James A. Spudich, Ph.D.

Professor of Biochemistry and Developmental Biology

We use a multifaceted approach to unravel the mechanism by which molecular motors transduce the chemical energy of ATP hydrolysis into mechanical motion. Work in my laboratory has focused on the myosin family of molecular motors, enzymes that generate the force and motions that underlie muscle contraction, cytokinesis in nonmuscle cells, cell movement, and membrane translocations in cells. We have established both in vitro motility assays and a cell system for functional and molecular genetic analyses of myosin. Using the cellular slime mold Dictyostelium, we provided genetic proof that myosin is required for cytokinesis of cells in suspension, changes in cell shape during morphogenesis, and capping of cell surface receptors. We also designed and developed in vitro assays for ATP-dependent movement of purified myosin on filaments reconstituted from purified actin. This assay has been extended to the single molecule level, using a variety of biophysical approaches. We are measuring directly the interaction of single myosin molecules with single actin filaments, examining both conventional myosin (myosin-II), found in muscle and in the contractile ring of dividing cells, and unconventional myosins such as myosin-V and myosin-VI (in collaboration with Drs. Mark Mooseker, Richard Cheney, and Lee Sweeney), found in nerve cells and other cells where membrane translocations are required.

Recent work:

It has long been hypothesized that the molecular motor myosin acts by binding to actin and swinging its light-chain binding region through a large angle to provide a ~10-nm step in motion coupled to changes in the nucleotide state at the active site. Direct dynamic measurements to date, however, have largely failed to reveal changes of that magnitude. We used a cysteine engineering approach to create a high resolution FRET-based sensor that reports a very large ~70-degree nucleotide dependent angle change of the light-chain binding region. The combination of steady-state and time-resolved (with Zygmunt Gryczynski and Joseph Lakowicz, Univ Maryland) fluorescence resonance energy transfer measurements unexpectedly reveals two distinct prestroke states. The measurements also show that bound Mg.ADP.Pi, and not bound Mg.ATP, induces the myosin to adopt the prestroke states.

It is thought that Switch II of myosin, kinesin and G-proteins plays a critical role in relating the nucleotide state to the protein conformation. We examined S456L myosin-II from Dictyostelium, a mutant of the Switch II region, whose mechanical activity is uncoupled from the chemical energy of ATP hydrolysis so that actin filament gliding velocities are only one-tenth that of wild type. The mutant myosin exhibits an extended strongly-bound state time and a shorter step size, which together account for the decrease in in vitro velocity.

Myosin-V is a molecular motor from brain that we showed moves processively along its actin track. With Mark Mooseker (Yale) and Richard Cheney (Univ North Carolina) we employed a feedback-enhanced optical trap to examine the stepping kinetics of this processive movement. By analyzing the distribution of time periods separating discrete ~36-nm mechanical steps, we characterized the number and duration of rate-limiting biochemical transitions preceding each such step. Based on this, we propose a model for myosin-V processivity involving a tightly coupled motor whose cycle time is limited by ADP release. In collaboration with Lee Sweeney (Univ Pennsylvania), we are characterizing a number of mutant forms of myosin-V, expressed in Baculovirus, as well as myosin-VI, a fascinating motor that moves in the opposite direction along an actin filament from all the other known myosins.