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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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Prolonged Lack of foci in WASP-/- T cells. CD4 T cells from WT mice were isolated and labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester, 5μM, pseudocolored green). CFSE labeled WT cells were mixed with WASP-/- T cells, incubated with surface coated with 2C11 and ICAM1 for 10 min (upper panels) or 1 hour (bottom panels), fixed and stained with Alexa568-phalloidin (pseudocolored red). The synaptic contacts cells were then imaged using spinning disc confocal microscope. Note that while WT cells (CFSE positive, green) maintain foci at 1 hour post activation, WASP-/- T cells (arrowheads) persistently lack foci. This lack of foci is clearly visible in more than 85% of WASP-/- T cells, both at 10 min and 1 hour (n>48).DOI:http://dx.doi.org/10.7554/eLife.04953.039
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fig12: Prolonged Lack of foci in WASP-/- T cells. CD4 T cells from WT mice were isolated and labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester, 5μM, pseudocolored green). CFSE labeled WT cells were mixed with WASP-/- T cells, incubated with surface coated with 2C11 and ICAM1 for 10 min (upper panels) or 1 hour (bottom panels), fixed and stained with Alexa568-phalloidin (pseudocolored red). The synaptic contacts cells were then imaged using spinning disc confocal microscope. Note that while WT cells (CFSE positive, green) maintain foci at 1 hour post activation, WASP-/- T cells (arrowheads) persistently lack foci. This lack of foci is clearly visible in more than 85% of WASP-/- T cells, both at 10 min and 1 hour (n>48).DOI:http://dx.doi.org/10.7554/eLife.04953.039

Mentions: We and others have previously shown that in WASP deficiency, the cell proliferation and cytokine secretion defects are of reduced amplitude, rather than delayed kinetics (Cannon et al. , 2004; Sims et al ., 2007; Zhang et al., 1999), since they manifest after prolonged culture of cells (30 min-24 hours). Our current data shows that the differences in F-actin foci between WT and WASP- /- or Arp2/3 inhibited cells are also of magnitude rather than kinetics. WASP-/- T cells fail to recover foci even after 1 hour of synapse formation (Author response image 5). Similarly in CK666 treatment, foci are permanently lost from the synapse even after prolonged incubation with the bilayer (Figure 6–figure supplement 1B), and do not recover even after 1 hour of incubation (data not shown). Thus, delayed kinetics of foci formation cannot explain the loss of foci in WASP-/- or CK666-treated cells.10.7554/eLife.04953.039Author response image 5.


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)

Prolonged Lack of foci in WASP-/- T cells. CD4 T cells from WT mice were isolated and labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester, 5μM, pseudocolored green). CFSE labeled WT cells were mixed with WASP-/- T cells, incubated with surface coated with 2C11 and ICAM1 for 10 min (upper panels) or 1 hour (bottom panels), fixed and stained with Alexa568-phalloidin (pseudocolored red). The synaptic contacts cells were then imaged using spinning disc confocal microscope. Note that while WT cells (CFSE positive, green) maintain foci at 1 hour post activation, WASP-/- T cells (arrowheads) persistently lack foci. This lack of foci is clearly visible in more than 85% of WASP-/- T cells, both at 10 min and 1 hour (n>48).DOI:http://dx.doi.org/10.7554/eLife.04953.039
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4355629&req=5

fig12: Prolonged Lack of foci in WASP-/- T cells. CD4 T cells from WT mice were isolated and labeled with CFSE (carboxyfluorescein diacetate, succinimidyl ester, 5μM, pseudocolored green). CFSE labeled WT cells were mixed with WASP-/- T cells, incubated with surface coated with 2C11 and ICAM1 for 10 min (upper panels) or 1 hour (bottom panels), fixed and stained with Alexa568-phalloidin (pseudocolored red). The synaptic contacts cells were then imaged using spinning disc confocal microscope. Note that while WT cells (CFSE positive, green) maintain foci at 1 hour post activation, WASP-/- T cells (arrowheads) persistently lack foci. This lack of foci is clearly visible in more than 85% of WASP-/- T cells, both at 10 min and 1 hour (n>48).DOI:http://dx.doi.org/10.7554/eLife.04953.039
Mentions: We and others have previously shown that in WASP deficiency, the cell proliferation and cytokine secretion defects are of reduced amplitude, rather than delayed kinetics (Cannon et al. , 2004; Sims et al ., 2007; Zhang et al., 1999), since they manifest after prolonged culture of cells (30 min-24 hours). Our current data shows that the differences in F-actin foci between WT and WASP- /- or Arp2/3 inhibited cells are also of magnitude rather than kinetics. WASP-/- T cells fail to recover foci even after 1 hour of synapse formation (Author response image 5). Similarly in CK666 treatment, foci are permanently lost from the synapse even after prolonged incubation with the bilayer (Figure 6–figure supplement 1B), and do not recover even after 1 hour of incubation (data not shown). Thus, delayed kinetics of foci formation cannot explain the loss of foci in WASP-/- or CK666-treated cells.10.7554/eLife.04953.039Author response image 5.

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