Charakterisierung des menschlichen zytoplasmatischen Dyneinmotors mittels biochemischer Methoden und Einzelmolekueldetektion
Zusammenfassung der Projektergebnisse
Mammalian cytoplasmic dyneins are vital motor proteins involved in a variety of cellular functions, including cell division, cell migration , and cargo transport toward the minus end of microtubules. Multiple devastating neurological disorders, such as lissencephaly and Perry syndrome, result from impaired motor functionality. Therefore, a detailed understanding of human dynein’s enzymatic mechanism could advance the development of drugs significantly. In addition, directed manipulation of this motor protein could prove useful in curing multiple viral infections, since viruses can hijack human dynein as a transportation means from the cell periphery to the nucleus. However, the biochemical and biophysical properties of human dynein remain poorly understood and only very recent advances have shed light on some ambiguous findings. Two major areas of intense scientific debate include the motor’s run length (processivity) in the absence of cofactors and the maximum achievable forces (stall forces). The presented research on human dynein investigated the motor’s ensemble and single-molecule characteristics in the absence of cofactors. Using an optical trapping instrument, human dynein’s forceproducing abilities were analyzed at various spring constants. By changing the optical trap’s stiffness (spring constant), the distances corresponding to a certain force can be modulated. This novel approach revealed a spring constant dependence of both the observed average detachment forces and distances. While the forces increased hyperbolically approximating a force plateau, the corresponding distances decayed in a hyperbolic fashion. We hypothesized that the force plateau corresponds to the motor’s intrinsic stall force (1.76 ± 0.03 pN at 0.1 mM ATP and ~1.89 ± 0.05 pN at 1.1 mM ATP, respectively) and that extrapolation to a spring constant of zero may provide human dynein’s average run length in the absence of load (90. ± 2 nm at 0.1 mM ATP and 107 ± 2 nm at 1.1 mM ATP, respectively). To test these hypotheses, we analyzed the highly processive motor protein kinesin (K560), whose run length is decreased upon addition of salt. Under high ionic strength conditions, kinesin showed a spring constant dependence analogous to human dynein’s behavior. The extrapolated force plateau at infinite spring constant coincided with the motor’s stall force observed under low ionic strength conditions, where the measured forces are independent of the spring constant. Thus, we suggest that a non-processive motor’s intrinsic stall force can be estimated by determining the average forces at various spring constants. Our experimental observations can be further substantiated by Monte-Carlo simulations supporting the hypothesis that the extrapolated distances at a spring constant of zero may correspond to the run length of the motor in the absence of load. We propose that the spread of published mammalian dynein stall forces in the 1 – 2 pN range can be explained by the use of different spring constants and of distinct assay conditions leading to a variable processivity. Our findings imply a potential force regulation mechanism by cofactors that tune dynein’s run length rather than changing its force generation abilities per se. The novel experimental approach of varying the optical trap’s spring constant can be used to analyze stall forces and run lengths of motors with limiting processivity. Moreover, the analysis of average forces at various spring constants does not require a definition of the stall time, but allows for an unbiased determination of the intrinsic stall forces using a much larger data set. As a result, this technique will likely prove useful in future molecular motor research.
Projektbezogene Publikationen (Auswahl)
- (2013) The yeast dynein Dyn2-Pac11 complex is a dynein dimerization/processivity factor: structural and single molecule characterization, Mol Biol Cell 24, 2362-2377
Rao, L., Romes, E. M., Nicholas, M. P., Brenner, S., Tripathy, A., Gennerich, A., and Slep, K
- (2015) Control of cytoplasmic dynein force production and processivity by its C-terminal domain. Nature Communications 6, Article number: 6206 (2015) (PDF: 8 S.)
Nicholas, M. P., Höök, P., Brenner, S., Wynne, C. L., Vallee, R. B., and Gennerich, A
(Siehe online unter https://doi.org/10.1038/ncomms7206) - (2015) Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains. Proceedings of the National Academy of Sciences (PNAS), published ahead of print May 4, 2015
Nicholas, M. P., Brenner, S., Berger, F., Cho, C., Rao, L., and Gennerich, A
(Siehe online unter https://doi.org/10.1073/pnas.1417422112) - Control of cytoplasmic dynein force production and processivity by its C-terminal domain. Nature Communications volume 6, Article number: 6206 (2015)
Brenner, S., Nicholas, M. P., Peter Höök, Caitlin L. Wynne, Richard B. Vallee, Gennerich, A
(Siehe online unter https://doi.org/10.1038/ncomms7206)
