Ratiometric imaging of the T-cell actin cytoskeleton reveals the nature of receptor-induced cytoskeletal enrichment.

The T-cell actin cytoskeleton mediates adaptive immune system responses to peptide antigens by physically directing the motion and clustering of T-cell receptors (TCRs) on the cell surface. When TCR movement is impeded by externally applied physical barriers, the actin network exhibits transient enrichment near the trapped receptors. The coordinated nature of the actin density fluctuations suggests that they are composed of filamentous actin, but it has not been possible to eliminate de novo polymerization at TCR-associated actin polymerizing factors as an alternative cause. Here, we use a dual-probe cytoskeleton labeling strategy to distinguish between stable and polymerizing pools of actin. Our results suggest that TCR-associated actin consists of a relatively high proportion of the stable cytoskeletal fraction and extends away from the cell membrane into the cell. This implies that actin enrichment at mechanically trapped TCRs results from three-dimensional bunching of the existing filamentous actin network.

Direct single molecule measurement of TCR triggering by agonist pMHC in living primary T cells.

T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering. DOI:http://dx.doi.org/10.7554/eLife.00778.001.

Modulation of T cell signaling by the actin cytoskeleton.

The actin cytoskeleton provides a dynamic framework to support membrane organization and cellular signaling events. The importance of actin in T cell function has long been recognized to go well beyond the maintenance of cell morphology and transport of proteins. Over the past several years, our understanding of actin in T cell activation has expanded tremendously, in part owing to the development of methods and techniques to probe the complex interplay between actin and T cell signaling. On the one hand, biochemical methods have led to the identification of many key cytoskeleton regulators and new signaling pathways, whereas, on the other, the combination of advanced imaging techniques and physical characterization tools has allowed the spatiotemporal investigation of actin in T cell signaling. All those studies have made a profound impact on our understanding of the actin cytoskeleton in T cell activation. Many previous reviews have focused on the biochemical aspects of the actin cytoskeleton. However, here we will summarize recent studies from a biophysical perspective to explain the mechanistic role of actin in modulating T cell activation. We will discuss how actin modulates T cell activation on multiple time and length scales. Specifically, we will reveal the distinct roles of the actin filaments in facilitating TCR triggering, orchestrating 'signalosome' assembly and transport, and establishing protein spatial organization in the immunological synapse.

Characterization of dynamic actin associations with T-cell receptor microclusters in primary T cells.

T cell triggering through T-cell antigen receptors (TCRs) results in spatial assembly of the receptors on multiple length scales. This assembly is mediated by the T cell actin cytoskeleton, which reorganizes in response to TCR phosphorylation and then induces the coalescence of TCRs into microclusters, followed by their unification into a micrometer-scale structure. The exact outcomes of the association of TCRs with a dynamic and fluctuating actin network across these length scales are not well characterized, but it is clear that weak and transient interactions at the single-molecule level sum to yield significant receptor rearrangements at the plasma membrane. We used the hybrid live cell-nanopatterned supported lipid bilayer system to quantitatively probe the actin-TCR interaction in primary T cells. A specialized tracking algorithm revealed that actin slows as it passes over TCR clusters in a direction-dependent manner with respect to the resistance against TCR motion. We also observed transient actin enrichments at sites corresponding to putative TCR clusters that far exceeded pure stochastic fluctuations and described an image time-autocorrelation analysis method to quantify these accumulations.

Myosin IIA modulates T cell receptor transport and CasL phosphorylation during early immunological synapse formation.

Activation of T cell receptor (TCR) by antigens occurs in concert with an elaborate multi-scale spatial reorganization of proteins at the immunological synapse, the junction between a T cell and an antigen-presenting cell (APC). The directed movement of molecules, which intrinsically requires physical forces, is known to modulate biochemical signaling. It remains unclear, however, if mechanical forces exert any direct influence on the signaling cascades. We use T cells from AND transgenic mice expressing TCRs specific to the moth cytochrome c 88-103 peptide, and replace the APC with a synthetic supported lipid membrane. Through a series of high spatiotemporal molecular tracking studies in live T cells, we demonstrate that the molecular motor, non-muscle myosin IIA, transiently drives TCR transport during the first one to two minutes of immunological synapse formation. Myosin inhibition reduces calcium influx and colocalization of active ZAP-70 (zeta-chain associated protein kinase 70) with TCR, revealing an influence on signaling activity. More tellingly, its inhibition also significantly reduces phosphorylation of the mechanosensing protein CasL (Crk-associated substrate the lymphocyte type), raising the possibility of a direct mechanical mechanism of signal modulation involving CasL.

Patterned two-photon photoactivation illuminates spatial reorganization in live cells.

Photoactivatable fluorescent proteins offer the possibility to optically tag and track the location of molecules in their bright state with high spatial and temporal resolution. Several reports of patterned photoactivation have emerged since the development of a photoactivatable variant of the green fluorescent protein (PaGFP) and the demonstration of two-photon activation of PaGFP. To date, however, there have been few methods developed to quantify the spatial reorganization of the photoactivated population. Here we report on the use of singular value decomposition (SVD) to track the time-dependent distribution of fluorophores after photoactivation. The method was used to describe live-cell actin cytoskeleton behavior in primary murine T-cells, in which a dynamic cytoskeleton is responsible for the reorganization of membrane proteins in response to antigen peptide recognition. The method was also used to observe immortalized simian kidney (Cos-7) cells, in which the cytoskeleton is more stable. Both cell types were transfected with PaGFP fused to the F-actin binding domain of utrophin (UtrCH). Photoactivation patterns were written in the samples with a pair of galvanometric scanning mirrors in circular patterns that were analyzed by transforming the images into a time series of radial distribution profiles. The time-evolution of the profiles was well-described by the first two SVD component states. For T-cells, we find that actin filaments are highly mobile. Inward transport from the photoactivation region was observed and occurred on a 1-2 s time scale, which is consistent with retrograde cycling. For Cos-7 cells, we find that the actin is relatively stationary and does not undergo significant centripetal flow as expected for a resting fibroblast. The combination of patterned photoactivation and SVD analysis offers a unique way to measure spatial redistribution dynamics within live cells.