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.
Highlights
Upon encountering antigen-presenting cells (APCs) displaying T cell receptor (TCR) ligands and adhesion molecules, T cells undergo a series of actin dependent shape changes and signaling steps
Utilizing a planar surface functionalized with anti-mouse CD3 antibody and ICAM1 as an antigenpresenting surface, we first compared the F-actin cytoskeleton in polyclonal primary CD4 T cells from either wild type (WT) or Wiscott Aldrich Syndrome protein (WASP) deficient C57BL/6J mice at the early spreading stage of immunological synapse formation
Assessment of the amount of newly polymerized actin in foci vs surrounding lamellar areas using the above-mentioned labeling and quantification method revealed that the F-actin foci incorporate fresh actin subunits (Figure 1B). This indicates that the foci are dynamically polymerizing structures, rather than sites where F-actin accumulates due to rearrangement of preexisting filaments
Summary
Upon encountering antigen-presenting cells (APCs) displaying T cell receptor (TCR) ligands and adhesion molecules, T cells undergo a series of actin dependent shape changes and signaling steps These ligands include MHC complexed with antigentic peptide (MHCp) for mouse monoclonal T cells, agonist anti-CD3 antibody for polyclonal mouse (2C11) or human (OKT3) T cells. TCR signaling is initiated at F-actin-based protrusions of polarized T cells (Valitutti et al, 1995; Negulescu et al, 1996) These early activation events lead to F-actin dependent formation of a broad immunological synapse (IS) (Wulfing and Davis, 1998; Grakoui et al, 1999) containing T cell antigen receptor (TCR) microclusters (MCs) (Bunnell et al, 2002; Campi et al, 2005).
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