Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) TCR-induced recruitment of NWASP and WAVE2 to IS. Mouse primary WT CD4 T cells were incubated with bilayer containing ICAM1 alone (−) or both ICAM1 and anti-CD3 (+) for 2 min at 37°C, fixed and immunostained for endogenous proteins. Stained cells were visualized using TIRF microscopy. The graph shows quantitation of antibody fluorescence at IS, where each point represents the value obtained from a single cell. n1 = 16, n2 = 54 (for WAVE2), n3 = 16, n4 = 78 (for NWASP); p1, p2 < 0.0001 . Each point represents average levels of indicated protein at synapse in a single cell. ( B ) The images shown are TIRF plane distributions of the indicated proteins. As elaborated in the magnified areas marked with white boundary in original ‘merge’ image, there is a lack of co-localization between either of these proteins and TCR MCs. Scale bar, 5 μm. Insets in ( B ) have been intensity scaled differently from original ‘Merge’ panel to highlight protein distribution with more clarity. DOI: http://dx.doi.org/10.7554/eLife.04953.007

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, Staining, Microscopy, Quantitation Assay, Fluorescence

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: Human CD4 T cells were incubated with culture media containing lentiviral particles carrying WASP shRNA or non-specific (control) shRNA for 48 hr ( A ) T cells transduced with WASP shRNA or control shRNA carrying lentiviral particles were incubated with endothelial monolayer for 10 min, fixed and processed for Alexa594-phalloidin (pseudo-colored green) and phospho-HS1 (pseudo-colored red) immuno-staining. The conjugates were then imaged using an EMCCD-coupled spinning disc confocal microscope. Each image represents a single confocal plane of T cell synapse, where the planar endothelial interface is in focus. The area outlined in ‘merge’ panels was further scaled and magnified to show the details with more clarity (bottom panels). The top panels show the image of the field of view in DIC (left image) or fluorescence settings. ( B ) A reduction in WASP levels results in defective phospho-HS1 accumulation at T cell-endothelial cell synapse. The upper graph shows quantitation of phalloidin intensity in the synaptic plane, while the lower graph shows phospho-HS1 levels in the same plane. For both the upper and lower graphs, n1 = 68, n2 = 29, p1 = 0.071, p2 < 0.0001 . This experiment was repeated twice with similar results. ( C ) Model of temporal sequence of events leading to F-actin foci formation and PLCγ signaling at the immunological synapse. Multiple pathways can result in actin polymerization and remodeling at the synaptic interface, contributing to F-actin organization in different SMAC zones. One such pathway involves WAVE2 recruitment by activated LFA1, followed by WAVE2 dependent Arp2/3 complex activation resulting in thick lamellipodial (dSMAC) and lamellar (pSMAC) F-actin meshworks. WAVE2-dependent F-actin pool is required for calcium-dependent calcium entry via the CRAC channel. Additional pathways including MyosinII-mediated actin remodeling is required for maintaining lamellar actin flow and directional persistence of microclusters (MCs) towards the cSMAC, and formin-mediated nucleation of F-actin promotes MTOC docking and stability of synapse. Another pool of F-actin or ‘F-actin foci’ is generated by the activity of WASP protein in the p- and dSMAC zones. Following TCR triggering, WASP is recruited at TCR signalosome via several possible mechanisms – such as via Vav, via NCK, via Zap70 and CrkL mediated WIP release and other effector mechanisms, and, through Fyn or PIP2 or PTP-PEST-binding at the plasma membrane (PM). Once activated, WASP recruits Arp2/3 complex to the MC, which then leads to actin branch nucleation and polymerization at the MC, over and above the local background actin. This process continues even during MC movement in the lamellar region, with a high F-actin turnover at the foci until its delivery to the cSMAC. In the foci, HS1 is recruited via binding both the Arp2/3 complex as well as F-actin, and is subsequently phosphorylated. As a consequence of early TCR signaling, PLCγ1 is also recruited to the MC signalosome, where it is stabilized via interactions with both F-actin, and foci residing HS1. F-actin foci dynamics in the proximity of the plasma membrane further support PLCγ1 phosphorylation, potentially by facilitating its interaction with PM-bound, upstream activators such as Itk. Phosphorylation of PLCγ1 by Itk then triggers phosphoinositide signaling, which in turn initiates calcium ion flux and NFAT1 activation. WASP deficiency or failure to activate Arp2/3 complex by WASPΔC mutant leads to selective loss of nucleation of foci at the MC. As a result, early signaling is not affected, however, both HS1 and PLCγ1 levels are severely reduced at the microcluster sites. The remaining PLCγ1 at synapse allows cell spreading and synapse formation, however, it is not sufficient to achieve calcium flux comparable to the control cells. Direct pharmacological inhibition of Arp2/3 complex using CK666 yields similar results; early TCR signaling is preserved while PLCγ1 phosphorylation and late signaling are severely perturbed. As actin polymerizing processes other than WASP also utilize Arp2/3 Complex, CK666-treated cells show a general reduction in lamellipodial and lamellar actin as well. However, the remaining F-actin levels are sufficient to support early TCR signaling. In contrast, total F-actin depolymerization at the synapse using CytochalasinD results in defects in early as well as late signaling, as has been reported in earlier studies. The image on the bottom shows a maximum intensity projection of synaptic contact interface of a human primary CD4 T cell, acquired using spinning disc confocal microscope. This cell was activated on a bilayer reconstituted with Alexa568 tagged anti-CD3 (red) and ICAM1 (unlabeled), for 2 min, fixed and stained for F-actin (green), and imaged. DOI: http://dx.doi.org/10.7554/eLife.04953.031

