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Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4

Nature Immunology volume 8, pages 257–267 (2007)Cite this article

Abstract

Cytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal 'exocytic vesicle'. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11+ recycling and Rab27+ late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte–target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells.

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Figure 1: Cytotoxic granules containing perforin and granzyme B do not localize together with hMunc13-4- or Rab27a-associated vesicles.
Figure 2: Rab27a localizes together with Rab7 on late endosomal structures.
Figure 3: The assembly of Rab11+ and Rab27a+ endosomal structures is directed by hMunc13-4.
Figure 4: Limited modulation of hMunc13-4 expression by CTL activation.
Figure 5: The MHD region of hMunc13-4 localizes on vesicular structures that are distinct from cytotoxic granules.
Figure 6: In vivo binding of hMunc13-4 with the active form of Rab27a in cytotoxic cells and characterization of a minimal interacting region of hMunc13-4.
Figure 7: Rab11–hMunc13-4–Rab27a endosomal structures and cytotoxic granules overlap only at the closest zone of cell-cell contact.
Figure 8: 'Exocytic' vesicles and cytotoxic granules assemble at the immunological synapse after hMunc13-4 expression.

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Acknowledgements

We thank B. Goud and J. Salamero (Institut Curie) for discussions and comments on the manuscript and for providing purified anti-Rab11 and pEGFPC1-Rab11; J. Gruenberg (University of Geneva) for providing pEGFC1-Rab7; and R. Micol for help with statistical analysis. Supported by the Institut National de la Santé et de la Recherche Médicale, Agence Nationale de la Recherche, Action Concerté du Ministère de l'Education Nationale et de la Recherche (BCMS103), and Ministère de l'Education Nationale de la Recherche et de la Technologie (M.M.M.).

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Authors and Affiliations

  1. Institut National de la Santé et de la Recherche Médicale Unité 768, Laboratoire du Développement Normal et Pathologique du Système Immunitaire, Paris, F-75015, France

    Mickaël M Ménager, Gaël Ménasché, Perrine Knapnougel, Chen-Hsuan Ho, Jérôme Feldmann, Alain Fischer & Geneviève de Saint Basile

  2. Université Paris Descartes, Faculté de Médecine René Descartes, Institut Fédératif de Recherche Necker Enfants-Malades (IFR95), Paris, F-75015, France

    Mickaël M Ménager, Gaël Ménasché, Perrine Knapnougel, Chen-Hsuan Ho, Mériem Garfa, Jérôme Feldmann, Alain Fischer & Geneviève de Saint Basile

  3. Centre National de la Recherche Scientifique UMR144, Institut Curie, Paris, F-75005, France

    Maryse Romao & Graça Raposo

  4. Assistance Publique-Hôpitaux de Paris, Hôpital Necker Enfants-Malades, Unité d'Immunologie-Hématologie Pédiatrique, Paris, F-75015, France

    Alain Fischer & Geneviève de Saint Basile

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  1. Mickaël M Ménager
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Contributions

M.M.M. and G.d.S.B. conceptualized and designed the research, analyzed the data and wrote the manuscript; G.d.S.B. supervised the research; M.M.M. did all experiments unless stated otherwise; G.M., J.F. and C.-H.H. contributed to experimental design and analysis; P.K. provided technical assistance; M.G. assisted in immunofluorescence analyses; M.R. and G.R. did the immunoelectron microscopy; and A.F. contributed to discussions and to the preparation of the manuscript.

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Correspondence to Geneviève de Saint Basile.

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Supplementary information

Supplementary Fig. 1

Tagged-hMunc13-4 and -Rab27a proteins overexpressed in CTLs are functional. (PDF 500 kb)

Supplementary Fig. 2

Tagged-hMunc13-4 expressed in CTLs colocalize with Rab11+ endosomal structures. (PDF 883 kb)

Supplementary Fig. 3

Mapping of the site of hMunc13-4 interacting with Rab27a. (PDF 398 kb)

Supplementary Fig. 4

Specificity of hMunc13-4 antibody. (PDF 894 kb)

Supplementary Video 1

3-D reconstruction of serial confocal sections taken through the CTL-target cell conjugate shown in Fig 8a (bottom) and rotated around x axis. Cytotoxic granules, labeled with perforin (red) and “exocytic vesicles” associating GFP-Rab11 (green) and unlabeled hMunc13-4 separately polarized toward the cell-cell contact site (blue arrow). Rotation around the x axis show the cytotoxic granules docked at the plasma membrane forming a ring around the small pool of “exocytic vesicles” reaching the cSMAC. (MOV 4464 kb)

Supplementary Video 2

3-D reconstruction of serial confocal sections taken through the CTL-target cell conjugate shown in Fig 8a (bottom) and rotated around y axis. Cytotoxic granules, labeled with perforin (red) and “exocytic vesicles” associating GFP-Rab11 (green) and unlabeled hMunc13-4 separately polarized toward the cell-cell contact site (blue arrow). Rotation around the y axis show the cytotoxic granules docked at the plasma membrane forming a ring around the small pool of “exocytic vesicles” reaching the cSMAC. (MOV 5019 kb)

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Ménager, M., Ménasché, G., Romao, M. et al. Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4. Nat Immunol 8, 257–267 (2007). https://doi.org/10.1038/ni1431

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