Abstract
Most kinesin motors move in only one direction along microtubules. Members of the kinesin-5 subfamily were initially described as unidirectional plus-end-directed motors and shown to produce piconewton forces. However, some fungal kinesin-5 motors are bidirectional. The force production of a bidirectional kinesin-5 has not yet been measured. Therefore, it remains unknown whether the mechanism of the unconventional minus-end-directed motility differs fundamentally from that of plus-end-directed stepping. Using force spectroscopy, we have measured here the forces that ensembles of purified budding yeast kinesin-5 Cin8 produce in microtubule gliding assays in both plus- and minus-end direction. Correlation analysis of pause forces demonstrated that individual Cin8 molecules produce additive forces in both directions of movement. In ensembles, Cin8 motors were able to produce single-motor forces up to a magnitude of ∼1.5 pN. Hence, these properties appear to be conserved within the kinesin-5 subfamily. Force production was largely independent of the directionality of movement, indicating similarities between the motility mechanisms for both directions. These results provide constraints for the development of models for the bidirectional motility mechanism of fission yeast kinesin-5 and provide insight into the function of this mitotic motor.
Highlights
Members of the kinesin-5 family are essential for correct spindle assembly and spindle function in most eukaryotic organisms
Beads coated with purified full-length Cin8-mGFP (Materials and Methods) were observed to bind to immobilized microtubules but, in contrast to control experiments, not to generate force that could displace the bead from the trap center, despite the experiments being conducted over a 100-fold trap-stiffness range (Fig. S3 b)
We have measured the increasing load generated by an ensemble of surface-immobilized Cin8 and Eg5 motors transporting a microtubule-attached microbead out of the center of an optical trap
Summary
Members of the kinesin-5 family are essential for correct spindle assembly and spindle function in most eukaryotic organisms. In vitro experiments with purified proteins showed that single Cin and Cut molecules preferentially moved toward the minus ends of individual microtubules [12,14,15,16] This atypical directionality of movement for an N-terminal kinesin was reversed when these motors acted as part of a larger team, either when immobilized on a glass surface at high densities in microtubule gliding experiments or when sliding two antiparallel microtubules relative to each other [12,16]. Recent mathematical modeling of fission-yeast spindle assembly and spindle-pole separation supports this idea and suggests that bidirectionality of kinesin-5 motors is essential for this process [24]
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