Altered Actin Centripetal Retrograde Flow in Physically Restricted Immunological Synapses

Cheng-Han Yu,Yoshihisa Kaizuka,Jay T Groves,Ronald D Vale,Hung-Jen Wu,Derya Unutmaz

doi:10.1371/journal.pone.0011878

Abstract

Antigen recognition by T cells involves large scale spatial reorganization of numerous receptor, adhesion, and costimulatory proteins within the T cell-antigen presenting cell (APC) junction. The resulting patterns can be distinctive, and are collectively known as the immunological synapse. Dynamical assembly of cytoskeletal network is believed to play an important role in driving these assembly processes. In one experimental strategy, the APC is replaced with a synthetic supported membrane. An advantage of this configuration is that solid structures patterned onto the underlying substrate can guide immunological synapse assembly into altered patterns. Here, we use mobile anti-CD3ε on the spatial-partitioned supported bilayer to ligate and trigger T cell receptor (TCR) in live Jurkat T cells. Simultaneous tracking of both TCR clusters and GFP-actin speckles reveals their dynamic association and individual flow patterns. Actin retrograde flow directs the inward transport of TCR clusters. Flow-based particle tracking algorithms allow us to investigate the velocity distribution of actin flow field across the whole synapse, and centripetal velocity of actin flow decreases as it moves toward the center of synapse. Localized actin flow analysis reveals that, while there is no influence on actin motion from substrate patterns directly, velocity differences of actin are observed over physically trapped TCR clusters. Actin flow regains its velocity immediately after passing through confined TCR clusters. These observations are consistent with a dynamic and dissipative coupling between TCR clusters and viscoelastic actin network.

Highlights

The formation of an immunological synapse, the intercellular junction between T cells and antigen presenting cells (APCs), involves formation of intermembrane protein complexes and micrometer length scale lateral reorganization within the interface [1,2,3]
We primarily focus on T cell receptor (TCR) coupling to actin network, and anti-CD3e/TCR ligations are the only intermembrane linkages between T cell and supported membranes
By analyzing translocation trajectories of both TCR clusters and actin speckles, we demonstrate their non-static associations in triggered T cell synapses

Summary

Introduction

The formation of an immunological synapse, the intercellular junction between T cells and antigen presenting cells (APCs), involves formation of intermembrane protein complexes and micrometer length scale lateral reorganization within the interface [1,2,3]. T cell receptor (TCR) engages the antigenic peptide loaded major histocompatibility complex II (pMHC) on the APC and forms sub-micrometer clusters during synapse formation. Under sufficiently high antigen stimulation, TCR clusters subsequently move inward to the center of the junction, forming the central supramolecular adhesion complex (c-SMAC). Interfering with their transport process has been shown to affect both TCR-specific tyrosine phosphorylation and intracellular calcium flux [8]. Forced movement of TCR clusters, driven from within the cell or externally, can alter TCR signaling behavior

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Results

Conclusion