Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

Dudenhöffer-Pfeifer, Monika; Schirra, Claudia; Pattu, Varsha; Halimani, Mahantappa; Maier-Peuschel, Monika; Marshall, Misty; Matti, Ulf; Becherer, Ute; Dirks, Jan; Jung, Martin; Lipp, Peter; Hoth, Markus; Sester, Martina; Krause, Elmar; Rettig, Jens

doi:10.1111/tra.12074

Cited by 30 publications

(25 citation statements)

References 52 publications

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“…Our finding that Munc13-4 is required for immobilizing dense granules offers new insights into the mechanisms of dense granule exocytosis and suggests that they exist in a “docked/tethered” state that is poised for release. This mechanism appears to be similarly used for lytic granules in cytotoxic T lymphocytes were loss of Munc13s increases granule mobility (57). Our data also account for the robust bleeding and hemostasis defect observed in the Unc13d Jinx mice that lack the protein (24, 26, 58, 59) and are congruent with the phenotype observed in humans harboring mutations in UNC13D , the human homologue of Munc13-4 (60, 61).…”

Section: Discussionmentioning

confidence: 99%

Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

Chicka

Ren

Richards

et al. 2016

Biochemical Journal

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The Munc13 family of exocytosis regulators has multiple Ca2+-binding, C2 domains. Here, we probed the mechanism by which Munc13-4 regulates in vitro membrane fusion and platelet exocytosis. We show that Munc13-4 enhances in vitro SNARE-dependent, proteoliposome fusion in a Ca2+- and phosphatidylserine (PS)-dependent manner that was independent of SNARE concentrations. Munc13-4-SNARE interactions, under the conditions used, were minimal in the absence or presence of Ca2+. However, Munc13-4 was able to bind and cluster liposomes harboring PS in response to Ca2+. Interestingly, Ca2+-dependent liposome binding/clustering and enhancement of proteoliposome fusion required both Munc13-4 C2 domains, but only the Ca2+-liganding aspartate residues of the C2B domain. Analytical ultracentrifugation measurements indicated that, in solution, Munc13-4 was a monomeric prolate ellipsoid with dimensions consistent with a molecule that could bridge two fusing membranes. To address the potential role of Munc13-4 as a tethering protein in platelets, we examined mepacrine-stained, dense granule mobility and secretion in platelets from wild-type and Munc13-4 null (Unc13dJinx) mice. In the absence of Munc13-4, dense granules were highly mobile in both resting and stimulated platelets, and stimulation-dependent granule release was absent. These observations suggest that dense granules are stably docked in resting platelets awaiting stimulation and that Munc13-4 plays a vesicle-stabilizing or tethering role in resting platelets and also in activated platelets in response to Ca2+. In summary, we show that Munc13-4 conveys Ca2+ sensitivity to platelet SNARE-mediated membrane fusion and reveal a potential mechanism by which Munc13-4 bridges and stabilizes apposing membranes destined for fusion. (246 words)

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Section: Discussionmentioning

confidence: 99%

Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

Chicka

Ren

Richards

et al. 2016

Biochemical Journal

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“…On the other hand, Munc13-4 is involved in regulated exocytosis in hematopoietic secretory cells, such as cytotoxic T lymphocytes (CTLs), platelets, neutrophils, and mast cells (7,12,27,30,33,35). It is also known that in neurons, chromaffin cells, and CTLs, multiple Munc13 isoforms are endogenously expressed, and play some redundant roles in the promotion of the docking and priming of secretory vesicles/granules (8,10,24,36). In mast cells, Munc13-4 is shown to be crucial for degranulation (11,35,38); however, the involvement of other Munc13 isoform(s) still remains unknown.…”

Section: Rt-pcr and Quantitative Real-time Rt-pcrmentioning

confidence: 99%

“…Upon multivesicular exocytosis in mast cells, late and recycling endosomes have been shown to fuse sequentially with the growing exocytic structures formed by Munc13-4-dependent SG-SG fusion (SG-derived exocytic structures) (38). Because the SGs in the mast cells themselves possess endosomal properties, especially those of late endo-DISCUSSION Munc13-1 has been recently found to be expressed together with Munc13-4 in CTLs, and both the proteins have been shown to positively regulate the priming process in lytic granule exocytosis, indicating their functional overlap (10). In the present study, we showed that Munc13-1, as well as Munc13-4, is endogenously expressed in the RBL-2H3 mast cell line; however, unexpectedly, we found that in contrast to its effect on CTLs, Munc13-1 inhibited the antigen-induced degranulation of the RBL-2H3 cells.…”

Section: Functional Relationship Between Munc13-1 and Munc13-4mentioning

confidence: 99%

<b>Inhibitory role of Munc13-1 in antigen-induced mast cell </b><b>degranulation </b>

2017

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Secretory granules (SGs) of mast cells are lysosome-related organelles that contain various inflammatory molecules such as histamine, which are stored in the cytoplasm. Mast cell degranulation is the regulated exocytosis of SGs in response to external stimuli, such as the antigen-mediated cross-linking of the high-affinity IgE receptor, FcεRI. Upon stimulation, SGs undergo priming to become fusion-competent prior to fusing with the plasma membrane, which is mediated by Munc13-4, one of the five members of the vesicle-priming Munc13 protein family. Although Munc13-4 is shown to be crucial for mast cell degranulation, the functional involvement of other Munc13 isoform(s) remains unknown. Herein, this was investigated using the RBL-2H3 mast cell line. We found that Munc13-1 and Munc13-4 are the only Munc13 isoforms that are expressed in the RBL-2H3 cells, and Munc13-1 is distributed in the cytoplasm, but highly concentrated on the late endosome and/or lysosome. Unexpectedly, antigen-induced degranulation was considerably increased by Munc13-1 knockdown, but decreased by its overexpression. Further, we found that the hypersecretion phenotype of the Munc13-1-knockdown cells was attenuated by simultaneous Munc13-4 knockdown. These results suggested that Munc13-1 has an inhibitory role in antigen-induced mast cell degranulation, which is performed in a Munc13-4-dependent manner.Mast cells are specialized secretory cells that play a central role in type I allergic reactions, as well as in certain innate and adaptive immune responses (1, 13). The antigen-mediated cross-linking of the highaffinity immunoglobulin E (IgE) receptor FcεRI triggers Ca 2+

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“…The green dot represents the starting position of the LG and the red dot represents the end position of the LG. The absence of the priming factor Munc13-4 leads to a significant increase in vesicle mobility in TIRFM (28). (C) Fluorescence profile of a fusing LG (left), a stationary LG (middle), and an LG that is undocking (right) as seen in TIRFM.…”

Section: Visualizing and Quantifying Lytic Granules At The Is By Tirfmmentioning

confidence: 99%

In the Crosshairs: Investigating Lytic Granules by High-Resolution Microscopy and Electrophysiology

et al. 2013

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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.

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Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

Cited by 30 publications

References 52 publications

Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

Role of Munc13-4 as a Ca2+-dependent tether during platelet secretion

<b>Inhibitory role of Munc13-1 in antigen-induced mast cell </b><b>degranulation </b>

In the Crosshairs: Investigating Lytic Granules by High-Resolution Microscopy and Electrophysiology

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