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With their longest dimension typically being less than 100 nm, molecularmotors are significantly below the optical-resolution limit. Despitesubstantial advances in fluorescence-based imaging methodologies, labeling withbeads remains critical for optical-trapping-based investigations of molecularmotors. A key experimental challenge in bead-based assays is that the number ofmotors on a bead is not well defined. Particularly for single-moleculeinvestigations, the probability of single versus multiple-motor events has notbeen experimentally investigated. Here, we used bead travel distance as anindicator of multiple-motor transport and determined the lower-boundprobability of bead transport by two or more motors. We limited the ATPconcentration to increase our detection sensitivity for multiple- versussingle-kinesin transport. Surprisingly, for all but the lowest motor numberexamined, our measurements exceeded estimations of a previous model by R2-fold.To bridge this apparent gap between theory and experiment, we derived aclosed-form expression for the probability of bead transport by multiplemotors, and constrained the only free parameter in this model using ourexperimental measurements. Our data indicate that kinesin extends to ~57 nmduring bead transport, suggesting that kinesin exploits its conformationalflexibility to interact with microtubules at highly curved interfaces such asthose present for vesicle transport in cells. To our knowledge, our findingsprovide the first experimentally constrained guide for estimating theprobability of multiple-motor transport in optical trapping studies. Theexperimental approach utilized here (limiting ATP concentration) may begenerally applicable to studies in which molecular motors are labeled withcargos that are artificial or are purified from cellular extracts.

Quantitative Determination of the Probability of Multiple-Motor Transport in Bead-Based Assays