Biological machines

Molecular motor teamwork

Synthetic muscles built from DNA nanotube scaffolds can be used to study how myosin motors work together to make real muscles function.

The generation of muscle force and velocity is achieved through the collective action of billions of nanoscale motors known as myosin molecules. Inside muscle tissue these molecular motors are arranged in a highly ordered pattern that is intimately linked to the mechanism of contraction, but how exactly the actions of individual myosins are coordinated to give rise to the emergent properties of muscle remains unclear1. Studying whole-muscle tissue to try to understand this coordination is too difficult because the measures produced represent the average behaviour of billions of motors, washing out the exquisite behaviour of a single motor. Conversely, while single-molecule assays can reveal important properties about an individual motor, they provide little insight into coordination between motors2. Writing in Nature Nanotechnology, Sivaraj Sivaramakrishnan and colleagues at the University of Michigan and Stanford University School of Medicine now report a new approach to bridge this knowledge gap in which DNA nanotubes are used to mechanically couple and manipulate myosin motors3.

In striated muscle, myosin motors are assembled as polymers that form 1.5–2.5-μm-thick filaments with a longitudinal motor spacing of 14.3 nm and a helical repeat of 42.9 nm; the researchers mimicked and manipulated this spacing by using 5-μm-thick DNA nanotubes (Fig. 1). The DNA nanotubes contain discrete numbers of myosin motors, and allow motor coordination to be probed by varying the number and spacing between motors, as well as the stiffness of their connection to the nanotube. Initial experiments used processive non-muscle myosin motors, myosin V and myosin VI, which normally operate as single motors to transport cargo within cells4,5. With the DNA nanotubes adhered to a glass coverslip surface, they measured the velocity that these motors could move their molecular track, actin. Consistent with earlier reports, which coupled multiple myosin V motors directly6 or through incorporation into a vesicle7, they observed a slower velocity than that observed for individual motors8. This suggests that mechanically coupling motors together negatively affects the function of an individual motor, possibly mediated through the intermolecular strain between motors. A second set of experiments demonstrated that increasing the space between motors by two- or even threefold had no further effect on velocity; a finding that is similar to the behaviour of non-processive muscle myosins9.

Figure 1: Building synthetic muscle to better understand contraction.
figure1

a, DNA nanotubes (right) are used to mimic the architecture of the myosin thick filament in muscle (left), which has a longitudinal motor spacing of 14.3 nm and a helical repeat of 42.9 nm. b, Myosin motors can be attached to the nanotubes to closely match the 14.3-nm spacing in muscle. The approach also allows the spacing and stiffness of the connection to the motor to be manipulated, providing unique insights into the mechanical coupling between motors. Arrow indicates direction of actin filament motion. Figure adapted from ref. 3, Nature Publishing Group.

To better understand inter-motor coupling, a stochastic computer model was formulated that represented the net stiffness of each actomyosin interaction (or 'cross-bridge') as a linear spring. A successful myosin step, leading to displacement of the actin filament, was only possible if the resisting load presented by the other attached motors was less than the maximum force (that is, stall force) that the individual motor could generate. Thus the load experienced by each individual motor is proportional to the net stiffness of each actomyosin interaction; in the model, as this load is increased the filament velocity will decrease. This predicts that actin filament velocity should be dependent on the stiffness of the connection between the motor and the DNA nanotube scaffold.

Sivaramakrishnan and colleagues directly tested this prediction by adding an additional flexible single-stranded DNA linker to connect the motor to the DNA nanotube. Consistent with their prediction, they found that decreasing the stiffness of the cross-bridge increased the velocity of the actin filaments driven by these motors, supporting the idea that actin filament motion, driven by an ensemble of molecular motors, is the result of the tensions experienced between motors that are elastically coupled. Indeed, the model suggests that if cross-bridge stiffness could be decreased even further the velocity of movement would approach that of a single motor, implying a complete uncoupling. The known load dependence of myosin's biochemical cycle10 is the likely governor of this characteristic decrease in velocity, with the additional cross-bridges and, therefore, stiffness slowing the rate of detachment. This property may confer an evolutionary advantage by allowing external force to be borne evenly by all the motors in the ensemble, allowing teams of motors to generate displacements over a broader range of forces.

The relevance of these findings to striated muscle was increased in a final set of experiments that took advantage of a recent breakthrough in molecular biology11 to incorporate expressed, cardiac myosin, a non-processive motor9, into the DNA nanotubes. Muscle myosin has a much lower duty ratio (5% of its biochemical cycle is spent strongly bound to actin) compared with processive myosins4 (>50%), accordingly it might be expected to behave quite differently in a team of motors. The motors in the DNA nanotubes moved actin twice as fast as individual cardiac myosins, the opposite of observations from the processive motors, but increasing the spacing between motors had no effect on velocity, similar to results from the processive motors. This increased velocity for teams of muscle myosins may reflect constraints on the number of cross-bridge attachments imposed by the nanotube architecture.

Collectively the work of Sivaramakrishnan and colleagues provides support for the notion that the velocity of an ensemble of myosin is influenced by the elastic coupling between the motors. The researchers suggest that large collections of motors may function as energy reservoirs in which the action of an individual motor increases the potential energy of the system that is then released at a steady rate to give rise to the observed average filament velocity. By further manipulating the kinetics of the motors and the architecture of the DNA nanotubes, this exciting technique may provide a method to directly test these ideas, leading to new insights into coordination between molecular motors working in an ensemble. The approach could be particularly helpful in bridging the gap between the single-molecule and whole-muscle behaviour.

References

  1. 1

    Walcott, S., Warshaw, D. M. & Debold, E. P. Biophys. J. 103, 501–510 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Finer, J. T., Simmons, R. M. & Spudich, J. A. Nature 368, 113–119 (1994).

    CAS  Article  Google Scholar 

  3. 3

    Hariadi, R. F. et al. Nature Nanotech. 10, 696–700 (2015).

    CAS  Article  Google Scholar 

  4. 4

    Moore, J. R. et al. J. Cell Biol. 155, 625–635 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Rock, R. S. et al. Proc. Natl Acad. Sci. USA 98, 13655–13659 (2001).

    CAS  Article  Google Scholar 

  6. 6

    Lu, H. et al. J. Biol. Chem. 287, 27753–27761 (2012).

    CAS  Article  Google Scholar 

  7. 7

    Nelson, S. R., Trybus, K. M. & Warshaw, D. M. Proc. Natl Acad. Sci. USA 111, E3986–E3995 (2014).

    CAS  Article  Google Scholar 

  8. 8

    Yildiz, A. et al. Science 300, 2061–2065 (2003).

    CAS  Article  Google Scholar 

  9. 9

    Harris, D. E. & Warshaw, D. M. J. Biol. Chem. 268, 14764–14768 (1993).

    CAS  Google Scholar 

  10. 10

    Veigel, C. et al. Nature Cell Biol. 5, 980–986 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Sommese, R. F. et al. Proc. Natl Acad. Sci. USA 110, 12607–12612 (2013).

    CAS  Article  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to Edward P. Debold.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Debold, E. Molecular motor teamwork. Nature Nanotech 10, 656–657 (2015). https://doi.org/10.1038/nnano.2015.175

Download citation

Further reading

Search

Find nanotechnology articles, nanomaterial data and patents all in one place. Visit Nano by Nature Research