Title:

Microtubule nucleation without a ring? Andreas Merdes Centre de Biologie Intégrative Université Paul Sabatier / Centre National de la Recherche Scientifique Toulouse, France andreas.merdes@univ-tlse3.fr Abstract:

The native -tubulin ring complex is an asymmetric, imperfect template for microtubule nucleation.

Main text:

-tubulin has a conserved role in microtubule nucleation, in particular during spindle formation. -tubulin assembles into multi-protein complexes with -tubulin complex proteins (GCPs): "-tubulin small complexes" (-TuSCs) are formed from two molecules of -tubulin and one copy each of GCP2 and GCP3. -TuSCs can form helical oligomers by lateral interaction, but these are of heterogeneous size and unstable (1). In fungi such as fission yeast, additional proteins such as Mto1/2 form a structural basis for the anchorage and lateral assembly of -TuSCs, to enable microtubule nucleation (2). In most eukaryotes, additional proteins co-assemble with -TuSCs into soluble "-tubulin-ring-complexes" (-TuRCs) of a fixed size of 2.2MDa.

Recent studies by three research groups have determined the composition and structure of native -TuRCs by cryo-electron microscopy, combined with crosslinking and mass-spectrometry (3,4,5,6): 14 GCPs are aligned laterally into a short helix, whereby the 14 th GCP overlaps with the first after a full helical turn. In a lateral view, the -TuRC resembles a cone, with the -tubulin molecules placed on top of the cone, at the C-terminal region of each GCP. Positions 1 to 8 in the complex are filled by four -TuSCs, whereas positions 9 to 14 are occupied by GCP4, GCP5, GCP4, GCP6, and a terminal -TuSC, respectively. The first four -TuSCs adopt a "closed conformation", resembling the geometry of a 13-protofilament microtubule wall, whereas the remaining GCPs are less closely aligned, leading to an asymmetry of the -TuRC, with the gamma-tubulin molecules in positions 9 to 14 being spaced too far apart to act as a perfect template for microtubule nucleation ("open conformation", Fig. 1A). Additional proteins were assigned to the -TuRC, such as MOZART 1, MOZART2, and actin, or an actin-related protein. Actin and two copies of MOZART1 are part of a "luminal bridge", together with N-terminal extensions of GCP6 and GCP3, spanning the inside basis of the cone. In this issue, Wieczorek et al. (7) provide the first insights into the role of this luminal bridge by reconstituting the -TuRC from 10 recombinant proteins, produced in baculovirus-infected insect cells. The structure of the reconstituted -TuRC resembles closely that of native -TuRC. In a next step, Wieczorek et al. reconstituted the complex without actin and MOZART1, to eliminate the luminal bridge (-TuRC LB ). This -TuRC LB still assembles into a partial helix, whereas the remaining part of the complex is disorganized, without any region of overlap. The partial helix is formed by eight GCPs whose identity cannot be resolved, but it is tempting to speculate whether they comprise the first four -TuSCs as seen in a regular -TuRC. Their helical assembly may reflect their inherent potential to oligomerize (1), but nevertheless requires the presence of the N-terminal extension of GCP6, since deletion of this domain, or complete loss of GCP6 prevents the assembly of GCPs into any higher order complexes (8).

Most interestingly, the loss of the luminal bridge does not interfere with microtubule nucleation, as -TuRC LB display nucleation kinetics similar to the full recombinant complex (7). This suggests that eight GCPs provide sufficient surface for the anchorage of tubulin dimers, to build a small nucleus for microtubule assembly. In fact, the idea of a minimal nucleus is supported by recent findings, demonstrating that the association of as few as four tubulin dimers may be sufficient for -TuRCmediated nucleation (6,9). Altogether, these data also illustrate how an asymmetric native -TuRC may function as a microtubule nucleator: starting with a minimal nucleus assembled from the surface of the "closed" part of the -TuRC, the microtubule cylinder can be completed by lateral addition of tubulin dimers. The shape of the -TuRC may then be rectified by allosteric effects of /-tubulin on the -TuRC conformation, or by stochastic switches of positions 9 to 14 into a "closed" state (Fig. 1B). Such a mechanism is of course inefficient compared to microtubule elongation at growing plus-ends, since a considerable kinetic barrier has to be overcome, but it is still more efficient than spontaneous nucleation in solution (6,9). -TuRC-dependent nucleation of microtubules might be boosted if regulatory proteins or post-translational modification of the -TuRC were able to alter its conformation directly into a "closed" state. An intact luminal bridge may contribute to this process, since it influences directly the conformation of positions 9 to 14 in the -TuRC (7). It can be speculated whether GCPs 4, 5, and 6 interact with regulatory proteins (5,6). Moreover, the exact role of nucleation activators such as Cdk5rap2 or other proteins carrying CM1 sequence motifs remains to be determined, since their addition to native -TuRC is insufficient to induce a "closed" conformation (3,5). Reconstituted -TuRCs, as developed by Wieczorek et al. (7) and by Zimmermann et al. (10), should pave the way for future analyses, since specific protein interactions can be manipulated by mutagenesis, or by the reconstitution of partial complexes. Microtubule nucleation from an asymmetric -tubulin ring complex. (A) -tubulin small complex (-TuSC), and -tubulin ring complex (-TuRC), viewed from the top. A "closed" conformation at positions 1 to 8 enables contacts between neighboring -tubulin molecules. GCPs in positions 9 to 14 are further spaced apart ("open"). Actin, MOZART1 (Mzt1), and an N-terminal extension of GCP6 are part of the luminal bridge. (B) Side views of -TuRCs in partly open and closed conformations. A minimal nucleus of tubulin dimers (/) can form at positions 1 to 8. Lateral association of additional dimers completes the cylinder, thus matching a perfect template for nucleation. GCPs in positions 9 to 14 undergo a conformational switch that might occur stochastically, or driven by allosteric effects of /-tubulin or regulatory proteins.

  Wieczorek et al. (2021. J. Cell Biol. doi: 10.1083/jcb.202009146) and Zimmermann et al. (2020. Sci. Adv. doi: 10.1126/sciadv.abe0894) have reconstituted a recombinant complex that allows to study structurefunction relationships and regulatory mechanisms.

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Acknowledgements

I thank Laurence Haren for critical reading of the manuscript, and for constructive comments. The work was funded in part by the Agence Nationale de la Recherche (project 13 BSV8 0007 01). The author declares no competing financial interests.