Jim Spudich
Department of Biochemistry, Stanford University, Stanford, CA 94305
A number of experiments have addressed unitary displacements induced by myosin, both in well ordered arrays within muscle myofibrils and at the single molecule level. However, none of these experiments have measured independently different points within a single actin filament. In fact, prior to the development of sensitive mechanical probes for measurement of single myosin molecule activity, it has been difficult to observe directly the correlations between actin filament motion on either side of a given myosin crossbridge. Such measurements are critical for eliminating certain classes of models of the energy transduction event.
In other systems, such as translocating DNA helicases or replication complexes which unwind DNA unidirectionally, motile enzymes induce distinct mechanical phenomena on either side of their attachment points. A class of models for actin-myosin based movement, most notably the actin powerstroke model of Schutt and Lindberg, involve similar assymmetric behaviors. Such models make clear predictions regarding spatial and temporal correlations between the motion of an actin filament at either side of an attached myosin crossbridge. Here, we describe an in vitro assay using a dual beam laser trap to test these assumptions by independently monitoring these actin filament segments while under the influence of a single attached crossbridge.
Conventional myosin plays a pivotal role in the cytoskeletal reorganization of nonmuscle cells necessary for cytokinesis, cell migration, and developmental morphology changes. Until now, myosin localization information during these dynamic processes has been confined to static snap-shots of fixed cells. We are now able to observe directly the dynamics of myosin movements in Dictyostelium discoideum. We have successfully expressed fully functional, green fluorescent protein-tagged Dictyostelium myosin heavy chain (GFP-myosin) in myosin null cells. Expression of GFP-myosin completely rescues all myosin null cell defects. Furthermore, GFP-myosin protein purified from these cells exhibits the same ATPase activities and in vitro motility behavior as wild-type myosin. The critical role of three phosphorylatable threonine residues in the tail domain of myosin is demonstrated in vivo using GFP-constructs of mutant myosins that have these threonine residues replaced with alanine or aspartate residues.