Phosphatidylinositol (4,5) Bisphosphate Controls T Cell Activation by Regulating T Cell Rigidity and Organization

Here we investigate the role of Phosphatidylinositol (4,5) bisphosphate (PIP2) in the physiological activation of primary murine T cells by antigen presenting cells (APC) by addressing two principal challenges in PIP2 biology. First, PIP2 is a regulator of cytoskeletal dynamics and a substrate for second messenger generation. The relative importance of these two processes needs to be determined. Second, PIP2 is turned over by multiple biosynthetic and metabolizing enzymes. The joint effect of these enzymes on PIP2 distributions needs to be determined with resolution in time and space. We found that T cells express four isoforms of the principal PIP2-generating enzyme phosphatidylinositol 4-phosphate 5-kinase (PIP5K) with distinct spatial and temporal characteristics. In the context of a larger systems analysis of T cell signaling, these data identify the T cell/APC interface and the T cell distal pole as sites of differential PIP2 turnover. Overexpression of different PIP5K isoforms, as corroborated by knock down and PIP2 blockade, yielded an increase in PIP2 levels combined with isoform-specific changes in the spatiotemporal distributions of accessible PIP2. It rigidified the T cell, likely by impairing the inactivation of Ezrin Moesin Radixin, delayed and diminished the clustering of the T cell receptor at the cellular interface, reduced the efficiency of T cell proximal signaling and IL-2 secretion. These effects were consistently more severe for distal PIP5K isoforms. Thus spatially constrained cytoskeletal roles of PIP2 in the control of T cell rigidity and spatiotemporal organization dominate the effects of PIP2 on T cell activation.


Introduction
Here we address roles of Phosphatidylinositol (4,5) bisphosphate (PIP 2 ) in T cell activation. Physiological T cell activation occurs in the cellular interaction between a T cell and an antigen presenting cell (APC). T cells polarize upon APC contact as driven by the cytoskeleton [1,2,3], yielding a complex organization of T cell signaling in dynamic and diverse spatiotemporal patterns [4,5,6]. Prominent is the sustained accumulation of the T cell receptor (TCR) at the center of the T cell/APC interface [5], an accumulation pattern that can be associated with efficient T cell activation [6,7]. A critical outcome of T cell activation is cytokine secretion, prominently that of the autocrine growth factor IL-2. PIP 2 is a central substrate for second messenger generation and a well-established regulator of cytoskeletal dynamics in many cell types [8,9]. Hydrolysis of PIP 2 by phospholipase Cc (PLCc) yields diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP 3 ) [10], two signaling intermediates critical for the induction of T cell IL-2 secretion. In cytoskeletal regulation, PIP 2 controls cytoskeletonplasma membrane adhesion, the activity of actin severing proteins, and assembly of endocytic vesicles [9,11,12]. Ezrin Radixin Moesin (ERM) proteins are a critical mediator of PIP 2 function in the regulation of cytoskeleton-plasma membrane adhesion, as binding of ERM to PIP 2 in the plasma membrane activates them to strengthen the association of the plasma membrane with the underlying cortical actin cytoskeleton [13,14]. A first general challenge in understanding the function of PIP 2 in any cell type is to determine whether the role of PIP 2 as a substrate for second messenger generation or cytoskeletal roles dominate the effects of PIP 2 on cellular activation. In other words, we had to investigate whether changes in PIP 2 levels primarily affected T cell activation through altered second messenger generation or through altered cytoskeletal dynamics. PIP 2 is turned over rapidly. The principal biosynthetic pathway of PIP 2 involves phosphorylation of phosphatidylinositol 4phosphate by the type I phosphatidylinositol 4-phosphate 5kinases (PIP5K) [15]. There are three PIP5K isoforms, a, b, and c The nomenclature for the a and b isoforms is switched between humans and mice. We use the more widely employed human nomenclature. The c isoform has multiple splice variants. The predominant isoforms are PIP5K c87 (also called c635) and c90 (c661) with the isoforms being denoted by their molecular weight (87 or 90 kDa) or the number of amino acids (635 or 661). PIP 2 is metabolized through hydrolysis by PLCc or phosphorylation by phosphatidylinositol 3-kinase (PI3K). Additionally, PIP 2 synthesized locally will be dissipated by diffusion [16] unless captured by scaffolding molecules [17]. PIP 2 is also dephosphorylated by phosphatidylinositol phosphatases [18]. A second key challenge in understanding roles of PIP 2 in any cell type is to gain comprehensive insight into how PIP 2 turnover is regulated by this complex group of pathways. As proteins that generate, metabolize, or function as effectors of PIP 2 often display