To understand the function of nonmuscle myosin heavy chain (NMHC) II-A, we have analyzed filament formation of different deletions of GFP-tagged NMHC II-A constructs in HeLa cells. A dominant active mutant of RhoA, L63RhoA, was used to induce stress fibers. Both GFP-tagged myosin II-A and II-B formed prominent myosin filaments following coexpression with L63RhoA, although the HeLa cells used in the study did not express endogenous myosin II-B protein. When GFP-tagged NMHC II-B and L63RhoA were introduced into the HeLa cells, the endogenous myosin II-A filaments detected by the antibody decreased compared with HeLa cells expressing L63RhoA alone, suggesting that Rho signaling might preferentially induce myosin II-B filament formation in this HeLa cell line. We also found that, following deletion of a portion of the motor domain (amino acids 1-591) or the nonhelical region of the tail (amino acids 1929-1961), the GFP-tagged truncated myosin was still capable of forming myosin filaments. However, a truncated form of NMHC II-A, deltaC170, in which 170 amino acids was deleted from the carboxy terminus, was not able to form myosin filaments. It was noted that the actin stress fibers induced by the expression of L63RhoA were partially disrupted by deltaC170. We reasoned that deltaC170, with its intact actin binding and ATPase domains, was still able to bind to actin filaments and hydrolyze ATP, but lost its ability to form myosin filaments, preventing the endogenous myosin from binding to actin and blocking normal actin stress fiber formation.We also studied the effect of Pitx2 expression on the organization of actomyosin cytoskeleton. GFP-tagged Pitx2 was ectopically expressed in HeLa cells using the tetracycline-inducible expression system. Expression of Pitx2 induced cell spreading and cell-cell contacts with reorganization of the actomyosin cytoskeleton. Pitx2 upregulated the expression of beta-catenin and N-cadherin, which were found to localize to sites of cell-cell contacts in a calcium-dependent manner. Furthermore, endogenous Rac was activated and the dominant-negative mutant of Rac1 blocked the phenotype induced by expression of Pitx2. Interestingly, the dominant-active, but not dominant-negative, mutant of RhoA blocked the phenotype, suggesting that a balance between Rho and Rac activity is important for development of the cell phenotype. Finally, expression of Pitx2 inhibits cell migration in a wound healing assay. In summary, Pitx2 is able to modulate the activity of the small GTPase proteins Rac and Rho, leading to changes in the actomyosin cytoskeleton, cell morphology and cell-cell contacts.