Munc13-4 Restricts Motility of Rab27a-expressing Vesicles to Facilitate Lipopolysaccharide-induced Priming of Exocytosis in Neutrophils

Abstract

LPS is an efficient sensitizer of the neutrophil exocytic response to a second stimulus. Although neutrophil exocytosis in response to pathogen-derived molecules plays an important role in the innate immune response to infections, the molecular mechanism underlying LPS-dependent regulation of neutrophil exocytosis is currently unknown. The small GTPase Rab27a and its effector Munc13-4 regulate exocytosis in hematopoietic cells. Whether Rab27a and Munc13-4 modulate discrete steps or the same steps during exocytosis also remains unknown. Here, using Munc13-4-and Rab27a-deficient neutrophils, we analyzed the mechanism of lipopolysaccharide-dependent vesicular priming to amplify exocytosis of azurophilic granules. We found that both Munc13-4 and Rab27a are necessary to mediate LPS-dependent priming of exocytosis. However, we show that LPS-induced mobilization of a small population of readily releasable vesicles is a Munc13-4-dependent but Rab27a-independent process. LPS-induced priming regulation could not be fully explained by secretory organelle maturation as the redistribution of the secretory proteins Rab27a or Munc13-4 in response to LPS treatment was minimal. Using total internal reflection fluorescence microscopy and a novel mouse model expressing EGFP-Rab27a under the endogenous Rab27a promoter but lacking Munc13-4, we demonstrate that Munc13-4 is essential for the mechanism of LPS-dependent exocytosis in neutrophils and unraveled a novel mechanism of vesicular dynamics in which Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharideinduced priming of exocytosis.

MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils

“…First, we determined the subcellular distribution of MUNC13-4 in relationship to that of flavocytochrome b 558 by cell fractionation and confocal microscopy. We found that endogenous MUNC13-4 colocalizes and cofractionates with organelles expressing gp91 phox and p22 MUNC13-4 regulates vesicular trafficking and has been proposed to regulate vesicular tethering (47), docking (30), and fusion (33) during exocytosis. To analyze whether the deficiency in MUNC13-4 is associated with abnormal distribution or mobilization of flavocytochrome b 558 -expressing organelles, we utilized TIRF microscopy and analyzed the localization of flavocytochrome b 558 -expressing granules in relationship to the plasma membrane in MUNC13-4 KO cells.…”

“…The cells were stored in unsolidified water-based mounting medium (refractive index, 1.37) or PBS until analyzed. TIRF microscopy experiments were performed using a 100ϫ 1.45 numerical aperture TIRF objective (Nikon) on a Nikon TE2000U microscope custom modified with a TIRF illumination module as described (30). Images were acquired on a 14-bit, cooled charge-coupled device camera (Hamamatsu) controlled through NIS-Elements software.…”

“…Because exocytosis is considered an important factor for NADPH oxidase activation (18) and MUNC13-4 regulates exocytosis in neutrophils (29,30), we sought to analyze a possible function for MUNC13-4 in ROS production regulation. First, we determined the subcellular distribution of MUNC13-4 in relationship to that of flavocytochrome b 558 by cell fractionation and confocal microscopy.…”

“…In neutrophils, MUNC13-4 regulates exocytosis of azurophilic (30) and tertiary (29) granules, but the increased susceptibility of patients with FHL3 to bacterial infections (34) suggests further defects in neutrophil function. Possible roles for MUNC13-4 in the control of the secretion of other neutrophil granules, ROS production, phagocytosis maturation, or NET formation have not been explored.…”

“…In neutrophils, RAB27A controls azurophilic granule exocytosis and regulates the activation of the NADPH oxidase at the plasma membrane (26 -28). The RAB27A effector MUNC13-4 is also involved in the regulation of granule exocytosis (27,29,30) and controls the priming of myeloperoxidase release in response to bacterial lipopolysaccharide (30), but its role in other neutrophil functions remains unknown. Importantly, defects in the rab27a or the unc13-4 genes are associated with immunodeficiencies in humans (31).…”