The process of cytoskeleton assembly and disassembly is essential for many critical phagocyte functions, including locomotion, phagocytosis, adhesion, egress from the vasculature, and cytokine production. Understanding the mechanisms by which cytoskeleton assembly is regulated and acts in turn to direct the functions of the phagocytic cell is at the core of understanding the mechanisms of augmentation of host defense at sites of inflammation. The molecular details of this regulation and it's influence on leukocyte activation has been difficult to study. This laboratory has developed a system which has significantly advanced the understanding of the role of the cytoskeleton in the mechanism of leukocyte activation. This lab has found that the pathway initiated by ligation of FcR by Ig6 in phagocytes increases [Ca2+]i by stimulating release of Ca2+ from intracellular stores through a novel IP3-independent pathway that requires the actin cytoskeleton. A specific actin-binding protein called l-plastin is required for this release of Ca2+. Our hypothesis is that l-plastin is an important link between cell surface receptors, such as FcR, and the cytoskeleton in phagocytes. In order to test this hypothesis, we will study the cell biology and biochemistry of l-plastin in phagocytes that naturally express the protein, as well as develop an in vitro transfection system to allow us to study the effects of altering or mutating l-plastin on l-plastin cell biology and biochemistry as well as IgG and integrin-mediated signal transduction. Actin binding, phosphorylation, and immunolocalization of l- plastin and IgG-mediated calcium responses and phagocytosis in phagocytic cells will be studied as models of normal l-plastin function. L-plastin function will be defined further by applying these assays to cell transfection systems using mutated forms of l-plastin. Cells that do not normally express l-plastin as well as mouse cell lines that express endogenous l-plastin will be transfected with the human form of l-plastin. The function of wild type human l-plastin in these cell lines will be validated by comparing l-plastin function and IgG-mediated responses in cells transfected with normal l-plastin and normal phagocytes.
| Jones, S L; Brown, E J (1996) FcgammaRII-mediated adhesion and phagocytosis induce L-plastin phosphorylation in human neutrophils. J Biol Chem 271:14623-30 |
