Direct observation of single kinesin molecules moving along microtubules

Abstract

Kinesin is a two-headed motor protein that powers organelle transport along microtubules. Many ATP molecules are hydrolysed by kinesin for each diffusional encounter with the microtubule. Here we report the development of a new assay in which the processive movement of individual fluorescently labelled kinesin molecules along a microtubule can be visualized directly; this observation is achieved by low-background total internal reflection fluorescence microscopy in the absence of attachment of the motor to a cargo (for example, an organelle or bead). The average distance travelled after a binding encounter with a microtubule is 600 nm, which reflects a approximately 1% probability of detachment per mechanical cycle. Surprisingly, processive movement could still be observed at salt concentrations as high as 0.3 M NaCl. Truncated kinesin molecules having only a single motor domain do not show detectable processive movement, which is consistent with a model in which kinesin's two force-generating heads operate by a hand-over-hand mechanism.

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

“…However, these monomeric kinesins do not exhibit processive movement when assayed as individual motors in a single molecule fluo-rescence motility assay (14) or a bead assay (15). A kinesin motor containing the complete motor and neck domains, on the other hand, forms a dimer and also exhibits processive movement (14,16). Collectively, these studies suggest that the dimeric structure of kinesin is not essential for force-generation per se, although it does appear to be required for processive movement.…”

“…Bacterial expression of the first 340 amino acids of the Drosophila kinesin heavy chain (which contains the core NH 2 -terminal globular motor domain and the first ϳ10 amino acids of the neck) produces a monomeric protein that generates directed motility when many motors are interacting simultaneously with a single microtubule in gliding motility assays (7,13). However, these monomeric kinesins do not exhibit processive movement when assayed as individual motors in a single molecule fluo-rescence motility assay (14) or a bead assay (15). A kinesin motor containing the complete motor and neck domains, on the other hand, forms a dimer and also exhibits processive movement (14,16).…”

Modeling transport of a pulse of radiolabeled organelles in a Drosophila unipolar motor neuron

“…The kinetic constants for fast axonal transport are expected to be at the high end of this range. Indeed, the average detachment rate from MTs for kinesin-1 is estimated to be 1 s −1 (Schnitzer et al [26], Vale et al [19]) while the average attachment rate for cytoplasmic dynein is estimated to be 1.5 s −1 (Carter and Cross [18], Vale et al [19]). However, the transition rate is expected to be smaller than these estimates because the transition involves detachment from MTs with plus-end-out orientation, switching of kinesin to dynein molecular motors, and then attachment to MTs with minus-end-out orientation.…”

“…It is assumed that radiolabeled organelles enter the primary neurite as a pulse; initially the concentration of radiolabeled organelles in the primary neurite is zero, then (at t * 1 = 0) the concentration of kinesin-driven organelles at x * 1 = 0 is suddenly increased to n * in and remains at this level until t * 1 = t * c and then it is suddenly dropped to zero. According to Carter and Cross [18] and Vale et al [19], kinesin-1 (conventional kinesin) walks to the MT plus-ends with an average velocity of 1 μm/s while according to King and Schroer [20] and Toba et al [21], cytoplasmic dynein walks to the MT minus-ends with approximately the same average velocity of 1 μm/s; therefore, for the ease of comparison between transport in axons and dendrites it is assumed that v * kin = v * dyn = v * .…”

Kinesin: a molecular motor with a spring in its step

“…The analysis is simplest when there is negligible ADP or P i in solution, as in the force-clamp experiment of Visscher et al (1999), in which case the two backward rate constants, k 32 and k 43 ( f ), may be taken to be zero. We may also simplify matters by neglecting dissociation of the kinesin-tubulin complex, since it has been shown experimentally that kinesin dimers can perform a hundred or more steps before they dissociate from a microtubule (Howard et al 1989;Block et al 1990;Svoboda et al 1993;Vale et al 1996). In that case, given that the ATP hydrolysis in figure 1c is tightly coupled to the kinesin stepping (Visscher et al 1999;Thomas et al 2001), the average velocity v = u 0 R. Since the Law of Mass Action requires that k 12 = K 12 [ATP], where K 12 is the molar rate constant for ATP-binding, we then find that the kinesin stepping velocity v obeys the Michaelis-Menten equation…”