understanding roles of PIP2 in any cell type is to gain comprehensive insight into how PIP2 turnover is regulated by this complex group of pathways. As proteins that generate, metabolize, or function as effectors of PIP2 often display distinct subcellular localization, the complex PIP2 turnover needs to be analyzed with resolution in time and space. In other words, we had to determine where and when PIP2 was synthesized and degraded during T cell activation. As we have already characterized spatiotemporal distributions of PIP2 hydrolysis by PLCc and of PIP2 phosphor- ylation by PIP3K at the T cell/APC interface as part of a larger systems analysis [6], we focus here on the spatiotemporal characteristics of PIP2 generation. Once it was better understood when and where PIP2 was turned over, it was important to elucidate how the spatiotemporal constraints of PIP2 turnover govern PIP2 function. In other words, we had to investigate how manipulation of PIP2 localization affected roles of PIP2 in T cell activation. During T cell activation, PIP2 is rapidly synthesized and hydrolyzed [19,20], even though the spatiotemporal characteristics of PIP2 synthesis are unknown. Cytoskeletal roles of PIP2 in T cell activation are also unresolved. They are likely prominent as PIP2 regulates polarization in many related cell types. In macrophages, PIP2 stabilizes actin at the phagocytic cup [21] and PIP5K c and a are critical in actin-dependent binding and internalization of particles [22]. In neutrophils, PIP5K b and c90 are critical for the turnover of the uropod, a posterior cell extension involved in adhesion in particular during the extravasation of blood cells from the vasculature [23,24,25]. In lymphocyte responses to chemo- kines, hydrolysis of PIP2 is critical in increasing cellular flexibility during extravasation as associated with inhibition of ERM activity [26]. Here we address the two general challenges of PIP2 biology, the balance between roles of PIP2 as a substrate for second messenger generation versus cytoskeletal roles and the complexity of PIP2 turnover with resolution in time and space with its functional consequences, in primary T cells. We found that PIP2 synthesis occurs with distinct spatiotemporal characteristics, as four PIP5K isoforms (abc87, and c90) displayed distinct dynamic localization during T cell activation. Overexpression in particular of the distal PIP5K isoforms, b and c90, yielded a general, yet modest increase in PIP2 levels combined with distinct isoform-specific changes in the spatiotemporal distributions of accessible PIP2. Such overex- pression, as corroborated by knock down and PIP2 blockade, established that PIP2 primarily controls T cell activation using spatially constrained cytoskeletal means, by regulating T cell rigidity and the spatiotemporal organization of T cell signaling with an emphasis on the T cell distal pole. Results Different PIP5Ks are enriched in distinct locations during T cell activation As a critical foundation for the understanding of the complexity of PIP2 turnover in T cells, we determined the spatiotemporal features of PIP2 synthesis. Primary T cells expressed all three PIP5K isoforms, a, b, and c with both the c87 and c90 splice variants of the c isoform (Fig. S1A), as determined by real time PCR. To elucidate the spatiotemporal distributions of PIP5K isoforms during the activation of primary T cells, we used in vitro primed primary T cells from 5C.C7 TCR transgenic mice. The 5C.C7 TCR recognizes peptide 83–102 of moth cytochrome C (MCC) as presented by the MHC II allele I-Ek. We retrovirally transduced T cells to express GFP-tagged PIP5K isoforms. We activated PIP5K expressing T cells with CH27 B cell lymphoma PIP2 Controls T Cell Rigidity and Organization APC incubated with a high concentration (10 mM) of the MCC agonist peptide. These were the default T cell activation conditions for this study. PIP5K c87 was rapidly recruited to the T cell/APC interface such that 1 min after interface formation 7468% of cell couples displayed PIP5K c87 interface accumulation (Figs. 1A, S1B). PIP5K c87 accumulation was transient such that 5 min after tight cell coupling only 27611% cell couples with interface accumu- lation remained (Fig. 1A). Interface accumulation of PIP5K a also occurred preferentially at the cellular interface, however, in a more sustained fashion (Figs. 1B, S1C). In contrast, PIP5K b accumulated almost exclusively at the distal pole (Figs. 1C, S1D). Within the first two minutes after cell coupling the c90 isoform preferentially accumulated at the distal pole (Figs. 1D, S1E). Subsequently however, a substantial portion of PIP5K c90 moved to the T cell/APC interface reaching about 40% cell couples with interface accumulation $5 min after tight cell coupling (Fig. 1D). Interface accumulation of PIP5K c87 without characterization of its spatiotemporal features has been described before [27]. The determination of the differential localization of the four PIP5K isoforms is a central contribution to the understanding of the complex regulation of PIP2 turnover in time and space, in particular in the context of our wider system analysis of T cell signaling [6], as discussed below. It also allowed us to manipulate PIP2 with spatial definition by overexpressing distinct PIP5K isoforms. For such studies we focused on the two distal PIP5K isoforms, c90 and b, as it of interest to understand how regulation of PIP2 levels at the distal pole, away from the location of T cell signaling at the interface, would impact T cell activation. PIP5K overexpression yields increased PIP2 levels and isoform-specific changes in the patterns of accessible PIP2 To address roles of PIP2 in T cell activation under consideration of PIP2 localization, we used primarily PIP5K overexpression (Fig. S2A–C), with knockdown and PIP2 blockade for corroboration. PIP5K overexpression modestly but significantly (p,0.005) increased cellular PIP2 levels by ,20% (Figs. 2A, S2D), as determined by immunostaining for PIP2. These data suggest that PIP2 levels were tightly controlled in T cells, similar to other cell types where PIP5K overexpression or deletion often affects PIP2 levels only modestly [22,28,29,30]. To assess consequences of PIP5K overexpression on PIP2 localization, we used the well-established PIP2-binding PLCd PH domain. As previously described [6], upon tight cell coupling 8365% of cell couples showed PLCd PH-GFP at the T cell/APC interface (Figs. 2B, S2E). Accumulation was transient, disappear- ing entirely at 7 min after tight cell coupling (Fig. 2B). Less than 1/ 3rd of the cell couples also displayed transient distal accumulation of PLCd PH-GFP within the first minute after tight cell coupling (Fig. 2B). Upon overexpression of PIP5K b (Fig. 2C, S2F), interface accumulation of PLCd PH-GFP was almost completely lost, not exceeding 25% of cell couples with such accumulation at any time (p,0.001 versus control between time points - 20 and 120 s). Instead, consistent accumulation of PLCd PH-GFP at the distal pole was observed in about 1/3rd of the cell couples at all time points (p,0.005 versus control at all time points $1 min)(Fig. 2C). This phenotype is consistent with the exclusive distal accumulation of PIP5K b itself (Fig. 1C). Overexpression of PIP5K c90 yielded significantly (p,0.01) increased accumulation of PLCd PH-GFP at the T cell/APC interface $3 min after its formation with as many as .50% of the cell couples showing such accumulation (Fig. 2D, S2G). This enhancement is consistent with translocation PLoS ONE | www.plosone.org 2 November 2011 | Volume 6 | Issue 11 | e27227