Speckled microtubules improve tracking in motor-protein gliding assays

Gliding assays of motor proteins such as kinesin, dynein and myosin are commonly carried out with fluorescently labeled microtubules or filamentous actin. In this paper, we show that speckled microtubules (MTs), prepared by copolymerizing 98% unlabeled tubulin with 2% rhodamine-labeled tubulin, can be localized to +/- 7.4 nm ( 24 measurements) in images acquired every 125 ms. If the speckled MTs move at about 800 nm s(-1), ten images are sufficient to determine their velocity to a precision of +/- 6.8 nm s(-1) (6 microtubules, 24 measurements). This velocity precision is four-fold better than manual methods for measuring the gliding velocity of uniformly labeled MTs by end-point localization. The improved velocity precision will permit the determination of velocity-force curves when one, two and three kinesin motors pull a single load in vitro.