Optical tweezers have greatly impacted the development of mechanobiology by enabling high precision sub-piconewton measurements of mechanical forces developed by force generating proteins, called molecular motors, at the single-molecule level. Molecular motors, such as kinesins hydrolyze ATP to generate force (10 pN) and transport in a directional manner intracellular cargoes along cytoskeletal filamentous tracks called microtubules. The force developed by kinesins have been mainly studied using the “single-bead” assay, where an optically trapped bead is pulled by a bead-attached kinesin molecule as it steps along a surface immobilized microtubule. This assay, besides forces parallel to the long axis of the filament on which the kinesin processes, forces perpendicular to the filament due to the bead interacting with the underlying microtubule. These perpendicular forces, which cannot be directly measured, can accelerate the detachment of the molecular motor from its filamentous track. An alternative approach is the “three-bead” assay, in which the vertical force component is minimized, and the total opposing force is mainly parallel to the microtubule. Experiments with kinesin sho
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