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Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells

Nature Immunology volume 10, pages 627–635 (2009)Cite this article

Abstract

The reorientation of the T cell microtubule-organizing center (MTOC) toward the antigen-presenting cell enables the directional secretion of cytokines and lytic factors. By single-cell photoactivation of the T cell antigen receptor, we show that MTOC polarization is driven by localized accumulation of diacylglycerol (DAG). MTOC reorientation was closely preceded first by production of DAG and then by recruitment of the microtubule motor protein dynein. Blocking DAG production or disrupting the localization of DAG impaired MTOC recruitment. Localized DAG accumulation was also required for cytotoxic T cell–mediated killing. Furthermore, photoactivation of DAG itself was sufficient to induce transient polarization. Our data identify a DAG-dependent pathway that signals through dynein to control microtubule polarity in T cells.

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Figure 1: Inhibition of PLC-γ blocks MTOC reorientation in helper T cells.
Figure 2: PMA inhibits MTOC reorientation, but Ca2+ blockade does not.
Figure 3: PMA impairs the maintenance of MTOC polarization, but ionomycin does not.
Figure 4: MTOC reorientation is spatially and temporally linked to the accumulation of DAG and dynein.
Figure 5: Accumulation of DAG and dynein 'predicts' MTOC reorientation.
Figure 6: Inhibition of DGKs disrupts DAG and dynein accumulation and impairs MTOC reorientation.
Figure 7: Localized DAG is sufficient to induce MTOC reorientation.
Figure 8: PMA and DGK II inhibit CTL mediated lysis but do not inhibit degranulation.

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Acknowledgements

We thank G. Altan-Bonnet (Memorial Sloan-Kettering Cancer Center) and O. Feinerman (Memorial Sloan-Kettering Cancer Center) for assistance with MATLAB programming, RMA-s cells and other reagents; T. Pentcheva-Hoang (Memorial Sloan-Kettering Cancer Center) and J. Allison (Memorial Sloan-Kettering Cancer Center) for CH12 cells and help with imaging experiments; Q. Li (Duke University) for helper T cell cDNA; W. Sha (University of California, Berkeley) and M.S. Kuhns (Stanford University) for pMSCV; R. Tsien (University of California, San Diego) and M.W. Davidson (Florida State University) for Tag-RFP-T; S.S. Yi and the Sloan-Kettering Institute Microchemistry Core Facility for peptide synthesis; D. Sant'Angelo, G. Altan-Bonnet, A. Hall, P. Abeyweera, D.A. Schaer and K. Pham for advice and critical reading of the manuscript; B. Driscoll for technical assistance; the laboratories of M.O. Li and B. Dupont for reagents and comments; and M.M. Davis for advice and support at the start of this project. Supported by the Spanish Ministry of Science and Innovation (E.M.), the Ministry of Education, Culture, Sports, Science and Technology of Japan (T.F.), the Searle Scholars Program (M.H.) and the Cancer Research Institute (M.H.).

Author information

Authors and Affiliations

  1. Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Emily J Quann, Ernesto Merino & Morgan Huse

  2. Department of Biomolecular Science, Toho University, Funabashi, Chiba, Japan

    Toshiaki Furuta

Authors
  1. Emily J Quann
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  2. Ernesto Merino
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Contributions

E.J.Q. and M.H. designed the experiments, collected the data and analyzed the results; E.M. helped develop the DGK studies; T.F. provided caged diC8; and M.H. wrote the manuscript with input from E.J.Q. and E.M.

Corresponding author

Correspondence to Morgan Huse.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–2 and Supplementary Movie Legends (PDF 1035 kb)

Supplementary Movie 1

Inhibition of PLC-γ blocks MTOC reorientation in TH cells. (MOV 1391 kb)

Supplementary Movie 2

The control compound U-73343 does not affect MTOC reorientation. (MOV 2226 kb)

Supplementary Movie 3

PMA inhibits MTOC reorientation. (MOV 2270 kb)

Supplementary Movie 4

Maintenance of MTOC polarization under control conditions. (MOV 756 kb)

Supplementary Movie 5

PMA impairs the maintenance of MTOC polarization. (MOV 525 kb)

Supplementary Movie 6

Ionomycin does not affect the maintenance of MTOC polarization. (MOV 417 kb)

Supplementary Movie 7

DAG accumulation precedes and is spatially correlated with MTOC reorientation. (MOV 216 kb)

Supplementary Movie 8

Dynein accumulation precedes and is spatially correlated with MTOC reorientation. (MOV 2517 kb)

Supplementary Movie 9

DAG accumulation precedes and is spatially correlated with dynein recruitment. (MOV 194 kb)

Supplementary Movie 10

DGK inhibition impairs the stable accumulation of DAG and the polarization of the MTOC. (MOV 2776 kb)

Supplementary Movie 11

DGK inhibition impairs the stable accumulation of dynein and the polarization of the MTOC. (MOV 399 kb)

Supplementary Movie 12

Localized DAG signaling is sufficient to induce transient reorientation of the MTOC. (MOV 3642 kb)

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Quann, E., Merino, E., Furuta, T. et al. Localized diacylglycerol drives the polarization of the microtubule-organizing center in T cells. Nat Immunol 10, 627–635 (2009). https://doi.org/10.1038/ni.1734

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