PIP2 Controls T Cell Rigidity and Organization Figure 6. PIP5K overexpression interferes with accumulation of the TCR at the center of the T cell/APC interface. A. 5C.C7 T cells were transduced to express TCRf-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 TCRf-GFP with the indicated patterns [6] relative to tight cell coupling. 45 cell couples were analyzed. B, C. Patterning data for 5C.C7 T cells expressing TCRf-GFP upon concurrent overexpression of PIP5K c90 (B) or b (C) are displayed as in (A). 36, 25 cell couples were analyzed per condition. Representative images for all three panels are given in Fig. S6A–C. Representative movies are given as Movies S12, S13, S14. doi:10.1371/journal.pone.0027227.g006 Discussion Here we have addressed two principal challenges in PIP2 biology in the activation of primary T cells. First, we found that changes in PIP2 levels affect T cell activation primarily through regulation of T cell rigidity and spatiotemporal organization, not through the role of PIP2 as a substrate in second messenger generation. Increased amounts of PIP2 did not yield increased IL-2 secretion, as to be expected for a central role of PIP2 as the substrate of PLCc, but they resulted in substantially less IL-2 secretion. This decrease matched a series of data generated here, as summarized in Table S1. Increased PIP2 yielded a more rigid T cell, a T cell with delayed and impaired clustering of the TCR at the center of the T cell/APC interface, and impaired proximal T cell signaling. Importantly, these observations have previously been linked using the same experimental system. In the activation of 5C.C7 T cells by APC plus peptide, TCR clustering is dependent on intact actin dynamics that also regulate cell shape [37,38] and central TCR clustering is related to efficient proximal signaling [6,7]. In addition, the effects of altered PIP2 generation were comparably spatially constrained. Increased generation of PIP2 at the T cell distal pole as opposed to at the T cell/APC interface consistently had the strongest effects. While all PIP5K isoforms controlled T cell rigidity at the interface, consistent with their shared role in regulating cellular PIP2 levels, only the distal isoforms also rigidified the distal pole. Initial recruitment of the TCR to distal pole, its release and its central accumulation were all more dependent on the distal PIP5K isoforms, as were proximal signaling and IL-2 secretion. The shared spatial constraint further strengthens the functional connections between T cell rigidity, organization, and function. Significantly, it links spatial features of PIP2 turnover to those of PIP2 function. Our data thus establish that PIP2 regulates T cell activation primarily through spatially constrained control of T cell rigidity and spatiotemporal organization, thus forming a first critical foundation for future more detailed work on roles of PIP2 in T cell activation. As a second general challenge, we have determined the spatiotemporal features of PIP2 synthesis as a critical part of a more comprehensive understanding of PIP2 turnover as resolved in time and space. Different PIP5K isoforms were enriched at the T cell/APC interface or the T cell distal pole with distinct local preferences and dynamics. As part of a larger analysis of the spatiotemporal organization of T cell signaling [6], the following picture emerges: The T cell/APC interface likely is the site of most intense PIP2 turnover. Not only are the two most abundant PIP5K isoforms (c87 and a) enriched there, but also two key enzymes in PIP2 metabolism, PLCc and PI3K. Based on the observed distributions of free PIP2, PIP2 synthesis dominated during the first minute of cell coupling, PIP2 metabolism thereafter. The distal pole in contrast is likely a site of slower PIP2 turnover. It displayed enrichment of only the less abundant PIP5K isoforms and none of PIP2 metabolizing enzymes. Nevertheless, initial PIP2 metabolism still seems active, as accumulation of accessible PIP2 after T cell/ APC coupling was only transient (Fig. 2B). However at later time points, overexpression of the distal PIP5K b isoform yielded stable accumulation of free PIP2 at the distal pole (Fig. 2C), indicative of slowed PIP2 turnover and something not seen at all at the T cell/ APC interface upon overexpression of interface-associated PIP5K isoforms. Slower turnover of distal PIP2 at later time points is consistent with the stable accumulation of the PIP2-binding ERM at the distal pole, as opposed to no or only transient accumulation at the T cell/APC interface [35]. This initial characterization of PIP2 turnover as resolved in time and space forms a second critical foundation for future more detailed work on roles of PIP2 in T cell activation. Materials and Methods Ethics Statement All mouse studies have been approved by the UT Southwestern Medical Center Institutional Animal Care and Use Committee under protocol 2010-0224 and were executed in accordance with the USDA Animal Welfare Act. Cells and reagents In vitro primed, primary T cells from 5C.C7 TCR transgenic mice were generated as described [37]. CH27 cells were used as APCs [37]. PIP5K-GFP fusion proteins were generated as fusions of GFP to the N-terminus of PIP5K. Actin-GFP, TCRf-GFP, PLCd PH-GFP have been described [6]. Retroviral transduction was performed as described [37]. T cells that retrovirally express fluorescent sensor proteins were FACS sorted for a consistent and low expression level. Through quantitative immnuoblotting with anti-GFP antibodies of cell extracts from the sorted T cells against dose responses of pure GFP, this sensor expression level was determined to be 5 mM [6]. For shRNA-mediated knockdown of PIP5K c, a RNA polymerase II-driven cassette for parallel expression of an shRNA hairpin and a marker protein [39] was cloned into the MMLV-based retroviral vector. For retroviral expression of PIP5K together with imaging sensors from the same mRNA, sensor translation was initiated on an EMCV-based internal ribosomal entry site. The following antibodies were used: antibodies against phospho-LAT Y191, phospho-PLCc Y783, Ezrin/Radixin/Moesin, phospho-Ezrin T567/Radixin T564/ Moesin T558 (Cell Signaling, Danvers, MA), PIP5K c (Epitomics, Burlingame, CA) antibodies against CD3 (2C11) and CD28 (BD Pharmingen), and polyclonal rabbit anti-PIP5K c90 [40] and mouse anti-PIP2 sera (a kind gift from K. Fukami, U. Tokyo). Alexa Fluor 594 phalloidin was from Molecular Probes (Eugene, OR). A protein transduction version of the PLCd PH domain was generated, purified under native conditions from E. coli by immobilized metal affinity chromatography, and applied to T cells in strict analogy to [41]. Image acquisition and image analysis Image acquisition and analysis were performed as described in great detail [6]. Briefly, T cell-APC interactions were imaged at 37uC. Every 20 seconds, 1 DIC and 21 fluorescence images that spanned 20 mm in the z-plane at 1 mm intervals were acquired. The acquisition and analysis software was Metamorph (Molecular Devices). The formation of a tight cell couple, time 0 in our analysis, was defined as either the first time point with a fully spread T cell-APC interface or 40 s after first membrane contact, PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27227