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Two-dimensional tracking of ncd motility by back focal plane interferometry.

Publication ,  Journal Article
Allersma, MW; Gittes, F; deCastro, MJ; Stewart, RJ; Schmidt, CF
Published in: Biophysical journal
February 1998

A technique for detecting the displacement of micron-sized optically trapped probes using far-field interference is introduced, theoretically explained, and used to study the motility of the ncd motor protein. Bead motions in the focal plane relative to the optical trap were detected by measuring laser intensity shifts in the back-focal plane of the microscope condenser by projection on a quadrant diode. This detection method is two-dimensional, largely independent of the position of the trap in the field of view and has approximately 10-micros time resolution. The high resolution makes it possible to apply spectral analysis to measure dynamic parameters such as local viscosity and attachment compliance. A simple quantitative theory for back-focal-plane detection was derived that shows that the laser intensity shifts are caused primarily by a far-field interference effect. The theory predicts the detector response to bead displacement, without adjustable parameters, with good accuracy. To demonstrate the potential of the method, the ATP-dependent motility of ncd, a kinesin-related motor protein, was observed with an in vitro bead assay. A fusion protein consisting of truncated ncd (amino acids 195-685) fused with glutathione-S-transferase was adsorbed to silica beads, and the axial and lateral motions of the beads along the microtubule surface were observed with high spatial and temporal resolution. The average axial velocity of the ncd-coated beads was 230 +/- 30 nm/s (average +/- SD). Spectral analysis of bead motion showed the increase in viscous drag near the surface; 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 nonhydrolyzable nucleotide adenylylimidodiphosphate.

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Published In

Biophysical journal

DOI

EISSN

1542-0086

ISSN

0006-3495

Publication Date

February 1998

Volume

74

Issue

2 Pt 1

Start / End Page

1074 / 1085

Related Subject Headings

  • Viscosity
  • Tubulin
  • Time Factors
  • Sensitivity and Specificity
  • Recombinant Fusion Proteins
  • Peptide Fragments
  • Models, Chemical
  • Microtubules
  • Microscopy, Interference
  • Lasers
 

Citation

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Allersma, M. W., Gittes, F., deCastro, M. J., Stewart, R. J., & Schmidt, C. F. (1998). Two-dimensional tracking of ncd motility by back focal plane interferometry. Biophysical Journal, 74(2 Pt 1), 1074–1085. https://doi.org/10.1016/s0006-3495(98)74031-7
Allersma, M. W., F. Gittes, M. J. deCastro, R. J. Stewart, and C. F. Schmidt. “Two-dimensional tracking of ncd motility by back focal plane interferometry.Biophysical Journal 74, no. 2 Pt 1 (February 1998): 1074–85. https://doi.org/10.1016/s0006-3495(98)74031-7.
Allersma MW, Gittes F, deCastro MJ, Stewart RJ, Schmidt CF. Two-dimensional tracking of ncd motility by back focal plane interferometry. Biophysical journal. 1998 Feb;74(2 Pt 1):1074–85.
Allersma, M. W., et al. “Two-dimensional tracking of ncd motility by back focal plane interferometry.Biophysical Journal, vol. 74, no. 2 Pt 1, Feb. 1998, pp. 1074–85. Epmc, doi:10.1016/s0006-3495(98)74031-7.
Allersma MW, Gittes F, deCastro MJ, Stewart RJ, Schmidt CF. Two-dimensional tracking of ncd motility by back focal plane interferometry. Biophysical journal. 1998 Feb;74(2 Pt 1):1074–1085.
Journal cover image

Published In

Biophysical journal

DOI

EISSN

1542-0086

ISSN

0006-3495

Publication Date

February 1998

Volume

74

Issue

2 Pt 1

Start / End Page

1074 / 1085

Related Subject Headings

  • Viscosity
  • Tubulin
  • Time Factors
  • Sensitivity and Specificity
  • Recombinant Fusion Proteins
  • Peptide Fragments
  • Models, Chemical
  • Microtubules
  • Microscopy, Interference
  • Lasers