Journal article
Two-Dimensional Tracking of ncd Motility by Back Focal Plane Interferometry
Biophysical journal, Vol.74(2), pp.1074-1085
1998
Handle:
https://hdl.handle.net/2376/107571
PMCID: PMC1302587
PMID: 9533719
Abstract
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 ∼10-
μs 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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Details
- Title
- Two-Dimensional Tracking of ncd Motility by Back Focal Plane Interferometry
- Creators
- Miriam W Allersma - Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109, USAFrederick Gittes - Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109, USAMichael J deCastro - Department of Bioengineering, University of Utah, Salt Lake City, Utah 84105 USARussell J Stewart - Department of Bioengineering, University of Utah, Salt Lake City, Utah 84105 USAChristoph F Schmidt - Department of Physics and Biophysics Research Division, University of Michigan, Ann Arbor, Michigan 48109, USA
- Publication Details
- Biophysical journal, Vol.74(2), pp.1074-1085
- Academic Unit
- Physics and Astronomy, Department of
- Publisher
- Elsevier Inc
- Identifiers
- 99900546991501842
- Language
- English
- Resource Type
- Journal article