The process of cytoskeleton assembly and disassembly is essential for many critical phagocyte functions including locomotion, adhesion, egress from the vasculature, phagocytosis, and cytokine or integrin-stimulated respiratory burst. Understanding the mechanisms by which cytoskeleton assembly is regulated and acts in turn to direct the function of host defense cells is at the core of a major mechanism for augmentation of host defense at sites of inflammation, and likely will lead to novel mechanisms for pharmacological intervention to enhance host defense. However, the molecular details of this regulation and of its influence on leukocyte activation have been difficult to study. My laboratory has recently done a series of experiments which significantly advance understanding of the role of the cytoskeleton in the biochemistry of leukocyte activation. We have found that the actin-associated cytoskeleton is required for a novel IP3-independent pathway for the release of Ca+2 from intracellular stores in phagocytes. This pathway is initiated by ligation of IgG Fc receptors and perhaps ligation of the integrin complement receptor CR3 as well. Furthermore, we have found that a specific actin binding protein called l- plastin is required for this increase in [Ca+2]i. L-plastin has characteristics which suggest it may play an important role in leukocyte activation. It is expressed only in leukocytes, its actin binding is affected by Ca+2, and it undergoes serine phosphorylation when leukocytes are activated. The overall experimental plan of this application is to test the hypothesis that l-plastin is an essential protein in phagocyte activation. For this, the proposal has three interdependent goals: to understand the cell biology and biochemistry of l-plastin in phagocytes; to determine the effects of alterations in plastin expression and plastin function on cytoskeleton and signal transduction in phagocytes; and to determine the effect of plasma membrane receptor mutations on l-plastin function. These studies will definitively test the role of l-plastin in leukocyte activation and will enhance understanding of IP3-independent Ca+2 release from intracellular stores in phagocytes. Interaction of l- plastin with actin likely represents a focused target for therapies to augment host defense and for treatment of autoimmune and allergic diseases as well.
