The two head domains of the budding yeast Kinesin-14 Kar3Vik1 bind adjacent protofilaments at the start of the motility cycle, followed by release of Vik1 from one protofilament to allow the motor’s powerstroke.

Kar3Cik1 is a

Background: Kar3Vik1 binds side-by-side microtubule protofilaments and utilizes a minus-end-directed powerstroke. Results: Microtubule collision occurs through Vik1 followed by Kar3 binding and ADP release, which destabilize Vik1 and generate the intermediate poised for ATP binding. Conclusion: The transient Kar3Vik1 two-head-bound state intermediate was identified.Significance: This study provides new insights into force generation by kinesin-14 motors.

Kar3Cik1 is a heterodimeric kinesin-14 from Saccharomyces cerevisiae involved in spindle formation during mitosis and karyogamy in mating cells. Kar3 represents a canonical kinesin motor domain that interacts with microtubules under the control of ATP-hydrolysis. In vivo, the localization and function of Kar3 is differentially regulated by its interacting stoichiometrically with either Cik1 or Vik1, two closely related motor homology domains that lack the nucleotide-binding site. Indeed, Vik1 structurally resembles the core of a kinesin head. Despite being closely related, Kar3Cik1 and Kar3Vik1 are each responsible for a distinct set of functions in vivo and also display different biochemical behavior in vitro. To determine a structural basis for their distinct functional abilities, we used cryo-electron microscopy and helical reconstruction to investigate the 3-D structure of Kar3Cik1 complexed to microtubules in various nucleotide states and compared our 3-D data of Kar3Cik1 with that of Kar3Vik1 and the homodimeric kinesin-14 Ncd from Drosophila melanogaster. Due to the lack of an X-ray crystal structure of the Cik1 motor homology domain, we predicted the structure of this Cik1 domain based on sequence similarity to its relatives Vik1, Kar3 and Ncd. By molecular docking into our 3-D maps, we produced a detailed near-atomic model of Kar3Cik1 complexed to microtubules in two distinct nucleotide states, a nucleotide-free state and an ATP-bound state. Our data show that despite their functional differences, heterodimeric Kar3Cik1 and Kar3Vik1 and homodimeric Ncd, all share striking structural similarities at distinct nucleotide states indicating a common mechanistic theme within the kinesin-14 family.

The first cell division in C. elegans is asymmetric. Asymmetric cell division requires correct positioning of the mitotic spindle. Prior to metaphase, the nuclear-centrosome complex, the precursor of the mitotic spindle, is positioned in the cell center. During anaphase, the spindle is displaced towards the posterior so that bisection of the spindle during cytokinesis leads to daughter cells of unequal sizes. Forces that center and position the spindle come from cortical force generators that pull on astral microtubules. In order to generate force, the cortex needs to provide a stiff anchoring platform. However, a role for the cortex in C. elegans has only been described with respect to polarity establishment. We perturbed the acto-myosin cortex by RNAi of non-muscle-myosin II (nmy-2) using conditions that allowed us to avoid disturbing polarity. Strikingly, in nmy-2(RNAi), membrane tubes are pulled from the plasma membrane into the cell. They were seen after RNAi against other actin cytoskeleton proteins and members of force generation complex, suggesting that the cortical force generators pull the invaginations, and a weakening of the cortex. As expected, we observed an increase in the variance of spindle position and orientation in nmy-2(RNAi). We used the oscillations of the centrosomes during anaphase as a reporter of spindle mechanics, and measured an increase in oscillations frequency but only a marginal decrease in amplitude. In order to understand this phenotype, we used our previously published model to analyze the results. Only by including the cortex into the model, we were able to fully describe the role of NMY-2. In summary, the occurrence of tubes after nmy-2(RNAi) strongly points towards a weakening of the cortex and the analysis of the spindle positioning suggests that the cortex provides a rigid platform for anchoring the force generators.