AmpA: A Link Between Actin Polymerization and Cell Migration, Cell Adhesion, and Endocytosis

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

AmpA: A Link Between Actin Polymerization and Cell Migration, Cell Adhesion, and Endocytosis

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

The adhesion modulation protein, AmpA localizes to an endocytic compartment and influences substrate adhesion, actin polymerization and endocytosis in vegetative Dictyostelium cells

<p>Abstract</p> <p>Background</p> <p>AmpA is a secreted 24Kd protein that has pleiotropic effects on <it>Dictyostelium</it> development. Null mutants delay development at the mound stage with cells adhering too tightly to the substrate. Prestalk cells initially specify as prespore cells and are delayed in their migration to the mound apex. Extracellular AmpA can rescue these defects, but AmpA is also necessary in a cell autonomous manner for anterior like cells (ALCs) to migrate to the upper cup. The ALCs are only 10% of the developing cell population making it difficult to study the cell autonomous effect of AmpA on the migration of these cells. AmpA is also expressed in growing cells, but, while it contains a hydrophobic leader sequence that is cleaved, it is not secreted from growing cells. This makes growing cells an attractive system for studying the cell autonomous function of AmpA.</p> <p>Results</p> <p>In growing cells AmpA plays an environment dependent role in cell migration. Excess AmpA facilitates migration on soft, adhesive surfaces but hinders migration on less adhesive surfaces. AmpA also effects the level of actin polymerization. Knockout cells polymerize less actin while over expressing cells polymerize more actin than wild type. Overexpression of AmpA also causes an increase in endocytosis that is traced to repeated formation of multiple endocytic cups at the same site on the membrane. Immunofluorescence analysis shows that AmpA is found in the Golgi and colocalizes with calnexin and the slow endosomal recycling compartment marker, p25, in a perinuclear compartment. AmpA is found on the cell periphery and is endocytically recycled to the perinuclear compartment.</p> <p>Conclusion</p> <p>AmpA is processed through the secretory pathway and traffics to the cell periphery where it is endocytosed and localizes to what has been defined as a slow endosomal recycling compartment. AmpA plays a role in actin polymerization and cell substrate adhesion. Additionally AmpA influences cell migration in an environment dependent manner. Wild type cells show very little variation in migration rates under the different conditions examined here, but either loss or over expression of AmpA cause significant substrate and environment dependent changes in migration.</p

Actin acting at the Golgi

The organization, assembly and remodeling of the actin cytoskeleton provide force and tracks for a variety of (endo)membrane-associated events such as membrane trafficking. This review illustrates in different cellular models how actin and many of its numerous binding and regulatory proteins (actin and co-workers) participate in the structural organization of the Golgi apparatus and in traf- ficking-associated processes such as sorting, biogenesis and motion of Golgi-derived transport carriers