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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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Lack of F-actin foci in the cSMAC of primary T cells and the Jurkat T cell line.AND CD4 T cell blasts were either incubated with bilayers containing Alexa568 tagged anti-CD3 and ICAM1 (far left panels) or were labeled with Alexa568-H57 Fab and incubated with MHCp/ICAM1 bilayers (center left panels), for 2 min. Cells were fixed and stained for F-actin using Alexa488-phalloidin (middle row, green) and visualized using spinning disc confocal microscopy. Each image is a maximum intensity projection of the bottom three planes that show high intensity signals from the bilayer. Note that TCR MCs (bottom row, red) in the F-actin depleted central zone of the cell (asterisk) exhibit no significant co-localization with actin foci. This phenomenon where central microclusters lack F-actin foci was observed in >90% cells exhibiting well-defined cSMAC (number of experiments >3). F-actin foci were not detected in Jurkat T cells (center right panel, and far right insets). Jurkat T cells were activated on bilayer containing Aexa568 tagged anti-CD3 (TCR, red, bottom row) and ICAM1 for 2 min and were fixed and stained with Alexa488-phalloidin (Actin, green, middle row). As highlighted in insets, visible F-actin enrichment is missing from the TCR MCs in these cells. Scale bars, 5 µm (AND T cell) and 10 µm (Jurkat T cell).DOI:http://dx.doi.org/10.7554/eLife.04953.014
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fig2s4: Lack of F-actin foci in the cSMAC of primary T cells and the Jurkat T cell line.AND CD4 T cell blasts were either incubated with bilayers containing Alexa568 tagged anti-CD3 and ICAM1 (far left panels) or were labeled with Alexa568-H57 Fab and incubated with MHCp/ICAM1 bilayers (center left panels), for 2 min. Cells were fixed and stained for F-actin using Alexa488-phalloidin (middle row, green) and visualized using spinning disc confocal microscopy. Each image is a maximum intensity projection of the bottom three planes that show high intensity signals from the bilayer. Note that TCR MCs (bottom row, red) in the F-actin depleted central zone of the cell (asterisk) exhibit no significant co-localization with actin foci. This phenomenon where central microclusters lack F-actin foci was observed in >90% cells exhibiting well-defined cSMAC (number of experiments >3). F-actin foci were not detected in Jurkat T cells (center right panel, and far right insets). Jurkat T cells were activated on bilayer containing Aexa568 tagged anti-CD3 (TCR, red, bottom row) and ICAM1 for 2 min and were fixed and stained with Alexa488-phalloidin (Actin, green, middle row). As highlighted in insets, visible F-actin enrichment is missing from the TCR MCs in these cells. Scale bars, 5 µm (AND T cell) and 10 µm (Jurkat T cell).DOI:http://dx.doi.org/10.7554/eLife.04953.014

Mentions: Since a fraction of TCR MC did not localize with foci, we sought to examine this population. High magnification microscopy of T cell synapses formed by mouse CD4 T cells on anti-CD3 and ICAM1 SLB provided insight into both synaptic and non-synaptic F-actin organization (Figure 2—figure supplement 4, Videos 1–2). As expected (Stinchcombe et al., 2006), the F-actin signal progressively decreases toward the synapse center where central TCR MC are devoid of F-actin foci (Figure 2—figure supplement 4, asterisks in the ‘Merge’ panel). To eliminate the possibility that the lack of foci on TCR MC in the nascent cSMAC is a consequence of cell fixation methodology, we examined foci in live T cells by transfecting them with LifeAct-GFP, a peptide construct that selectively labels F-actin (Riedl et al., 2008). Since primary mouse T cells showed poor survival after transfection, we used human primary CD4 T cells that exhibit higher viability (Chicaybam et al., 2013). In live cell synapses, LifeAct-GFP was observed to form foci on TCR MCs, which continued to co-migrate until the delivery of MCs to the cSMAC (Figure 2—figure supplement 5, Video 3). Analysis of kymographs revealed that the average TCR MC speed was 5.66 ± 2.2 μm/min, average Foci speed was 6.63 ± 3.1 μm/min (p = 0.21, ns). The TCR signals persisted in the cSMAC, whereas the GFP foci were extinguished, consistent with continual nucleation of F-actin at the MC site (Figure 1A) until it reaches the cSMAC, where TCR signals are known to be terminated (Vardhana et al., 2010; Choudhuri et al., 2014).Video 1.AND CD4 T cells were incubated with bilayer containing ICAM1 and Alexa568 tagged anti-CD3 (red) for 2 min, fixed and stained with Alexa488-phalloidin (green).


