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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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F-actin foci on microdots are not enriched in phospholipid membranes.(A) F-actin enrichment at the microdot is not a consequence of cell morphology around the microdot. Human CD4 T cells were labeled with CM-DiI according to manufacturer's protocol and were then incubated with glass coverslip coated with patterned Alexa647 tagged anti-CD3 and ICAM1 for 5 min, fixed and stained with Alexa488-phalloidin. Cells were then imaged using TIRF microscopy. The graphs on the bottom panels show line-scan profiles of anti-CD3 (TCR, red, left), actin (Green, center) and DiI (blue, right) acquired from 70 different microdots. The TCR, actin and DiI intensities were measured from identical pixel positions for a given microdot, were then normalized by the lowest pixel intensity per microdot for a given fluorescent channel, and plotted. Note that while actin shows enrichment at the microdot site, DiI is not enriched at the same position. (B) The same procedure as described above, except with the use of Cy3-anti- LFA1 Fab fragments instead of DiI, was carried out in T cells, and the normalized line-scan intensities were plotted as average value per pixel position ±SEM. The graph represents mean of normalized intensities across 25 pixels, acquired using 74 different microdots. In both these experiments (A, B), n > 20.DOI:http://dx.doi.org/10.7554/eLife.04953.025
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fig5s2: F-actin foci on microdots are not enriched in phospholipid membranes.(A) F-actin enrichment at the microdot is not a consequence of cell morphology around the microdot. Human CD4 T cells were labeled with CM-DiI according to manufacturer's protocol and were then incubated with glass coverslip coated with patterned Alexa647 tagged anti-CD3 and ICAM1 for 5 min, fixed and stained with Alexa488-phalloidin. Cells were then imaged using TIRF microscopy. The graphs on the bottom panels show line-scan profiles of anti-CD3 (TCR, red, left), actin (Green, center) and DiI (blue, right) acquired from 70 different microdots. The TCR, actin and DiI intensities were measured from identical pixel positions for a given microdot, were then normalized by the lowest pixel intensity per microdot for a given fluorescent channel, and plotted. Note that while actin shows enrichment at the microdot site, DiI is not enriched at the same position. (B) The same procedure as described above, except with the use of Cy3-anti- LFA1 Fab fragments instead of DiI, was carried out in T cells, and the normalized line-scan intensities were plotted as average value per pixel position ±SEM. The graph represents mean of normalized intensities across 25 pixels, acquired using 74 different microdots. In both these experiments (A, B), n > 20.DOI:http://dx.doi.org/10.7554/eLife.04953.025

Mentions: Since CK666 treatment can rapidly eliminate foci, we utilized CK666 to probe the kinetics of foci formation at individual TCR MCs. Given the small size and large lamellar actin background at the primary T cell synapse as well as highly mobile nature of MCs, this analysis would be extremely challenging to perform in the bilayer system. Therefore, we activated T cells on micron-scale anti-CD3 dots adsorbed on a glass surface where the remaining space was coated with ICAM1 in an attempt to restrict TCR signaling to a known, micron scale location in an artificial immunological synapse. Due to their aforementioned performance in time-lapse studies after transfection, human CD4 T cells were utilized for LifeAct-GFP transfection and live imaging on anti-CD3 microdots. Incubation with the patterned surface triggered rapid actin polymerization at the anti-CD3 microdot, reflected in LifeAct-GFP accumulation (Figure 5C–E). This enrichment of LifeAct at the microdot was not due to the topography of the cell contact interface (Owen et al., 2013), since a global membrane labeling dye, DiI, and integrin LFA1 were not enriched at these sites (Figure 5—figure supplement 2A,B). Within 5 s of CK666 treatment, pre-existing LifeAct enrichment was lost from the microdot site (Figure 5C–EVideo 5) and fresh microdot contact events failed to elicit LifeAct-GFP enrichment in the presence of CK666 (Video 5). These data confirm that the TCR engagement sites dictate Arp2/3 complex-dependent F-actin foci formation at the synapse, de novo. Furthermore, ligand microdots can spatially pattern F-actin foci by predefining the sites of TCR engagement, and thus provide a suitable platform for spatially isolating TCR dependent mechanisms.Video 5.Human CD4 T cells were transfected with LifeAct-GFP plasmid (green) and incubated with ICAM1-coated glass substrate with 1 µm anti-CD3 printed dots (red) on stage at 37°C and imaged live.


