Looking back on the launch of single molecule biophysics

Molecular motors play a key role in controlling movement in living cells. A well-known example is myosin, originally described as a key player in muscle contraction, but which is involved in many cell biological phenomena. Detailed knowledge of the function of myosin and its interaction with its partner molecule, actin has been obtained in recent years by studying single molecules of these important proteins. Here, James Spudich of Stanford University looks back to the time when he and his colleagues Steven Chu (Stanford) and Bob Simmons (King’s College London) with HFSP support successfully employed methods normally used to study individual atoms to study large biological molecules, thus helping to launch the field of single molecule biophysics.

About the author: James A. Spudich is the Douglass M. and Nola Leishman Professor of Cardiovascular Disease in the Department of Biochemistry at Stanford University School of Medicine. He has had a long-standing interest in the mechanisms underlying the role of the molecular motor myosin in cell biological phenomena such as cell motility, cytokinesis and muscle contraction. He has pioneered the development of single molecule methods for studying biological macromolecules. He was one of the founders, together with Stephen Chu, of the interdisciplinary Bio-X program at Stanford University.

In 1984 my laboratory fulfilled a long-sought dream of developing a quantitative in vitro motility assay, initially using Nitella actin filaments as tracks along which myosin-coated beads move.  Shortly thereafter, using purified proteins, my graduate student Steve Kron measured the velocity of movement of actin filaments over a lawn of myosin molecules bound to a glass microscope cover slip (1).  This assay provided immediate payoff by showing that the globular head domain of myosin is the motor domain (2).

All efforts could now be directed toward understanding how the globular head of myosin converts the chemical energy of ATP hydrolysis into mechanical movement. Inthe late 1980s and early 1990s, however, my colleagues and I realized that this assay was not sufficient to understand fundamental aspects of the motor behavior such as the stroke or step size produced for every ATP hydrolysis, and the maximum force that the molecular motor was capable of generating.  We needed a way to watch a single molecule of myosin go through one cycle of ATP hydrolysis and measure these parameters directly.

In 1986 Ashkin et al reported that micron sized objects could be trapped and held in position in solution by a laser beam (3).  We decided to try using this method to levitate and position an actin filament over a single myosin molecule in a microscope, and so achieve our goal of watching a single molecule go through its energy transduction cycle.  The idea was simple really, and was a modification of the already established Kron in vitro motility assay.  But we needed the help of a physicist!

The advances that ensued depended heavily on grant support from the Human Frontier Science Program, which generously funded our project on “Single molecule mechanics using optical tweezers” during a critical period for our research, from 1993 to 1996.

A coauthor on the Ashkin et al paper (3), Steve Chu, was then at Stanford.  He and I were from two different worlds.  He was a pure physicist, trapping atoms while he was at the Bell labs.  I am fundamentally a biochemist, with a love for physics that I maintain from my undergraduate years at the University of Illinois.  Importantly, we were both receptive to new adventures and forging new and unique ties.  This turned out to be pivotal for the future of our respective research programs and that of many others.  But more than that, our scientific partnership became a paradigm for the way interdisciplinary bioscience can be practiced.

As I said, I and my colleagues in the molecular motor world needed a way to watch a single molecule of myosin go through one cycle of ATP hydrolysis and to measure the stroke or step size produced as well as the maximum force that the molecular motor was capable of generating.  Steve Chu, on the other hand, was fascinated by biology, had already made a name for himself in the world of the physics of atom trapping (for which he received a Nobel Prize in 1997), and was eager to apply physics to biological problems.  He would begin that by examining the detailed properties of DNA, the cell’s genetic machinery.

Steve and I did something that in retrospect was very unusual.  My graduate student Jeff Finer and sabbatical visitor to my laboratory Bob Simmons, who had previously designed feedback circuits and the like for measurement of forces and displacements produced by myosin at the muscle fiber level, went to Steve’s lab space in the Varian Physics Lab across Campus Drive West from the Biochemistry Department in the Beckman Center. In Varian, Finer and Simmons built the first dual beam, 3-bead assay laser trap that we had dreamed about, with inputs from Steve Chu and members of his group.  We were able to build an improved second generation dual-beam laser trap in my laboratory, which proved critical for the success of our molecular motor studies (see Figure).

 

Figure.  Jeff Finer, an MD-PhD student, 1993.  Finer attended electronics courses while taking anatomy in the medical school in order to single-handedly build an improved second dual-beam optical trap in my laboratory in the Biochemistry Department. The second trap, made possible by HFSP funding, had better signal to noise than the prototype instrument, which was critical for our myosin measurements. The first trap in the Varian Physics Lab was then freed up for DNA experiments carried out by Steve Chu’s laboratory. The second trap had an open optical path rather than a microscope frame, allowing for improved optics in multiple ways and better stabilization and therefore less vibration. Filters were improved to allow better fluorescent imaging and simultaneous imaging of the actin filaments and the beads.  A second quadrant detector, second acousto-optic deflector, and a second set of feedback electronics were added. Also new software allowed the automated stretching of filaments that used information from both detectors.  

In exchange for Steve’s accommodating us in the Varian Physics Lab, I gave space in my laboratory in the Biochemistry Department to several of Steve Chu’s physics students, including Steve Quake (a Stanford undergraduate student in Physics at that time) and Tom Perkins.  They had free reign in the Department of Biochemistry, interacting productively with the faculty, and probably more importantly with the very bio-oriented students and postdoctoral fellows in the Department.  They quickly mastered working with DNA, and both Steve Quake and Tom Perkins have gone on to important work in biophysics and bioengineering.