distinct subcellular localization, the complex PIP 2 turnover needs to be analyzed with resolution in time and space. In other words, we had to determine where and when PIP 2 was synthesized and degraded during T cell activation. As we have already characterized spatiotemporal distributions of PIP 2 hydrolysis by PLCc and of PIP 2 phosphorylation by PIP3K at the T cell/APC interface as part of a larger systems analysis [6], we focus here on the spatiotemporal characteristics of PIP 2 generation. Once it was better understood when and where PIP 2 was turned over, it was important to elucidate how the spatiotemporal constraints of PIP 2 turnover govern PIP 2 function. In other words, we had to investigate how manipulation of PIP 2 localization affected roles of PIP 2 in T cell activation.
During T cell activation, PIP 2 is rapidly synthesized and hydrolyzed [19,20], even though the spatiotemporal characteristics of PIP 2 synthesis are unknown. Cytoskeletal roles of PIP 2 in T cell activation are also unresolved. They are likely prominent as PIP 2 regulates polarization in many related cell types. In macrophages, PIP 2 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 chemokines, hydrolysis of PIP 2 is critical in increasing cellular flexibility during extravasation as associated with inhibition of ERM activity [26].
Here we address the two general challenges of PIP 2 biology, the balance between roles of PIP 2 as a substrate for second messenger generation versus cytoskeletal roles and the complexity of PIP 2 turnover with resolution in time and space with its functional consequences, in primary T cells. We found that PIP 2 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 PIP 2 levels combined with distinct isoform-specific changes in the spatiotemporal distributions of accessible PIP 2 . Such overexpression, as corroborated by knock down and PIP 2 blockade, established that PIP 2 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.

Different PIP5Ks are enriched in distinct locations during T cell activation
As a critical foundation for the understanding of the complexity of PIP 2 turnover in T cells, we determined the spatiotemporal features of PIP 2 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-E k . We retrovirally transduced T cells to express GFP-tagged PIP5K isoforms. We activated PIP5K expressing T cells with CH27 B cell lymphoma 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 accumulation 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 PIP 2 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 PIP 2 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 PIP 2 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 PIP 2 levels and isoform-specific changes in the patterns of accessible PIP 2 To address roles of PIP 2 in T cell activation under consideration of PIP 2 localization, we used primarily PIP5K overexpression ( Fig.  S2A-C), with knockdown and PIP 2 blockade for corroboration. PIP5K overexpression modestly but significantly (p,0.005) increased cellular PIP 2 levels by ,20% (Figs. 2A, S2D), as determined by immunostaining for PIP 2 . These data suggest that PIP 2 levels were tightly controlled in T cells, similar to other cell types where PIP5K overexpression or deletion often affects PIP 2 levels only modestly [22,28,29,30].
To assess consequences of PIP5K overexpression on PIP 2 localization, we used the well-established PIP 2 -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, disappearing entirely at 7 min after tight cell coupling (Fig. 2B). Less than 1/ 3 rd 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 1/3 rd 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 a 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 PIP 2 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 PIP 2 levels without substantial changes in PIP 2 localization (Figs. 2A, S2H, S2I). We also reduced PIP 2 generation and access with knockdown of the PIP5K c isoforms (Fig. S2J) and PIP 2 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 PIP 2 levels together with isoformspecific changes in PIP 2 localization. Isoform-specific effects of PIP5K overexpression thus will identify location-dependent roles of PIP 2 . 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 PIP 2 . 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. Corroborating 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 PIP 2 , in particular distal PIP 2 , as a substantial regulator of T cell activation. Second, they are critical in understanding a potential role of PIP 2 as second messenger precursor. If PIP 2 as a substrate for PLCc was central for mediating effects of PIP 2 turnover on T cell activation, mass action dictates that increased PIP 2 generation should yield increased second messenger levels and thus increased amounts of IL-2. As our findings run contrary to this expectation, roles of PIP 2 as a second messenger substrate were surprisingly minor.
