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Owing to their wide spectrum of in vivo functions, motor proteins, such as kinesin-1, show great potential for application as nanomachines in engineered environments. When attached to a substrate surface, these motors are envisioned to shuttle cargo that is bound to reconstituted microtubules--one component of the cell cytoskeleton--from one location to another. One potentially serious problem for such applications is, however, the rotation of the microtubules around their longitudinal axis. Here we explore this issue by labelling the gliding microtubules with quantum dots to simultaneously follow their sinusoidal side-to-side and up-and-down motion in three dimensions with nanometre accuracy. Microtubule rotation, which originates from the kinesin moving along the individual protofilaments of the microtubule, was not impeded by the quantum dots. However, pick-up of large cargo inhibited the rotation but did not affect the velocity of microtubule gliding. Our data show that kinesin-driven microtubules make flexible, responsive and effective molecular shuttles for nanotransport applications.

Kinesin-1 motor proteins walk parallel to the protofilament axes of microtubules as they step from one tubulin dimer to the next. Is protofilament tracking an inherent property of processive kinesin motors, like kinesin-1, and what are the structural determinants underlying protofilament tracking? To address these questions, we investigated the tracking properties of the processive kinesin-8, Kip3. Using in vitro gliding motility assays, we found that Kip3 rotates microtubules counterclockwise around their longitudinal axes with periodicities of ∼1 μm. These rotations indicate that the motors switch protofilaments with a bias toward the left. Molecular modeling suggests 1), that the protofilament switching may be due to kinesin-8 having a longer neck linker than kinesin-1, and 2), that the leftward bias is due the asymmetric geometry of the motor neck linker complex.

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Single-molecule experiments have been used with great success to explore the mechanochemical cycles of processive motor proteins such as kinesin-1, but it has proven difficult to apply these approaches to nonprocessive motors. Therefore, the mechanochemical cycle of kinesin-14 (ncd) is still under debate. Here, we use the readout from the collective activity of multiple motors to derive information about the mechanochemical cycle of individual ncd motors. In gliding motility assays we performed 3D imaging based on fluorescence interference contrast microscopy combined with nanometer tracking to simultaneously study the translation and rotation of microtubules. Microtubules gliding on ncd-coated surfaces rotated around their longitudinal axes in an [ATP]-and [ADP]-dependent manner. Combined with a simple mechanical model, these observations suggest that the working stroke of ncd consists of an initial small movement of its stalk in a lateral direction when ADP is released and a second, main component of the working stroke, in a longitudinal direction upon ATP binding.kinesin | microtubule | gliding motility assay | fluorescence interference contrast microscopy | mathematical modeling S ingle-molecule techniques, in particular single-molecule fluorescence (1) and optical tweezers (2), have greatly advanced our understanding of motor proteins. Such experiments normally rely on the processivity of the motor, which makes it possible to observe multiple consecutive working cycles (i.e., steps). Although single-molecule mechanical measurements on nonprocessive motors can be performed with optical tweezers (3, 4), they require a sophisticated statistical analysis (5, 6) and, if the motor is to be studied under load, advanced feedback (7) or force-clamp mechanisms (8). The minus-end directed C-terminal kinesin-14, ncd, is such a nonprocessive motor. It has been shown to play an essential role in spindle focusing (9-11) and to slide and cross-link microtubules (12, 13).For the working stroke of a single ncd motor a lever arm model has been suggested (14, 15). However, as reviewed in ref. 16 the triggering event for the working stroke is still under debate. On the one hand, it has been suggested that the working stroke is connected to the ADP release (14, 17). On the other hand, it has been proposed to be coupled to the binding of ATP to the microtubule (MT)-ncd complex (4,15,18). In another study, a twostage displacement of the ncd stalk has been proposed where stalk displacement is initiated by binding of ncd to the MT and completed upon ATP binding (19). However, even though a variety of approaches including X-ray crystallography (15), EM (20-23), optical trapping (4, 24), FRET (19), and computational modeling (25) have been used to study the coupling between nucleotide states and mechanical states of ncd, the exact mechanism remains to be elucidated.Alternatively, nonprocessive motors can be studied in gliding motility assays (26) albeit with the disadvantage that single-motor behavior is difficult to access. On an ncd...

Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by kinesin-1 and kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.

Recent developments in optical microscopy and nanometer tracking have greatly improved our understanding of cytoskeletal motor proteins. Using fluorescence microscopy, dynamic interactions are now routinely observed in vitro on the level of single molecules mainly using a geometry, where fluorescently labeled motors move on surface-immobilized filaments. In this chapter, we review recent methods related to single-molecule kinesin motility assays. In particular, we aim to provide practical advice on: how to set up the assays, how to acquire high-precision data from fluorescently labeled kinesin motors and attached quantum dots, and how to analyze data by nanometer tracking.

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