Contraction of Dictyostelium Ghosts Reconstituted with Myosin II

Abstract

Cytoskeletons, or 'Triton ghosts, ' that contained mainly actin and myosin II were prepared from Dictyostelium discoideum amoebae by extraction with Triton X-100. The Triton ghosts contracted immediately upon addition of ATP. However, under high-salt conditions in the presence of ATP, they did not contract but released myosin II. Almost all of the applied myosin II became associated with ghosts when myosin-free Triton ghosts, prepared in this way, were incubated with purified actin and then with myosin II from Dictyostelium. Immunofluorescence microscopy demonstrated that the associated myosin was localized in the cortical actin layer of the ghosts. Furthermore, the ghosts reconstituted with purified myosin resumed ATP-dependent contraction. Skeletal muscle myosin could also restore contractility to ghosts from which myosin had been extracted. The amount of myosin II necessary for the contraction of the ghosts was calculated by two methods. Less than 10% of the myosin II in intact cells was necessary for the contraction. These results show that myosin II is responsible for the contraction of the Dictyostelium cytoskeleton.