To assess whether PIP 2 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 overexpression 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 PIP 2 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.

PIP 2 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 PIP 2 do not substantially affect T cell activation through a role of PIP 2 as a second messenger substrate, we next addressed cytoskeletal roles of PIP 2 . We started with the control of cellular rigidity, as this is a common function of PIP 2 [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 PIP 2 blockade altered neither the interface diameter at T cell/APC coupling nor uropod retraction. Effects of PIP5K gamma knockdown of PIP 2 levels were limited (Fig. S2J) and blockade of PIP 2 by the PLCd PH domain was 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 partial (Fig. S2K), providing potential technical explanations for the more limited nature of these effects. A possible biological explanation is that T cell flexibility might have already been maximized upon T cell activation with a strong stimulus, thus preventing further enhancement upon interference with PIP 2 generation or access. Our data on T cell rigidity are corroborated by data on actin spreading to the edge of the T cell/APC interface ( Fig. S4C-L). Importantly, effects of PIP 2 on T cell rigidity and IL-2 matched. Increased T cell rigidity should interfere with T cell activation by impairing tight APC coupling and thus extensive receptor ligand engagement. Accordingly, increased PIP 2 generation in parallel made T cells more rigid and interfered with IL-2 secretion. Interestingly, local features of PIP 2 turnover contributed substantially, as effects of overexpression of distal PIP5K isoforms were more dramatic both in the control of T cell rigidity, involving uropod retraction, and IL-2 secretion.

PIP5K overexpression impairs ERM protein dephosphorylation upon T cell activation
T cell rigidification could be caused by altered ERM protein function, as ERM proteins link the plasma membrane to the cortical actin cytoskeleton in a PIP 2 -dependent fashion. ERM protein activity requires threonine phosphorylation at residues T567 of Ezrin and T558 of Moesin [33,34,35,36]. We therefore determined ERM threonine phosphorylation during T cell activation. TCR engagement was provided by a-CD3 plus a-CD28 antibodies, as the determination of T cell ERM phosphorylation in T cell/APC couples, where both cell types contain ERM proteins, is challenging. Due to limiting cell numbers, these experiments were restricted to overexpression of PIP5K c90 and PIP5K c87 as a non-distal control. In non-transduced T cells, ERM phosphorylation dropped significantly (p,0.001) to 3569% of the level of non-stimulated T cells within 2 min of tight cell coupling (Fig. 5). This reduction is indicative of the increased T cell flexibility required to execute the T cell shape changes associated with T cell activation. However, upon overexpression of PIP5K c90, ERM phosphorylation dropped significantly less (c90)(p#0.01) to only 6665% of the level of non-stimulated T cells (Fig. 5). Overall ERM expression was not altered by PIP5K overexpression (Fig. S5A). We thus suggest as a mechanism of PIP5K-dependent T cell rigidification that elevated PIP 2 generation inhibits ERM inactivation upon T cell activation. Upon knockdown of PIP5K c, T cell stimulation triggered slightly enhanced reduction Figure 2. PIP5K overexpression alters PIP 2 levels and localization. A. PIP 2 levels were determined by immunofluorescence staining of 5C.C7 T cells transduced with different GFP-tagged PIP5K isoforms as indicated. PIP 2 levels of T cells with PIP5K overexpression (grey bars) and control (black bars) are normalized to control and are given with standard errors for two independent experiments each. An asterisks indicates statistical significance versus control with p,0.005. 100-200 cells were analyzed per experiment. A representative PIP 2 staining experiment is shown as Fig.  S2D. B. 5C.C7 T cells were transduced to express PLCd PH-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 PLCd PH-GFP with the indicated patterns [6] relative to tight cell coupling. 