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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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Arp2/3 complex inhibition and synaptic dynamics of Zap70 and PLCγ1 in live cells.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
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fig6s3: Arp2/3 complex inhibition and synaptic dynamics of Zap70 and PLCγ1 in live cells.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

Mentions: To directly visualize the involvement of F-actin foci in PLCγ1 recruitment and stabilization at the TCR signalosome in live cells, we transiently overexpressed PLCγ1-YFP in human CD4 T cells, and examined its distribution at the synapse on SLB presenting anti-CD3 and ICAM1 in real time. PLCγ1 localized with TCR microclusters in live cell synapse, and this distribution was lost upon treatment of cells with CK666 (Figure 6—figure supplement 3). As a control, the association of human Zap70-GFP with MC was maintained after CK666 treatment (Figure 6—figure supplement 3). However, due the mobile nature of MC, it was challenging to follow individual cluster before and after CK666 treatment in this setting. We thus chose to utilize anti-CD3 microdots to activate human T cells. Human CD4 T cells expressing PLCγ1-YFP were incubated with micron-size immobile anti-CD3 dots, and visualized using live cell imaging immediately after cell attachment. Similar to F-actin foci assembly at the microdot, PLCγ1 enrichment was also visible at the microdot sites. CK666 treatment of cells led to a reduction of PLCγ1 enrichment at microdots (Figure 6I,J). These data show that TCR MC associated F-actin foci assist in sustained accumulation of PLCγ1 at the TCR MC.


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)

Arp2/3 complex inhibition and synaptic dynamics of Zap70 and PLCγ1 in live cells.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
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Related In: Results  -  Collection

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fig6s3: Arp2/3 complex inhibition and synaptic dynamics of Zap70 and PLCγ1 in live cells.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
Mentions: To directly visualize the involvement of F-actin foci in PLCγ1 recruitment and stabilization at the TCR signalosome in live cells, we transiently overexpressed PLCγ1-YFP in human CD4 T cells, and examined its distribution at the synapse on SLB presenting anti-CD3 and ICAM1 in real time. PLCγ1 localized with TCR microclusters in live cell synapse, and this distribution was lost upon treatment of cells with CK666 (Figure 6—figure supplement 3). As a control, the association of human Zap70-GFP with MC was maintained after CK666 treatment (Figure 6—figure supplement 3). However, due the mobile nature of MC, it was challenging to follow individual cluster before and after CK666 treatment in this setting. We thus chose to utilize anti-CD3 microdots to activate human T cells. Human CD4 T cells expressing PLCγ1-YFP were incubated with micron-size immobile anti-CD3 dots, and visualized using live cell imaging immediately after cell attachment. Similar to F-actin foci assembly at the microdot, PLCγ1 enrichment was also visible at the microdot sites. CK666 treatment of cells led to a reduction of PLCγ1 enrichment at microdots (Figure 6I,J). These data show that TCR MC associated F-actin foci assist in sustained accumulation of PLCγ1 at the TCR MC.

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