Noncanonical interaction with microtubules via the N-terminal nonmotor domain is critical for the functions of a bidirectional kinesin.

Several kinesin-5 motors (kinesin-5s) exhibit bidirectional motility. The mechanism of such motility remains unknown. Bidirectional kinesin-5s share a long N-terminal nonmotor domain (NTnmd), absent in exclusively plus-end-directed kinesins. Here, we combined in vivo, in vitro, and cryo-electron microscopy (cryo-EM) studies to examine the impact of NTnmd mutations on the motor functions of the bidirectional kinesin-5, Cin8. We found that NTnmd deletion mutants exhibited cell viability and spindle localization defects. Using cryo-EM, we examined the structure of a microtubule (MT)-bound motor domain of Cin8, containing part of its NTnmd. Modeling and molecular dynamic simulations based on the cryo-EM map suggested that the NTnmd of Cin8 interacts with the C-terminal tail of ß-tubulin. In vitro experiments on subtilisin-treated MTs confirmed this notion. Last, we showed that NTnmd mutants are defective in plus-end-directed motility in single-molecule and antiparallel MT sliding assays. These findings demonstrate that the NTnmd, common to bidirectional kinesin-5s, is critical for their bidirectional motility and intracellular functions.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Intracellular functions and motile properties of bi-directional kinesin-5 Cin8 are regulated by neck linker docking.

In this study, we analyzed intracellular functions and motile properties of neck-linker (NL) variants of the bi-directional S. cerevisiae kinesin-5 motor, Cin8. We also examined - by modeling - the configuration of H-bonds during NL docking. Decreasing the number of stabilizing H-bonds resulted in partially functional variants, as long as a conserved backbone H-bond at the N-latch position (proposed to stabilize the docked conformation of the NL) remained intact. Elimination of this conserved H-bond resulted in production of a non-functional Cin8 variant. Surprisingly, additional H-bond stabilization of the N-latch position, generated by replacement of the NL of Cin8 by sequences of the plus-end directed kinesin-5 Eg5, also produced a nonfunctional variant. In that variant, a single replacement of N-latch asparagine with glycine, as present in Cin8, eliminated the additional H-bond stabilization and rescued the functional defects. We conclude that exact N-latch stabilization during NL docking is critical for the function of bi-directional kinesin-5 Cin8.

Mechanisms by Which Kinesin-5 Motors Perform Their Multiple Intracellular Functions.

Bipolar kinesin-5 motor proteins perform multiple intracellular functions, mainly during mitotic cell division. Their specialized structural characteristics enable these motors to perform their essential functions by crosslinking and sliding apart antiparallel microtubules (MTs). In this review, we discuss the specialized structural features of kinesin-5 motors, and the mechanisms by which these features relate to kinesin-5 functions and motile properties. In addition, we discuss the multiple roles of the kinesin-5 motors in dividing as well as in non-dividing cells, and examine their roles in pathogenetic conditions. We describe the recently discovered bidirectional motility in fungi kinesin-5 motors, and discuss its possible physiological relevance. Finally, we also focus on the multiple mechanisms of regulation of these unique motor proteins.

Drag-induced directionality switching of kinesin-5 Cin8 revealed by cluster-motility analysis.

Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of directionality switching of mitotic kinesin-5 motors challenged the long-standing paradigm that individual kinesin motors are characterized by an intrinsic directionality. The underlying mechanism, however, remains unexplained. Here, we studied clustering-induced directionality switching of the bidirectional kinesin-5 Cin8. Based on the characterization of single-molecule and cluster motility, we developed a model that predicts that directionality switching of Cin8 is caused by an asymmetric response of its active motion to opposing forces, referred to as drag. The model shows excellent quantitative agreement with experimental data obtained under high and low ionic strength conditions. Our analysis identifies a robust and general mechanism that explains why bidirectional motor proteins reverse direction in response to seemingly unrelated experimental factors including changes in motor density and molecular crowding, and in multimotor motility assays.