Limits...
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.

Show MeSH

Related in: MedlinePlus

ILP F-actin and signaling in T-EC immunological synapse is dependent on Arp2/3 activity.(A) Visualization of loss of F-actin in ILPs following CK666 treatment. T cells were incubated with membrane-YFP (green) expressing endothelial cells in the presence of DMSO (control, left) or CK666 (right) for 5 min, then fixed, permeabilized and stained with Alexa594-phalloidin (‘Actin’, pink). Note that F-actin rich protrusions are missing at the CK666-treated T cell interface. Scale bar, 5 µm. (B) Loss of ILPs correlated with reduced phospho-HS1 at the T cell-endothelial cell synaptic interface. T cells were incubated with endothelial cell layer, as described in ‘Materials and methods’ for 5 min and were then fixed and stained with Alexa594-phalloidin (‘Actin’, green), phospho-HS1 (red) and anti-CD3 (blue). Cells were then imaged using confocal microscopy. The insets show single T cells marked in the ‘Merge’ image. Scale bar, 10 µm. (C) The intensity of the indicated proteins was then analyzed in the synaptic plane without and with CK666 treatment. n1 = 58, n2 = 71, p1 = 0.031, p2, p3 < 0.0001.DOI:http://dx.doi.org/10.7554/eLife.04953.032
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4355629&req=5

fig7s1: ILP F-actin and signaling in T-EC immunological synapse is dependent on Arp2/3 activity.(A) Visualization of loss of F-actin in ILPs following CK666 treatment. T cells were incubated with membrane-YFP (green) expressing endothelial cells in the presence of DMSO (control, left) or CK666 (right) for 5 min, then fixed, permeabilized and stained with Alexa594-phalloidin (‘Actin’, pink). Note that F-actin rich protrusions are missing at the CK666-treated T cell interface. Scale bar, 5 µm. (B) Loss of ILPs correlated with reduced phospho-HS1 at the T cell-endothelial cell synaptic interface. T cells were incubated with endothelial cell layer, as described in ‘Materials and methods’ for 5 min and were then fixed and stained with Alexa594-phalloidin (‘Actin’, green), phospho-HS1 (red) and anti-CD3 (blue). Cells were then imaged using confocal microscopy. The insets show single T cells marked in the ‘Merge’ image. Scale bar, 10 µm. (C) The intensity of the indicated proteins was then analyzed in the synaptic plane without and with CK666 treatment. n1 = 58, n2 = 71, p1 = 0.031, p2, p3 < 0.0001.DOI:http://dx.doi.org/10.7554/eLife.04953.032

Mentions: While SLB and solid-phase adsorbed antigen presentation systems allowed for superior optical resolution of the synapse, it is important to further test the validity of predictions from these reductionist models in more physiological T cell-APC interactions. Thus we utilized an activated cultured endothelial cells (EC) based planar APC system, where a flat synaptic interface is formed that is ideal for en-face visualization (Sage et al., 2012). Using this system, F-actin and HS1 rich ‘invadopod-like-protrusions’ (ILPs) are observed at TCR MC-like features in the periphery of the synapse (Sage et al., 2012). We found that ILPs were associated with the Y397 phosphorylated form of HS1, similar to F-actin foci (Figure 7A). Furthermore, when pre-formed T cell-EC conjugates were treated with CK666, there was concomitant loss of ILPs and phospho-HS1, indicating that these structures also require Arp2/3-dependent continuous polymerization of F-actin, and support local phospho-HS1 levels (Figure 7—figure supplement 1A–C). Since CK666-treatment also led to a reduction in total synaptic F-actin, which could be a consequence of inhibition of the EC cytoskeleton (Figure 7—figure supplement 1A–C), we utilized specific shRNA-mediated reduction in WASP levels in T cells, and examined foci (ILPs), total F-actin and phospho-HS1 levels at the T cell-EC conjugate interface (Figure 7A,B). WASP silenced T cells display selective loss of both ILPs as well as phospho-HS1, while maintaining total F-actin levels (Figure 7A–C). Therefore, the F-actin foci that we have primarily been characterized using SLB in this study, are the counterparts of ILPs in the cell conjugate system, and are necessary for efficient T cell activation. Thus, F-actin foci may represent a cytoskeletal module that functions to optimally support TCR distal signaling (Figure 7C) in diverse antigen presenting contexts.10.7554/eLife.04953.031Figure 7.ILP signaling in T-EC immunological synapse is WASP dependent.


