Ménager MM, Ménasché G, Romao M et al.Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4. Nat Immunol. 8:257-267

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.
Nature Immunology (Impact Factor: 20). 04/2007; 8(3):257-67. DOI: 10.1038/ni1431
Source: PubMed


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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Available from: Mickael Menager, May 04, 2014
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    • "After polarizing to the IS and before degranulation, lytic granules dock at the IS and fuse with the plasma membrane (Figure 1n).26, 120, 124, 125 Docking is likely mediated primarily by Munc 13-4 and Rab27a, both of which are recruited to lytic granules following activating receptor ligation.126 In CTL, lytic granules become fully mature only at late stages of the exocytic pathway, when Munc13-4 and Rab27a associate with perforin- and granzyme-containing lysosomal components.127 Upon interacting with Rab27a, Munc13-4 may also regulate the interaction between vesicle and target soluble NSF Attachment Protein REceptor (SNARE) required for lytic granule fusion with the plasma membrane.123 "
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    ABSTRACT: Natural killer (NK) cell-mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps, which give rise to an extensive array of cellular checkpoints in accessing NK cell-mediated cytolytic defense. Appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death. Here we highlight recent discoveries in the formation of the NK cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process.Immunology and Cell Biology advance online publication, 21 January 2014; doi:10.1038/icb.2013.96.
    Immunology and Cell Biology 01/2014; 92(3). DOI:10.1038/icb.2013.96 · 4.15 Impact Factor
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    • "In the case of Rab27a and Munc13-4, in-depth studies using confocal imaging and high-resolution electron microscopy (EM) identified their precise function in LG fusion (8, 9). In addition, new insight on LG maturation was gained by elucidating the requirement of Munc13-4 for this process (10). Munc13-4 is an effector of Rab27a (11), and later studies using high-resolution total internal reflection fluorescence microscopy (TIRFM) showed that the Munc13-4 Rab27 complex is required for tethering LG at the plasma membrane (12). "
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    ABSTRACT: Cytotoxic T lymphocytes (CTLs) form an integral part of the adaptive immune system. Their main function is to eliminate bacteria- and virus-infected target cells by releasing perforin and granzymes (the lethal hit) contained within lytic granules (LGs), at the CTL-target-cell interface [the immunological synapse (IS)]. The formation of the IS as well as the final events at the IS leading to target-cell death are both highly complex and dynamic processes. In this review we highlight and discuss three high-resolution techniques that have proven invaluable in the effort to decipher key features of the mechanism of CTL effector function and in particular lytic granule maturation and fusion. Correlative light and electron microscopy allows the correlation between organelle morphology and localization of particular proteins, while total internal reflection fluorescence microscopy (TIRFM) enables the study of lytic granule dynamics at the IS in real time. The combination of TIRFM with patch-clamp membrane capacitance measurements finally provides a tool to quantify the size of fusing LGs at the IS.
    Frontiers in Immunology 11/2013; 4:411. DOI:10.3389/fimmu.2013.00411
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    • "In this framework, the ability of cytolytic effectors to execute multiple killing cycles in a short time period (86–88) is thought to depend both on the release of a fraction of lytic granules (77) and on a continuum refilling of the granule store through newly synthesized cytotoxic mediators (89). Whether secretory lysosome retrieval could facilitate recycling and reusing of cytotoxic machinery components thus contributing to the serial killing potential, is not fully understood. "
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    ABSTRACT: Target cell recognition by cytotoxic lymphocytes implies the simultaneous engagement and clustering of adhesion and activating receptors followed by the activation of an array of signal transduction pathways. The cytotoxic immune synapse represents the highly specialized dynamic interface formed between the cytolytic effector and its target that allows temporal and spatial integration of signals responsible for a defined sequence of processes culminating with the polarized secretion of lytic granules. Over the last decades, much attention has been given to the molecular signals coupling receptor ligation to the activation of cytolytic machinery. Moreover, in the last 10 years the discovery of genetic defects affecting cytotoxic responses greatly boosted our knowledge on the molecular effectors involved in the regulation of discrete phases of cytotoxic process at post-receptor levels. More recently, the use of super resolution and total internal reflection fluorescence imaging technologies added new insights on the dynamic reorganization of receptor and signaling molecules at lytic synapse as well as on the relationship between granule dynamics and cytoskeleton remodeling. To date we have a solid knowledge of the molecular mechanisms governing granule movement and secretion, being not yet fully unraveled the machinery that couples early receptor signaling to the late stage of synapse remodeling and granule dynamics. Here we highlight recent advances in our understanding of the molecular mechanisms acting in the activation of cytolytic machinery, also discussing similarities and differences between Natural killer cells and cytotoxic CD8(+) T cells.
    Frontiers in Immunology 11/2013; 4:390. DOI:10.3389/fimmu.2013.00390
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