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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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Test for co-localization of foci and MCs.(A) An example of image analysis carried out to assess co-localization between F-actin foci and TCR/ICAM1, using 50% rank-filter based processing, as described in Figure 1–figure supplement 1A (B) TCR MC and F-actin foci co-localization is significantly higher than chance overlap. To measure chance level co-localization, the actin images from Figure 1 were shifted by 5 pixels in x and y dimensions (schematic on top), and analyzed for co-localization with respect to the unshifted TCR (left graph) or ICAM1 (right graph) images. Note that while pixel shift results in significant reduction TCR-F-actin foci co-localization (left graph, p < 0.0001), levels of ICAM1/F-actin foci association are unaffected (right graph, p = 0.53).DOI:http://dx.doi.org/10.7554/eLife.04953.011
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fig2s1: Test for co-localization of foci and MCs.(A) An example of image analysis carried out to assess co-localization between F-actin foci and TCR/ICAM1, using 50% rank-filter based processing, as described in Figure 1–figure supplement 1A (B) TCR MC and F-actin foci co-localization is significantly higher than chance overlap. To measure chance level co-localization, the actin images from Figure 1 were shifted by 5 pixels in x and y dimensions (schematic on top), and analyzed for co-localization with respect to the unshifted TCR (left graph) or ICAM1 (right graph) images. Note that while pixel shift results in significant reduction TCR-F-actin foci co-localization (left graph, p < 0.0001), levels of ICAM1/F-actin foci association are unaffected (right graph, p = 0.53).DOI:http://dx.doi.org/10.7554/eLife.04953.011

Mentions: To understand the mechanism underlying formation of the F-actin foci, we determined if they were associated with TCR MCs. In murine polyclonal CD4 T cells activated on anti-CD3 and ICAM1 containing bilayers, as in Figure 1, TCR MC localized with F-actin foci (Figure 2A, upper panels, arrows). Similar co-localization of F-actin foci with TCR MC was observed with monoclonal AND TCR transgenic T cells stimulated with MHCp and ICAM1 (Figure 2A, lower panels). The tracking of TCR with H57 Fab allowed accurate quantification of co-localization between TCR and F-actin. After 1 min of cell attachment, 40 ± 1.7% of TCR MCs co-localized with F-actin foci in these cells (Figure 2—figure supplement 1A, ‘Materials and methods’, Figure 2B), significantly above chance level co-localization (Figure 2—figure supplement 1B), whereas only 15 ± 0.07% of total ICAM1 exhibited association with actin foci (Figure 2B), which was not different from chance. When examined in Human primary CD4 T cells as well, F-actin foci formed in WASP-dependent manner (Figure 2—figure supplement 2A,B), and TCR MCs, but not ICAM1 MCs, were associated with F-actin foci (TCR co-localization with F-actin foci was 34.5% ± 2.0, n = 76, co-localization of ICAM1 with F-actin foci was 13.1% ± 0.8, n = 66) (Figure 2C,D). Furthermore, TCR-activation led to HS1 phosphorylation at the T cell synapse, as shown previously (Figure 2—figure supplement 3A) (Hutchcroft et al., 1998; Gomez et al., 2006), and the phospho-HS1 co-localized with F-actin foci in both TCR-activation stimuli (Figure 2—figure supplement 3B). This indicated that TCR-MCs associated foci exist in a variety of primary CD4 T cells including mouse and human primary T cells, stimulated with diverse TCR-triggering contexts, namely anti-CD3 and peptide-MHC complexes.10.7554/eLife.04953.010Figure 2.F-actin foci co-localize with TCR MC and not ICAM1.


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)

Test for co-localization of foci and MCs.(A) An example of image analysis carried out to assess co-localization between F-actin foci and TCR/ICAM1, using 50% rank-filter based processing, as described in Figure 1–figure supplement 1A (B) TCR MC and F-actin foci co-localization is significantly higher than chance overlap. To measure chance level co-localization, the actin images from Figure 1 were shifted by 5 pixels in x and y dimensions (schematic on top), and analyzed for co-localization with respect to the unshifted TCR (left graph) or ICAM1 (right graph) images. Note that while pixel shift results in significant reduction TCR-F-actin foci co-localization (left graph, p < 0.0001), levels of ICAM1/F-actin foci association are unaffected (right graph, p = 0.53).DOI:http://dx.doi.org/10.7554/eLife.04953.011
© Copyright Policy
Related In: Results  -  Collection

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

fig2s1: Test for co-localization of foci and MCs.(A) An example of image analysis carried out to assess co-localization between F-actin foci and TCR/ICAM1, using 50% rank-filter based processing, as described in Figure 1–figure supplement 1A (B) TCR MC and F-actin foci co-localization is significantly higher than chance overlap. To measure chance level co-localization, the actin images from Figure 1 were shifted by 5 pixels in x and y dimensions (schematic on top), and analyzed for co-localization with respect to the unshifted TCR (left graph) or ICAM1 (right graph) images. Note that while pixel shift results in significant reduction TCR-F-actin foci co-localization (left graph, p < 0.0001), levels of ICAM1/F-actin foci association are unaffected (right graph, p = 0.53).DOI:http://dx.doi.org/10.7554/eLife.04953.011
Mentions: To understand the mechanism underlying formation of the F-actin foci, we determined if they were associated with TCR MCs. In murine polyclonal CD4 T cells activated on anti-CD3 and ICAM1 containing bilayers, as in Figure 1, TCR MC localized with F-actin foci (Figure 2A, upper panels, arrows). Similar co-localization of F-actin foci with TCR MC was observed with monoclonal AND TCR transgenic T cells stimulated with MHCp and ICAM1 (Figure 2A, lower panels). The tracking of TCR with H57 Fab allowed accurate quantification of co-localization between TCR and F-actin. After 1 min of cell attachment, 40 ± 1.7% of TCR MCs co-localized with F-actin foci in these cells (Figure 2—figure supplement 1A, ‘Materials and methods’, Figure 2B), significantly above chance level co-localization (Figure 2—figure supplement 1B), whereas only 15 ± 0.07% of total ICAM1 exhibited association with actin foci (Figure 2B), which was not different from chance. When examined in Human primary CD4 T cells as well, F-actin foci formed in WASP-dependent manner (Figure 2—figure supplement 2A,B), and TCR MCs, but not ICAM1 MCs, were associated with F-actin foci (TCR co-localization with F-actin foci was 34.5% ± 2.0, n = 76, co-localization of ICAM1 with F-actin foci was 13.1% ± 0.8, n = 66) (Figure 2C,D). Furthermore, TCR-activation led to HS1 phosphorylation at the T cell synapse, as shown previously (Figure 2—figure supplement 3A) (Hutchcroft et al., 1998; Gomez et al., 2006), and the phospho-HS1 co-localized with F-actin foci in both TCR-activation stimuli (Figure 2—figure supplement 3B). This indicated that TCR-MCs associated foci exist in a variety of primary CD4 T cells including mouse and human primary T cells, stimulated with diverse TCR-triggering contexts, namely anti-CD3 and peptide-MHC complexes.10.7554/eLife.04953.010Figure 2.F-actin foci co-localize with TCR MC and not ICAM1.

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