Rho is a small GTP-binding protein that regulates actin stress fibers and focal adhesions, playing a key role in cell migration and adhesion. The study investigates how Rho is regulated by cell adhesion, the cytoskeleton, and soluble factors like lysophosphatidic acid (LPA). Using a new assay to detect GTP-bound Rho, the researchers found that cell adhesion to the extracellular matrix (ECM) leads to a negative feedback loop that inhibits Rho activity. In serum-starved cells, LPA induces transient Rho activation, while suspended cells show sustained activation. Suspended cells also exhibit higher Rho activity than adherent cells in the presence of serum. The study also shows that cytochalasin D and colchicine can activate Rho despite their opposing effects on stress fibers and focal adhesions. These findings suggest that ECM, cytoskeletal structures, and soluble factors all contribute to Rho regulation. The results indicate that Rho activity is regulated by a complex interplay between cell adhesion, cytoskeletal dynamics, and soluble factors, with implications for cell migration and tissue homeostasis.Rho is a small GTP-binding protein that regulates actin stress fibers and focal adhesions, playing a key role in cell migration and adhesion. The study investigates how Rho is regulated by cell adhesion, the cytoskeleton, and soluble factors like lysophosphatidic acid (LPA). Using a new assay to detect GTP-bound Rho, the researchers found that cell adhesion to the extracellular matrix (ECM) leads to a negative feedback loop that inhibits Rho activity. In serum-starved cells, LPA induces transient Rho activation, while suspended cells show sustained activation. Suspended cells also exhibit higher Rho activity than adherent cells in the presence of serum. The study also shows that cytochalasin D and colchicine can activate Rho despite their opposing effects on stress fibers and focal adhesions. These findings suggest that ECM, cytoskeletal structures, and soluble factors all contribute to Rho regulation. The results indicate that Rho activity is regulated by a complex interplay between cell adhesion, cytoskeletal dynamics, and soluble factors, with implications for cell migration and tissue homeostasis.