A severe limitation for us to proceed with the trap development and the myriad experiments that became possible as a result of having it, was lack of funding for such projects. With Bob Simmons, then back at King’s College in London as Principal Investigator, we applied and got funding for three years from the Human Frontier Science Program.

Further improvements in the dual beam trap both at Stanford and in London involved a trip to King’s College London for 5 weeks by Jeff Finer and a return trip to Stanford by Bob Simmons.  The dual beam laser trap technology resulted quite quickly in multiple high profile papers, thanks in no small part to the HFSP (e.g., 4-15). 

The 1996 Biophysics Journal paper (10) provided the first detailed description of the dual beam optical trap assay for single molecule analyses, and drew a lot of attention from my scientific colleagues, including competitors, around the world.  In all cases, we invited them to visit, take photographs, sent them home with a complete parts list, and then gave expert advice from a distance to help them set up their own dual-beam system.  And so single molecule analyses spread very quickly.

The union of physics and biology certainly had been happening in other laboratories, but our work funded by the HFSP was a very successful example of such a collaboration.  What was unusual about our collaboration was that our students spent a significant amount of time working in each other’s environments, with constant interactions with ‘native speakers’ of the ‘new language’ that they wanted to learn.

We proposed to the provost and to the deans at Stanford University that such multidisciplinary interactions could be happening throughout the campus, not only between physicists and biochemists, but between physicists, all biologists, chemists, all engineers, computational scientists and clinical scientists.  Our proposal was that the global program should facilitate interactions throughout the campus (the Bio-X Program), and a hub should be built in the geographic center of the three Schools initially involved: the School of Medicine, the School of Humanities and Sciences, and the School of Engineering.  All three Schools are contiguous on the campus and the geographic center was a parking lot on the medical school side of Campus Drive West.  That lot is now occupied by the 225,000 square foot building known as the Clark Center, a very open building designed by the English architect Norman Foster, with funding from James H. Clark.  The Clark Center houses faculty from all the different disciplines mentioned, whose home Departments for the most part are elsewhere on the campus.  Many of our faculty colleagues were excited about the concept and participated actively in bringing this grass roots effort to a reality.

Bio-X has been wildly successful in fostering broad interdisciplinary interactions that have importantly advanced knowledge in bioscience, bioengineering and biomedicine and large amounts of new grant monies have been awarded to the university for interdisciplinary research. The Stanford Bio-X Program has ignited the development of similar programs elsewhere, and the world of interdisciplinary sciences continues to advance!

I am very grateful for this opportunity to thank the HFSP for their support from 1993-1996, without which what I describe above may never have happened.

 

Key references

  1. Kron, J., and Spudich, J.A. (1986).  Fluorescent actin filaments move on myo­sin fixed to a glass surface.  Proc. Natl. Acad. Sci. USA 83:6272-6276.
  2. Toyoshima, Y.Y., Kron, S.J., McNally, E.M., Niebling, K.R., Toyoshima, C., and Spudich, J.A. (1987).  Myosin subfragment-1 is sufficient to move actin fila­ments In VitroNature 328:536-539.
  3. Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E., and Chu, S. (1986).  Observation of a single-beam gradient force optical trap for dielectric particles. Opt Lett.11:288-290.
  4. Simmons, R.M., Finer, J.T., Warrick, H.M., Kralik, B., Chu, S., and Spudich, J.A. (1993).  Force on single actin filaments in a motility assay measured with an optical trap. Adv Exp Med Biol 332:331-336.
  5. Finer, J.T., Simmons, R.M., and Spudich, J.A. (1994).  Single myosin molecule mechanics:  Piconewton forces and nanometre steps.  Nature 368:113-119.
  6. Perkins TT, Smith DE, Chu S. (1994). Direct observation of tube-like motion of a single polymer chain.   Science. 264:819-822.
  7. Perkins, T.T., Quake, S.R., Smith, D.E., and Chu, S. (1994).  Relaxation of a single DNA molecule observed by optical microscopy.  Science 264:822-826.
  8. Perkins, T.T., Smith, D.E., Larson, R.G., and Chu, S. (1995).  Stretching of a single tethered polymer in a uniform flow.  Science 268:83-87.
  9. Finer, J.T., Mehta, A.D., and Spudich, J.A. (1995).  Characterization of Single Actin-Myosin Interactions.  Biophys. J.  68:291s-297s.
  10. Simmons, R.M., Finer, J.T., Chu, S., and Spudich, J.A. (1996).  Quantitative Measurements of Force and Displacement Using an Optical Trap.  Biophys. J. 70:1813-1822.
  11. Quake, S.R., Babcock, H., and Chu, S. (1997). The dynamics of partially extended single molecules of DNA. Nature 388:151-154.
  12. Perkins, T.T., Smith, D.E., and Chu, S. (1997).  Single polymer dynamics in an elongational flow. Science 276:2016-2021.
  13. Tskhovrebova, L., Trinick, J., Sleep, J.A., and Simmons, R.M. (1997). Elasticity and unfolding of single molecules of the giant muscle protein titin.  Nature 387:308-312.
  14. Mehta, A.D., Finer, J.T., and Spudich, J.A. (1997).  Detection of Single-Molecule Interactions Using Correlated Thermal Diffusion.  Proc. Natl. Acad. Sci. USA 94:7927-7931.
  15. Mehta, A.D., Rief, M., Spudich, J.A., Smith, D.A., and Simmons, R.M. (1999).  Single-molecule biomechanics with optical methods.  Science 283:1689-1695.