PIP2 Controls T Cell Rigidity and Organization Figure 2. PIP5K overexpression alters PIP2 levels and localization. A. PIP2 levels were determined by immunofluorescence staining of 5C.C7 T cells transduced with different GFP-tagged PIP5K isoforms as indicated. PIP2 levels of T cells with PIP5K overexpression (grey bars) and control (black bars) are normalized to control and are given with standard errors for two independent experiments each. An asterisks indicates statistical significance versus control with p,0.005. 100–200 cells were analyzed per experiment. A representative PIP2 staining experiment is shown as Fig. S2D. B. 5C.C7 T cells were transduced to express PLCd PH-GFP and activated with CH27 APCs and 10 mM MCC agonist peptide. The graph shows 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. 60 cell couples were analyzed. C, D. Similar to B, the panels show patterning data for 5C.C7 T cells expressing PLCd PH-GFP with concurrent overexpression of PIP5K b (C) or c90 (D). 41, 26 cell couples were analyzed per condition. Representative images for panels B–D are given in Fig. S2E– G. Representative movies have been published (c87 [6]) or are given as Movies S4, S5, S6. doi:10.1371/journal.pone.0027227.g002 partial (Fig. S2K), providing potential technical explanations for the more limited nature of these effects. A possible biological explanation is that T cell flexibility might have already been maximized upon T cell activation with a strong stimulus, thus preventing further enhancement upon interference with PIP2 generation or access. Our data on T cell rigidity are corroborated by data on actin spreading to the edge of the T cell/APC interface (Fig. S4C–L). Importantly, effects of PIP2 on T cell rigidity and IL- 2 matched. Increased T cell rigidity should interfere with T cell activation by impairing tight APC coupling and thus extensive receptor ligand engagement. Accordingly, increased PIP2 gener- ation in parallel made T cells more rigid and interfered with IL-2 secretion. Interestingly, local features of PIP2 turnover contributed substantially, as effects of overexpression of distal PIP5K isoforms were more dramatic both in the control of T cell rigidity, involving uropod retraction, and IL-2 secretion. PIP5K overexpression impairs ERM protein dephosphorylation upon T cell activation T cell rigidification could be caused by altered ERM protein function, as ERM proteins link the plasma membrane to the cortical actin cytoskeleton in a PIP2-dependent fashion. ERM protein activity requires threonine phosphorylation at residues T567 of Ezrin and T558 of Moesin [33,34,35,36]. We therefore determined ERM threonine phosphorylation during T cell activation. TCR engage- ment was provided by a-CD3 plus a-CD28 antibodies, as the determination of T cell ERM phosphorylation in T cell/APC couples, where both cell types contain ERM proteins, is challenging. Due to limiting cell numbers, these experiments were restricted to overexpression of PIP5K c90 and PIP5K c87 as a non-distal control. In non-transduced T cells, ERM phosphorylation dropped signifi- cantly (p,0.001) to 3569% of the level of non-stimulated T cells within 2 min of tight cell coupling (Fig. 5). This reduction is indicative of the increased T cell flexibility required to execute the T cell shape changes associated with T cell activation. However, upon overex- pression of PIP5K c90, ERM phosphorylation dropped significantly less (c90)(p#0.01) to only 6665% of the level of non-stimulated T cells (Fig. 5). Overall ERM expression was not altered by PIP5K overexpression (Fig. S5A). We thus suggest as a mechanism of PIP5K-dependent T cell rigidification that elevated PIP2 generation inhibits ERM inactivation upon T cell activation. Upon knockdown of PIP5K c, T cell stimulation triggered slightly enhanced reduction Figure 3. Manipulation of PIP5K expression affects IL-2 secretion and proximal signal transduction. A, B. 5C.C7 T cells were activated by CH27 APCs and 10 mM MCC agonist peptide for 16 h upon manipulation of PIP5K expression and PIP2 blockade as indicated. Cell culture supernatants were analyzed for IL-2 by ELISA. A representative IL-2 ELISA is given in (A). Changes in IL-2 secretion upon manipulation of PIP5K expression and PIP2 blockade relative to non-transduced T cells are given with standard errors in (B) on a logarithmic scale to comparably display reduction and enhancement. ‘GFP’ indicates retroviral expression of GFP as a control. One/two asterisks indicate significance versus non-transduced control with p,0.05/0.005, respectively. Data from 3–6 independent experiments are given. When the agonist peptide concentration during T cell activation was reduced to a limiting concentration, 0.1 mM, knockdown of PIP5K c still did not result in impaired IL-2 secretion (Fig. S3). C, D. 5C.C7 T cells were activated by CH27 APCs and 10 mM MCC agonist peptide for 2 min upon manipulation of PIP5K expression as indicated. T cell/APC extracts were blotted for LAT Y191 and PLCc Y783. A representative blot is given in (C). Changes in LAT and PLCc phosphorylation upon manipulation of PIP5K expression relative to non-transduced T cells are given with standard errors in (D) on a logarithmic scale to comparably display reduction and enhancement. Data from 3–4 independent experiments are given. doi:10.1371/journal.pone.0027227.g003 PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27227