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)

F-actin foci on microdots are not enriched in phospholipid membranes.(A) F-actin enrichment at the microdot is not a consequence of cell morphology around the microdot. Human CD4 T cells were labeled with CM-DiI according to manufacturer's protocol and were then incubated with glass coverslip coated with patterned Alexa647 tagged anti-CD3 and ICAM1 for 5 min, fixed and stained with Alexa488-phalloidin. Cells were then imaged using TIRF microscopy. The graphs on the bottom panels show line-scan profiles of anti-CD3 (TCR, red, left), actin (Green, center) and DiI (blue, right) acquired from 70 different microdots. The TCR, actin and DiI intensities were measured from identical pixel positions for a given microdot, were then normalized by the lowest pixel intensity per microdot for a given fluorescent channel, and plotted. Note that while actin shows enrichment at the microdot site, DiI is not enriched at the same position. (B) The same procedure as described above, except with the use of Cy3-anti- LFA1 Fab fragments instead of DiI, was carried out in T cells, and the normalized line-scan intensities were plotted as average value per pixel position ±SEM. The graph represents mean of normalized intensities across 25 pixels, acquired using 74 different microdots. In both these experiments (A, B), n > 20.DOI:http://dx.doi.org/10.7554/eLife.04953.025
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
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fig5s2: F-actin foci on microdots are not enriched in phospholipid membranes.(A) F-actin enrichment at the microdot is not a consequence of cell morphology around the microdot. Human CD4 T cells were labeled with CM-DiI according to manufacturer's protocol and were then incubated with glass coverslip coated with patterned Alexa647 tagged anti-CD3 and ICAM1 for 5 min, fixed and stained with Alexa488-phalloidin. Cells were then imaged using TIRF microscopy. The graphs on the bottom panels show line-scan profiles of anti-CD3 (TCR, red, left), actin (Green, center) and DiI (blue, right) acquired from 70 different microdots. The TCR, actin and DiI intensities were measured from identical pixel positions for a given microdot, were then normalized by the lowest pixel intensity per microdot for a given fluorescent channel, and plotted. Note that while actin shows enrichment at the microdot site, DiI is not enriched at the same position. (B) The same procedure as described above, except with the use of Cy3-anti- LFA1 Fab fragments instead of DiI, was carried out in T cells, and the normalized line-scan intensities were plotted as average value per pixel position ±SEM. The graph represents mean of normalized intensities across 25 pixels, acquired using 74 different microdots. In both these experiments (A, B), n > 20.DOI:http://dx.doi.org/10.7554/eLife.04953.025
Mentions: Since CK666 treatment can rapidly eliminate foci, we utilized CK666 to probe the kinetics of foci formation at individual TCR MCs. Given the small size and large lamellar actin background at the primary T cell synapse as well as highly mobile nature of MCs, this analysis would be extremely challenging to perform in the bilayer system. Therefore, we activated T cells on micron-scale anti-CD3 dots adsorbed on a glass surface where the remaining space was coated with ICAM1 in an attempt to restrict TCR signaling to a known, micron scale location in an artificial immunological synapse. Due to their aforementioned performance in time-lapse studies after transfection, human CD4 T cells were utilized for LifeAct-GFP transfection and live imaging on anti-CD3 microdots. Incubation with the patterned surface triggered rapid actin polymerization at the anti-CD3 microdot, reflected in LifeAct-GFP accumulation (Figure 5C–E). This enrichment of LifeAct at the microdot was not due to the topography of the cell contact interface (Owen et al., 2013), since a global membrane labeling dye, DiI, and integrin LFA1 were not enriched at these sites (Figure 5—figure supplement 2A,B). Within 5 s of CK666 treatment, pre-existing LifeAct enrichment was lost from the microdot site (Figure 5C–EVideo 5) and fresh microdot contact events failed to elicit LifeAct-GFP enrichment in the presence of CK666 (Video 5). These data confirm that the TCR engagement sites dictate Arp2/3 complex-dependent F-actin foci formation at the synapse, de novo. Furthermore, ligand microdots can spatially pattern F-actin foci by predefining the sites of TCR engagement, and thus provide a suitable platform for spatially isolating TCR dependent mechanisms.Video 5.Human CD4 T cells were transfected with LifeAct-GFP plasmid (green) and incubated with ICAM1-coated glass substrate with 1 µm anti-CD3 printed dots (red) on stage at 37°C and imaged live.

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