James A. Spudich

Ph.D., Stanford, 1968.
Professor of Biochemistry; Professor of Developmental Biology

Email:
Web: http://biochem.stanford.edu/spudich/

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.

Select Publications (2004 - present)

Robinson, D.N. and Spudich, J.A. (2004).  Mechanics and Regulation of Cytokinesis.  Current Opinion in Cell Biology  16: 1-7.

Altman, D., Sweeney, H.L., and Spudich, J.A. (2004).  The Mechanism of Myosin VI Translocation and Its Load-Induced Anchoring.  Cell 116: 737-749.

Ökten, Z., Churchman, L.S., Rock, R.S., and Spudich, J.A. (2004). Myosin VI Walks Hand-Over-Hand along Actin.  Nature Struct Mol Biol.  11: 884-887.

Hostetter, D., Rice, S., Dean, S., Altman, D., McMahon, P.M., Sutton, S., Tripathy, A., and Spudich, J.A.  (2004).  Dictyostelium Myosin Bipolar Thick Filament Formation: Importance of Charge and Specific Domains of the Myosin Rod.  PLoS Biol. 2: 1880-1892.

Spudich, J.A. (2004). Two Important Polymers Cross Paths. Proc Natl Acad Sci USA 101:15825-15826

Lakshmikanth, G.S., Warrick, H.M., and Spudich, J.A. (2004).  A Mitotic Kinesin-like Protein Required for Normal Karyokinesis, Myosin Localization to the Furrow, and Cytokinesis in Dictyostelium.  Proc Natl Acad Sci USA 101: 16519-16524.

Churchman, L.S., Ökten, Z., Rock, R.S., Dawson, J.F., and Spudich, J.A. (2005).  Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time.  Proc Natl Acad Sci USA 102: 1419-1423.

Rock, R.S., Ramamurthy,B., Dunn,A.R., Beccafico,S., Rami, B.R., Morris,C., Spink,B.J., Franzini-Armstrong,C., Spudich, J.A., and H. Lee Sweeney, H.L. (2005).  A Flexible Domain is Essential for the Large Step Size and Processivity of Myosin VI.  Molecular Cell 17: 603-609.

Dean, S.O., Rogers, S.L., Stuurman, N., Vale, R.D. and Spudich, J.A.  (2005).   Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis.  Proc Natl Acad Sci U S A  102:13473-13478.

Purcell, T.J., Sweeney, H. L. and Spudich, J.A. (2005).  A force-dependent state controls the coordination of processive myosin V.  Proc Natl Acad Sci U S A  102:13873-13878.

Altman, D., Goswami, D., Spink, B., Hasson, T., Spudich, J.A. and Mayor, S.  (2005).  Myosin VI is an oligomer on endocytic vesicles in vivo.Submitted.

Last Updated: 2/23/06