Figure S6 PIP2 manipulation interferes with accumula- tion of the TCR at the center of the T cell/APC interface. A. A representative interaction of a TCRf-GFP-transduced 5C.C7 T cell with a CH27 APC in the presence of 10 mM MCC peptide is shown at the indicated time points (in minutes) relative to the time of tight cell coupling as in Fig. S1B. A movie covering the entire time frame is given as Movie S12. B–I. Representative images and patterning data for 5C.C7 T cells expressing TCRf-GFP upon concurrent overexpression of PIP5K c90 (B), b (C), c87 (D, E), a knockdown cassette for PIP5K c (F, G), or T cell treatment with 5 mM tat PLCd PH (H, I) are displayed as in Fig. S1B (images) or Fig. 6A (patterning data). Movies covering the entire time frame are given as Movies S13, S14, S15, S16, S17. 32–56 cell couples were analyzed per condition. (EPS) Table S1 PIP2 controls T cell activation by regulating T cell rigidity and spatiotemporal organization. (DOC) Movie S1 A representative interaction of sensor-transduced 5C.C7 T cell with a CH27 B cell lymphoma APC in the presence of 10 mM MCC agonist peptide is shown in this and all subsequent movies. Movies were acquired using time-lapse epifluorescence microscopy. Differential interference contrast (DIC) images are shown on top, with matching top-down, maximum projections of 3- dimensional sensor fluorescence data on the bottom. The sensor fluorescence intensity is displayed in a rainbow-like false-color scale (increasing from blue to red). 20 s intervals in movie acquisition are played back as 2 frames per second. The individual captions list for each movie the figure it refers to, the sensor and any non-fluorescent additional proteins expressed by the 5C.C7 T cell, the time of T cell/APC couple formation given both as the movie frame number and the displayed movie time (in parentheses), and noteworthy features of the movie. Movie S1 refers to Fig. S1C, the 5C.C7 T cells are transduced with PIP5K a-GFP, cell coupling occurs in frame 3 (1s), and prominent are a large lamellum upon cell coupling and transient interface accumulation of PIP5K a-GFP. (MOV) Movie S2 Refers to Fig. S1D, the 5C.C7 T cells are transduced with PIP5K b-GFP, cell coupling occurs in frame 7 (3s), and prominent are a small lamellum upon cell coupling and sustained distal accumulation of PIP5K b-GFP. (MOV) Movie S3 Refers to Fig. S1E, the 5C.C7 T cells are transduced with PIP5K c90-GFP, cell coupling occurs in frame 5 (2s), and prominent are a small lamellum upon cell coupling and initial distal accumulation of PIP5K c90-GFP followed by late interface accumulation. (MOV) Movie S4 Refers to Fig. S2H, the 5C.C7 T cells are transduced with PLCd PH-GFP and PIP5K c87, cell coupling occurs in frame 9 (4s), and prominent is transient lamellal accumulation of PLCd PH-GFP. The T cell transits to an adjacent APC during the movie. Only data from the 1st T cell/APC interaction are included in the analysis. (MOV) Movie S5 Refers to Fig. S2G, the 5C.C7 T cells are transduced with PLCd PH-GFP and PIP5K c90, cell coupling occurs in frame 5 (2s), and prominent is transient interface accumulation of PLCd PH-GFP. It is better sustained than that in the control T cell/APC interactions. (MOV) PIP2 Controls T Cell Rigidity and Organization Movie S6 Refers to Fig. S2F, the 5C.C7 T cells are transduced with PLCd PH-GFP and PIP5K b, cell coupling occurs in frame 2 (0s), and prominent is sustained distal accumulation of PLCd PH- GFP. (MOV) Movie S7 Contains two T cell/APC interactions, as indicated by the positions of the respective T cells relative to the APC. Movie S7 refers to Fig. S4A, the 5C.C7 T cells are transduced with TCRj-GFP and PIP5K c87, cell coupling cannot be timed precisely, and prominent is that in particular for the T cell on top a lamellum mediating cell coupling cannot be detected. (MOV) Movie S8 Refers to Fig. S4C, the 5C.C7 T cells are transduced with actin-GFP, cell coupling occurs in frame 2 (0s), and prominent is initial peripheral actin-GFP interface accumulation followed by variable patterns with diminishing intensity. (MOV) Movie S9 Refers to Fig. S4F, the 5C.C7 T cells are transduced with actin-GFP and PIP5K b, cell coupling occurs in frame 6 (2s), and prominent is transient diffuse actin-GFP interface accumula- tion. (MOV) Movie S10 Refers to Fig. S4I, the 5C.C7 T cells are transduced with actin-GFP and sh PIP5K c, cell coupling occurs in frame 4 (1s), and prominent is early peripheral actin-GFP interface accumulation that is sustained in various patterns. (MOV) Movie S11 Refers to Fig. S4K, the 5C.C7 T cells are transduced with actin-GFP and treated with 5 mM tat PLCd PH, cell coupling occurs in frame 3 (1s), and prominent is transient lamellal and diffuse actin-GFP interface accumulation. (MOV) Movie S12 Refers to Fig. S6A, the 5C.C7 T cells are transduced with TCRj-GFP, cell coupling occurs in frame 7 (3s), and prominent is rapid and sustained TCRj-GFP accumulation at the center of the T cell/APC interface. (MOV) Movie S13 Refers to Fig. S6B, the 5C.C7 T cells are transduced with TCRj-GFP and PIP5K c90, cell coupling occurs in frame 6 (2s), and prominent is initial distal with delayed central TCRj- GFP accumulation. (MOV) Movie S14 Refers to Fig. S6C, the 5C.C7 T cells are transduced with TCRj-GFP and PIP5K b, cell coupling occurs in frame 5 (2s), and prominent are delayed weak central TCRj- GFP interface accumulation and subsequent removal of TCRf- GFP from the interface with accumulation in internal structures, likely vesicles. (MOV) Movie S15 Refers to Fig. S6D, the 5C.C7 T cells are transduced with TCRj-GFP and PIP5K c87, cell coupling occurs in frame 4 (1s), and prominent is delayed accumulation of TCRj-GFP at the center of the T cell/APC interface. (MOV) Movie S16 Refers to Fig. S6F, the 5C.C7 T cells are transduced with TCRj-GFP and sh PIP5K c, cell coupling occurs in frame 3 (1s), and prominent is transient accumulation of TCRj-GFP at the center of the T cell/APC interface. (MOV) PLoS ONE | www.plosone.org 13 November 2011 | Volume 6 | Issue 11 | e27227