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)

Lack of F-actin foci in the cSMAC of primary T cells and the Jurkat T cell line.AND CD4 T cell blasts were either incubated with bilayers containing Alexa568 tagged anti-CD3 and ICAM1 (far left panels) or were labeled with Alexa568-H57 Fab and incubated with MHCp/ICAM1 bilayers (center left panels), for 2 min. Cells were fixed and stained for F-actin using Alexa488-phalloidin (middle row, green) and visualized using spinning disc confocal microscopy. Each image is a maximum intensity projection of the bottom three planes that show high intensity signals from the bilayer. Note that TCR MCs (bottom row, red) in the F-actin depleted central zone of the cell (asterisk) exhibit no significant co-localization with actin foci. This phenomenon where central microclusters lack F-actin foci was observed in >90% cells exhibiting well-defined cSMAC (number of experiments >3). F-actin foci were not detected in Jurkat T cells (center right panel, and far right insets). Jurkat T cells were activated on bilayer containing Aexa568 tagged anti-CD3 (TCR, red, bottom row) and ICAM1 for 2 min and were fixed and stained with Alexa488-phalloidin (Actin, green, middle row). As highlighted in insets, visible F-actin enrichment is missing from the TCR MCs in these cells. Scale bars, 5 µm (AND T cell) and 10 µm (Jurkat T cell).DOI:http://dx.doi.org/10.7554/eLife.04953.014
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fig2s4: Lack of F-actin foci in the cSMAC of primary T cells and the Jurkat T cell line.AND CD4 T cell blasts were either incubated with bilayers containing Alexa568 tagged anti-CD3 and ICAM1 (far left panels) or were labeled with Alexa568-H57 Fab and incubated with MHCp/ICAM1 bilayers (center left panels), for 2 min. Cells were fixed and stained for F-actin using Alexa488-phalloidin (middle row, green) and visualized using spinning disc confocal microscopy. Each image is a maximum intensity projection of the bottom three planes that show high intensity signals from the bilayer. Note that TCR MCs (bottom row, red) in the F-actin depleted central zone of the cell (asterisk) exhibit no significant co-localization with actin foci. This phenomenon where central microclusters lack F-actin foci was observed in >90% cells exhibiting well-defined cSMAC (number of experiments >3). F-actin foci were not detected in Jurkat T cells (center right panel, and far right insets). Jurkat T cells were activated on bilayer containing Aexa568 tagged anti-CD3 (TCR, red, bottom row) and ICAM1 for 2 min and were fixed and stained with Alexa488-phalloidin (Actin, green, middle row). As highlighted in insets, visible F-actin enrichment is missing from the TCR MCs in these cells. Scale bars, 5 µm (AND T cell) and 10 µm (Jurkat T cell).DOI:http://dx.doi.org/10.7554/eLife.04953.014
Mentions: Since a fraction of TCR MC did not localize with foci, we sought to examine this population. High magnification microscopy of T cell synapses formed by mouse CD4 T cells on anti-CD3 and ICAM1 SLB provided insight into both synaptic and non-synaptic F-actin organization (Figure 2—figure supplement 4, Videos 1–2). As expected (Stinchcombe et al., 2006), the F-actin signal progressively decreases toward the synapse center where central TCR MC are devoid of F-actin foci (Figure 2—figure supplement 4, asterisks in the ‘Merge’ panel). To eliminate the possibility that the lack of foci on TCR MC in the nascent cSMAC is a consequence of cell fixation methodology, we examined foci in live T cells by transfecting them with LifeAct-GFP, a peptide construct that selectively labels F-actin (Riedl et al., 2008). Since primary mouse T cells showed poor survival after transfection, we used human primary CD4 T cells that exhibit higher viability (Chicaybam et al., 2013). In live cell synapses, LifeAct-GFP was observed to form foci on TCR MCs, which continued to co-migrate until the delivery of MCs to the cSMAC (Figure 2—figure supplement 5, Video 3). Analysis of kymographs revealed that the average TCR MC speed was 5.66 ± 2.2 μm/min, average Foci speed was 6.63 ± 3.1 μm/min (p = 0.21, ns). The TCR signals persisted in the cSMAC, whereas the GFP foci were extinguished, consistent with continual nucleation of F-actin at the MC site (Figure 1A) until it reaches the cSMAC, where TCR signals are known to be terminated (Vardhana et al., 2010; Choudhuri et al., 2014).Video 1.AND CD4 T cells were incubated with bilayer containing ICAM1 and Alexa568 tagged anti-CD3 (red) for 2 min, fixed and stained with Alexa488-phalloidin (green).

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