60 cell couples were analyzed. C, D. Similar to B, the panels show patterning data for 5C.C7 T cells expressing PLCd PH-GFP with concurrent overexpression of PIP5K b (C) or c90 (D). 41, 26 cell couples were analyzed per condition. Representative images for panels B-D are given in Fig. S2E-G. Representative movies have been published (c87 [6]) or are given as Movies S4, S5, S6. doi:10.1371/journal.pone.0027227.g002 Figure 3. Manipulation of PIP5K expression affects IL-2 secretion and proximal signal transduction. A, B. 5C.C7 T cells were activated by CH27 APCs and 10 mM MCC agonist peptide for 16 h upon manipulation of PIP5K expression and PIP 2 blockade as indicated. Cell culture supernatants were analyzed for IL-2 by ELISA. A representative IL-2 ELISA is given in (A). Changes in IL-2 secretion upon manipulation of PIP5K expression and PIP 2 blockade relative to non-transduced T cells are given with standard errors in (B) on a logarithmic scale to comparably display reduction and enhancement. 'GFP' indicates retroviral expression of GFP as a control. One/two asterisks indicate significance versus non-transduced control with p,0.05/0.005, respectively. Data from 3-6 independent experiments are given. When the agonist peptide concentration during T cell activation was reduced to a limiting concentration, 0.1 mM, knockdown of PIP5K c still did not result in impaired IL-2 secretion (Fig. S3). C, D. 5C.C7 T cells were activated by CH27 APCs and 10 mM MCC agonist peptide for 2 min upon manipulation of PIP5K expression as indicated. T cell/APC extracts were blotted for LAT Y191 and PLCc Y783. A representative blot is given in (C). Changes in LAT and PLCc phosphorylation upon manipulation of PIP5K expression relative to non-transduced T cells are given with standard errors in (D) on a logarithmic scale to comparably display reduction and enhancement. Data from 3-4 independent experiments are given. doi:10.1371/journal.pone.0027227.g003 in ERM phosphorylation to 1967% of the prestimulation levels ( Fig. 5, not significantly different from control however) as consistent with unimpaired T cell flexibility. A reduced frequency of T cell coupling and impaired F-actin polymerization could be ruled out as alternate mechanisms of PIP 2 action (Fig. S5B, C).

The spatiotemporal organization of T cell signaling depends on PIP 2
To understand how T cell rigidity may be linked to T cell signaling, we addressed effects of PIP 2 on TCR clustering. TCR clustering as a representative element of the system wide For all cell couples with persistent interface accumulation of TCRf-GFP it was determined whether preceding cell coupling occurred with (e.g. Figs. S1C, S2E, and S6A) or without (Fig. S4A, Movie S7) the formation of a visible lamellum. Of all T cell/APC couples with persistent TCRf-GFP interface accumulation, the percentage of cell couples without visible lamellum upon cell coupling is given with standard errors upon manipulation of PIP5K expression, as indicated. An asterisk indicates statistical significance with p,0.001 relative to control. 25-56 cell couples were analyzed per condition. C. 5C.C7 T cells were activated with CH27 APCs and 10 mM MCC agonist peptide. The percentage of cell couples with a visible uropod is given with standard errors relative to tight cell coupling upon manipulation of PIP5K expression and PIP 2 blockade, as indicated. 17-68 cell couples were analyzed per condition. doi:10.1371/journal.pone.0027227.g004 spatiotemporal organization of T cell signaling [6], is dependent of cytoskeletal dynamics [37,38], and is thus likely to be influenced by ERM-regulated cytoskeleton plasma membrane interactions. Upon tight cell coupling, the TCR is recruited to the T cell/ APC interface with a preference for the interface center. In the activation of 5C.C7 T cells by APCs such central clustering is related to the efficiency of T cell signaling [6,7]. Moreover, TCR clustering has a strong distal component as in about half of the cell couples the TCR is recruited transiently to the T cell distal pole [6] (Figs. 6A, S6A). Rapid release of the TCR from the distal pole is associated with increasing interface accumulation.
