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

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

Inhibition of myosin IIA abolishes intracellular Ca²⁺ influx.

<p>The ratio of Fura-2 fluorescence emission intensity in response to 340 nm and 380 nm excitation (340/380) is proportional to intracellular [Ca²⁺]. (A) Fura-2 340/380 emission ratios are plotted against the cell stimulation time for four representative cells pretreated with either DMSO or ML-7. (B) Fura-2 340/380 emission ratios of control cells (n = 1602) and cells pretreated with ML-7 (n = 2187) are plotted against time on a color scale and organized along the y-axis according to the summed calcium influx.</p

Myosin IIA transiently drives TCR translocation and actin retrograde flow in immunological synapse formation.

<p>T cell receptors (TCRs) were labeled with H57 αTCR F_ab (Alexa Fluor dyes) and imaged starting from the initial cell-bilayer contact (<i>t</i> = 0 sec). (A) Trajectories of all TCR microclusters (Alexa Fluor 594) show their centripetal movement at the cell periphery and highly confined motion at the central area of the immunological synapse. Color bar corresponds to the elapsed time after the initial cell-bilayer contact. Data are representative of 6 independent experiments. (B) Inhibition of myosin IIA changes the time-dependence of TCR microcluster translocation. Time-averaged radial velocities (t)>) of TCRs (Alexa Fluor 643) in individual cells are plotted against the elapsed time after the initial cell-bilayer contact in the presence of DMSO control, blebbistatin, or ML-7. Data are representative of 5 independent experiments. (C) Inhibition of myosin IIA changes the time-dependence of actin retrograde flow during the immunological synapse formation. Time-averaged radial velocities (t)>) of tracked EGFP-UtrCH in individual cells are plotted against the elapsed time after the initial cell-bilayer contact in the presence of DMSO control, ML-7, or ML7 with jasplakinolide. Data are representative of 4 independent experiments. Error bars in (B) and (C) represent standard errors.</p

Physical constraints on TCR microcluster translocation impede myosin IIA movements.

<p>(A) TIRF, reflection interference contrast microscopy (RICM), and bright field (BF) images of T cells expressing EGFP-myosin on unpatterned or patterned bilayers. Scale bars: 5 µm. (B) Time-averaged radial velocities (t)>) of EGFP-myosin in individual cells are plotted against the elapsed time (<i>t</i>) after the initial cell-bilayer contact (<i>t</i> = 0). Data are representative of 2 independent experiments.</p

Inhibition of myosin only affects morphology of the early immunological synapse.

<p>Total internal reflection fluorescence (TIRF) images of TCRs labeled with H57 αTCR F_ab (Alexa Fluor 594) and ICAM-1 (Alexa Fluor 488) are shown. Cells were pretreated with DMSO, blebbistatin, or ML-7, and fixed at (A) 3 min and (B) 10 min after interacting with bilayers. Data are representative of 3 independent experiments. Scale bars: 5 µm.</p

Inhibition of myosin IIA reduces phosphorylation of CasL.

<p>(A) TIRF images of TCR and pCasL from T cells fixed at 1.5 min and 3 min and pretreated with DMSO (control), blebbistatin or ML-7. (B) Fluorescence intensities of pCasL in IS are shown normalized to those in control cells. Each column in Panel (A) and (B) is an averaged value from approximately 200 cells. Data were reproduced in 2 independent experiments. Scale bars: 5 µm.</p