Importantly, despite the fact that Munc13-4 regulates trafficking of Rab27a-positive organelles in neutrophils, the defects in Rab11-positive vesicle trafficking observed here cannot be explained by a putative impairment in Rab27a function, because Rab27a and Rab11 localize entirely at different intracellular compartments in neutrophils, even under stimulated conditions (Fig

Importantly, despite the fact that Munc13-4 regulates trafficking of Rab27a-positive organelles in neutrophils, the defects in Rab11-positive vesicle trafficking observed here cannot be explained by a putative impairment in Rab27a function, because Rab27a and Rab11 localize entirely at different intracellular compartments in neutrophils, even under stimulated conditions (Fig. Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. Keywords: docking, intracellular trafficking, NADPH oxidase, neutrophil, Rab, Munc13-4, Rab11, Rab27, recycling endosome, super-resolution microscopy == Introduction == Rab GTPases are membrane organizers that regulate the specificity of subcellular compartments and trafficking of these compartments through interactions with specific effectors (1). In particular, Rab11, a ubiquitously expressed GTPase, has been implicated in the regulation of several intracellular mechanisms including recycling endosome trafficking, transport of cargo from the sorting endosomes and the trans-Golgi network to the endosomal recycling compartment and regulation of trafficking during cytokinesis (2). In addition , the Rab11 family of GTPases, comprised by Rab11a, Rab11b and Rab11c/Rab25, are known to regulate several cellular functions including exocytosis (3), phagocytosis (4), and cell-cell communication (5). Rab11 functions are regulated by a broad TRPC6-IN-1 range of molecular interactors and effectors (2), and proteomic analyses have identified TRPC6-IN-1 a large array of structurally diverse molecules that interact with Rab11. The most studied family of Rab11 effectors, FIPs (Rab11 family-interacting proteins), 4comprises five members. RAB11FIP3 Rip11, FIP2, and RCP are characterized by the presence of a conserved Rab11-binding domain at their C terminus and a C2 domain at their N terminus, and FIP3 and FIP4 are characterized by the presence of EF-hand TRPC6-IN-1 domains but lack C2 domains (extensively reviewed in Ref. 6). Despite the increasing knowledge in the function of Rab11 and effectors in the regulation of cellular processes, it is accepted that the mechanism of Rab11 recruitment to specific sets of membranes and the possible cross-regulation of Rab11 functions by multiple effectors require further investigation (2). In particular, although both the mobilization of recycling endosomes and exocytosis would require the ultimate fusion of Rab11-containg vesicles with the plasma membrane, the molecular mechanisms regulating these processes remain elusive, and the Rab11 effectors mediating fusion are not fully identified. Munc13-4 is a 120-kDa factor expressed in several cells and tissues with secretory functions. It is characterized by the presence of two C2-domains at the N and C terminus and two central Munc-homology domains (MHC) (7). The importance of these domains in the interaction of Munc13-4 with SNARE proteins to mediate membrane fusion has been recently described (8), and Munc13-4 is considered a regulator of vesicle tethering and membrane fusion (9). Several studies have shown that Munc13-4 is an essential regulator of lytic granule exocytosis at the immunological synapse (10), neutrophil granule secretion (11, 12), and platelet-dense granule exocytosis (13). In fact , Munc13-4 deficiency is associated with the human severe immunodeficiency FHL3 caused by the inefficiency of immune cells to mediate killing of pathogens and infected cells (9). Based on the similarity of this phenotype with that observed in Rab27a-deficient patients along with pull-down studies, protein-protein interaction, and functional assays, Munc13-4 was identified as a Rab27a-specific effector (14). Despite shared roles in regulating common secretory pathways, Munc13-4 regulates many cellular functions in a Rab27a-independent manner including phagosomal maturation in neutrophils (15), the differential secretory response to stimuli in NK cells (16) and the regulation of readily releasable secretory granules in granulocytes (12). It is therefore unclear how these discrete molecular events during.