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

Yu Y, Fay NC, Smoligovets AA, Wu HJ, Groves JT - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California, United States of America.

ABSTRACT
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.

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Myosin IIA transiently drives TCR translocation and actin retrograde flow in immunological synapse formation.T cell receptors (TCRs) were labeled with H57 αTCR Fab (Alexa Fluor dyes) and imaged starting from the initial cell-bilayer contact (t = 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 (<V(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 (<V(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.
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pone-0030704-g001: Myosin IIA transiently drives TCR translocation and actin retrograde flow in immunological synapse formation.T cell receptors (TCRs) were labeled with H57 αTCR Fab (Alexa Fluor dyes) and imaged starting from the initial cell-bilayer contact (t = 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 (<V(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 (<V(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.

Mentions: During antigen recognition, TCR-pMHC complexes undergo a series of spatial translocations including: local clustering and long range transport to the center of the IS [6]–[10]. To explore the role of myosin IIA in these steps, we imaged fluorescently labeled TCRs at the cell-bilayer interface and tracked their movements with a custom tracking algorithm that implements an intensity gradient method to find centers of non-spherical fluorescent objects. Essentially, the entire ensemble of TCR microclusters within each individual cell (∼100 microclusters) was imaged and tracked with ∼50 nm spatial resolution and ∼50 ms temporal resolution over the course of IS formation. In control cells, TCR trajectories reveal coordinated centripetal movement in pSMAC region and the cell periphery following the initial cell-bilayer contact, but more confined motion at the center (cSMAC) (Figure 1A). Pharmacological inhibition of myosin IIA by blebbistatin (100 µM) and ML-7 (20 µM) does not alter the clustering of TCRs, but leads to much less directed motion of the microclusters (Figure S1). For a more quantitative measure, we analyzed the time-dependence of TCR translocation during IS formation. Averaged radial velocities, <V(t)>, are plotted against time, t, on a single cell basis. <V(t)> is defined negative for centripetal movements and positive for movements toward the cell periphery. In control cells, translocation of TCR microclusters varies significantly as IS formation proceeds (Figure 1B). After the initial cell-bilayer contact (t = 0 sec) microclusters undergo very rapid centripetal movement (<V(t)>max≈−70 nm/sec) for approximately 2 min and then maintain a reduced yet constant speed (<V(t)>≈−15 nm/sec) for an additional 3–5 min until the central accumulation of TCRs stabilizes. By contrast, TCR microclusters in cells pretreated with blebbistatin or ML-7 do not exhibit the rapid initial centripetal movement, but move at an almost constant velocity (<V(t)>≈−10 to −15 nm/sec) throughout the entire time course of IS formation (Figure 1B). The loss of the initial rapid component of centripetal movement indicates that myosin IIA is transiently involved in TCR transport and the slower movement in the presence of myosin inhibitors suggests a secondary driving force, presumably actin polymerization. In control experiments in which we simultaneously imaged TCR translocation and cell edge movement, we confirm that TCR microclusters, which move almost one order of magnitude faster than the cell membrane contraction (−5 nm/sec), are actively driven by forces from myosin IIA instead of the global cell movement (Figure S2).


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

Yu Y, Fay NC, Smoligovets AA, Wu HJ, Groves JT - PLoS ONE (2012)

Myosin IIA transiently drives TCR translocation and actin retrograde flow in immunological synapse formation.T cell receptors (TCRs) were labeled with H57 αTCR Fab (Alexa Fluor dyes) and imaged starting from the initial cell-bilayer contact (t = 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 (<V(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 (<V(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275606&req=5

pone-0030704-g001: Myosin IIA transiently drives TCR translocation and actin retrograde flow in immunological synapse formation.T cell receptors (TCRs) were labeled with H57 αTCR Fab (Alexa Fluor dyes) and imaged starting from the initial cell-bilayer contact (t = 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 (<V(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 (<V(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.
Mentions: During antigen recognition, TCR-pMHC complexes undergo a series of spatial translocations including: local clustering and long range transport to the center of the IS [6]–[10]. To explore the role of myosin IIA in these steps, we imaged fluorescently labeled TCRs at the cell-bilayer interface and tracked their movements with a custom tracking algorithm that implements an intensity gradient method to find centers of non-spherical fluorescent objects. Essentially, the entire ensemble of TCR microclusters within each individual cell (∼100 microclusters) was imaged and tracked with ∼50 nm spatial resolution and ∼50 ms temporal resolution over the course of IS formation. In control cells, TCR trajectories reveal coordinated centripetal movement in pSMAC region and the cell periphery following the initial cell-bilayer contact, but more confined motion at the center (cSMAC) (Figure 1A). Pharmacological inhibition of myosin IIA by blebbistatin (100 µM) and ML-7 (20 µM) does not alter the clustering of TCRs, but leads to much less directed motion of the microclusters (Figure S1). For a more quantitative measure, we analyzed the time-dependence of TCR translocation during IS formation. Averaged radial velocities, <V(t)>, are plotted against time, t, on a single cell basis. <V(t)> is defined negative for centripetal movements and positive for movements toward the cell periphery. In control cells, translocation of TCR microclusters varies significantly as IS formation proceeds (Figure 1B). After the initial cell-bilayer contact (t = 0 sec) microclusters undergo very rapid centripetal movement (<V(t)>max≈−70 nm/sec) for approximately 2 min and then maintain a reduced yet constant speed (<V(t)>≈−15 nm/sec) for an additional 3–5 min until the central accumulation of TCRs stabilizes. By contrast, TCR microclusters in cells pretreated with blebbistatin or ML-7 do not exhibit the rapid initial centripetal movement, but move at an almost constant velocity (<V(t)>≈−10 to −15 nm/sec) throughout the entire time course of IS formation (Figure 1B). The loss of the initial rapid component of centripetal movement indicates that myosin IIA is transiently involved in TCR transport and the slower movement in the presence of myosin inhibitors suggests a secondary driving force, presumably actin polymerization. In control experiments in which we simultaneously imaged TCR translocation and cell edge movement, we confirm that TCR microclusters, which move almost one order of magnitude faster than the cell membrane contraction (−5 nm/sec), are actively driven by forces from myosin IIA instead of the global cell movement (Figure S2).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley, California, United States of America.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus