ArticlebioRxiv : the preprint server for biology2025
Dimeric Cin8 motors have an inherent plus-end bias and weak inter-head coordination.
Article in bioRxiv : the preprint server for biology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
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.
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