Neutrophils contain four types of secretory organelles that hold specialized proteins essential for their microbicidal activity. Unrestricted release of the toxic content of their granules would be injurious to the host. Exocytosis of these granules is hierarchical and differentially regulated. This correlates with the role the cargo proteins play in the processes of adhesion, migration, chemotaxis, phagocytosis and production of reactive oxygen species. The mechanism of vesicle exocytosis in neutrophils is poorly understood. Rab27a is a small GTPase associated with secretory processes. Its role in granulocytes is unknown. Genetic defects in Rab27a are associated with the human immunodeficiency Griscelli syndrome in which neutrophil function may be altered. On the basis of extensive evidence from our laboratory, we propose the following hypotheses: 1) Rab27a and its effectors JFC1 and Munc13-4 play a central role in exocytosis in neutrophils and in associated functions that depend on granular protein mobilization;2) granules implicated in cargo release into the surrounding milieu are molecularly and mechanistically different from those involved in cargo release into the phagosome;3) JFC1 provides the specificity required to target Rab27a-containing vesicles to the docking site at the plasma membrane during exocytosis while Munc13-4 regulates the fusion process. To understand the mechanisms of Rab27a-dependent vesicular trafficking in granulocytes, we propose three specific aims: 1) To examine the role that Rab27a plays in exocytosis and phagocytosis in granulocytes, using Rab27a-deficient mice;2) To identify the mechanisms that regulate the docking of Rab27a-containing vesicles in granulocytes by electron and evanescence fluorescence microscopy using JFC1-deficient cells;3) To identify the cellular and biochemical events regulated by the Rab27a-effector Munc13-4. We plan to elucidate the mechanism underlying vesicle fusion and exocytosis using neutrophils from Munc13-4-deficient mice. The results of the proposed research plan should uncover the molecular mechanisms of vesicle docking, fusion and secretion in neutrophils and lead to effective molecular strategies for the treatment of inflammatory diseases.
Neutrophils are white cells that circulate in the blood. They play an essential role in protecting us from microbial infections through the production and release of substances that are toxic to the microorganisms. These toxic products, when released in large amounts or in an uncontrolled manner, can be harmful to the host (humans). The study of the mechanism that controls the release of toxic products by neutrophils will lead to the design of therapeutic strategies for the control of diseases like arthritis and other inflammatory disorders.
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