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, shRNA, Control, Transduction, Immunostaining, Microscopy, Fluorescence, Quantitation Assay, Sequencing, Activation Assay, Generated, Activity Assay, Binding Assay, Clinical Proteomics, Membrane, Phospho-proteomics, Mutagenesis, Inhibition, Staining

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) Barbed-end decoration of freshly polymerized actin filaments reveals the dynamic behavior in WT (top panel) and WASP−/− (bottom panel) T cell synapses. CD4 T cells isolated from WT and Was−/− C57BL/6J mice were processed for barbed end labeling (fresh actin) to identify the actin incorporation sites within 1 min of polymerization, as well as total F-actin labeling, as described in ‘Materials and methods’. Arrowheads indicate the sites of F-actin foci in both ‘Total’ as well as ‘Fresh’ F-actin images. ( B ) The graph shows average incorporation of Rhodamine actin per pixel within the foci or the surrounding lamellar pixels (Fresh F-actin); and a ratio of Rhodamine and Alexa488 actin intensities in foci or surrounding pixels, in the WT cells. The foci areas in the total F-actin image were identified and outlined by intensity rank-based filtering as described in ‘Materials and methods’. The synaptic area outside the foci was defined as lamellar surround. These outlined areas were then analyzed in both ‘fresh F-actin’, and ‘total F-actin’ raw images and per pixel intensity is plotted in the graph. Note that while the rate of polymerization (Fresh/Total) ratio is not altered, there is higher incorporation of actin at foci sites (Fresh). n = 20 cells, p1 < 0.0001, p2 = 0.181 (Wilcoxon nonparametric pairwise comparison). ( C ) The graph shows total F-actin intensity, fresh F-actin intensity, or the ratio of the two at the synapse, each point represents value obtained from single cell, n in WT = 33, n in WASP−/− = 35; p1 = 0.06 , p2 = 0.059 , p3 = 0.0008 . Scale bars, 5 μm. ( D ) WASP is critical for F-actin foci generation. Freshly purified CD4 T cells from WT 129 (top left), Was−/− 129 (bottom left), Wasl−/− (top right), or Was/− Wasl− /− 129 (bottom right) mice were activated on SLB containing ICAM1 and anti-CD3 antibody, fixed and stained with Alexa488-phalloidin. Note that only the lack of WASP, and not N-WASP, causes loss of actin foci. n1 = 54, n2 = 45, n3 = 53, n4 = 28. p1 = 0.30, p2 < 0.0001, p3 < 0.0001 . ( E ) F-actin foci in CD4 T cells freshly purified from wild type (WT) mouse (left) or from Hcls1−/− mouse (center) or Hcls1−/− Was−/− (right) C57BL/6J mice were incubated with bilayer containing anti-CD3 antibody and ICAM1 for 2 min. Cells were then processed for F-actin staining (Alexa488-phalloidin) and visualized. Note that, while there is a minor alteration in F-actin foci in HS1−/− T cells, there is a gross deficit in HS1−/− WASP−/− double knockout T cells. The graph shows the quantification of total intensity of F-actin foci at the synapse in individual T cells derived from the indicated backgrounds. n1 = 48, n2 = 70, n3 = 64, p1 = 0.031, p2 = 0.0001 . DOI: http://dx.doi.org/10.7554/eLife.04953.003

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Isolation, End Labeling, Labeling, Comparison, Purification, Staining, Incubation, Double Knockout, Derivative Assay

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) HS1 silencing and F-actin foci. AND CD4 T cell blasts were electroporated with non-specific (control siRNA oligos) or oligos targeting HS1. The depletion of protein was assessed using western blotting (top left, gel). Cells electroporated with control or HS1 siRNA oligos were incubated with bilayers for 2 min at 37°C, fixed, and stained with Alexa488-phalloidin, and then imaged using TIRF illumination. The images (right) show F-actin distribution in their respective backgrounds. The graph shows quantification of F-actin foci in control or HS1 depleted cells. n1 = 70, n2 = 100, p = 0.0268 . ( B ) WASP determines phospho-HS1 levels at the synapse. CD4 T cells purified from WT-129 mouse, Was−/ −, or Wasl−/− 129 mice were incubated with bilayer containing ICAM1 and anti-CD3 for 2 min, and were fixed and stained with anti-phospho-HS1 antibody. The TIRF images were quantified for phospho-HS1 levels. Each dot represents average phospho-HS1 intensity per cell. n1 = 42, n2 = 43, n3 = 46, n4 = 27. p1 = 0.637, p2 , p3 < 0.0001 . DOI: http://dx.doi.org/10.7554/eLife.04953.008