Overexpression of PIP5K b and c90 interfered with TCR accumulation at the T cell/APC interface within the first minute of tight cell coupling (Figs. 6B, C, S6B, C). For example upon overexpression of PIP5K c90, interface accumulation of the TCR in any pattern and in a central pattern at 1 min was reduced from 7966% and 4968% of control T cell/APC couples with such accumulation to 4268% and 1766%, respectively (p,0.005) (Fig. 6B). Delayed interface accumulation was accompanied by delayed TCR release from the distal pole. For example at 2 min no distal pole accumulation was observed in control T cell/APC couples any more, while in cell couples with T cell overexpression of PIP5K c90 or b 3368% and 865% of cell couples, respectively, still displayed distal accumulation of the TCR (p#0.01) (Fig. 6B, C). The phenotype upon PIP5K b overexpression was most complex, as both the inducible recruitment of the TCR to the distal pole and the subsequent release were defective (Fig. 6C). Intriguingly, the severity of the defects in the early spatiotemporal organization of T cell signaling upon overexpression of the different PIP5K isoforms matched the degree of impairment of IL-2 secretion, in that distal PIP5K isoforms were most effective. Effects of overexpression of PIP5K c87 (Fig. S6D, E), PIP5K c knockdown (Fig. S6F, G), and PIP 2 blockade (Fig.  S6H, I) were less severe to not significant, again consistent with data on IL-2 secretion.
The TCR localization experiments tie our data together into a consistent scenario of how cytoskeletal roles of PIP 2 control T cell activation: Increased rigidity of the T cell upon PIP5K overexpression (Figs. 4, S4) could trap the TCR, in particular at the distal pole (Fig. 6), thus delaying and impairing the formation of a TCR-anchored signaling complex at the center of the T cell/ APC interface (Fig. 6). A smaller interface would also limit receptor engagement. Less central TCR clustering and receptor engagement is linked to less efficient proximal signaling in 5C.C7 T cell/APC couples [6,7], as also seen here (Fig. 3C, D), and thus to less IL-2 secretion (Fig. 3A, B).

Discussion
Here we have addressed two principal challenges in PIP 2 biology in the activation of primary T cells. First, we found that changes in PIP 2 levels affect T cell activation primarily through regulation of T cell rigidity and spatiotemporal organization, not through the role of PIP 2 as a substrate in second messenger generation. Increased amounts of PIP 2 did not yield increased IL-2 secretion, as to be expected for a central role of PIP 2 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 PIP 2 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 PIP 2 generation were comparably spatially constrained. Increased generation of PIP 2 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 PIP 2 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 PIP 2 turnover to those of PIP 2 function. Our data thus establish that PIP 2 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 PIP 2 in T cell activation.
As a second general challenge, we have determined the spatiotemporal features of PIP 2 synthesis as a critical part of a more comprehensive understanding of PIP 2 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 PIP 2 turnover. Not only are the two most abundant PIP5K isoforms (c87 and a) enriched there, but also two key enzymes in PIP 2 metabolism, PLCc and PI3K. Based on the observed distributions of free PIP 2 , PIP 2 synthesis dominated during the first minute of cell coupling, PIP 2 metabolism thereafter. The distal pole in contrast is likely a site of slower PIP 2 turnover. It displayed enrichment of only the less abundant PIP5K isoforms and none of PIP 2 metabolizing enzymes. Nevertheless, initial PIP 2 metabolism still seems active, as accumulation of accessible PIP 2 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 PIP 2 at the distal pole (Fig. 2C), indicative of slowed PIP 2 turnover and something not seen at all at the T cell/ APC interface upon overexpression of interface-associated PIP5K isoforms. Slower turnover of distal PIP 2 at later time points is consistent with the stable accumulation of the PIP 2 -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 PIP 2 turnover as resolved in time and space forms a second critical foundation for future more detailed work on roles of PIP 2 in T cell activation.

