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A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells

Abstract

Polarization of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell (APC) is driven by the accumulation of diacylglycerol (DAG) at the immunological synapse (IS). The mechanisms that couple DAG to the MTOC are not known. By single-cell photoactivation of the T cell antigen receptor (TCR), we found that three distinct isoforms of protein kinase C (PKC) were recruited by DAG to the IS in two steps. PKC-ɛ and PKC-η accumulated first in a broad region of membrane, whereas PKC-θ arrived later in a smaller zone. Functional experiments indicated that PKC-θ was required for MTOC reorientation and that PKC-ɛ and PKC-η operated redundantly to promote the recruitment of PKC-θ and subsequent polarization responses. Our results establish a previously uncharacterized role for PKC proteins in T cell polarity.

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Figure 1: PKC-θ, PKC-ɛ and PKC-η accumulate at the region of TCR activation before MTOC reorientation.
Figure 2: Distinct accumulation patterns and kinetics of nPKC proteins.
Figure 3: Recruitment of nPKC proteins correlates with more PKC activity.
Figure 4: PKC activity is required for MTOC polarization.
Figure 5: PKC-θ and either PKC-ɛ or PKC-η are required for MTOC polarization.
Figure 6: Defective MTOC polarization in PKC-θ-deficient T cells.
Figure 7: PKC-θ recruitment requires PKC-ɛ and PKC-η.
Figure 8: TCR-induced cytokine production requires PKC-θ, PKC-ɛ and PKC-η.

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Acknowledgements

We thank D. Littman (New York University) for PKC-θ-deficient mice; W. Marks and the Memorial Sloan-Kettering Cancer Center Mouse Genetics Core Facility for assistance with generating the C1-GFP mice; R. Wedlich-Soldner (International Max Planck Research School) for Lifeact-RFP; A. Hall (Memorial Sloan-Kettering Cancer Center), S. Rotenberg (Queens College) and M.O. Li (Memorial Sloan-Kettering Cancer Center) for reagents and advice; E. Alonzo for assistance with functional studies; N. Bantilan for technical support; S.S. Yi and the Memorial Sloan-Kettering Cancer Center Microchemistry Core Facility for peptide synthesis; A. Hall for critical reading of the manuscript; and members of the Huse and Li laboratories for helpful comments. Supported by the Searle Scholars Program (M.H.), the Cancer Research Institute (M.H.) and the US National Institutes of Health (Ruth L. Kirschstein National Research Service Award to E.J.Q., R01-AI087644 to M.H. and R01-AI083408 to G.A.-B.).

Author information

Authors and Affiliations

Authors

Contributions

E.J.Q. and M.H. designed the experiments; E.J.Q. collected and analyzed the data; X.L. did the Marcksl1 experiments; G.A.-B. assisted with MATLAB programming and data analysis; and M.H. wrote the manuscript (with help from E.J.Q. and G.A.-B.).

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Correspondence to Morgan Huse.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 2492 kb)

Supplementary Video 1

PKC-θ accumulates in the region of TCR stimulation prior to MTOC reorientation. (MOV 2666 kb)

Supplementary Video 2

PKC-ɛ accumulates in the region of TCR stimulation prior to MTOC reorientation. (MOV 2515 kb)

Supplementary Video 3

PKC-η accumulates in the region of TCR stimulation prior to MTOC reorientation. (MOV 1649 kb)

Supplementary Video 4

PKC-η and PKC-θ display distinct spatiotemporal patterns of recruitment. (MOV 5617 kb)

Supplementary Video 5

PKC-ɛ and PKC-θ display similar spatiotemporal patterns of recruitment. (MOV 2539 kb)

Supplementary Video 6

Marcksl1 is depleted from the region of TCR stimulation prior to MTOC reorientation. (MOV 3146 kb)

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Quann, E., Liu, X., Altan-Bonnet, G. et al. A cascade of protein kinase C isozymes promotes cytoskeletal polarization in T cells. Nat Immunol 12, 647–654 (2011). https://doi.org/10.1038/ni.2033

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