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Control, Western Blot, Incubation, Staining, Purification

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: AND CD4 T cell blasts were incubated with bilayers reconstituted with ICAM1 alone (left image) or both ICAM1 and MHCp (right image) for 2 min at 37°C, and then fixed and immunostained for phosphorylated HS1. The images show phospho-HS1 (p-HS1) signal in the cells acquired in the TIRF field. The graph (far right) shows quantitation of total p-HS1 intensities, where each point represents integrated intensity per cell in the TIRF field. n1 = 33, n2 = 33, p < 0.0001 . ( B ) Phospho-HS1 localizes to F-actin foci and TCR MCs. Mouse AND CD4 T cell blasts (left panels) or primary CD4 T cells (panels on the right) were incubated with bilayer containing ICAM1 and MHCp (for blasts) or ICAM1 and anti-CD3 (right panels) for 2 min, fixed and stained with phospho-Y397 HS1 antibody (red) and Alexa488-phalloidin (green). The area marked in ‘Merge’ image has been further enlarged in insets; the arrows indicate F-actin foci sites that localize with phospho-HS1. DOI: http://dx.doi.org/10.7554/eLife.04953.013

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, Quantitation Assay, Staining

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) WASP deficient T cells exhibit impaired TCR-induced PLCγ1-Y783 phosphorylation. CD4 T cells freshly isolated from C57BL/6J WT or Was −/− mice were activated with ICAM1 alone, or both ICAM1 and anti-CD3 for 2 min. Cells were fixed and immunostained for phospho-PLCγ1 (image not shown), and visualized using confocal microscopy. The images show phospho-PLCγ1 staining in the bottom section (synapse plane) of WT (left) or WASP−/− (right) cells. The graph on the left shows phospho-PLCγ1 levels in the synapse planes in the cells in WASP deficiency background. n1 = 104, n2 = 60, n3 = 94, p < 0.0001 . ( B ) Assessment of total cellular PLCγ1 phosphorylation in WASP−/− T cells using western blot. Freshly isolated WT or WASP−/− CD4 T cells were incubated with anti-CD3/CD28 beads for 5 min, lysed, and the lysates were analyzed using western blotting. Note that, in WASP−/− T cells TCR-induced PLCγ1 phosphorylation is defective, while total PLCγ1 is comparable to the WT cells. HS1 phosphorylation was included as a control that exhibits diminished phosphorylation in WASP−/− T cells. These experiments were repeated twice with similar results. ( C ) Arp2/3 activation by WASP is essential for F-actin foci generation and optimal phospho-PLCγ1 at the synapse. Human CD4 T cells were transfected with GFP-WASP (WT), GFP-WASPΔC, or GFP-WASP291F (shown in the schematic on the left) for 16 hr and were then incubated with anti-CD3/ICAM1-reconstituted bilayers for 2 min, fixed and stained with Alexa568-phalloidin and anti-phospho-PLCγ1 antibody. The images show the GFP (green), F-actin (blue) and phospho-PLCγ1 (red) distribution at the synapse for WT (left) and WASPΔC (right) T cells. ( D ) The graph shows levels of GFP-tagged constructs at the synapse, analyzed and obtained via 50% rank filtering of images shown in ( D ), as described in ‘Materials and methods’, as well as the quantitation of total synaptic F-actin, foci and phospho-PLCγ1 in the same cells, normalized to mean values obtained for WT cells. n1 = 23, n2 = 27, n3 = 33 , n4 = 27 p values, p < 0.0001 between WT and WASPΔC cells for foci and phospho-PLCγ1 levels, and between untransfected and cells expressing GFP tagged constructs for ‘GFP puncta’. For all other comparisons, p > 0.05 . DOI: http://dx.doi.org/10.7554/eLife.04953.009

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Phospho-proteomics, Isolation, Confocal Microscopy, Staining, Western Blot, Incubation, Control, Activation Assay, Transfection, Construct, Quantitation Assay, Expressing

