whichever occurred first. A region of sensor accumulation was defined by an average fluorescence intensity of .135% of the background cellular fluorescence. To classify spatial accumulation features, six mutually exclusive interface patterns were used: central, invagination, diffuse, lamellal, asymmetric and peripheral, as defined by strict geometrical constraints (Table 2 and actin data in Figure S12 in [6]). Distal accumulation was scored indepen- dently. A T cell was scored to have a uropod as long as an inversion of curvature of the plasma membrane could be detected at the distal pole in the DIC images. To ensure the reliability of this analysis, data were routinely analyzed by two investigators independently. PIP2 staining 5C.C7 T cells expressing different GFP-tagged PIP5K isoforms were FACS sorted for matching numbers of GFP positive and negative T cells, using the same sort windows as in all other experiments. Both populations were mixed, adhered to a poly d- lysine-coated cover slip, and stained for PIP2 as established [28]. Briefly, cells were fixed in 4% Paraformaldehyde, permeabilized with 0.5% Saponin, and stained with an anti-PIP2 antiserum and an anti-mouse antibody conjugated to Alexa 568. Signal was .20- fold above non-specific staining background. Three-dimensional images of the stained cells were acquired and 100–200 cells per experiment were analyzed for PIP2 staining intensity, separately in the same field for GFP positive and negative cells: T cells were identified by intensity thresholding and the integrated Alexa 568 fluorescence was measured as a readout of total cellular PIP2 amounts. Biochemical and functional assays To determine PIPI5K mRNA levels, cDNA from primary 5C.C7 T cells was analyzed by real time PCR using SYBR Green labeling and the Applied Biosystems 7300 real time PCR system with b2-microglobulin as the quantification standard. cDNA was prepared using the RNA STAT-60 reagent (Tel Test, Inc.) and SuperScript reverse transcriptase (Invitrogen) according to man- ufacturer’s instructions. Phosphorylation of LAT and PLCc was determined by Western blotting of cell extracts from T cell/APC couples, as described [6]. The phosphorylation of ERM proteins was similarly determined after T cell stimulation for 2 min with 10 mg/ml a-CD3 and a-CD28 and secondary antibody cross- linking. ERM and PIP5K c expression were determined similarly in cell extracts from non-stimulated cells. Staining with Alexa 594 phalloidin was performed according to the instructions provided by the manufacturer. IL-2 was measured in T cell/APC culture supernatants after 16 h of cell contact using the OptEIA kit from DB Biosciences according to the instructions provided by the manufacturer as scaled down to as few as 10,000 sorted T cells. Supporting Information Figure S1 Different PIP5K isoforms display distinct spatiotemporal patterns. A. The mRNA abundance of different PIP5K isoforms in 5C.C7 T cells was determined by real time PCR with b2 microglobulin mRNA as a standard and is given relative to the amount of b2 microglobulin mRNA with standard errors as indicated. Averages of 5–6 independent experiments are given. PIP5K b mRNA could be detected only once. B–E. Representative interactions of 5C.C7 T cells transduced with PIP5K c87-GFP (B), a-GFP (C), b-GFP (D), and c90-GFP (E) with CH27 APCs in the presence of 10 mM MCC peptide are shown at the indicated time points (in minutes) relative to the time of tight cell coupling. Differential interference PIP2 Controls T Cell Rigidity and Organization contrast (DIC) images are shown on top, with top-down, maximum projections of 3-dimensional GFP fluorescence data at the bottom. GFP fluorescence intensity is displayed in a rainbow- like false-color scale (increasing from blue to red). Movies covering the entire time frames are in [6](for PIP5K c87) and as Movies S1, S2, S3. (EPS) Figure S2 PIP5K overexpression alters PIP2 levels and localization. A, B. To determine spatiotemporal distributions of PIP5K-GFP isoforms, 5C.C7 T cells transduced to express a PIP5K-GFP isoform were FACS-sorted into a 5-fold range of expression centered at 360.6 mM that is minimally required for detection by fluorescence microscopy [6]. (A) To determine such PIP5K-GFP expression relative to endogenous PIP5K, cell extracts from 5C.C7 T cells expressing GFP fusions with PIP5K c 87 or c 90 and from matching numbers of non-transduced control cells were blotted for both PIP5K c isoforms (on top) or for PIP5K c90 only (on the bottom). Representative blots are shown. A suitable antibody against the b isoform is not available. (B) Based on previous calibration of GFP fluorescence intensity as a function of GFP expression [6](experimental procedures) endogenous PIP5K c expression was calculated using the ratio of band intensities for PIP5K c-GFP and PIP5K c and is given with standard errors based on at least 2 independent experiments. C. In many experiments throughout this manuscript non-fluorescent PIP5K isoforms were overexpressed alongside imaging sensors using the same retroviral vector backbone used for the expression of the GFP-tagged PIP5K isoforms only. T cells expressing non- fluorescent PIP5K and a sensor where then sorted for low sensor expression, as established [6]. This resulted in expression levels of overexpressed non-fluorescent PIP5K similar to that of PIP5K- GFP: Cell extracts from 5C.C7 T cells expressing PIP5K c87-GFP or PIP5K c87 together with TCRf-GFP and from matching numbers of non-transduced control cells were blotted for both PIP5K c isoforms. One representative blot is shown. D. A representative PIP2 staining experiment is shown. FACS-sorted T cells expressing PIP5K b-GFP and non-transduced control cells were mixed in equal numbers, fixed, and stained with anti-PIP2 antiserum followed by an Alexa 568-conjugated secondary antibody. A series of matching bright field, GFP, and Alexa 568 images is shown. E. A representative interaction of a PLCd PH- GFP-transduced 5C.C7 T cell with a CH27 APC in the presence of 10 mM MCC peptide is shown as in Fig. S1B. A movie covering the entire time frame is in [6]. F–H. Similar to E, the panels show representative interactions for 5C.C7 T cells expressing PLCd PH- GFP with concurrent overexpression of PIP5K b (F), PIP5K c90 (G), or PIP5K c87 (H). Movies covering the entire time frame are given as Movies S4, S5, S6. I. Similar to Fig. 2B, the panel shows patterning data for 5C.C7 T cells expressing PLCd PH-GFP with concurrent overexpression of PIP5K c87. Given is the percentage accumulation of PLCd PH-GFP with standard errors with the indicated patterns [6] relative to tight cell coupling. 29 cell couples were analyzed. J. For shRNA-mediated knockdown of the PIP5K c isoforms, we used a retrovirally-expressed hairpin that targets both the c87 and the c90 isoforms. shRNA-mediated knockdown reduced PIP5K c90 expression by 56621% (p = 0.05). A representative Western blot is shown. Knockdown of combined PIP5K c87/c90 was less, not reaching significance any more. PIP2 levels as determined by immunofluorescence similar to Fig. 2A were reduced by about 5% (not reaching statistical significance), consistent with tight regulation of PIP2 levels. K. To block PIP2, PLCd PH was employed as an E. coli expressed protein transduction reagent (‘tatPLCd PH’). Effects of tatPLCd PH on T cell activation were highly dose-dependent, consistent with tight PLoS ONE | www.plosone.org 11 November 2011 | Volume 6 | Issue 11 | e27227