Centrosome polarization delivers secretory granules to the immunological synapse (original) (raw)

Nature volume 443, pages 462–465 (2006)Cite this article

An Erratum to this article was published on 09 November 2006

Abstract

Cytotoxic T lymphocytes (CTLs) destroy virally infected and tumorigenic cells by releasing the contents of specialized secretory lysosomes—termed ‘lytic granules’—at the immunological synapse formed between the CTL and the target1. On contact with the target cell, the microtubule organizing centre of the CTL polarizes towards the target2,3 and granules move along microtubules in a minus-end direction towards the polarized microtubule organizing centre. However, the final steps of secretion have remained unclear. Here we show that CTLs do not require actin or plus-end microtubule motors for secretion, but instead the centrosome moves to and contacts the plasma membrane at the central supramolecular activation cluster of the immunological synapse. Actin and IQGAP1 are cleared away from the synapse, and granules are delivered directly to the plasma membrane. These data show that CTLs use a previously unreported mechanism for delivering secretory granules to the immunological synapse, with granule secretion controlled by centrosome delivery to the plasma membrane.

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Acknowledgements

We thank J. Neefjes for the RILP complementary DNA, B. Imhof for actin–GFP, A. Thrasher for lentiviral vectors, M. Simon for anti-granzyme A antibodies, and K. Gull for anti-cenexin antibodies. J. Kaufman, P. A. van der Merwe and K. Gull provided many helpful discussions. This research was funded by the Wellcome Trust. Funding for the Dunn School Bio-imaging facility was provided by the Edward Abraham and Wellcome Trusts. G.M.G. is the recipient of a Royal Society Wolfson Research Merit Award. Author Contributions J.C.S. carried out all experiments except for RILP analysis (G.B.), tomographic reconstruction (E.M. and S.F.) and CTL culture (G.M.G.). J.C.S. and G.M.G. analysed the data and wrote the manuscript with contributions from all authors.

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Authors and Affiliations

  1. Sir William Dunn School of Pathology, South Parks Road, OX1 3RE, Oxford, UK
    Jane C. Stinchcombe, Giovanna Bossi & Gillian M. Griffiths
  2. Wellcome Trust Centre for Human Genetics, Roosevelt Drive, OX3 7BN, Oxford, UK
    Endre Majorovits & Stephen Fuller

Authors

  1. Jane C. Stinchcombe
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  2. Endre Majorovits
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  3. Giovanna Bossi
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  4. Stephen Fuller
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  5. Gillian M. Griffiths
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Corresponding author

Correspondence toGillian M. Griffiths.

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Supplementary information

Supplementary Information guide

This file contains Supplementary Methods, Supplementary Figure 1 and Supplementary Movie Legends 1–10. (DOC 659 kb)

Supplementary Movie 1A

3D reconstruction, rotated around the yz axis, of serial confocal images through a CTL conjugated to a target cell showing CTL centrioles (red) polarized right up to the plasma membrane at the contact site between the CTL and its target (shown in green) during killing of the target cell. (MOV 1358 kb)

Supplementary Movie 1B

3D reconstruction, rotated around the xz axis, of serial confocal images through a CTL conjugated to a target cell showing CTL centrioles (red) polarised right up to the plasma membrane at the contact site between the CTL and its target (shown in green) during killing of the target cell. (MOV 1213 kb)

Supplementary Movie 2

Tomogram generated from a -65-+65° tilt series of an EM section across the contact site between a CTL and its target showing polarisation of the CTL centrosome to the membrane and distribution of the lytic granules, loaded with an electron dense reaction product, at the contact site during target cell killing (AVI 682 kb)

Supplementary Movie 3

Model generated from the tomogram shown in S2 highlighting the distribution of the polarised centrosome (blue), lytic granules (grey), microtubules (red), Golgi complex (green) in the CTL at the contact site. (AVI 1540 kb)

Supplementary Movie 4A

3D reconstruction, rotated around the yz axis, of serial confocal images taken through a CTL conjugated to a target cell showing the CTL centrosome (blue) contacts the plasma membrane within the pSMAC (green) with the leading centriole on the edge of the cSMAC (red). (MOV 854 kb)

Supplementary Movie 4B

3D reconstruction, rotated around the xz axis, of serial confocal images taken through a CTL conjugated to a target cell showing the CTL centrosome (blue) contacts the plasma membrane within the pSMAC (green) with the leading centriole on the edge of the cSMAC (red). (MOV 734 kb)

Supplementary Movie 5A

3D reconstruction, rotated around the yz axis, of serial confocal images through a CTL conjugated to a target cell showing the CTL centrosome (blue) contacts the plasma membrane at the boundary between the cSMAC (red) and the site of lytic granule (green) docking and secretion. (MOV 696 kb)

Supplementary Movie 5B

3D reconstruction, rotated around the xz axis, of serial confocal images through a CTL conjugated to a target cell showing the CTL centrosome (blue) contacts the plasma membrane at the boundary between the cSMAC (red) and the site of lytic granule (green) docking and secretion. (MOV 763 kb)

Supplementary Movie 6

3D reconstruction, rotated around the yx axis, of serial confocal images through a single CTL showing the steady state distribution of IQGAP1 (red), actin (blue) and CD11a (green) in a CTL before it encounters a target cell. (MOV 3333 kb)

Supplementary Movie 7

3D reconstruction, rotated around the yz axis, of serial confocal images through a CTL conjugated to a target cell showing IQGAP1 (red) and actin (blue) cleared from the contact site to outside the pSMAC (CD11a, green) on encounter with a target cell and formation of an immunological synapse. (MOV 4500 kb)

Supplementary Movie 8

3D reconstruction, rotated around the yz axis, of serial confocal images through a CTL conjugated to a target cell showing IQGAP1 (red) present across the whole contact site area in a conjugate where the CTL centrosome (blue) is not polarised and CD11a (green) is not organised into a pSMAC. (MOV 3642 kb)

Supplementary Movie 9

3D reconstruction, rotated around the yz axis, of serial confocal images through a CTL conjugated to a target cell showing IQGAP1 (red) cleared from the contact site in a conjugate where the CTL centrosome (blue) has polarised to the plasma membrane and CD11a (green) is organised into a pSMAC. (MOV 3543 kb)

Supplementary Movie 10

3D reconstruction, rotated around the yz axis, of serial confocal images through a CTL conjugated to a target cell showing IQGAP1 (green) and actin (blue) cleared from the contact site during docking of lytic granules (red) and targeted secretion at the secretory domain of the immunological synapse. (MOV 6704 kb)

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Stinchcombe, J., Majorovits, E., Bossi, G. et al. Centrosome polarization delivers secretory granules to the immunological synapse.Nature 443, 462–465 (2006). https://doi.org/10.1038/nature05071

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Cytotoxic T lymphocytes are an important part of the immune system, killing targets by releasing the content of secretory lysosomes ('lytic' granules) at the immunological synapse. It's generally assumed that, by analogy to melanocytes, lytic granules travel by short plus-end directed microtubules from the microtubule organizing centre to the actin cytoskeleton in order to reach the immunological synapse. But no. Cytotoxic T lymphocytes have now been found to be independent of actin or plus-end microtubule motors, instead using a novel mechanism controlled by movement of the centrosome to deliver the lethal lytic granules.