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) Images from were analyzed for phospho-PLCγ1 and F-actin foci chance level co-localization, after pixel shifts of phospho-PLCγ1 images by 5 pixels, as performed earlier, in . Note that both in DMSO (control) as well as CK666 treated cells, a shift of 5 pixels (x and y) in phospho-PLCγ1 image results in significant reduction in co-localization, indicating that loss of overlap in CK666 treatment is not just due to reduction in total phospho-PLCγ1 levels. ( B ) Characterization of the effect of CK666 vs Cytochalasin D (CytoD) on total synaptic F-actin, F-actin foci, phospho-Zap70 and phospho-PLCγ1 levels. Mouse CD4 T cells were treated with 100 µM CK666 or 5 µM CytoD for indicated time duration, were activated on anti-CD3 and ICAM1 containing surface for 2 min in the presence of inhibitors, fixed and processed for immunofluorescence of the indicated molecules. Note that, while both CK666 as well as CytoD treatments lead to loss of F-actin foci, CytoD treatment causes a greater reduction in total F-actin and phospho-Zap70 levels. For the left graph, for total actin and F-actin foci, n1 = 123, n2 = 118, n3 = 100, n4 = 87; for phospho-Zap70, n1 = 49, n2 = 47, n3 = 53, n4 = 42; for phospho-PLCγ1, n1 = 78, n2 = 67, n3 = 48, n4 = 42. For total phospho-Zap70, p > 0.90 , and for the rest of the datasets, p < 0.0001 . In the CytoD graph, for total actin and F-actin foci, n1 = 128, 67, 88; for phospho-Zap70, n1 = 80, n2 = 27, n3 = 57; for phospho-PLCγ1 n1 = 52, n2 = 54, n3 = 31. For all of the data across treatment durations , p < 0.0001 . ( C ) CK666-treated or untreated AND T cells were incubated with anti-CD3/ICAM1 containing bilayer, and stained with phospho-HS1 antibody (left graph) or pan-HS1 antibody (right graph). Cells were then imaged, and images were quantified for total synaptic phospho-HS1 (left graph) or total synaptic HS1 (right graph) intensities per cell. Each point on the graph represents integrated intensity of the indicated protein per cell. In the left graph, n1 = 47, n2 = 18, p < 0.0001; for the graph on the right, n1 = 42, n2 = 59, p < 0.0001 . DOI: http://dx.doi.org/10.7554/eLife.04953.028

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Control, Immunofluorescence, Incubation, Staining

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: Human CD4 T cells were incubated with culture media containing lentiviral particles carrying WASP shRNA or non-specific (control) shRNA for 48 hr ( A ) T cells transduced with WASP shRNA or control shRNA carrying lentiviral particles were incubated with endothelial monolayer for 10 min, fixed and processed for Alexa594-phalloidin (pseudo-colored green) and phospho-HS1 (pseudo-colored red) immuno-staining. The conjugates were then imaged using an EMCCD-coupled spinning disc confocal microscope. Each image represents a single confocal plane of T cell synapse, where the planar endothelial interface is in focus. The area outlined in ‘merge’ panels was further scaled and magnified to show the details with more clarity (bottom panels). The top panels show the image of the field of view in DIC (left image) or fluorescence settings. ( B ) A reduction in WASP levels results in defective phospho-HS1 accumulation at T cell-endothelial cell synapse. The upper graph shows quantitation of phalloidin intensity in the synaptic plane, while the lower graph shows phospho-HS1 levels in the same plane. For both the upper and lower graphs, n1 = 68, n2 = 29, p1 = 0.071, p2 < 0.0001 . This experiment was repeated twice with similar results. ( C ) Model of temporal sequence of events leading to F-actin foci formation and PLCγ signaling at the immunological synapse. Multiple pathways can result in actin polymerization and remodeling at the synaptic interface, contributing to F-actin organization in different SMAC zones. One such pathway involves WAVE2 recruitment by activated LFA1, followed by WAVE2 dependent Arp2/3 complex activation resulting in thick lamellipodial (dSMAC) and lamellar (pSMAC) F-actin meshworks. WAVE2-dependent F-actin pool is required for calcium-dependent calcium entry via the CRAC channel. Additional pathways including MyosinII-mediated actin remodeling is required for maintaining lamellar actin flow and directional persistence of microclusters (MCs) towards the cSMAC, and formin-mediated nucleation of F-actin promotes MTOC docking and stability of synapse. Another pool of F-actin or ‘F-actin foci’ is generated by the activity of WASP protein in the p- and dSMAC zones. Following TCR triggering, WASP is recruited at TCR signalosome via several possible mechanisms – such as via Vav, via NCK, via Zap70 and CrkL mediated WIP release and other effector mechanisms, and, through Fyn or PIP2 or PTP-PEST-binding at the plasma membrane (PM). Once activated, WASP recruits Arp2/3 complex to the MC, which then leads to actin branch nucleation and polymerization at the MC, over and above the local background actin. This process continues even during MC movement in the lamellar region, with a high F-actin turnover at the foci until its delivery to the cSMAC. In the foci, HS1 is recruited via binding both the Arp2/3 complex as well as F-actin, and is subsequently phosphorylated. As a consequence of early TCR signaling, PLCγ1 is also recruited to the MC signalosome, where it is stabilized via interactions with both F-actin, and foci residing HS1. F-actin foci dynamics in the proximity of the plasma membrane further support PLCγ1 phosphorylation, potentially by facilitating its interaction with PM-bound, upstream activators such as Itk. Phosphorylation of PLCγ1 by Itk then triggers phosphoinositide signaling, which in turn initiates calcium ion flux and NFAT1 activation. WASP deficiency or failure to activate Arp2/3 complex by WASPΔC mutant leads to selective loss of nucleation of foci at the MC. As a result, early signaling is not affected, however, both HS1 and PLCγ1 levels are severely reduced at the microcluster sites. The remaining PLCγ1 at synapse allows cell spreading and synapse formation, however, it is not sufficient to achieve calcium flux comparable to the control cells. Direct pharmacological inhibition of Arp2/3 complex using CK666 yields similar results; early TCR signaling is preserved while PLCγ1 phosphorylation and late signaling are severely perturbed. As actin polymerizing processes other than WASP also utilize Arp2/3 Complex, CK666-treated cells show a general reduction in lamellipodial and lamellar actin as well. However, the remaining F-actin levels are sufficient to support early TCR signaling. In contrast, total F-actin depolymerization at the synapse using CytochalasinD results in defects in early as well as late signaling, as has been reported in earlier studies. The image on the bottom shows a maximum intensity projection of synaptic contact interface of a human primary CD4 T cell, acquired using spinning disc confocal microscope. This cell was activated on a bilayer reconstituted with Alexa568 tagged anti-CD3 (red) and ICAM1 (unlabeled), for 2 min, fixed and stained for F-actin (green), and imaged. DOI: http://dx.doi.org/10.7554/eLife.04953.031

