The Rab27a Effectors JFC1/Slp1 and Munc13‐4 Regulate Exocytosis of Neutrophil Granules

Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Traffic (Impact Factor: 4.35). 11/2008; 9(12):2151-64. DOI: 10.1111/j.1600-0854.2008.00838.x
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Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene-targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13-4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.

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Available from: Bruce Beutler, Oct 01, 2015
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    • "Munc13-4 (also known as UNC13D) is highly expressed in neutrophils and interacts with Rab27, which is the Rab that direct granules to dock at the plasma membrane. Munc13-4 tethers granules to the plasma membrane for exocytosis via two calcium-sensitive lipid bind C2 domains (Figure 4) (113). Neutrophil subfractionation revealed that high levels of Munc13-4 rapidly translocate from the cytosol to granules upon stimulation with a secretagogue like fMLF (114). "
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    ABSTRACT: Neutrophils are part of a family of granulocytes that, together with eosinophils and basophils, play an essential role in innate immunity. Neutrophils are the most abundant circulating leukocytes and are vital for rapid immune responses, being recruited to sites of injury or infection within minutes, where they can act as specialized phagocytic cells. However, another prominent function of neutrophils is the release of pro-inflammatory compounds, including cytokines, chemokines, and digestive enzymes, which are stored in intracellular compartments and released through regulated exocytosis. Hence, an important feature that contributes to rapid immune responses is capacity of neutrophils to synthesize and store pre-formed pro-inflammatory mediators in specialized intracellular vesicles and thus no new synthesis is required. This review will focus on advancement in three topics relevant to neutrophil secretion. First, we will examine what is known about basal level pro-inflammatory mediator synthesis, trafficking, and storage in secretory compartments. Second, we will review recent advancements in the mechanisms that control vesicle mobilization and the release of pre-formed mediators. Third, we will examine the upregulation and de novo synthesis of pro-inflammatory mediators by neutrophils engaged at sites of infection.
    Frontiers in Immunology 09/2014; 5:448. DOI:10.3389/fimmu.2014.00448
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    • "This mechanism increases the number of adhesion molecules at the plasma membrane. Mobilization of secretory organelles that contain the integrin subunit CD11b, a molecule that plays an important role in neutrophil adhesion to the activated endothelium during the innate immune response, was not affected in Rab27a-or Munc13-4-KO neutrophils (Johnson et al., 2010a), (Brzezinska et al., 2008). Further analysis using Rab27b-KO and Rab27a/b-double KO neutrophils demonstrated that the Rab27 family of small GTPases is not necessary for CD11b upregulation (Johnson et al., 2010a). "
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    ABSTRACT: Neutrophils are central regulators of the innate immune response and help shape the adaptive immune response. Malfunction and unregulated neutrophil activation leads to disease and inflammation. During the host response to infection, neutrophils display several mechanisms of defense mediated by their arsenal of granular proteins. Regulation of granular trafficking, docking and fusion is at the core of the neutrophil defense response to pathogens. The small GTPase Rab27a has emerged as a central regulator of the neutrophil response through its tight control of vesicular trafficking and degranulation. This review focuses on the latest research that has led to the characterization of Rab27a as an essential regulator of neutrophil function.
    Cellular Microbiology 06/2014; 16(9). DOI:10.1111/cmi.12328 · 4.92 Impact Factor
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    • "Developmental Cell 27, 215–226, October 28, 2013 ª2013 Elsevier Inc. 223 Immunostaining of Neutrophils Neutrophils were placed onto the coverslips for 15 min before stimulation. The cells were then fixed with 4% paraformaldehyde and permeabilized with 0.01% saponin and blocked with 2% BSA in PBS (Brzezinska et al., 2008). Neutrophils were stained with a STK24 antibody (3723, Cell Signaling, 1:100) and/or biotinylated MPO antibody (HM1051BT, Hycult Biotech, 1:100), followed by Alexa-488-Goat-anti-Rabbit (Invitrogen, 1:300 for the STK antibody) and APC-streptavidin (BD, 1:300 for the MPO antibody) staining using a previously described protocol (Xu et al., 2010). "
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    ABSTRACT: Neutrophil degranulation plays an important role in acute innate immune responses and is tightly regulated because the granule contents can cause tissue damage. However, this regulation remains poorly understood. Here, we identify the complex of STK24 and CCM3 as being an important regulator of neutrophil degranulation. Lack of either STK24 or CCM3 increases the release of a specific granule pool without affecting other neutrophil functions. STK24 appears to suppress exocytosis by interacting and competing with UNC13D C2B domain for lipid binding, whereas CCM3 has dual roles in exocytosis regulation. Although CCM3 stabilizes STK24, it counteracts STK24-mediated inhibition of exocytosis by recruiting STK24 away from the C2B domain through its Ca(2+)-sensitive interaction with UNC13D C2A domain. This STK24/CCM3-regulated exocytosis plays an important role in the protection of kidneys from ischemia-reperfusion injury. Together, these findings reveal a function of the STK24 and CCM3 complex in the regulation of ligand-stimulated exocytosis.
    Developmental Cell 10/2013; 27(2):215-26. DOI:10.1016/j.devcel.2013.09.021 · 9.71 Impact Factor
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