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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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Barbed-end labeling of polymerizing actin filaments in Human CD4 T cells. T cells were incubated with coverslips coated with anti-CD3 and ICAM1 for 5 min, and then processed for barbed end labeling and visualized using TIRF microscopy as described in Methods and Figure 1A. The images show total F-actin (Alexa488-phalloidin, left image) and barbed ends (Rhodamine-phalloidin or ‘fresh F-actin’, middle image) labeled within 1 min of actin polymerization at the synapse. The foci areas in the total F-actin image were identified and outlined by intensity rank-based filtering. These outlined areas were then analyzed in both ‘fresh F-actin’ (barbed end labeled), and ‘total F-actin’ images. The average intensity per pixel within foci regions (‘F-actin foci’), and outside the foci area (lamellipodial surround), was measured and plotted (bottom right graph). The bottom left graph shows the normalization of fresh F-actin (graph on the right) with total F-actin in the same area. n=17 cells, for right graph, p<0.0001, for the graph on the left, p=0.9.DOI:http://dx.doi.org/10.7554/eLife.04953.036
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fig9: Barbed-end labeling of polymerizing actin filaments in Human CD4 T cells. T cells were incubated with coverslips coated with anti-CD3 and ICAM1 for 5 min, and then processed for barbed end labeling and visualized using TIRF microscopy as described in Methods and Figure 1A. The images show total F-actin (Alexa488-phalloidin, left image) and barbed ends (Rhodamine-phalloidin or ‘fresh F-actin’, middle image) labeled within 1 min of actin polymerization at the synapse. The foci areas in the total F-actin image were identified and outlined by intensity rank-based filtering. These outlined areas were then analyzed in both ‘fresh F-actin’ (barbed end labeled), and ‘total F-actin’ images. The average intensity per pixel within foci regions (‘F-actin foci’), and outside the foci area (lamellipodial surround), was measured and plotted (bottom right graph). The bottom left graph shows the normalization of fresh F-actin (graph on the right) with total F-actin in the same area. n=17 cells, for right graph, p<0.0001, for the graph on the left, p=0.9.DOI:http://dx.doi.org/10.7554/eLife.04953.036

Mentions: Activation of TCR transgenic CD4 T cells using agoinst peptide-MHC is the most physiological mode of stimulation with SLB as both TCR and CD4 are engaged with natural ligands. This system has been routinely utilized for high resolution characterization of cytoskeletal organization at T cell immune synapse (Beemiller et al., 2012; Hashimoto-Tane et al., 2011; Kumari et al., 2012). Using siRNA-mediated silencing, we found that the mechanism of foci formation was indeed WASP dependent in this system (30% reduction in foci upon partial WASP silencing, data not shown), and was comparable to foci induction in cells activated using anti-CD3 (Results section, paragraph 1, 27% reduction in foci in T cells activated using anti-CD3, p=0.0025 (Figure 1–figure supplement 3); 30% reduction in foci in T cells activated using MHCp, p=0.0005 (data removed now), upon partial WASP silencing]. We have now replaced the MHCp data with anti-CD3 data, for a more coherent presentation of results. We continued to employ MHCp-based activation for most colocalization experiments in the paper, as this system provided cleaner staining with some antibodies, including the mouse monoclonal anti-Arp3 antibody (Figure 5A), which would otherwise cross-react with agonist 2C11 (hamster in origin) antibody, and NWASP and WAVE2 antibodies (Author response image 2). In addition, since this system utilized activated CD4 T cells, which are larger and more suitable for transfection procedures than primary T cells, we were able to achieve higher transfection and survival rates for siRNA experiments, and better spatial resolution for colocalization experiments.10.7554/eLife.04953.036Author response image 2.


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)

Barbed-end labeling of polymerizing actin filaments in Human CD4 T cells. T cells were incubated with coverslips coated with anti-CD3 and ICAM1 for 5 min, and then processed for barbed end labeling and visualized using TIRF microscopy as described in Methods and Figure 1A. The images show total F-actin (Alexa488-phalloidin, left image) and barbed ends (Rhodamine-phalloidin or ‘fresh F-actin’, middle image) labeled within 1 min of actin polymerization at the synapse. The foci areas in the total F-actin image were identified and outlined by intensity rank-based filtering. These outlined areas were then analyzed in both ‘fresh F-actin’ (barbed end labeled), and ‘total F-actin’ images. The average intensity per pixel within foci regions (‘F-actin foci’), and outside the foci area (lamellipodial surround), was measured and plotted (bottom right graph). The bottom left graph shows the normalization of fresh F-actin (graph on the right) with total F-actin in the same area. n=17 cells, for right graph, p<0.0001, for the graph on the left, p=0.9.DOI:http://dx.doi.org/10.7554/eLife.04953.036
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Related In: Results  -  Collection

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fig9: Barbed-end labeling of polymerizing actin filaments in Human CD4 T cells. T cells were incubated with coverslips coated with anti-CD3 and ICAM1 for 5 min, and then processed for barbed end labeling and visualized using TIRF microscopy as described in Methods and Figure 1A. The images show total F-actin (Alexa488-phalloidin, left image) and barbed ends (Rhodamine-phalloidin or ‘fresh F-actin’, middle image) labeled within 1 min of actin polymerization at the synapse. The foci areas in the total F-actin image were identified and outlined by intensity rank-based filtering. These outlined areas were then analyzed in both ‘fresh F-actin’ (barbed end labeled), and ‘total F-actin’ images. The average intensity per pixel within foci regions (‘F-actin foci’), and outside the foci area (lamellipodial surround), was measured and plotted (bottom right graph). The bottom left graph shows the normalization of fresh F-actin (graph on the right) with total F-actin in the same area. n=17 cells, for right graph, p<0.0001, for the graph on the left, p=0.9.DOI:http://dx.doi.org/10.7554/eLife.04953.036
Mentions: Activation of TCR transgenic CD4 T cells using agoinst peptide-MHC is the most physiological mode of stimulation with SLB as both TCR and CD4 are engaged with natural ligands. This system has been routinely utilized for high resolution characterization of cytoskeletal organization at T cell immune synapse (Beemiller et al., 2012; Hashimoto-Tane et al., 2011; Kumari et al., 2012). Using siRNA-mediated silencing, we found that the mechanism of foci formation was indeed WASP dependent in this system (30% reduction in foci upon partial WASP silencing, data not shown), and was comparable to foci induction in cells activated using anti-CD3 (Results section, paragraph 1, 27% reduction in foci in T cells activated using anti-CD3, p=0.0025 (Figure 1–figure supplement 3); 30% reduction in foci in T cells activated using MHCp, p=0.0005 (data removed now), upon partial WASP silencing]. We have now replaced the MHCp data with anti-CD3 data, for a more coherent presentation of results. We continued to employ MHCp-based activation for most colocalization experiments in the paper, as this system provided cleaner staining with some antibodies, including the mouse monoclonal anti-Arp3 antibody (Figure 5A), which would otherwise cross-react with agonist 2C11 (hamster in origin) antibody, and NWASP and WAVE2 antibodies (Author response image 2). In addition, since this system utilized activated CD4 T cells, which are larger and more suitable for transfection procedures than primary T cells, we were able to achieve higher transfection and survival rates for siRNA experiments, and better spatial resolution for colocalization experiments.10.7554/eLife.04953.036Author response image 2.

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