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, shRNA, Control, Transduction, Immunostaining, Microscopy, Fluorescence, Quantitation Assay, Sequencing, Activation Assay, Generated, Activity Assay, Binding Assay, Clinical Proteomics, Membrane, Phospho-proteomics, Mutagenesis, Inhibition, Staining

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) Visualization of loss of F-actin in ILPs following CK666 treatment. T cells were incubated with membrane-YFP (green) expressing endothelial cells in the presence of DMSO (control, left) or CK666 (right) for 5 min, then fixed, permeabilized and stained with Alexa594-phalloidin (‘Actin’, pink). Note that F-actin rich protrusions are missing at the CK666-treated T cell interface. Scale bar, 5 µm. ( B ) Loss of ILPs correlated with reduced phospho-HS1 at the T cell-endothelial cell synaptic interface. T cells were incubated with endothelial cell layer, as described in ‘Materials and methods’ for 5 min and were then fixed and stained with Alexa594-phalloidin (‘Actin’, green), phospho-HS1 (red) and anti-CD3 (blue). Cells were then imaged using confocal microscopy. The insets show single T cells marked in the ‘Merge’ image. Scale bar, 10 µm. ( C ) The intensity of the indicated proteins was then analyzed in the synaptic plane without and with CK666 treatment. n1 = 58, n2 = 71, p1 = 0.031, p2, p3 < 0.0001 . DOI: http://dx.doi.org/10.7554/eLife.04953.032

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, Membrane, Expressing, Control, Staining, Confocal Microscopy

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) WASP deficient T cells exhibit impaired TCR-induced PLCγ1-Y783 phosphorylation. CD4 T cells freshly isolated from C57BL/6J WT or Was −/− mice were activated with ICAM1 alone, or both ICAM1 and anti-CD3 for 2 min. Cells were fixed and immunostained for phospho-PLCγ1 (image not shown), and visualized using confocal microscopy. The images show phospho-PLCγ1 staining in the bottom section (synapse plane) of WT (left) or WASP−/− (right) cells. The graph on the left shows phospho-PLCγ1 levels in the synapse planes in the cells in WASP deficiency background. n1 = 104, n2 = 60, n3 = 94, p < 0.0001 . ( B ) Assessment of total cellular PLCγ1 phosphorylation in WASP−/− T cells using western blot. Freshly isolated WT or WASP−/− CD4 T cells were incubated with anti-CD3/CD28 beads for 5 min, lysed, and the lysates were analyzed using western blotting. Note that, in WASP−/− T cells TCR-induced PLCγ1 phosphorylation is defective, while total PLCγ1 is comparable to the WT cells. HS1 phosphorylation was included as a control that exhibits diminished phosphorylation in WASP−/− T cells. These experiments were repeated twice with similar results. ( C ) Arp2/3 activation by WASP is essential for F-actin foci generation and optimal phospho-PLCγ1 at the synapse. Human CD4 T cells were transfected with GFP-WASP (WT), GFP-WASPΔC, or GFP-WASP291F (shown in the schematic on the left) for 16 hr and were then incubated with anti-CD3/ICAM1-reconstituted bilayers for 2 min, fixed and stained with Alexa568-phalloidin and anti-phospho-PLCγ1 antibody. The images show the GFP (green), F-actin (blue) and phospho-PLCγ1 (red) distribution at the synapse for WT (left) and WASPΔC (right) T cells. ( D ) The graph shows levels of GFP-tagged constructs at the synapse, analyzed and obtained via 50% rank filtering of images shown in ( D ), as described in ‘Materials and methods’, as well as the quantitation of total synaptic F-actin, foci and phospho-PLCγ1 in the same cells, normalized to mean values obtained for WT cells. n1 = 23, n2 = 27, n3 = 33 , n4 = 27 p values, p < 0.0001 between WT and WASPΔC cells for foci and phospho-PLCγ1 levels, and between untransfected and cells expressing GFP tagged constructs for ‘GFP puncta’. For all other comparisons, p > 0.05 . DOI: http://dx.doi.org/10.7554/eLife.04953.009

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Phospho-proteomics, Isolation, Confocal Microscopy, Staining, Western Blot, Incubation, Control, Activation Assay, Transfection, Construct, Quantitation Assay, Expressing

