The regulation of actin filament dynamics in neutrophils and macrophages allows cells to crawl to sites of infection and ingest invading pathogens. The present grant focuses on the most abundant actin regulatory protein in macrophages, CapG.
Aim 1 will explore the functional consequences of knocking out CapG in mice. CapG-null mice have profound defects in the ability of their macrophages, neutrophils and dendritic cells to move and take in foreign material. In order to determine how CapG functionally relates to other proteins in the cell, the compensatory changes in other macrophage proteins will be assessed by 2-D gel electrophoresis and microsequencing, as well as by gene microarray analysis. The functional significance of these adaptive protein changes will be assessed by over-expressing the identified proteins and by lowering their concentrations by RNA interference. Their ability to bind to CapG will be assessed by pull-down and yeast-two hybrid assays. Loss of CapG results in increased susceptibility to infection by the intracellular bacterium Listeria, indicating a defect in cell-mediated immunity. Levels of the inflammatory mediators IL-2 and interferon-gamma will be measured. CD4 and CDS lymphocyte responses, macrophage and dendritic cell antigen processing and communication with CD4 lymphocytes, killer cell lysis of target cells, and B cell antibody production will be examined. CapG is expressed in phagocytes at far higher concentrations than required to regulate actin assembly, raising the possibility that CapG may serve other functions. The effects of over-expressing CapG on the survival of different cell lines will be examined. Apoptosis of CapG-null neutrophils and macrophages will be compared to wild-type cells.
Aim 2 will analyze structure-function relationships in CapG gain-of-function mutant proteins. Actin filament severing and capping are critical steps for macrophage movement. PCR mutagenesis has created a series of gain-of-function CapG severing proteins and crystallographic studies have revealed their structure. Pyrenyl actin and analytical centrifugation are being used to define the structure-function relationship of these vital processes. Investigations of CapG promise to provide new insights into cell motility, innate and cell-mediated immunity, and the mechanisms underlying cell survival. These studies may provide new strategies for defending against infections, controlling auto-immune diseases and regulating the timing of cell death. ? ? ?
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