A Nonmotor Microtubule Binding Site in Kinesin-5 Is Required for Filament Crosslinking and Sliding

Abstract

Summary Kinesin-5, a widely conserved motor protein required for assembly of the bipolar spindle in vertebrates, forms homotetramers with two pairs of motor domains positioned at opposite ends of a dumbbell shaped molecule [1–3]. It has long been assumed that this configuration of motor domains is the basis of kinesin-5’s ability to drive relative sliding of microtubules [2, 4, 5]. Recently, it was suggested that in addition to the N-terminal motor domain, kinesin-5 also has a non-motor microtubule binding site in its C-terminus [6]. However, it is not known how the non-motor domain contributes to motor activity, or how a kinesin-5 tetramer utilizes a combination of four motor and four non-motor microtubule binding sites for its microtubule organizing functions. Here we show, in single molecule assays, that kinesin-5 homotetramers require the non-motor C-terminus for crosslinking and relative sliding of two microtubules. Remarkably, this domain enhances kinesin-5’s microtubule binding without substantially reducing motor activity. Our results demonstrate that tetramerization of kinesin-5’s low-processivity motor domains is not sufficient for microtubule sliding, as the motor domains alone are unlikely to maintain persistent microtubule crosslinks. Rather, kinesin-5 utilizes non-motor microtubule binding sites to tune its microtubule attachment dynamics, enabling it to efficiently align and sort microtubules during metaphase spindle assembly and function.

Prime movers: the mechanochemistry of mitotic kinesins

“…KIF11 is built from two homodimers arranged in an antiparallel manner so that identical pairs of heads project from each end of the tetramer, forming a microtubule cross-linker 52,54,55 . The C-terminal tip of the KIF11 tail also contributes to microtubule binding 56 . KIF11 can therefore crosslink the microtubules that project from one centrosome towards the other.…”

Kinesin-5 Kip1 is a bi-directional motor that stabilizes microtubules and tracks their plus-ends in vivo

“…For example, it had been demonstrated for the plus-end tracking Drosophila kinesin-13 KLP10A and KLP59C, that a ring-structure formation required for the plus-end tracking, was favored specifically by the ATP-bound state (Moores et al, 2003;Varga et al, 2006). Finally, it has been recently shown that a non-motor MT binding site in kinesin-5 is required for MT crosslinking (Weinger et al, 2011) and that the tail of Kip3 promotes its accumulation at the MT plus-ends (Su et al, 2011). Therefore, the tail of Kip1 may be involved in plus-end MT tracking, in addition to the role of its motor domain.…”

TPX2 Inhibits Eg5 by Interactions with Both Motor and Microtubule

“…Eg5 has been shown to exhibit diffusive behavior on microtubules at physiological salt concentration (22,28). To determine whether mammalian Eg5 present in diluted cell extracts showed similar diffusive behavior, we added increasing concentrations of KCl to the motility buffer and examined motor behavior.…”