2-DIMENSIONAL TRACKING OF NCD MOTILITY BY BACK FOCAL-PLANE INTERFEROMETRY

A technique for detecting the displacement of micron-sized optically t rapped probes using far-field interference is introduced, theoreticall y explained, and used to study the motility of the nod motor protein. Bead motions in the focal plane relative to the optical trap were dete cted by measuring laser intensity shifts in the back-focal plane of th e microscope condenser by projection on a quadrant diode. This detecti on method is two-dimensional, largely independent of the position of t he trap in the field of view and has similar to 10-mu S time resolutio n. The high resolution makes it possible to apply spectral analysis to measure dynamic parameters such as local viscosity and attachment com pliance. A simple quantitative theory for back-focal-plane detection w as derived that shows that the laser intensity shifts are caused prima rily by a far-field interference effect. The theory predicts the detec tor response to bead displacement, without adjustable parameters, with good accuracy. To demonstrate the potential of the method, the ATP-de pendent motility of nod, a kinesin-related motor protein, was observed with an in vitro bead assay. A fusion protein consisting of truncated nod (amino acids 195-685) fused with glutathione-S-transferase was ad sorbed to silica beads, and the axial and lateral motions of the beads along the microtubule surface were observed with high spatial and tem poral resolution. The average axial velocity of the nod-coated beads w as 230 +/- 30 nm/s (average +/- SD). Spectral analysis of bead motion showed the increase in viscous drag near the surface; we also found th at any elastic constraints of the moving motors are much smaller than the constraints due to binding in the presence of the nonhydrolyzable nucleotide adenylylimidodiphosphate.