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-PIP 2 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, 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 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 independently. 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.

PIP 2 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 dlysine-coated cover slip, and stained for PIP 2 as established [28]. Briefly, cells were fixed in 4% Paraformaldehyde, permeabilized with 0.5% Saponin, and stained with an anti-PIP 2 antiserum and an anti-mouse antibody conjugated to Alexa 568. Signal was .20fold above non-specific staining background. Three-dimensional images of the stained cells were acquired and 100-200 cells per experiment were analyzed for PIP 2 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 PIP 2 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 manufacturer'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 crosslinking. 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 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 rainbowlike 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 PIP 2 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 nonfluorescent 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 PIP 2 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-PIP 2 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. PIP 2 levels as determined by immunofluorescence similar to Fig. 2A were reduced by about 5% (not reaching statistical significance), consistent with tight regulation of PIP 2 levels. K. To block PIP 2 , 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 regulation of PIP 2 effector functions. While at 1 mM tatPLCd PH substantial effects could not be found, at .5 mM tatPLCd PH T cell coupling upon APC contact was reduced by .65% (p,0.001) from 5065% to less than 20%. This was most likely caused by a.95% decrease (p,0.001) in the percentage of T cells with overt migratory polarity. We therefore used tatPLCd PH at 5 mM, the maximal concentration allowing effective cell coupling. To directly assess the efficacy of 5 mM tat PLCd PH, we tested its ability to compete with PLCd PH-GFP FACS-sorted to a concentration of 2 mM. Similar to Fig. 2B, the panel shows patterning data for 5C.C7 T cells expressing PLCd PH-GFP with concurrent PIP 2 blockade by T cell pretreatment with 5 mM tat PLCd PH. Given is the percentage of cell couples with standard errors that displayed accumulation of PLCd PH-GFP with the indicated patterns [6] relative to tight cell coupling. Only 14 cell couples could be analyzed. Preincubation of T cells with 5 mM tatPLCd PH made interface PLCd PH-GFP accumulation moderately but significantly more transient with reduced accumulation at the time of tight cell couple formation (8365% to 50613%, p,0.005) and 80-120 s thereafter (6064% to 4368%, p = 0.05), thus defining the efficacy of tatPLCd PH. To distinguish between effects of PIP5K b and c90 in uropod retraction, we counteracted PIP5K overexpression by parallel expression of PLCd PH-GFP. Only PIP5K b-overexpressing T cells still showed delayed uropod retraction: 5C.C7 T cells transduced with PLCd PH-GFP were activated with CH27 APCs and 10 mM MCC agonist peptide. The percentage of cell couples with a visible uropod is given with standard errors relative to tight cell coupling upon manipulation of PIP5K expression, as indicated. 19-74 cell couples were analyzed per condition. The most severe effect of PIP5K b overexpression on uropod retraction is consistent with its exclusive distal localization (Fig. 1C). C, F, I, K. As T cell spreading (Fig. 4A) is actin-driven, we assessed changes in T cell actin dynamics upon manipulation of PIP 2 generation and access. Representative interactions of actin-GFP-transduced 5C.C7 T cells with CH27 APCs in the presence of 10 mM MCC peptide are shown as in Fig.  S1B for a control (C), upon overexpression of PIP5K b (F), upon PIP5K c knockdown (I), or upon T cell pretreatment with 5 mM tat PLCd PH (K). Movies covering the entire time frame are given as Movies S8, S9, S10, S11. D, E, G, H, J, L. 5C.C7 T cells were transduced to express actin-GFP 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 actin-GFP with the indicated patterns [6] relative to tight cell coupling upon manipulation of the expression of PIP5K and PIP 2 blockade as indicated. 