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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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WASP family members NWASP and WAVE2 are not associated with F-actin foci.(A) TCR-induced recruitment of NWASP and WAVE2 to IS. Mouse primary WT CD4 T cells were incubated with bilayer containing ICAM1 alone (−) or both ICAM1 and anti-CD3 (+) for 2 min at 37°C, fixed and immunostained for endogenous proteins. Stained cells were visualized using TIRF microscopy. The graph shows quantitation of antibody fluorescence at IS, where each point represents the value obtained from a single cell. n1 = 16, n2 = 54 (for WAVE2), n3 = 16, n4 = 78 (for NWASP); p1, p2 < 0.0001. Each point represents average levels of indicated protein at synapse in a single cell. (B) The images shown are TIRF plane distributions of the indicated proteins. As elaborated in the magnified areas marked with white boundary in original ‘merge’ image, there is a lack of co-localization between either of these proteins and TCR MCs. Scale bar, 5 μm. Insets in (B) have been intensity scaled differently from original ‘Merge’ panel to highlight protein distribution with more clarity.DOI:http://dx.doi.org/10.7554/eLife.04953.007
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fig1s4: WASP family members NWASP and WAVE2 are not associated with F-actin foci.(A) TCR-induced recruitment of NWASP and WAVE2 to IS. Mouse primary WT CD4 T cells were incubated with bilayer containing ICAM1 alone (−) or both ICAM1 and anti-CD3 (+) for 2 min at 37°C, fixed and immunostained for endogenous proteins. Stained cells were visualized using TIRF microscopy. The graph shows quantitation of antibody fluorescence at IS, where each point represents the value obtained from a single cell. n1 = 16, n2 = 54 (for WAVE2), n3 = 16, n4 = 78 (for NWASP); p1, p2 < 0.0001. Each point represents average levels of indicated protein at synapse in a single cell. (B) The images shown are TIRF plane distributions of the indicated proteins. As elaborated in the magnified areas marked with white boundary in original ‘merge’ image, there is a lack of co-localization between either of these proteins and TCR MCs. Scale bar, 5 μm. Insets in (B) have been intensity scaled differently from original ‘Merge’ panel to highlight protein distribution with more clarity.DOI:http://dx.doi.org/10.7554/eLife.04953.007

Mentions: We next assessed the role of other WASP-family proteins—NWASP and WAVE2—in generating F-actin foci in WT cells. NWASP (WASL) is a close homolog of WASP and is capable of compensating for WASP's function in T cell development (Cotta-de-Almeida et al., 2007). However, primary CD4 T cells from Wasl−/− mice (Figure 1D) formed immunological synapses with F-actin foci similar to T cells from WT mice. WAVE2 is required for immunological synapse formation itself (Nolz et al., 2006), thus we could not examine synaptic foci in WAVE2 deficient T cells. Instead, we determined the localization of NWASP and WAVE2 in the immunological synapse in order to gain further insight into their possible role in foci formation. Activation of T cells with anti-CD3 and ICAM1 recruited NWASP and WAVE2 to the IS (Figure 1—figure supplement 4A) (Nolz et al., 2006), but the recruited proteins failed to co-localize with F-actin foci (Figure 1—figure supplement 4B, NWASP coloc. = 9.48% ± 0.76 n = 78; WAVE2 coloc. = 14.36% ± 1.25, n = 54). We were unable to identify suitable reagents for staining of endogenous WASP in the immunological synapse. These results demonstrate that NWASP does not contribute to F-actin foci and suggest that WAVE2 is unlikely to contribute directly to formation of F-actin foci.


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)

WASP family members NWASP and WAVE2 are not associated with F-actin foci.(A) TCR-induced recruitment of NWASP and WAVE2 to IS. Mouse primary WT CD4 T cells were incubated with bilayer containing ICAM1 alone (−) or both ICAM1 and anti-CD3 (+) for 2 min at 37°C, fixed and immunostained for endogenous proteins. Stained cells were visualized using TIRF microscopy. The graph shows quantitation of antibody fluorescence at IS, where each point represents the value obtained from a single cell. n1 = 16, n2 = 54 (for WAVE2), n3 = 16, n4 = 78 (for NWASP); p1, p2 < 0.0001. Each point represents average levels of indicated protein at synapse in a single cell. (B) The images shown are TIRF plane distributions of the indicated proteins. As elaborated in the magnified areas marked with white boundary in original ‘merge’ image, there is a lack of co-localization between either of these proteins and TCR MCs. Scale bar, 5 μm. Insets in (B) have been intensity scaled differently from original ‘Merge’ panel to highlight protein distribution with more clarity.DOI:http://dx.doi.org/10.7554/eLife.04953.007
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fig1s4: WASP family members NWASP and WAVE2 are not associated with F-actin foci.(A) TCR-induced recruitment of NWASP and WAVE2 to IS. Mouse primary WT CD4 T cells were incubated with bilayer containing ICAM1 alone (−) or both ICAM1 and anti-CD3 (+) for 2 min at 37°C, fixed and immunostained for endogenous proteins. Stained cells were visualized using TIRF microscopy. The graph shows quantitation of antibody fluorescence at IS, where each point represents the value obtained from a single cell. n1 = 16, n2 = 54 (for WAVE2), n3 = 16, n4 = 78 (for NWASP); p1, p2 < 0.0001. Each point represents average levels of indicated protein at synapse in a single cell. (B) The images shown are TIRF plane distributions of the indicated proteins. As elaborated in the magnified areas marked with white boundary in original ‘merge’ image, there is a lack of co-localization between either of these proteins and TCR MCs. Scale bar, 5 μm. Insets in (B) have been intensity scaled differently from original ‘Merge’ panel to highlight protein distribution with more clarity.DOI:http://dx.doi.org/10.7554/eLife.04953.007
Mentions: We next assessed the role of other WASP-family proteins—NWASP and WAVE2—in generating F-actin foci in WT cells. NWASP (WASL) is a close homolog of WASP and is capable of compensating for WASP's function in T cell development (Cotta-de-Almeida et al., 2007). However, primary CD4 T cells from Wasl−/− mice (Figure 1D) formed immunological synapses with F-actin foci similar to T cells from WT mice. WAVE2 is required for immunological synapse formation itself (Nolz et al., 2006), thus we could not examine synaptic foci in WAVE2 deficient T cells. Instead, we determined the localization of NWASP and WAVE2 in the immunological synapse in order to gain further insight into their possible role in foci formation. Activation of T cells with anti-CD3 and ICAM1 recruited NWASP and WAVE2 to the IS (Figure 1—figure supplement 4A) (Nolz et al., 2006), but the recruited proteins failed to co-localize with F-actin foci (Figure 1—figure supplement 4B, NWASP coloc. = 9.48% ± 0.76 n = 78; WAVE2 coloc. = 14.36% ± 1.25, n = 54). We were unable to identify suitable reagents for staining of endogenous WASP in the immunological synapse. These results demonstrate that NWASP does not contribute to F-actin foci and suggest that WAVE2 is unlikely to contribute directly to formation of F-actin foci.

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