Organizing Microtubule Asters
Microtubules (MTs) are a critical component of the cytoskeleton, playing a role in a range of processes from cell polarization to division. MTs are polymers of αβ-tubulin that grow and shrink from a rapidly changing plus-end and a more slowly changing minus-end to form two primary architectures – linear bundles and radial asters. MT organization is aided by MT-associated proteins (MAPs). Asters, which are key to the formation of the bipolar mitotic spindle, assemble primarily around centrosomes; however, a number of MAPs can also facilitate aster formation, among them the kinesin-14 family. Kinesin-14 proteins are dimers of subunits comprising a C-terminal motor domain that uses the power of ATP to enable movement along a preformed MT and an N-terminal tail. In most in vitrostudies, kinesin-14 family members have tended to form, slide, and sort MT bundles rather than assemble asters. These observations led Vanderbilt Basic Sciences investigator Marija Zanic and her collaborators Ryoma Ohi and Melanie Ohi (University of Michigan) to hypothesize that kinesin-14-dependent aster formation requires the presence of soluble tubulin dimers. They tested this hypothesis using the human kinesin-14 HSET expressed as a GFP conjugate. They showed that incubation of HSET-GFP with soluble tubulin and ATP/GTP resulted in aster formation. In contrast, addition of HSET-GFP with preformed MTs led to bundle formation. HSET-GFP diffused randomly on the surface of preformed MTs but became processive after soluble tubulin was added. As a result, HSET-GFP moved to the minus-end of the MT and stayed there, behavior expected to promote aster formation. Immunoprecipitation studies revealed that HSET-GFP directly binds to tubulin via an interaction requiring the N-terminal tail. Incubation of HSET-GFP and tubulin led to formation of heterogeneous clusters that moved processively along MTs. Similarly, artificial clusters formed by binding HSET-GFP to nanoparticles moved processively on MTs. Studies with intact cells revealed that conditions resulting in increased levels of HSET or soluble tubulin led to formation of multiple asters independently of centrosomes. The results suggest that HSET and soluble tubulin form aggregates that can move along preformed MTs, carrying another MT to the minus-end to allow aster formation. Further research will focus on the exact structure of the HSET-tubulin aggregates. The work is published in the journal Nature Communications[S. R. Norriset al. Nat. Commun, (2018) 9, 2659].