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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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Related in: MedlinePlus

Inhibition of myosin only affects morphology of the early immunological synapse.Total internal reflection fluorescence (TIRF) images of TCRs labeled with H57 αTCR Fab (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.
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pone-0030704-g002: Inhibition of myosin only affects morphology of the early immunological synapse.Total internal reflection fluorescence (TIRF) images of TCRs labeled with H57 αTCR Fab (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.

Mentions: The role of myosin IIA in IS formation has been controversial in previous studies [15], [23], [24]. Since our fluorescence tracking data suggests that TCR microclusters are able to translocate in the absence of myosin IIA, we studied whether myosin is required for the spatial organization at the IS. TCR and ICAM-1 in control cells exhibit the characteristic “bull's eye” pattern, where the TCR microclusters accumulate at the center while LFA-1 bound to ICAM-1 localizes at the periphery (Figure 2). Inhibition of myosin leads to a more dispersed distribution of TCR microclusters in cells fixed at 3 min after stimulation, but the difference becomes negligible when cells are fixed at 10 min (Figure 2 and Figure S4). The overall ring-like distribution of ICAM-1 in the pSMAC is not affected by myosin inhibition. This demonstrates that myosin IIA influences the time frame of IS formation, but is not required for the superficial appearance of the final pattern. It is important to recognize that these observations do not suggest that the cells arrive at the same final state with and without myosin, as detailed below. Rather, it reveals that the large scale spatial organization of the IS probably offers insufficient information from which to judge the more subtle internal state of the cell.


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)

Inhibition of myosin only affects morphology of the early immunological synapse.Total internal reflection fluorescence (TIRF) images of TCRs labeled with H57 αTCR Fab (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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275606&req=5

pone-0030704-g002: Inhibition of myosin only affects morphology of the early immunological synapse.Total internal reflection fluorescence (TIRF) images of TCRs labeled with H57 αTCR Fab (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.
Mentions: The role of myosin IIA in IS formation has been controversial in previous studies [15], [23], [24]. Since our fluorescence tracking data suggests that TCR microclusters are able to translocate in the absence of myosin IIA, we studied whether myosin is required for the spatial organization at the IS. TCR and ICAM-1 in control cells exhibit the characteristic “bull's eye” pattern, where the TCR microclusters accumulate at the center while LFA-1 bound to ICAM-1 localizes at the periphery (Figure 2). Inhibition of myosin leads to a more dispersed distribution of TCR microclusters in cells fixed at 3 min after stimulation, but the difference becomes negligible when cells are fixed at 10 min (Figure 2 and Figure S4). The overall ring-like distribution of ICAM-1 in the pSMAC is not affected by myosin inhibition. This demonstrates that myosin IIA influences the time frame of IS formation, but is not required for the superficial appearance of the final pattern. It is important to recognize that these observations do not suggest that the cells arrive at the same final state with and without myosin, as detailed below. Rather, it reveals that the large scale spatial organization of the IS probably offers insufficient information from which to judge the more subtle internal state of the cell.

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