PIP2 Controls T Cell Rigidity and Organization Figure 1. Different PIP5K isoforms display distinct spatiotemporal patterns. 5C.C7 T cells were transduced to express GFP-tagged PIP5K isoforms 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 PIP5K c87-GFP (A), a-GFP (B), b-GFP (C), and c90-GFP (D) with the indicated patterns [6] relative to tight cell coupling. Representative images are given in Fig. S1B–E. Representative movies have been published (c87 [6]) or are given as Movies S1, S2, S3. 34–59 cell couples were analyzed per condition. doi:10.1371/journal.pone.0027227.g001 of PIP5K c90 to the interface during this time (Fig. 1D). Interestingly, the early distal presence of PIP5K c90 did not trigger distal accumulation of PLCd PH-GFP, consistent with a lack of activators of PIP5K c90 or rapid turnover of PIP2 at the distal pole at that time. For corroboration we used overexpression of the interface-localized PIP5K c87 isoform. Such overexpression yielded a comparable increase in cellular PIP2 levels without substantial changes in PIP2 localization (Figs. 2A, S2H, S2I). We also reduced PIP2 generation and access with knockdown of the PIP5K c isoforms (Fig. S2J) and PIP2 blockade with the PLCd PH domain as a protein transduction reagent (Fig. S2K). In summary, PIP5K overexpression yielded a modest but significant (p,0.005) increase in PIP2 levels together with isoform- specific changes in PIP2 localization. Isoform-specific effects of PIP5K overexpression thus will identify location-dependent roles of PIP2. Additional roles of PIP5K isoforms, such as in scaffolding as recently described for PIP5K a [31], cannot be ruled out. PIP5K overexpression impairs IL-2 secretion and proximal T cell signaling IL-2 secretion is a key outcome of T cell activation. We therefore determined its dependence on PIP2. Overexpression of the distal PIP5K isoforms c90 or b reduced IL-2 secretion to 4464% and 3063% of IL-2 secretion of control T cells, respectively (Fig. 3A, B)(p,0.005). In contrast, knockdown of PIP5K c and 5 mM tatPLCd PH modestly but significantly (p,0.05) enhanced IL-2 secretion by 2669% (1862% in a second set of experiments, Fig. S3) and 29610%, respectively. Corrob- orating these data, complete loss of PIP5K c90 leads to an increase in IL-2 secretion by about 40% [27]. The comparatively modest size of this enhancement is discussed below. Changes in IL-2 secretion upon overexpression of the interface-associated PIP5K isoform c87 were not significant (Fig. 3B). These data are significant, first, as they establish PIP2, in particular distal PIP2, as a substantial regulator of T cell activation. Second, they are critical in understanding a potential role of PIP2 as second messenger precursor. If PIP2 as a substrate for PLCc was central for mediating effects of PIP2 turnover on T cell activation, mass action dictates that increased PIP2 generation should yield increased second messenger levels and thus increased amounts of IL-2. As our findings run contrary to this expectation, roles of PIP2 as a second messenger substrate were surprisingly minor. To assess whether PIP2 could function within minutes of T cell coupling to APCs, the time of drastic cytoskeletal rearrangements, of maximal biochemical signaling activity, and of the translocation of transcription factors to the nucleus [6], we addressed phosphorylation of linker of activated T cells (LAT) and phospholipase Cc (PLCc). Due to limiting cell numbers, the determination of tyrosine phosphorylation of LAT (Y191) and PLCc (Y783) in T cell/APC extracts was restricted to overex- pression of PIP5K c90 and PIP5K c87 as a non-distal control. LAT and PLCc phosphorylation were significantly decreased (p,0.05) upon overexpression of PIP5K c90 by 4666% and 3966% (Fig. 3C, D). In contrast, LAT phosphorylation was enhanced by 110640% upon knockdown of PIP5K c (p,0.05). Changes upon overexpression of PIP5K c87 were not significant (Fig. 3D). Expression of total LAT and PLCc has previously been shown to not vary during early primary T cell activation [32]. Importantly, effects of PIP2 manipulation on proximal signaling and IL-2 secretion were extensively matched, consistent with the suggestion that reduced efficiency of proximal T cell signaling was a key contributor to decreased IL-2 secretion. PIP2 regulates T cell spreading and uropod retraction upon APC contact As the data on IL-2 secretion suggest that changes in the amounts and/or localization of PIP2 do not substantially affect T cell activation through a role of PIP2 as a second messenger substrate, we next addressed cytoskeletal roles of PIP2. We started with the control of cellular rigidity, as this is a common function of PIP2 [15]. Overexpression of PIP5K b, c87, or c90 yielded smaller initial T cell/APC interface diameters. To account for variable T cell size, we determined the interface diameter relative to the diameter of the T cell body. In control T cells at the time of tight cell coupling, the interface diameter was 1.0260.03 times that of the T cell body (Fig. 4A). It was significantly reduced to 0.8460.02 to 0.9060.03 times the diameter of the cell body upon overexpression of each of the PIP5K isoforms (p,0.01 versus control, no significant differences between PIP5K isoforms), indicative of increased T cell rigidity. Impaired T cell spreading became even more evident in experiments to assess TCR clustering at the T cell/APC interface. In these experiments the majority of productive T cell/APC couples displayed accumulation of the TCR at the center of the T cell/APC interface, as discussed in detail below. In control T cells, such accumulation was invariably preceded by spreading of a clearly visible wide lamellum against the APC [6]. However, upon overexpression of PIP5K isoforms, we observed TCR clustering without a visible lamellum and the formation of a wide T cell/ APC interface (Fig. S4A) in a substantial portion of the cell couples. As we could not determine the precise time of cell coupling and its associated interface diameter in these cell couples any more, we used an alternate measurement: When we analyzed all T cell/APC couples with eventual interface accumulation of the TCR, upon overexpression of PIP5K b, c87, or c90, 2469% to 4168% of the cell couples did not display a lamellum upon cell coupling (p,0.001 versus control, no significant differences between PIP5K isoforms)(Figs. 4B, S4A), indicative of most severely increased T cell rigidity. As a second cell shape change upon APC coupling, the T cell retracts its uropod. Overexpression of PIP5K b or c90 specifically delayed uropod retraction (Figs. 4C, S4B), another indication of increased T cell rigidity, this time focused on the T cell distal pole. In control cells at 2 min after tight cell coupling, only 963% of cell couples still displayed a visible uropod. This percentage was significantly (p,0.001) increased upon overexpression of PIP5K b or c90 to 6569% and 5069%. In summary, overexpression of PIP5K isoforms substantially interfered with T cell shape changes upon APC contact, both at the interface and at the distal pole, indicative of increased T cell rigidity. PIP5K c knockdown or PIP2 blockade altered neither the interface diameter at T cell/APC coupling nor uropod retraction. Effects of PIP5K gamma knockdown of PIP2 levels were limited (Fig. S2J) and blockade of PIP2 by the PLCd PH domain was PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27227