Abstract
Advanced techniques in single-molecule optical microscopy have contributed greatly to our current view on the dynamics of motor proteins. While so far most studies have been limited to the 2-D imaging on a CCD-camera chip, a complete understanding of motor protein function requires insight in how motor proteins move in 3-D on the lattice of cytoskeletal filaments.Here, we report a novel and versatile method to study the interactions of motor proteins with cytoskeletal filaments in 3-D with nanometer accuracy. We sparsely label reconstituted microtubules with quantum dots and use fluorescence microscopy to image their longitudinal and rotational movement over reflective silicon surfaces coated with motor proteins. We determine the 2-D xy-positions of the QDs with sub-pixel accuracy by nanometer tracking and combine this data with simultaneous height measurements based on fluorescence-interference contrast microscopy. We use this technique (i) to investigate the stability of the paths of cooperating processive kinesin-1 motors and (ii) to study the asymmetry in the powerstrokes of non-processive microtubule motors.
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