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

Kumari S, Depoil D, Martinelli R, Judokusumo E, Carmona G, Gertler FB, Kam LC, Carman CV, Burkhardt JK, Irvine DJ, Dustin ML - Elife (2015)

Bottom Line: Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling.These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation.We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

View Article: PubMed Central - PubMed

Affiliation: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin 'foci'. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

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WASP regulates calcium ion signaling via generation of F-actin foci.(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
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fig4: WASP regulates calcium ion signaling via generation of F-actin foci.(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

Mentions: Having found that TCR MC formation and proximal signaling is independent of WASP, we investigated further TCR-distal signaling events that would lead to WASP-dependent calcium ion flux, as reported previously in human and mouse T cells (Zhang et al., 2002; Calvez et al., 2011). PLCγ1 is one of the key effector molecules regulating calcium ion flux downstream of TCR-activation, which mediates inositol-1,3,4-trisphosphate synthesis in the plasmamembrane, thereby facilitating calcium ion release from endoplasmic reticulum stores (DeBell et al., 1992; Babich et al., 2012). In addition, there is evidence that PLCγ1 can bind to F-actin (Carrizosa et al., 2009). We therefore reasoned that F-actin foci may locally enrich PLCγ1 at the TCR MC, supporting PLCγ1 interaction with TCR signalosome effectors such as Itk and LAT, thereby promoting its phosphorylation and activation (Braiman et al., 2006). We therefore assessed the role of WASP in PLCγ1 activation in WT and WASP−/− T cells. WASP−/− T cells exhibited significantly reduced phospho-PLCγ1 levels at the synapse (Figure 4A). The reduction in phospho-PLCγ1 levels was not due to a general decrease in total PLCγ1 levels in the WASP−/− T cells, as the levels of total cellular PLCγ1 were comparable in WT and WASP−/− whole T cell lysates (Figure 4B). Thus, a loss of WASP-driven F-actin foci is correlated with impairment of PLCγ1 activation at the synapse.10.7554/eLife.04953.009Figure 4.WASP regulates calcium ion signaling via generation of F-actin foci.


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

Kumari S, Depoil D, Martinelli R, Judokusumo E, Carmona G, Gertler FB, Kam LC, Carman CV, Burkhardt JK, Irvine DJ, Dustin ML - Elife (2015)

WASP regulates calcium ion signaling via generation of F-actin foci.(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
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fig4: WASP regulates calcium ion signaling via generation of F-actin foci.(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
Mentions: Having found that TCR MC formation and proximal signaling is independent of WASP, we investigated further TCR-distal signaling events that would lead to WASP-dependent calcium ion flux, as reported previously in human and mouse T cells (Zhang et al., 2002; Calvez et al., 2011). PLCγ1 is one of the key effector molecules regulating calcium ion flux downstream of TCR-activation, which mediates inositol-1,3,4-trisphosphate synthesis in the plasmamembrane, thereby facilitating calcium ion release from endoplasmic reticulum stores (DeBell et al., 1992; Babich et al., 2012). In addition, there is evidence that PLCγ1 can bind to F-actin (Carrizosa et al., 2009). We therefore reasoned that F-actin foci may locally enrich PLCγ1 at the TCR MC, supporting PLCγ1 interaction with TCR signalosome effectors such as Itk and LAT, thereby promoting its phosphorylation and activation (Braiman et al., 2006). We therefore assessed the role of WASP in PLCγ1 activation in WT and WASP−/− T cells. WASP−/− T cells exhibited significantly reduced phospho-PLCγ1 levels at the synapse (Figure 4A). The reduction in phospho-PLCγ1 levels was not due to a general decrease in total PLCγ1 levels in the WASP−/− T cells, as the levels of total cellular PLCγ1 were comparable in WT and WASP−/− whole T cell lysates (Figure 4B). Thus, a loss of WASP-driven F-actin foci is correlated with impairment of PLCγ1 activation at the synapse.10.7554/eLife.04953.009Figure 4.WASP regulates calcium ion signaling via generation of F-actin foci.

Bottom Line: Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling.These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation.We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

View Article: PubMed Central - PubMed

Affiliation: Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, United States.

ABSTRACT
Wiscott Aldrich Syndrome protein (WASP) deficiency results in defects in calcium ion signaling, cytoskeletal regulation, gene transcription and overall T cell activation. The activation of WASP constitutes a key pathway for actin filament nucleation. Yet, when WASP function is eliminated there is negligible effect on actin polymerization at the immunological synapse, leading to gaps in our understanding of the events connecting WASP and calcium ion signaling. Here, we identify a fraction of total synaptic F-actin selectively generated by WASP in the form of distinct F-actin 'foci'. These foci are polymerized de novo as a result of the T cell receptor (TCR) proximal tyrosine kinase cascade, and facilitate distal signaling events including PLCγ1 activation and subsequent cytoplasmic calcium ion elevation. We conclude that WASP generates a dynamic F-actin architecture in the context of the immunological synapse, which then amplifies the downstream signals required for an optimal immune response.

Show MeSH
Related in: MedlinePlus