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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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Effect of CK666 on TCR-induced Zap70 and PLCγ phosphorylation in human T cells.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
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fig6s2: Effect of CK666 on TCR-induced Zap70 and PLCγ phosphorylation in human T cells.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

Mentions: We have shown above that WASP's foci forming ability is required for PLCγ1 activation (Figure 4B). In order to test whether the independent elimination of foci by perturbation of Arp2/3 complex would phenocopy this effect, we chose to deplete foci using CK666 and monitor the consequences of this treatment on early TCR signaling, PLCγ1 activation and subsequent calcium ion signaling. CK666 treatment, which resulted in severe inhibition of F-actin foci (Figure 5B, Figure 6—figure supplement 1B), did not diminish TCR-proximal activation events. While fewer T cells formed synapses after CK666 treatment (37% reduction), upon adhesion, F-actin dependent processes including formation of TCR MC and exclusion of CD45 phosphatase from MCs (Campi et al., 2005; Varma et al., 2006) (Figure 6A), and the phosphorylation of Zap70 on Y319 (Figure 6B) proceeded normally in CK666-treated cells. In addition, other TCR-activation dependent signaling events leading to calcium flux, such as the phosphorylation of LAT on Y171 and SLP76 on Y145 were not altered in CK666-treated cells in comparison to control treated cells (data not shown). However, a severe defect was observed in the levels of TCR-induced PLCγ1 phosphorylation on Y783 after CK666 treatment (Figure 6C,D), where a drop was observed in both the number and intensity of phospho-PLCγ1 clusters at the synapse (Figure 6C). Furthermore, while phospho-PLCγ1 localized with F-actin foci in control cells, treatment with CK666 led to loss of foci-associated phospho-PLCγ1 (Figure 6E, Figure 6—figure supplement 1A). Upon attenuation of F-actin foci, total PLCγ1 levels were also reduced at the synapse, matching the decline in phospho-PLCγ1 levels (Figure 6F) and indicating that the impaired levels of phospho-PLCγ1 at the whole cell level may be accounted for by the reduction in PLCγ1 enrichment at the TCR signalosome. When assaying signaling events downstream of PLCγ1 activation, such as TCR-induced calcium ion elevation in the cytoplasm and nuclear mobilization of NFAT1, CK666-treated cells showed significant reduction in these processes (Figure 6G,H). Although a reduction in lamellipodial and lamellar actin was observed in CK666 treated cells (mean total F-actin 42% reduced), this reduction was relatively lower than foci depletion (mean 67% reduced) or under global F-actin depletion (CytoD, mean F-actin 90% reduced), and did not affect early TCR signaling (Figure 6—figure supplement 1B). We thus conclude that WASP and Arp2/3 dependent foci play a non-redundant role in PLCγ1 activation and calcium ion signaling at the synapse. In line with this result, CK666-treated cells exhibited a loss of phospho-HS1, as well as HS1, at the synapse (Figure 6—figure supplement 1C), similar to that observed in WASP−/− T cells (Figure 1E). Similar to the TCR-distal signaling defects observed in CK666 treated mouse CD4 T cells described above, CK666-treated human CD4 T cells also displayed a reduction in phospho-PLCγ1, but not in phospho-Zap70 (Figure 6—figure supplement 2), indicating that foci-dependent PLCγ1 activation cascade is conserved in T cells from diverse origins.10.7554/eLife.04953.027Figure 6.Arp2/3 inhibition leads to defective TCR-distal signaling.


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)

Effect of CK666 on TCR-induced Zap70 and PLCγ phosphorylation in human T cells.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
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fig6s2: Effect of CK666 on TCR-induced Zap70 and PLCγ phosphorylation in human T cells.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
Mentions: We have shown above that WASP's foci forming ability is required for PLCγ1 activation (Figure 4B). In order to test whether the independent elimination of foci by perturbation of Arp2/3 complex would phenocopy this effect, we chose to deplete foci using CK666 and monitor the consequences of this treatment on early TCR signaling, PLCγ1 activation and subsequent calcium ion signaling. CK666 treatment, which resulted in severe inhibition of F-actin foci (Figure 5B, Figure 6—figure supplement 1B), did not diminish TCR-proximal activation events. While fewer T cells formed synapses after CK666 treatment (37% reduction), upon adhesion, F-actin dependent processes including formation of TCR MC and exclusion of CD45 phosphatase from MCs (Campi et al., 2005; Varma et al., 2006) (Figure 6A), and the phosphorylation of Zap70 on Y319 (Figure 6B) proceeded normally in CK666-treated cells. In addition, other TCR-activation dependent signaling events leading to calcium flux, such as the phosphorylation of LAT on Y171 and SLP76 on Y145 were not altered in CK666-treated cells in comparison to control treated cells (data not shown). However, a severe defect was observed in the levels of TCR-induced PLCγ1 phosphorylation on Y783 after CK666 treatment (Figure 6C,D), where a drop was observed in both the number and intensity of phospho-PLCγ1 clusters at the synapse (Figure 6C). Furthermore, while phospho-PLCγ1 localized with F-actin foci in control cells, treatment with CK666 led to loss of foci-associated phospho-PLCγ1 (Figure 6E, Figure 6—figure supplement 1A). Upon attenuation of F-actin foci, total PLCγ1 levels were also reduced at the synapse, matching the decline in phospho-PLCγ1 levels (Figure 6F) and indicating that the impaired levels of phospho-PLCγ1 at the whole cell level may be accounted for by the reduction in PLCγ1 enrichment at the TCR signalosome. When assaying signaling events downstream of PLCγ1 activation, such as TCR-induced calcium ion elevation in the cytoplasm and nuclear mobilization of NFAT1, CK666-treated cells showed significant reduction in these processes (Figure 6G,H). Although a reduction in lamellipodial and lamellar actin was observed in CK666 treated cells (mean total F-actin 42% reduced), this reduction was relatively lower than foci depletion (mean 67% reduced) or under global F-actin depletion (CytoD, mean F-actin 90% reduced), and did not affect early TCR signaling (Figure 6—figure supplement 1B). We thus conclude that WASP and Arp2/3 dependent foci play a non-redundant role in PLCγ1 activation and calcium ion signaling at the synapse. In line with this result, CK666-treated cells exhibited a loss of phospho-HS1, as well as HS1, at the synapse (Figure 6—figure supplement 1C), similar to that observed in WASP−/− T cells (Figure 1E). Similar to the TCR-distal signaling defects observed in CK666 treated mouse CD4 T cells described above, CK666-treated human CD4 T cells also displayed a reduction in phospho-PLCγ1, but not in phospho-Zap70 (Figure 6—figure supplement 2), indicating that foci-dependent PLCγ1 activation cascade is conserved in T cells from diverse origins.10.7554/eLife.04953.027Figure 6.Arp2/3 inhibition leads to defective TCR-distal signaling.

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