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) Images from were analyzed for phospho-PLCγ1 and F-actin foci chance level co-localization, after pixel shifts of phospho-PLCγ1 images by 5 pixels, as performed earlier, in . Note that both in DMSO (control) as well as CK666 treated cells, a shift of 5 pixels (x and y) in phospho-PLCγ1 image results in significant reduction in co-localization, indicating that loss of overlap in CK666 treatment is not just due to reduction in total phospho-PLCγ1 levels. ( B ) Characterization of the effect of CK666 vs Cytochalasin D (CytoD) on total synaptic F-actin, F-actin foci, phospho-Zap70 and phospho-PLCγ1 levels. Mouse CD4 T cells were treated with 100 µM CK666 or 5 µM CytoD for indicated time duration, were activated on anti-CD3 and ICAM1 containing surface for 2 min in the presence of inhibitors, fixed and processed for immunofluorescence of the indicated molecules. Note that, while both CK666 as well as CytoD treatments lead to loss of F-actin foci, CytoD treatment causes a greater reduction in total F-actin and phospho-Zap70 levels. For the left graph, for total actin and F-actin foci, n1 = 123, n2 = 118, n3 = 100, n4 = 87; for phospho-Zap70, n1 = 49, n2 = 47, n3 = 53, n4 = 42; for phospho-PLCγ1, n1 = 78, n2 = 67, n3 = 48, n4 = 42. For total phospho-Zap70, p > 0.90 , and for the rest of the datasets, p < 0.0001 . In the CytoD graph, for total actin and F-actin foci, n1 = 128, 67, 88; for phospho-Zap70, n1 = 80, n2 = 27, n3 = 57; for phospho-PLCγ1 n1 = 52, n2 = 54, n3 = 31. For all of the data across treatment durations , p < 0.0001 . ( C ) CK666-treated or untreated AND T cells were incubated with anti-CD3/ICAM1 containing bilayer, and stained with phospho-HS1 antibody (left graph) or pan-HS1 antibody (right graph). Cells were then imaged, and images were quantified for total synaptic phospho-HS1 (left graph) or total synaptic HS1 (right graph) intensities per cell. Each point on the graph represents integrated intensity of the indicated protein per cell. In the left graph, n1 = 47, n2 = 18, p < 0.0001; for the graph on the right, n1 = 42, n2 = 59, p < 0.0001 . DOI: http://dx.doi.org/10.7554/eLife.04953.028

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Control, Immunofluorescence, Incubation, Staining