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)

ILP F-actin and signaling in T-EC immunological synapse is dependent on Arp2/3 activity.(A) Visualization of loss of F-actin in ILPs following CK666 treatment. T cells were incubated with membrane-YFP (green) expressing endothelial cells in the presence of DMSO (control, left) or CK666 (right) for 5 min, then fixed, permeabilized and stained with Alexa594-phalloidin (‘Actin’, pink). Note that F-actin rich protrusions are missing at the CK666-treated T cell interface. Scale bar, 5 µm. (B) Loss of ILPs correlated with reduced phospho-HS1 at the T cell-endothelial cell synaptic interface. T cells were incubated with endothelial cell layer, as described in ‘Materials and methods’ for 5 min and were then fixed and stained with Alexa594-phalloidin (‘Actin’, green), phospho-HS1 (red) and anti-CD3 (blue). Cells were then imaged using confocal microscopy. The insets show single T cells marked in the ‘Merge’ image. Scale bar, 10 µm. (C) The intensity of the indicated proteins was then analyzed in the synaptic plane without and with CK666 treatment. n1 = 58, n2 = 71, p1 = 0.031, p2, p3 < 0.0001.DOI:http://dx.doi.org/10.7554/eLife.04953.032
© Copyright Policy
Related In: Results  -  Collection

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

fig7s1: ILP F-actin and signaling in T-EC immunological synapse is dependent on Arp2/3 activity.(A) Visualization of loss of F-actin in ILPs following CK666 treatment. T cells were incubated with membrane-YFP (green) expressing endothelial cells in the presence of DMSO (control, left) or CK666 (right) for 5 min, then fixed, permeabilized and stained with Alexa594-phalloidin (‘Actin’, pink). Note that F-actin rich protrusions are missing at the CK666-treated T cell interface. Scale bar, 5 µm. (B) Loss of ILPs correlated with reduced phospho-HS1 at the T cell-endothelial cell synaptic interface. T cells were incubated with endothelial cell layer, as described in ‘Materials and methods’ for 5 min and were then fixed and stained with Alexa594-phalloidin (‘Actin’, green), phospho-HS1 (red) and anti-CD3 (blue). Cells were then imaged using confocal microscopy. The insets show single T cells marked in the ‘Merge’ image. Scale bar, 10 µm. (C) The intensity of the indicated proteins was then analyzed in the synaptic plane without and with CK666 treatment. n1 = 58, n2 = 71, p1 = 0.031, p2, p3 < 0.0001.DOI:http://dx.doi.org/10.7554/eLife.04953.032
Mentions: While SLB and solid-phase adsorbed antigen presentation systems allowed for superior optical resolution of the synapse, it is important to further test the validity of predictions from these reductionist models in more physiological T cell-APC interactions. Thus we utilized an activated cultured endothelial cells (EC) based planar APC system, where a flat synaptic interface is formed that is ideal for en-face visualization (Sage et al., 2012). Using this system, F-actin and HS1 rich ‘invadopod-like-protrusions’ (ILPs) are observed at TCR MC-like features in the periphery of the synapse (Sage et al., 2012). We found that ILPs were associated with the Y397 phosphorylated form of HS1, similar to F-actin foci (Figure 7A). Furthermore, when pre-formed T cell-EC conjugates were treated with CK666, there was concomitant loss of ILPs and phospho-HS1, indicating that these structures also require Arp2/3-dependent continuous polymerization of F-actin, and support local phospho-HS1 levels (Figure 7—figure supplement 1A–C). Since CK666-treatment also led to a reduction in total synaptic F-actin, which could be a consequence of inhibition of the EC cytoskeleton (Figure 7—figure supplement 1A–C), we utilized specific shRNA-mediated reduction in WASP levels in T cells, and examined foci (ILPs), total F-actin and phospho-HS1 levels at the T cell-EC conjugate interface (Figure 7A,B). WASP silenced T cells display selective loss of both ILPs as well as phospho-HS1, while maintaining total F-actin levels (Figure 7A–C). Therefore, the F-actin foci that we have primarily been characterized using SLB in this study, are the counterparts of ILPs in the cell conjugate system, and are necessary for efficient T cell activation. Thus, F-actin foci may represent a cytoskeletal module that functions to optimally support TCR distal signaling (Figure 7C) in diverse antigen presenting contexts.10.7554/eLife.04953.031Figure 7.ILP signaling in T-EC immunological synapse is WASP dependent.

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