A single class II myosin modulates T cell motility and stopping, but not synapse formation

Abstract

Upon encountering an antigen, motile T cells stop crawling, change morphology and ultimately form an 'immunological synapse'. Although myosin motors are thought to mediate various aspects of this process, the molecules involved and their exact roles are not defined. Here we show that nonmuscle myosin heavy chain IIA, or MyH9, is the only class II myosin expressed in T cells and is associated with the uropod during crawling. MyH9 function is required for maintenance of the uropod and for T cell motility but is dispensable for synapse formation. Phosphorylation of MyH9 in its multimerization domain by T cell receptor–generated signals indicates that inactivation of this motor may be a key step in the 'stop' response during antigen recognition.

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Figure 1: MyH9 is the predominant class II myosin motor expressed in T cells.
Figure 2: MyH9 is enriched and recycled at the uropod of crawling T cells and repolarizes to the T cell–APC interface during synapse formation.
Figure 3: MyH9 motor activity is required for T cell polarity.
Figure 4: MyH9 function is necessary for T cell crawling.
Figure 5: Pharmacological inhibition of MyH9 does not prevent T cell-B cell coupling.
Figure 6: MyH9 inhibition does not prevent relocalization of immunological synapse and cytoskeletal components.
Figure 7: MyH9 is phosphorylated at threonine residues in response to TCR recognition.

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Acknowledgements

We thank A. Weiss and S. Reck-Peterson (University of California at San Francisco) for comments on the manuscript, R.S. Adelstein (National Heart, Lung and Blood Institute, National Institutes of Health) for kindly providing us with the human MyH9-GFP fusion construct and for the MyH14 specific antibody, and S. Jiang for expert technical assistance with cell sorting. J.J. was supported in part by a fellowship from the Fondazione Italiana per la Ricerca sul Cancro. This work was supported by a grant from the US National Institutes of Health (RO1-AI52116-01) and from the Howard Hughes Medical Institute Biomedical Research Support Program (#5300246).

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Correspondence to Matthew F Krummel.

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Supplementary information

Supplementary Fig. 1 (PDF 61 kb)

Supplementary Fig. 2 (PDF 45 kb)

Supplementary Fig. 3 (PDF 72 kb)

Supplementary Fig. 4 (PDF 44 kb)

41590_2004_BFni1065_MOESM5_ESM.mov

DIC (top) and fluorescence (bottom) images of an MyH9-GFP transfected D10 T cell clone crawling from left to right within the field have been recorded every 5 seconds. (MOV 2992 kb)

Supplementary Video 1

DIC (top) and fluorescence (bottom) images of an MyH9-GFP transfected D10 T cell clone crawling from left to right within the field have been recorded every 5 seconds. (MOV 2992 kb)

41590_2004_BFni1065_MOESM6_ESM.mov

MyH9 polarization to the synapse. Simultaneous DIC (left), Calcium concentrations (as measured by Fura2-AM displayed in pseudo-color scale, middle), and MyH9-GFP fluorescence (right) images of a transfected D10 T cell clone during coupling with Ag-pulsed APCs were recorded every 30 seconds. (MOV 4107 kb)

Supplementary Video 2

MyH9 polarization to the synapse. Simultaneous DIC (left), Calcium concentrations (as measured by Fura2-AM displayed in pseudo-color scale, middle), and MyH9-GFP fluorescence (right) images of a transfected D10 T cell clone during coupling with Ag-pulsed APCs were recorded every 30 seconds. (MOV 4107 kb)

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Jacobelli, J., Chmura, S., Buxton, D. et al. A single class II myosin modulates T cell motility and stopping, but not synapse formation. Nat Immunol 5, 531–538 (2004). https://doi.org/10.1038/ni1065

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