29-60 cell couples were analyzed per condition. In non-transduced control T cells, actin rapidly and transiently spread to the periphery of the T cell/APC interface (C, D). At the time of tight cell couple formation 6866% of cell couples displayed peripheral actin accumulation (D). The percentage of cell couples with peripheral accumulation rapidly declined to 1365% at 3 min. Overexpression of PIP5K b, c87, or c90 all interfered with actin spreading to the interface periphery (E-H). The frequency of peripheral accumulation was reduced with a concomitant increase in diffuse patterning. For example upon overexpression of PIP5K c90, at the time of tight cell coupling the percentage of cell couples with peripheral actin-GFP accumulation was reduced from 6866% to 3866% (p,0.01), whereas diffuse accumulation was increased from 1665% to 3566% (p = 0.01)(D, G). Such differences were significant (p,0.05) at most time points between tight cell coupling and 2 min thereafter in the comparison of control T cells with T cell overexpressing each of the PIP5K isoforms. Knockdown of PIP5K c promoted peripheral actin accumulation by making it more sustained (I, J). From 1 to 5 min after tight cell coupling the percentage of cell couples with peripheral actin accumulation was significantly (p,0.05) enhanced at each time point in the knockdown T cells compared to control. Upon blocking access to PIP 2 with 5 mM tatPLCd PH (K, L), actin dynamics were largely unaltered, consistent with intact T cell spreading (Fig. 4A). (EPS) Figure S5 PIP5K overexpression does not impair ERM expression, T cell coupling, or F-actin amounts. A. To assess whether manipulation of PIP5K expression altered ERM expression, 5C.C7 T cell extracts were blotted for ERM proteins. ERM expression with standard errors upon overexpression/ knockdown of the indicated PIP5K isoforms is given relative to non-transduced control cells. B. T cell spreading is a TCRdependent process. Therefore, impaired T cell spreading could be the consequence of impaired TCR engagement. The ability of a T cell to form a cell couple upon initial APC contact is the most immediate readout of TCR engagement upon APC contact, occurring in seconds. The percentage of 5C.C7 T cells that form a tight cell couple upon contact with CH27 APCs in the presence of 10 mM MCC peptide is given with standard errors upon overexpression of PIP5K as indicated. 61-101 cell couples were analyzed per condition. An investigation of the modest differences in cell coupling upon PIP5K overexpression is beyond the scope of this study. However, because cell coupling is not reduced, it can be safely concluded that TCR engagement was not impaired. C. T cell rigidification could be caused by altered cellular F-actin amounts. We therefore determined F-actin amounts by phalloidin staining prior and past TCR engagement. T cell activation was mediated by antibodies, as the determination of T cell F-actin amounts in T cell/ APC couples, where both cell types contain F-actin, is challenging. 5C.C7 T cells were activated with a-CD3 and a-CD28 antibodies for 2 min upon manipulation of PIP5K expression and PIP 2 blockade as indicated. F-actin contents were determined by FACS analysis of Phalloidin-stained T cells. Mean intensity of Phalloidin staining is displayed with standard errors relative to that of nontransduced, non-stimulated T cells. Data from three independent experiments are given. Consistent with actin-driven T cell spreading upon APC contact, T cell stimulation with a-CD3 and a-CD28 triggered a significant (p,0.001) increase in T cell F-actin contents in control cells by 2363%. PIP5K overexpression and knockdown did not substantially alter T cell F-actin amounts relative to control neither prior nor post T cell stimulation. PIP 2 blockade modestly increased F-actin amounts prior to T cell activation (p,0.05 as indicated with an asterisk) but not thereafter. An investigation of this effect is beyond the scope of this study. (EPS) Figure S6 PIP 2 manipulation interferes with accumulation 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) 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 3dimensional 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 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 accumulation.

(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) Movie S17 Refers to Fig. S6H, the 5C.C7 T cells are transduced with TCRj-GFP and treated with 5 mM tat PLCd PH, cell coupling occurs in frame 7 (3s), and prominent is sustained central TCRj-GFP interface accumulation without initial distal accumulation. (MOV) Author Contributions