cell couples were analyzed per condition. doi:10.1371/journal.pone.0027227.g004 in ERM phosphorylation to 1967% of the prestimulation levels (Fig. 5, not significantly different from control however) as consistent with unimpaired T cell flexibility. A reduced frequency of T cell coupling and impaired F-actin polymerization could be ruled out as alternate mechanisms of PIP2 action (Fig. S5B, C). The spatiotemporal organization of T cell signaling depends on PIP2 To understand how T cell rigidity may be linked to T cell signaling, we addressed effects of PIP2 on TCR clustering. TCR clustering as a representative element of the system wide PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27227