Abstract
The role of non-muscle myosin IIA (heavy chain encoded by the non-muscle myosin heavy chain 9 gene, Myh9) in immunological synapse formation is controversial. We have addressed the role of myosin IIA heavy chain protein (MYH9) in mouse T cells responding to MHC-peptide complexes and ICAM-1 in supported planar bilayers – a model for immunological synapse maturation. We found that reduction of MYH9 expression levels using Myh9 siRNA in proliferating mouse CD4+ AND T cell receptor (TCR) transgenic T cells resulted in increased spreading area, failure to assemble the central and peripheral supramolecular activation clusters (cSMAC and pSMAC), and increased motility. Surprisingly, TCR microcluster speed was reduced marginally, however TCR microclusters dissipated prior to forming a cSMAC. TCR microclusters formed in the Myh9 siRNA-treated T cells showed reduced phosphorylation of the Src family kinase (SFK) activation loop and displayed reduced cytoplasmic calcium ion (Ca2+) elevation. In addition, Myh9 siRNA-treated cells displayed reduced phosphorylation of the Cas-L substrate domain – a force-dependent SFK substrate – which was observed in control siRNA-treated cells in foci throughout the immunological synapse except the cSMAC. Cas-L exhibited TCR ligation-dependent induction of phosphorylation. These results provide further evidence that T cell activation is modulated by intrinsic force-generating systems and can be viewed as a mechanically responsive process influenced by MYH9.
Highlights
T cells are highly migratory cells that cycle through the blood, lymph, and peripheral lymphoid organs, scanning antigen presenting cells (APCs) in search of their cognate antigen (Miller et al, 2004)
We have addressed the role of myosin IIA heavy chain protein (MYH9) in mouseT cells responding to MHC-peptide complexes and ICAM-1 in supported planar bilayers – a model for immunological synapse maturation
We found that reduction of MYH9 expression levels using Myh9 siRNA in proliferating mouse CD4+ AND T cell receptor (TCR) transgenic T cells resulted in increased spreading area, failure to assemble the central and peripheral supramolecular activation clusters, and increased motility
Summary
T cells are highly migratory cells that cycle through the blood, lymph, and peripheral lymphoid organs, scanning antigen presenting cells (APCs) in search of their cognate antigen (Miller et al, 2004). T cells initiate signaling within seconds of contacting an APC, but the immunological synapse undergoes a maturation process over a period of a few minutes to form three supramolecular activation clusters (SMACs; Monks et al, 1998; Freiberg et al, 2002). In the last stage of synapse maturation the large TCR microclusters reach the center and undergo a transformation from individual microclusters into a stable central SMAC (cSMAC) that lacks active signaling in a mechanism dependent upon the endosomal complexes required for transport (ESCRT) component TSG101 (Varma et al, 2006; Vardhana et al, 2010). The role of cytoskeletal transport in this process is well established (Mossman et al, 2005; Varma et al, 2006; DeMond et al, 2008; Hashimoto-Tane et al, 2011), but the role of the actin based motor myosin IIA (MYH9, encoded by the gene Myh9) has been controversial
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