Microtubule minus-end aster organization is driven by processive HSET-tubulin clusters

Abstract

16Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two 17 architectures: bundles and asters. Although both architectures are critical for cellular function, 18 the molecular pathways that drive aster formation are poorly understood. Here, we study aster 19 formation by human minus-end directed kinesin-14 (HSET/KIFC1). We show that HSET is 20 incapable of forming asters from pre-formed, non-growing MTs, but rapidly forms MT asters in 21 the presence of soluble tubulin. HSET binds soluble (non-polymer) tubulin via its N-terminal tail 22 domain to form heterogeneous HSET-tubulin "clusters" containing multiple motors. Cluster 23 formation induces motor processivity and rescues the formation of asters from non-growing 24MTs. We then show that excess soluble tubulin stimulates aster formation in HeLa cells 25 overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT 26 bundle and aster formation in a manner governed by the availability of soluble tubulin. 27 28 65 processivity is essential for promoting aster formation. Finally, we present data suggesting that 66HSET's aster-forming behavior can be activated by excess soluble tubulin in mitotic HeLa cells. 67Our work establishes a general principle of aster formation by MT-crosslinking motor proteins, 68and demonstrates how motor regulation on a molecular level dictates the formation of higher-69 order MT structures. 70 71Results 72 Aster and bundle formation by HSET is context-dependent 73To determine if HSET, like its Xenopus homologue XCTK2, could form MT asters, we 74 expressed and purified four HSET truncations tagged with an N-terminal EGFP (Fig. 1a, 75 Supplementary Fig. S1a) for use in self-organization assays 17 . We verified that each construct 76 is a dimer by comparing their stepwise photobleaching traces 28 to EGFP-XMCAK, a known 77 dimer 29 (Supplementary Fig. S1b-c). When we combined 100 nM full-length EGFP-HSET, 20 78 µ M tubulin, and saturating ATP/GTP at 37°C, we observed MT aster formation which reached a 79 steady state within ~10 minutes (Fig. 1b, Supplementary Fig. S1d, and Supplementary Movie 80 1), consistent with published studies using Ncd multimers 7 or XCTK2 17 . Deletion of either the 81 N-terminal tail (EGFP-HSETΔTail), or the conserved C-terminal motor domain (EGFP-82HSETΔMotor) eliminated aster formation (Fig. 1b, middle). When the motor domain was 83 replaced by another copy of the N-terminal tail domain (EGFP-HSET-DoubleTail, see 17 ), we 84 observed bundle formation, but no MT asters (Fig. 1b, bottom). The entire HSET molecule is 85 thus required for aster formation of growing MTs. 86We next asked whether HSET was capable of forming asters from preformed, non-87 growing MTs stabilized by GMPCPP 30 . When we combined 100 nM full-length EGFP-HSET 88 with preformed MTs (1

Intracellular cargo transport by single-headed kinesin motors

“…This is consistent with previous work showing that weak motor-microtubule interactions are sufficient for nonprocessive axonemal dynein motors to move microtubules (32). Furthermore, some dimeric motors display little to no processivity as individual motors but can generate continuous motion when working in teams (33)(34)(35)(36). Our results extend these findings and suggest that nonprocessive motors, whether monomeric or dimeric, can drive continuous cargo transport as long as high-force generation is not required.…”

Two Kinesin-14A Motors Oligomerize to Drive Poleward Microtubule Convergence for Acentrosomal Spindle Morphogenesis in Arabidopsis thaliana

“…Different from the dependence of spindle pole organization on ATK1-ATK5 association-, the human HSET motor contributes to the minus end organization of microtubules into asters through the formation of HSET-tubulin clusters (Norris et al, 2018). The HSET-tubulin clusters MT minus end-directed processive motility which is probably required for aster formation.…”

Theory of antiparallel microtubule overlap stabilization by motors and diffusible crosslinkers

“…System C was simulated for a motor with the characteristics of kinesin-14: v 0 ∼ 0.2 µm/s, f s ∼ 5 pN [10] and D 1 ∼ 0.1 µm 2 /s [2], [14] (Fig. 3a, dots).…”