The Role of the Small GTPase Rab27a in the Regulated Secretion of Granulocytes.

Neutrophils kill microorganisms using microbicidal products that they release into the phagosome or the extracellular space. Mature neutrophils contain four types of exocytosable storage organelles: azurophilic granules that contain myeloperoxidase, specific granules, gelatinase granules and secretory vesicles. Since the uncontrolled release of the contents of these organelles is potentially harmful, granule exocytosis must be tightly regulated. However, the secretory machinery utilized by neutrophils is poorly characterized. Here, we evaluate the role of Rab27a and its effector JFC1 in the regulated secretion of granulocytes. First, we show that JFC1 and Rab27a are highly expressed in human neutrophils. Furthermore, in HL-60 promyelocytic cells, the expression of JFC1 and Rab27a dramatically increased when they are differentiated to neutrophil-like granulocytes by DMSO treatment, supporting a role for these proteins in the secretory machinery of granulocytes. We found that while Rab27a distribution is restricted to the membrane fraction after cell fractionation, JFC1 is distributed between the membrane fraction and the cytosol. The localization of Rab27a in neutrophils was confirmed by immuno-electron microscopy. Colocalization of endogenous JFC1 and Rab27a was observed by immunofluorescence analysis. After cell fractionation, JFC1 and Rab27a were mainly associated with the tertiary granules (γ fraction) which are enriched in MMP-9 and cytochrome b558. A small proportion of the myeloperoxidase-positive granules were also detected in this fraction. Both Rab27a and JFC1 colocalized with VAMP2, a marker of secretory vesicles. The introduction of the plasma membrane binding domain (C2A domain) of JFC1 into permeabilized neutrophils significantly decreased exocytosis, however, neither Rab27a nor JFC1 integrated to the phagolysosome when neutrophils were exposed to opsonized particles, suggesting that the granules implicated in cargo release towards the surrounding milieu are molecularly and mechanistically different from those involved in their release towards the phagolysosome.