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ABSTRACT
Kinesin and cytoplasmic dynein are microtubule-based motor proteins that actively transport material throughout the cell. Microtubules can intersect at a variety of angles both near the nucleus and at the cell periphery, and the behavior of molecular motors at these intersections has implications for long-range transport efficiency and accuracy. To test motor function at microtubule intersections, crossovers were arranged in vitro using flow to orient successive layers of filaments. Single kinesin and cytoplasmic dynein-dynactin molecules fused with green-fluorescent protein, and artificial bead cargos decorated with multiple motors, were observed while they encountered intersections. Single kinesins tend to cross intersecting microtubules, whereas single dynein-dynactins have a more varied response. For bead cargos, kinesin motion is independent of motor number. Dynein beads with high motor numbers pause, but their actions become more varied as the motor number decreases. These results suggest that regulating the number of active dynein molecules could change a motile cargo into one that is anchored at an intersection, consistent with dynein's proposed transport and tethering functions in the cell.
INTRODUCTION
Kinesin-1 and cytoplasmic dynein are the major cytoplasmic motors responsible for long-range transport in many cell types. Kinesin walks along microtubules toward the plus ends, facilitating material transport from the cell interior toward the cortex. Dynein transports material toward the microtubule minus ends, moving from the cell periphery to the cell interior.
Although both proteins are microtubule-based transport motors, they are structurally distinct. Conventional kinesin is primarily a homodimer of heavy chains that each fold into a compact motor head ~4 nm in diameter. Crystal structures show that ATP hydrolysis at a single catalytic site causes con format ional changes in the head (1). These structural changes alter the motor head's affinity for the microtubule and lead to nanometer-scale motions of a short linker region that extends from the globular head. The C-terminus of kinesin heavy chain is a dimerization domain that mediates the assembly of the two-headed motor. Alternating and coordinated ATP hydrolysis at each of the two heads causes the kinesin to step processively and robustly along the microtubule (2). Kinesin's C-terminal tail can bind to cargo directly or via two light chains that have been implicated in regulation of kinesin by autoinhibition (1,3).
Dynein is also composed primarily...