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: ( A ) Formation of TCR MCs or CD45 exclusion does not require F-actin foci. AND CD4 T cell blasts labeled with Alexa568-H57 Fab (to assess TCR clustering), or Alexa488-CD45 Fab (for CD45 exclusion) at 4⁰C, were then incubated with bilayers containing ICAM1 and MHCp for 2 min, fixed (to assess TCR clustering) or visualized live (for CD45 exclusion) using TIRF microscopy. The images were processed to assess TCR clustering, or CD45 and TCR colocalization using rank filter based filtering, as described in ‘Materials and methods’ section. For the bars showing TCR cluster intensities per cell, n1 = 45, n2 = 28, p = 0.6; for CD45 co-localization, n1 = 26, n2 = 13, p = 0.09 . ( B ) Phosphorylation of TCR-proximal molecule Zap70 is not reduced in the cells treated with CK666. T cells were treated with DMSO or CK666 for 10 min, then incubated with surface containing ICAM1/anti-CD3 for 3 min, and processed for Y319-phospho-Zap70 and imaged using TIRF. The images were quantified to obtain the synaptic levels phospho-Zap70, and plotted as normalized to mean value of the ‘control’ cells. In the graph shown here, n1 = 34, n2 = 38, p = 0.16 . ( C ) Synaptic phospho-PLCγ1 levels are reduced in cells lacking F-actin foci. DMSO (control, top panel) or CK666 (bottom panel) treated AND CD4 T cell blasts were incubated with bilayer containing anti-CD3 and ICAM1 for 2 min, fixed and stained with Alexa488-phalloidin (green) and phospho-Y783-PLCγ1 (red), and visualized using TIRF microscopy. In these images, total synaptic levels of phospho-PLCγ1 were assessed (top left, graph). These images were also analyzed using integrated morphometry and total number of phospho-PLCγ1 events per cell (c, top right, graph) or intensity distribution of the events across the population of T cells (bottom right, histogram) were measured. Note that CK666 treatment leads to a uniform reduction in phospho-PLCγ1 events across different intensity ranges (c, bottom histogram). In all graphs in c , n1 = 50, n2 = 67, p < 0.0001 ( D ) Total cellular levels of phospho-PLCγ1 and PLCγ1 with or without treatment of cells with CK666. CD4 T cell blasts were treated with 100 μM CK666 or DMSO (control), and then incubated with anti-CD3/CD28 beads for 5 min in the presence of the inhibitor, lysed and analyzed using western blotting. ( E ) In CK666-treated cells, there is a substantial reduction in synaptic phospho-PLCγ1 co-localized with F-actin foci. Colocalization analysis was performed on images from ( C ), to estimate phospho-PLCγ1 and F-actin colocalization, as described in ‘Materials and methods’. ( F ) Pan- PLCγ1 levels at the synapse are reduced in CK666-treated cells. T cells were processed for PLCγ1 immunofluorescence and imaged as described above. In the graph, each dot represents PLCγ1 intensity per cell in the TIRF images. n1 = 29, n2 = 34, p < 0.0001 . ( G ) Defective calcium ion mobilization in CK666 treated cells. AND CD4 T cell blasts were loaded with Fluo4, and treated with DMSO or 100 µM CK666 for 1 min, and then imaged live on anti-CD3 and ICAM1, to monitor calcium ion flux (‘Materials and methods’). Each point on the graph (far right) represents mean value of the baseline corrected fluorescence ±SEM (n = 30 cells). ( H ) Nuclear translocation of NFAT1 in cells treated with CK666. DMSO (control, top panels) or CK666-treated (bottom panels) AND CD4 T cell blasts were incubated with glass coverslips coated with ICAM1 alone (left), or ICAM1 and anti-CD3 (right) for 10 min at 37°C, fixed and immunostained for NFAT1 (red), and stained with Alexa488-phalloidin (green). Cells were subsequently imaged using confocal microscopy. The middle sections from the z-stack of the images of the cells were used to quantitate nuclear levels of NFAT1, by outlining phalloidin-free central area (nucleus) of the cell. The graph (right) shows nuclear NFAT intensity in the nuclear area, in a single section per cell. n1 = 98, n2 = 40, n3 = 156, n4 = 31. p -values, p1 = 0.92, p2, p3 < 0.0001 . ( I ) PLCγ1 dynamics at TCR microdots. Human CD4 T cells transiently expressing PLCγ1-YFP (PLC, green) were incubated with glass coverslips patterned with Alexa647 tagged anti-CD3 microdots (TCR, red) and coated with ICAM1 and imaged live using TIRF microscopy. The graphs on the bottom show linescan intensity profiles of PLCγ1 and anti-CD3 (TCR) across the pixels marked in the corresponding image in the top panels. The middle panel shows the intensity profiles during the addition of CK666, and the right panel shows the profiles 30 s after the addition of CK666. The graph in ( J ) shows anti-CD3 (TCR) and PLCγ1-YFP profiles obtained from at least 30 different microdots from >12 cells in each control and CK666 treatment background. PLCγ1-YFP expressing human T cells were incubated with patterned substrate for (1 + 4) min in the presence of DMSO (Control) or CK666, fixed and imaged using TIRF microscope. The CK666 was added after 1 min of incubation of cells with the substrate. The graph shows mean value of pixel intensities calculated from the linescan profiles. The pixel intensities within a linescan were normalized to lowest pixel intensity within that linescan prior to calculating mean value across different linescans. For a given treatment background and linescan profile, TCR and PLCγ1 intensities were obtained from the identical pixel positions. Scale bar, 5 µm. DOI: http://dx.doi.org/10.7554/eLife.04953.027

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Labeling, Incubation, Microscopy, Phospho-proteomics, Control, Staining, Western Blot, Immunofluorescence, Fluorescence, Translocation Assay, Confocal Microscopy, Expressing

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: Freshly purified human CD4 T cells were treated with DMSO (control) or CK666, and incubated with bilayer reconstituted with anti-CD3 and ICAM1 for 2 min, in presence of the inhibitor. Cells were then fixed and stained with Alexa488 phalloidin (Actin, green) and either of anti-phospho Zap70 (upper panels) or anti-phospho-PLCγ1 (Lower panels) antibodies, and subsequently imaged using TIRFM. The graph represents phospho-Zap70 (upper graph) or phospho-PLCγ1 levels at synapse, normalized to the mean value in control cells. For upper graph, n1 = 49 n2 = 47, p = 0.976; for the lower graph, n1 = 78, n2 = 67, p = 0.004. DOI: http://dx.doi.org/10.7554/eLife.04953.029

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Purification, Control, Incubation, Staining

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: Human CD4 T cells were transfected with Zap70-GFP ( A ) or PLCγ1-GFP ( B ) for 16 hr, were then incubated with bilayers containing ICAM1 and Alexa568-OKT3 and imaged live at a rate of 1 frame per 5 s. The images represent maximum intensity projection of five frames (25 s of imaging) immediately before (-CK666) or after (+CK666) CK666 treatment onstage. The projections have been scaled to an identical extent before (upper panels) and after (lower panels) CK666 treatment. An area in the ‘Merge’ panel has been further magnified in the insets (rightmost panels). The insets have been scaled differently from the original ‘Merge’ panels (but similarly between + CK666 and–CK666 cases), to visualize local protein distribution in better details. Note that while Zap70 localization to mobile TCR MC is maintained after CK666 treatment, PLCγ1 localization is severely reduced. DOI: http://dx.doi.org/10.7554/eLife.04953.030

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Transfection, Incubation, Imaging

