Granule mobilization and degranulation are important aspects of neutrophil function in acute inflammation and host-defense against microbial infection. When neutrophils are activated, their cytoplasmic granules are mobilized and transported to the plasma membrane for exocytosis and for fusion with phagosomes. There is compelling evidence that microtubules have a role in the translocation of neutrophil granules as they do in the directed movement of organelles in other types of cells. Although recent studies have revealed much about the biochemical machinery of microtubule-based organelle translocation systems, basic questions concerning the control mechanisms which govern these systems remain unanswered. These questions are particularly relevant to an understanding of neutrophil degranulation and exocytosis because these processes are highly regulated. This proposal will examine the molecular basis for regulated granule- microtubule interactions in human neutrophils. Studies will be directed at defining reversible modifications to ATPase proteins (e.g., kinesin and cytoplasmic dynein) that propel organelles along microtubules in resting and activated neutrophils. Studies will also examine post- translational modifications of tubulin that occur in response to neutrophil activation and their effects on granule-microtubule interactions. Kinesin and kinesin-associated proteins will be isolated from resting and activated human neutrophils by affinity chromatography using antibodies produced against peptides based on different regions of the amino acid sequence of Drosophila kinesin. Purified proteins will then be analyzed for neutrophil activation-induced changes in phosphorylation and electrophoretic mobility as well as changes in microtubule-activated ATPase activity and in their capacity to move microtubules in an in vitro system. Analogous studies will also be carried out in which cytoplasmic dynein and dynein-associated proteins will be isolated by immunoaffinity chromatography and examined for post- translational modifications associated with neutrophil activation. Tubulin phosphorylation, as well as tyrosination, acetylation, and glutamylation of the alpha-tubulin subunit, in the microtubules of resting and activated neutrophils will also be examined. Neutrophil activation-dependent changes in the relative proportions of tubulin isoforms will be determined by quantitative immunoblotting. Microtubules, polymerized in vitro from different isoforms of tubulin, will be examined for their capacity to enhance the ATPase activity of purified neutrophil kinesin and dynein and for their binding affinity for neutrophil granules isolated by density gradient centrifugation. It is anticipated that these studies will expand our understanding of basic mechanisms that control neutrophil degranulation and could define potential targets for the design of novel anti-inflammatory agents.
