The ampA gene encodes a secreted protein that modulates cell adhesion, actin polymerization, endocytosis, and cell migration. AmpA is secreted into the supernatant during development, and remains cell associated during growth. AmpA loss in growing cells results in an increase in cell adhesion, and a reduction in F actin. Over expression of AmpA reduces adhesion and increases F actin. As a result of these changes in the cytoskeleton and in adhesion, I have shown AmpA influences cell migration. AmpA knockout cells are defective in migration on top of agar compared to wild type. AmpA over expressing cells migrate better than wild type on top of agar. This defect in the knockout can be rescued by placing the cells in a 3D environment where they migrate under agar. Knockout cells migrate better than wild type under these conditions and over expressing cells migrate about the same as wild type. In order to visualize actin dynamics in live cells, wild type, AmpA KO and AmpA over-expressing strains were created containing an actin binding domain fused to GFP. In stationary cells, over expressers make more actin rich endosomal cups, which form repeatedly in the same area of the membrane, leading to an increased rate of endocytosis. Knockout cells have significantly reduced F-actin, but make relatively normal endosomal cups. In order to determine how AmpA affects these processes, localization analyses were performed. Immuno-fluorescence analysis using AmpA tagged to the Tap tag and to mRFP show that AmpA is localized in possible vesicles throughout the cell and also show some localization with calnexin, an ER marker, at discrete sites surrounding the nucleus. These sites co-localize with p25, a marker for an endosomal recycling compartment. AmpA can also be found at the cell periphery under certain staining conditions. Some vesicles should be secretory vesicles, but results indicate that AmpA on the surface is endocytosed back into the cells. This indicates that AmpA is likely bound to the membrane through an interaction with its receptor where it could possibly regulate cell adhesion and actin polymerization. It is actively endocytosed and proceeds through the membrane recycling pathway
