regulation of PIP2 effector functions. While at 1 mM tatPLCd PH substantial effects could not be found, at .5 mM tatPLCd PH T cell coupling upon APC contact was reduced by .65% (p,0.001) from 5065% to less than 20%. This was most likely caused by a.95% decrease (p,0.001) in the percentage of T cells with overt migratory polarity. We therefore used tatPLCd PH at 5 mM, the maximal concentration allowing effective cell coupling. To directly assess the efficacy of 5 mM tat PLCd PH, we tested its ability to compete with PLCd PH-GFP FACS-sorted to a concentration of 2 mM. Similar to Fig. 2B, the panel shows patterning data for 5C.C7 T cells expressing PLCd PH-GFP with concurrent PIP2 blockade by T cell pretreatment with 5 mM tat PLCd PH. Given is the percentage of cell couples with standard errors that displayed accumulation of PLCd PH-GFP with the indicated patterns [6] relative to tight cell coupling. Only 14 cell couples could be analyzed. Preincubation of T cells with 5 mM tatPLCd PH made interface PLCd PH-GFP accumulation moderately but signifi- cantly more transient with reduced accumulation at the time of tight cell couple formation (8365% to 50613%, p,0.005) and 80–120 s thereafter (6064% to 4368%, p = 0.05), thus defining the efficacy of tatPLCd PH. (EPS) Figure S3 Manipulation of PIP5K expression affects IL- 2 secretion. To determine whether reduced PIP2 generation would interfere with T cell activation at limiting activation conditions, 5C.C7 T cells were activated by CH27 APCs in the presence of 0.1–10 mM MCC peptide as indicated for 16 h upon knockdown of PIP5K c. Cell culture supernatants were analyzed for IL-2 by ELISA and data are displayed similar to Fig. 3B. Data from 3 independent experiments are given. (EPS) Figure S4 PIP5K overexpression rigidifies T cells. A. 5C.C7 T cells transduced with TCRf-GFP and PIP5K c87 were activated with CH27 APCs and 10 mM MCC agonist peptide. Two interactions of such T cells binding to the same APC at the center of the image with no (top) or a very small (bottom) visible lamellum are shown as in Fig. S1B. The time of tight cell coupling could only be guessed. A movie covering the entire time frame is given as Movie S7. B. To distinguish between effects of PIP5K b and c90 in uropod retraction, we counteracted PIP5K overexpression by parallel expression of PLCd PH-GFP. Only PIP5K b-overexpressing T cells still showed delayed uropod retraction: 5C.C7 T cells transduced with PLCd PH-GFP 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, as indicated. 19–74 cell couples were analyzed per condition. The most severe effect of PIP5K b overexpression on uropod retraction is consistent with its exclusive distal localization (Fig. 1C). C, F, I, K. As T cell spreading (Fig. 4A) is actin-driven, we assessed changes in T cell actin dynamics upon manipulation of PIP2 generation and access. Representative interactions of actin-GFP-transduced 5C.C7 T cells with CH27 APCs in the presence of 10 mM MCC peptide are shown as in Fig. S1B for a control (C), upon overexpression of PIP5K b (F), upon PIP5K c knockdown (I), or upon T cell pretreatment with 5 mM tat PLCd PH (K). Movies covering the entire time frame are given as Movies S8, S9, S10, S11. D, E, G, H, J, L. 5C.C7 T cells were transduced to express actin-GFP and activated with CH27 APCs and 10 mM MCC agonist peptide. The graphs show the percentage of cell couples with standard errors that displayed accumulation of actin-GFP with the indicated patterns [6] relative to tight cell coupling upon manipulation of the expression of PIP5K and PIP2 blockade as indicated. 29–60 cell couples were analyzed per PIP2 Controls T Cell Rigidity and Organization condition. In non-transduced control T cells, actin rapidly and transiently spread to the periphery of the T cell/APC interface (C, D). At the time of tight cell couple formation 6866% of cell couples displayed peripheral actin accumulation (D). The percentage of cell couples with peripheral accumulation rapidly declined to 1365% at 3 min. Overexpression of PIP5K b, c87, or c90 all interfered with actin spreading to the interface periphery (E–H). The frequency of peripheral accumulation was reduced with a concomitant increase in diffuse patterning. For example upon overexpression of PIP5K c90, at the time of tight cell coupling the percentage of cell couples with peripheral actin-GFP accumulation was reduced from 6866% to 3866% (p,0.01), whereas diffuse accumulation was increased from 1665% to 3566% (p = 0.01)(D, G). Such differences were significant (p,0.05) at most time points between tight cell coupling and 2 min thereafter in the comparison of control T cells with T cell overexpressing each of the PIP5K isoforms. Knockdown of PIP5K c promoted peripheral actin accumulation by making it more sustained (I, J). From 1 to 5 min after tight cell coupling the percentage of cell couples with peripheral actin accumulation was significantly (p,0.05) enhanced at each time point in the knockdown T cells compared to control. Upon blocking access to PIP2 with 5 mM tatPLCd PH (K, L), actin dynamics were largely unaltered, consistent with intact T cell spreading (Fig. 4A). (EPS) Figure S5 PIP5K overexpression does not impair ERM expression, T cell coupling, or F-actin amounts. A. To assess whether manipulation of PIP5K expression altered ERM expression, 5C.C7 T cell extracts were blotted for ERM proteins. ERM expression with standard errors upon overexpression/ knockdown of the indicated PIP5K isoforms is given relative to non-transduced control cells. B. T cell spreading is a TCR- dependent process. Therefore, impaired T cell spreading could be the consequence of impaired TCR engagement. The ability of a T cell to form a cell couple upon initial APC contact is the most immediate readout of TCR engagement upon APC contact, occurring in seconds. The percentage of 5C.C7 T cells that form a tight cell couple upon contact with CH27 APCs in the presence of 10 mM MCC peptide is given with standard errors upon overexpression of PIP5K as indicated. 61–101 cell couples were analyzed per condition. An investigation of the modest differences in cell coupling upon PIP5K overexpression is beyond the scope of this study. However, because cell coupling is not reduced, it can be safely concluded that TCR engagement was not impaired. C. T cell rigidification could be caused by altered cellular F-actin amounts. We therefore determined F-actin amounts by phalloidin staining prior and past TCR engagement. T cell activation was mediated by antibodies, as the determination of T cell F-actin amounts in T cell/ APC couples, where both cell types contain F-actin, is challenging. 5C.C7 T cells were activated with a-CD3 and a-CD28 antibodies for 2 min upon manipulation of PIP5K expression and PIP2 blockade as indicated. F-actin contents were determined by FACS analysis of Phalloidin-stained T cells. Mean intensity of Phalloidin staining is displayed with standard errors relative to that of non- transduced, non-stimulated T cells. Data from three independent experiments are given. Consistent with actin-driven T cell spreading upon APC contact, T cell stimulation with a-CD3 and a-CD28 triggered a significant (p,0.001) increase in T cell F-actin contents in control cells by 2363%. PIP5K overexpression and knockdown did not substantially alter T cell F-actin amounts relative to control neither prior nor post T cell stimulation. PIP2 blockade modestly increased F-actin amounts prior to T cell activation (p,0.05 as indicated with an asterisk) but not thereafter. An investigation of this effect is beyond the scope of this study. (EPS) PLoS ONE | www.plosone.org 12 November 2011 | Volume 6 | Issue 11 | e27227