Journal: eLife

Article Title: Actin foci facilitate activation of the phospholipase C-γ in primary T lymphocytes via the WASP pathway

doi: 10.7554/eLife.04953

Figure Lengend Snippet: Human CD4 T cells were incubated with culture media containing lentiviral particles carrying WASP shRNA or non-specific (control) shRNA for 48 hr ( A ) T cells transduced with WASP shRNA or control shRNA carrying lentiviral particles were incubated with endothelial monolayer for 10 min, fixed and processed for Alexa594-phalloidin (pseudo-colored green) and phospho-HS1 (pseudo-colored red) immuno-staining. The conjugates were then imaged using an EMCCD-coupled spinning disc confocal microscope. Each image represents a single confocal plane of T cell synapse, where the planar endothelial interface is in focus. The area outlined in ‘merge’ panels was further scaled and magnified to show the details with more clarity (bottom panels). The top panels show the image of the field of view in DIC (left image) or fluorescence settings. ( B ) A reduction in WASP levels results in defective phospho-HS1 accumulation at T cell-endothelial cell synapse. The upper graph shows quantitation of phalloidin intensity in the synaptic plane, while the lower graph shows phospho-HS1 levels in the same plane. For both the upper and lower graphs, n1 = 68, n2 = 29, p1 = 0.071, p2 < 0.0001 . This experiment was repeated twice with similar results. ( C ) Model of temporal sequence of events leading to F-actin foci formation and PLCγ signaling at the immunological synapse. Multiple pathways can result in actin polymerization and remodeling at the synaptic interface, contributing to F-actin organization in different SMAC zones. One such pathway involves WAVE2 recruitment by activated LFA1, followed by WAVE2 dependent Arp2/3 complex activation resulting in thick lamellipodial (dSMAC) and lamellar (pSMAC) F-actin meshworks. WAVE2-dependent F-actin pool is required for calcium-dependent calcium entry via the CRAC channel. Additional pathways including MyosinII-mediated actin remodeling is required for maintaining lamellar actin flow and directional persistence of microclusters (MCs) towards the cSMAC, and formin-mediated nucleation of F-actin promotes MTOC docking and stability of synapse. Another pool of F-actin or ‘F-actin foci’ is generated by the activity of WASP protein in the p- and dSMAC zones. Following TCR triggering, WASP is recruited at TCR signalosome via several possible mechanisms – such as via Vav, via NCK, via Zap70 and CrkL mediated WIP release and other effector mechanisms, and, through Fyn or PIP2 or PTP-PEST-binding at the plasma membrane (PM). Once activated, WASP recruits Arp2/3 complex to the MC, which then leads to actin branch nucleation and polymerization at the MC, over and above the local background actin. This process continues even during MC movement in the lamellar region, with a high F-actin turnover at the foci until its delivery to the cSMAC. In the foci, HS1 is recruited via binding both the Arp2/3 complex as well as F-actin, and is subsequently phosphorylated. As a consequence of early TCR signaling, PLCγ1 is also recruited to the MC signalosome, where it is stabilized via interactions with both F-actin, and foci residing HS1. F-actin foci dynamics in the proximity of the plasma membrane further support PLCγ1 phosphorylation, potentially by facilitating its interaction with PM-bound, upstream activators such as Itk. Phosphorylation of PLCγ1 by Itk then triggers phosphoinositide signaling, which in turn initiates calcium ion flux and NFAT1 activation. WASP deficiency or failure to activate Arp2/3 complex by WASPΔC mutant leads to selective loss of nucleation of foci at the MC. As a result, early signaling is not affected, however, both HS1 and PLCγ1 levels are severely reduced at the microcluster sites. The remaining PLCγ1 at synapse allows cell spreading and synapse formation, however, it is not sufficient to achieve calcium flux comparable to the control cells. Direct pharmacological inhibition of Arp2/3 complex using CK666 yields similar results; early TCR signaling is preserved while PLCγ1 phosphorylation and late signaling are severely perturbed. As actin polymerizing processes other than WASP also utilize Arp2/3 Complex, CK666-treated cells show a general reduction in lamellipodial and lamellar actin as well. However, the remaining F-actin levels are sufficient to support early TCR signaling. In contrast, total F-actin depolymerization at the synapse using CytochalasinD results in defects in early as well as late signaling, as has been reported in earlier studies. The image on the bottom shows a maximum intensity projection of synaptic contact interface of a human primary CD4 T cell, acquired using spinning disc confocal microscope. This cell was activated on a bilayer reconstituted with Alexa568 tagged anti-CD3 (red) and ICAM1 (unlabeled), for 2 min, fixed and stained for F-actin (green), and imaged. DOI: http://dx.doi.org/10.7554/eLife.04953.031

Article Snippet: HS1 antibody (D5A9), Phospho-Y397 HS-1 antibody (D12C1), phospho-Y319 Zap70/Y352 Syk (affinity purified antisera #2704), PLCγ1 (D9H10), phospho-Y783 PLCγ1 (#2821), NFAT1 (D43B1), phospho-Y416 SFK (#6943), phospho-Y171 LAT (#3581) and WAVE2 (D2C8) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Incubation, shRNA, Control, Transduction, Immunostaining, Microscopy, Fluorescence, Quantitation Assay, Sequencing, Activation Assay, Generated, Activity Assay, Binding Assay, Clinical Proteomics, Membrane, Phospho-proteomics, Mutagenesis, Inhibition, Staining