Skip to main content Accessibility help

High Resolution Two-Dimensional Detection of Ncd Motility with Optical Tweezers

  • M. W. Allersma (a1), F. Gittes (a1), M. J. deCastro (a2), R. J. Stewart (a2) and C. F. Schmidt (a1)...


The ATP-dependent motility of the kinesin-related non claret disjunctional (ncd) mechanoenzyme was observed in an in vitro bead motility assay using optical tweezers in combination with a new two-dimensional displacement detection method. The detection technique is based on observing the far-field interference pattern formed in the back focal plane (BFP) of the microscope condenser by the illuminating laser focus and the light scattered from the trapped dielectric bead. The ability to observe the two-dimensional motion, with high temporal and spatial resolution, and in a manner largely independent of position in the microscope field-of-view, is the particular advantage of this detection method. In the assay, a fusion protein (GST-N195) of truncated ncd and glutathione-Stransferase was adsorbed to silica beads and the axial and lateral motions of the beads along the microtubule surface were observed. The average axial velocity of the ncd coated beads was 230 ± 30 nm/s (± std. dev.). Spectral analysis of bead motion showed an increase in viscous drag near the surface. Furthermore, we also found that any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the non-hydrolyzable nucleotide adenylylimido-diphosphate (AMP-PNP).



Hide All
[1] Moore, J.D., and Endow, S.A., Bioessays. 18, 207 (1996).
[2] S.T., Brady, Nature. 317, 73 (1985).
[3] Berliner, E., Young, E.C., Anderson, K., Mahtani, H.K., and Gelles, J., Nature. 373, 718 (1995).
[4] Ray, S., Meyhöfer, E., Milligan, R.A., and Howard, J., J. Cell Biol. 121, 1083 (1993).
[5] Sablin, E.P., Kull, F.J., Cooke, R., Vale, R.D., and Fletterick, R.J., Nature. 380, 555 (1996).
[6] McDonald, H.B. and Goldstein, L.S., Cell. 61, 991 (1990).
[7] Walker, R.A., Salmon, E. D., and Endow, S. A., Nature. 347, 780 (1990).
[8] Allersma, M.W., Gittes, F., deCastro, M.J., Stewart, R.J., Schmidt, C.F., Biophys. J. 74, 1074 (1998).
[9] Gittes, F., and Schmidt, C.F., Opt. Lett. 1, 4 (1998).
[10] Gittes, F., and Schmidt, C.F., in Methods in Cell Biology, vol 55.(Academic Press, San Diego, 1997), pp. 129156.
[11] Stewart, R.J., Thaler, J.P., and Goldstein, L.S., Proc. Natl. Acad. Sci. U S A. 90, 5209 (1993).
[12] Williams, R.C. Jr., and Lee, J.C., in Methods in Enzymology, vol 85 Pt B, (Academic Press, San Dieog, 1982) pp. 376–85.
[13] Happel, J., and Brenner, H., Low Reynolds Number Hydrodynamics with Special Applications to Particulate Media, (Kluwer Academic, Dordrecht, The Netherlands, 1983).


Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed