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

Abstract

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.

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

“…To correlate between the intracellular phenotypes of the NL variants and their motor functions, we examined their activity in vitro. In these assays, we characterized the activity of GFP-tagged full-length NL variants, overexpressed and purified from S. cerevisiae cells, on fluorescently labeled GMPCPP-stabilized MTs ( Pandey et al, 2021 ; Shapira et al, 2017 ). We first compared the MT affinities of the NL variants by determining the average number of MT-attached motors per MT length at equal motor protein concentrations.…”

“…molecules of Cin8 from a total population of moving Cin8 particles, based on their fluorescence intensities(Pandey et al, 2021) (see Materials and methods). By following the fluorescence intensity of Cin8 particles as a function of time, we observed single events of intensity decrease, of ~45 arbitrary intensity units (a.u.…”

The microtubule plus-end tracking protein Bik1 is required for chromosome congression

“…Likewise, Bik1 might be needed for Cin8 to reach the plus-ends of long kinetochore microtubules that extend beyond bundled kinetochore microtubules. Cin8 is a bidirectional kinesin and has been shown to preferentially move in a minus-end directed manner as single motors (Gerson-Gurwitz et al, 2011; Pandey et al, 2021; Roostalu et al, 2011). Directional switching has been shown to occur when Cin8 motors are clustered (Pandey et al, 2021; Roostalu et al, 2011), and other kinesin-5 motors switch directionality in response to steric crowding on MTs (Britto et al, 2016).…”

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

“…This study further proposed that since the ability to move in both directions is an intrinsic property of Cin8 tetramers [33,55,147], specific intermolecular interactions between Cin8 tetramers in a cluster control the directionality of this motor [62]. Based on the characterization of single-molecule and cluster motility, a recently developed model predicts that directionality switching of the bidirectional Cin8 is caused by an asymmetric response of its active motion to opposing forces, referred to as drag [148]. The model showed excellent quantitative agreement with experimental data obtained under high and low ionic strength conditions.…”

“…The model showed excellent quantitative agreement with experimental data obtained under high and low ionic strength conditions. This recent analysis identified a robust and general mechanism that explains why bidirectional motor proteins reverse direction in response to experimental conditions, including changes in motor density and molecular crowding and in multimotor motility assays [148].…”