Dimeric Cin8 motors have an inherent plus-end bias and weak inter-head coordination

Kinesin-5 motors are bipolar tetramers that crosslink and slide antiparallel microtubules during mitotic spindle assembly. Fungal kinesin-5 motors, such as Cin8, exhibit bidirectional motility, switching between minus- and plus-end-directed stepping in response to environmental conditions; however, the molecular basis of this directional switching remains unclear. To better understand the origin of this bidirectional behavior, we investigated the motility and ATPase kinetics of two Cin8 dimers, created by fusing the motor domains to a stable coiled-coil domain from kinesin-1. To investigate the role of the proximal neck coiled-coil region in coordinating motor activity, we compared Cin8 dimers that included or lacked the first four heptads of the Cin8 neck-coil domain. By analyzing the stepping kinetics, microtubule residence times, and directional switching dynamics, we found that these Cin8 dimers move processively with a net plus-end directionality along with undirected movements, behaviors that mimic the plus-ended motility state of wild-type Cin8. However, fast minus-ended motility seen in wild-type Cin8 tetramers was not observed in the dimers. The instantaneous velocity distributions and ATPase rates were inconsistent with the undirected movement being solely due to passive diffusion, suggesting that they reflect random bidirectional stepping. Fewer undirected movements were seen on yeast microtubules, their native physiological substrate, compared to on bovine microtubules. Replacing the Cin8 neck-coil domain with a stable coiled-coil led to faster plus-end stepping, fewer undirected movements, a reduction in the microtubule binding duration, and enhanced coupling between ATP hydrolysis and plus-end stepping. Our results suggest that the native Cin8 neck coil confers flexibility between the two motor domains that contributes to bidirectional stepping, and that sustained minus-end movement requires regions outside the motor domain. Statement of SignificanceThe kinesin-5 family of motors, which contain two pairs of heads located on either end of a long stalk domain, power mitotic spindle formation by sliding antiparallel microtubules. The yeast kinesin-5, Cin8 moves to microtubule minus-ends under specific conditions, breaking the dogma that N-terminal kinesins move to microtubule plus-ends. To gain insight into how Cin8 changes its walking direction, we analyzed engineered Cin8 dimers, models of one half of full-length Cin8. The dimers step erratically, consistent with stepping in both directions, but have a plus-end bias. We find evidence that the two heads poorly coordinate, which contrasts with other kinesins, and suggest that the stepping direction may be regulated by altering the degree of inter-head coordination.