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PO4-3-40

Indispensable role of formin-dependent cortical F-actin remodeling in TCR signaling

[Speaker] Yoshichika Katsura:1
[Co-author] Dean Thumkeo:1, Yukako Nishimura:2, Pakorn Kanchanawong:2, Shuh Narumiya:1
1:Drug Discovery Medicine, Graduate school of Medicine, Kyoto University, Japan, 2:Mechanobiology Institute, National University of Singapore, Singapore

T cell is one of the important cell of our acquired immune system. It is known that T cell forms immunological synapse upon the recognition of antigen presented by the antigen presenting cell (APC). This process is mediated by T cell receptor (TCR), and critical for T cell differentiation and function. It was shown previously cytoskeletal actin plays an indispensable role in TCR signaling which is a cascade of proteins phosphorylation. However, the molecular mechanism how F-actin exerts its function and is regulated in TCR signaling remains largely unknown.
 In this work, we focused on formin, an actin binding protein with nucleation and polymerization activity, in TCR signaling. We investigated the contribution of formin to TCR signaling by utilizing a formin inhibitor, SMIFH2. We found that although phosphorylation of ZAP-70 was not significantly affected, the phosphorylation of LAT, a substrate of ZAP-70, was strongly suppressed in naive CD8 T cell upon SMIFH2 treatment. We further observed phosphorylated ZAP-70 and LAT cellular localization and found that phosphorylated ZAP-70 colocalized with phosphorylated LAT on the cell membrane in TCR-stimulated control cells, suggesting that LAT is phosphorylated by phosphorylated ZAP-70 on the cell membrane. In addition, we examined TCR stimulation dependent F-actin dynamics and found that TCR stimulation induced F-actin remodeling proximal to phosphorylated LAT in a formin-dependent manner. These results together suggest that phosphorylation of LAT on the cell membrane is achieved by the colocalization of phosphorylated ZAP-70 and LAT driven by formin-dependent cortical F-actin remodeling upon TCR stimulation.
 In order to identify which formin protein(s) is (are) responsible for these phenotypes, we further analyzed the expression of formin proteins and found that mDia1 and mDia3 expression was relatively high in naive CD8 T cell. Moreover, we found both mDia1 and mDia3 are localized to immunological synapse upon TCR stimulation in the reconstructed stimulating bilayer system. Furthermore, depletion of mDia1/3 in naive CD8 T cell resulted in significant reduction of TCR-stimulation dependent IL-2 production and cell proliferation. These results together therefore suggested that mDia1 and mDia3 are the important formin proteins playing an indispensable role in TCR signaling-dependent T cell activation.

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