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.

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.

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.

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. While several kinesin motors have now been shown to exhibit contextdependent directionality that can be altered under diverse experimental conditions, the underlying mechanism remains unknown. Here, we studied clustering-induced directionality switching of the mitotic kinesin-5 Cin8, using a fluorescence-based single-molecule motility assay combined with biophysical theory. Based on the detailed characterization of the motility of single motors and clusters of Cin8, we developed a predictive molecular model, that quantitatively agrees with experimental data. This combined approach allowed us to quantify the response of Cin8 motors to external forces as well as the interactions between Cin8 motors, and thereby develop a detailed understanding of the molecular mechanism underlying directionality switching. The main insight is that directionality switching is caused by a single feature of Cin8: an asymmetric response of active motion to forces that oppose motion, here referred to as drag. This general mechanism explains why bidirectional motor proteins are capable of reversing direction in response to seemingly unrelated experimental factors including clustering, changes in the ionic strength of the buffer, increased motor density and molecular crowding, and in motility assays. Significance Statement:Kinesin-5 motor proteins perform essential functions in chromosome segregation during mitotic cell division. Surprisingly, several kinesin-5 motors have the ability to reverse directionality under different experimental conditions, which contradicts the long-standing paradigm that individual kinesin motors are characterized by an intrinsic directionality. The mechanism underlying this ability to switch directionality has remained elusive. Here, we combine fluorescence-based motility assays and theoretical modeling to analyze cluster-sizedependent motility of the bidirectional kinesin-5 Cin8. Our results show that bidirectional motors can switch directionality because they exhibit an asymmetric response of active motion to drag. This mechanism explains multiple seemingly unrelated experimental factors that have been shown to cause directionality switching of kinesin motors.

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