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

Stephen R Norris,Marija Zanic,Amanda L Erwin,Ryoma Ohi,Melanie D Ohi,Claire E Strothman,Seungyeon Jung,Prashant Singh

doi:10.1038/s41467-018-04991-2

Stephen R Norris, Marija Zanic + Show 6 more

Open Access

https://doi.org/10.1038/s41467-018-04991-2

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Abstract

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Although both architectures are critical for cellular function, the molecular pathways that drive aster formation are poorly understood. Here, we study aster formation by human minus-end-directed kinesin-14 (HSET/KIFC1). We show that HSET is incapable of forming asters from preformed, nongrowing MTs, but rapidly forms MT asters in the presence of soluble (non-MT) tubulin. HSET binds soluble (non-MT) tubulin via its N-terminal tail domain to form heterogeneous HSET-tubulin clusters containing multiple motors. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We then show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. We propose a model where HSET can toggle between MT bundle and aster formation in a manner governed by the availability of soluble (non-MT) tubulin.

Highlights

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters
When the motor domain was replaced by another copy of the N-terminal tail domain (EGFP-HSET-DoubleTail, see19), we observed bundle formation, but no MT asters (Fig. 1b, bottom)
After using fluorescence intensity analysis to confirm that ~2–3 motors were present on QDots (2.3 for enhanced green fluorescent protein (EGFP)-HSET and 3.4 for EGFP-HSETΔTail, on average) (Supplementary Figure 5a), we investigated their motility by total internal reflection fluorescence (TIRF) (Fig. 5a and Supplementary Movie 7)

Summary

Introduction

Higher-order structures of the microtubule (MT) cytoskeleton are comprised of two architectures: bundles and asters. Cluster formation induces motor processivity and rescues the formation of asters from nongrowing MTs. We show that excess soluble (non-MT) tubulin stimulates aster formation in HeLa cells overexpressing HSET during mitosis. With the exception of templated structures (e.g., the axoneme), MT structures are built from two basic architectures[7]: (i) MT bundles, which can exist in either antiparallel or parallel configurations, and (ii) MT asters, which are radial arrays of MTs focused at a pole Both architectures are represented in the mitotic spindle. The minus-end-directed kinesin-14 XCTK2 is capable of driving aster formation from growing MTs, but unlike dynein, XCTK2 requires no additional cofactors[19]. Our work establishes a general principle of aster formation by MT-cross-linking motor proteins, and demonstrates how motor regulation on a molecular level dictates the formation of higher-order MT structures

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