Yeast kinesin-8 depolymerizes microtubules in a length-dependent manner

Abstract

The microtubule cytoskeleton and the mitotic spindle are highly dynamic structures, yet their sizes are remarkably constant, thus indicating that the growth and shrinkage of their constituent microtubules are finely balanced. This balance is achieved, in part, through kinesin-8 proteins (such as Kip3p in budding yeast and KLP67A in Drosophila) that destabilize microtubules. Here, we directly demonstrate that Kip3p destabilizes microtubules by depolymerizing them--accounting for the effects of kinesin-8 perturbations on microtubule and spindle length observed in fungi and metazoan cells. Furthermore, using single-molecule microscopy assays, we show that Kip3p has several properties that distinguish it from other depolymerizing kinesins, such as the kinesin-13 MCAK. First, Kip3p disassembles microtubules exclusively at the plus end and second, remarkably, Kip3p depolymerizes longer microtubules faster than shorter ones. These properties are consequences of Kip3p being a highly processive, plus-end-directed motor, both in vitro and in vivo. Length-dependent depolymerization provides a new mechanism for controlling the lengths of subcellular structures.

The Kinesin-8 Kip3 Switches Protofilaments in a Sideward Random Walk Asymmetrically Biased by Force

“…We determined the speed by linear fits to the tracked x position. The speed was 40 5 2 nm/s (N ¼ 52, mean 5 SE unless noted otherwise), consistent with previous reports (24,28,29). With the trap turned on and no load applied in any direction (zero-force feedback), the speed was 39 5 2 nm/s (N ¼ 90, zero-force data point in Fig.…”

“…Instead of transporting cargo, Kip3 depolymerizes microtubules and thereby regulates their dynamics and length (24)(25)(26). To do so, the motor needs to reach the microtubule end and thus, ideally, should have two features: 1) it should be highly processive-taking many steps without dissociating from the microtubule; and 2) it should be able to bypass obstacles.…”

“…To do so, the motor needs to reach the microtubule end and thus, ideally, should have two features: 1) it should be highly processive-taking many steps without dissociating from the microtubule; and 2) it should be able to bypass obstacles. Whereas the high processivity of Kip3, with an average run length of up to 12 mm (24,27), has been shown to be due to a weakly bound slip state (28,29), in addition to a second microtubule binding site at the motor's tail (27), the obstacle-bypassing capability has only been proposed (22). To bypass an obstacle, the motor should have the ability to switch microtubule protofilaments, as suggested by microtubule rotations seen in motor-ensemble gliding assays (22).…”

Prime movers: the mechanochemistry of mitotic kinesins

“…KIF18A operates at kinetochores 106 , whereas KIF18B is found on astralmicrotubules 107,108 . Some of the members of the kinesin-8 family are highly processive, and initial work on S. cerevisiae Kip3 indicated that it has depolymerase activity 49,109 . However a depolymerase activity for KIF18 from non-budding yeast is controversial (for review see 110 ).…”

Rings, bracelets, sleeves, and chevrons: new structures of kinetochore proteins

“…In fission yeast, the Dam1 complex is required only if kinesin-8 is absent, suggesting kinesin-8 performs a redundant function (20). Kinesin-8 is a plus-end directed motor that acts as a length-dependent depolymerase (21). Its ability to substitute for the Dam1 complex is especially interesting considering that, evolutionarily, the kinesin-8s are the next closely related clade to the kinesin-13s (see below) and may possess mechanistic similarities (21)(22)(23).…”