Eukaryotic-like microtubules and dynamic instability of Asgard archaeal tubulins

Structure and dynamics of a four-protofilament microtubule from Heimdallarchaeales α/β-tubulin

Eukaryotic microtubules are typically 13-protofilament tubes assembled from α/β-tubulin heterodimers that combine mechanical rigidity with dynamic instability. Homologous tubulins have been identified in Asgard archaea, the closest prokaryotic relatives to eukaryotes. Here, we characterize a heterodimeric α/β-tubulin system from Heimdallarchaeales. Biochemical reconstitution shows that Heim–α/β-tubulin forms a heterodimer that undergoes guanosine 5′-triphosphate–dependent polymerization with coupled nucleotide hydrolysis. Cryo–electron microscopy reveals that the polymers are composed of four-protofilament tubules, with microtubule-like lattices formed by conserved longitudinal interfaces and ball-and-socket lateral contacts. Single-filament imaging demonstrates intrinsic kinetic polarity and dynamic instability, while liposome encapsulation shows that microtubule growth generates forces sufficient to deform membranes. Despite their reduced protofilament number, Heim–α/β-microtubules share key structural and dynamic features with eukaryotic microtubules but exhibit lower bending stiffness and polymerization force. Thus, microtubule-like polymers can form from a range of protofilament numbers, with reduced architectures potentially adapted to small cellular dimensions and lower mechanical loads. Together, our results indicate expansion in microtubule protofilament number during eukaryogenesis.

An architectural switch in the evolution of the γ-tubulin ring complex

Canonical microtubules contain 13-protofilaments and are templated by the γ-tubulin ring complex (γ-TuRC). However, some eukaryotes assemble non-canonical microtubules, like the 11-protofilament structures found in Caenorhabditis elegans. How γ-TuRCs adapt to template alternative microtubule geometries is unclear. Here, we present the cryo-electron microscopy structure of the C. elegans γ-TuRC (γ-TuRCCe), revealing a cone-shaped assembly consistent with an 11-protofilament template. While the complex incorporates the conserved subunits actin, GCP2 and GCP3, γ-TuRCCe replaces GCP4-6 with a divergent 4-spoked assembly containing additional copies of GCP2 and the nematode-specific proteins GTAP-1 and GTAP-2. Structures of nucleotide-free γ-TuRCCe subcomplexes reveal partial γ-tubulin unfolding, suggesting nucleotide binding stabilizes eukaryotic tubulins. Remarkably, reconstituted 4-spoked assemblies can multimerize into ∼13-fold symmetric microtubule nucleation templates in vitro, contrasting with the native complex’s 11-protofilament architecture. Our work defines the structural blueprint of an 11-protofilament microtubule template and shows how divergent γ-tubulin components are repurposed to accommodate non-canonical microtubule lattices.

The extraordinary robustness of mitotic spindle assembly to microtubule dynamics plasticity revealed by end-binding proteins and tubulin tagging

Microtubules are dynamic, conserved cytoskeletal filaments that are essential for all eukaryotic cells. Microtubule dynamic properties are primarily characterized by measuring how fast they grow and shrink (growth and shrinkage rates) and how often they switch between assembly and disassembly (catastrophe and rescue frequencies). These four parameters measured for individual filaments can inform on the behavior of the entire microtubule network at the cell level. By comparing microtubule dynamics in Caenorhabditis elegans one-cell embryos using different genetically-encoded fluorescent probes, we observed an unexpected high variability in these parameters. Microtubule dynamics parameters were consistently higher in C. elegans strains expressing a fluorescently labelled microtubule end-binding protein than in strains relying on tubulin labelling, with microtubule growth rates differing by nearly a factor of two between the two conditions. This discrepancy was not limited to C. elegans, as we observed a similar effect in embryos of the tunicate Phallusia mammillata. Despite this, spindle size and assembly timing were only mildly affected. However, embryos expressing labelled end-binding protein exhibited higher frequency of mitotic defects and perturbed embryonic development upon exposure to various stresses such as elevated temperature or a compromised spindle assembly checkpoint. Thus, our work reveals both the remarkable robustness of mitotic spindle assembly in response to extreme microtubule dynamics plasticity, and the requirement for strict control of microtubule dynamics across successive early embryonic divisions. Our findings should also serve as a cautionary note when using tagged end-binding proteins to measure microtubule dynamics.

Intrafilament nucleotide exchange in a prokaryotic actin homolog

Mutating the interprotofilament interface allows microtubules to assemble in GDP

Microtubule dynamic instability, driven by GTP turnover, allows microtubules in cells to reorganise themselves adaptively. GTP promotes the assembly of tubulin into microtubules, but exactly how it does so is controversial. In some models, GTP mainly supports the assembly of tubulin into single protofilaments, as in prokaryotic tubulins. In others, GTP mainly supports the formation of lateral bonds between protofilaments. To investigate, we mutated the interprotofilament interface in human α1bβ3 and α1bβ4b tubulins, whose sequences diverge markedly in this region. We find that transplanting the α1bβ3 M-loop or its binding pocket into α1bβ4b tubulin creates tubulins that assemble in 1 mM GDP. We accordingly propose that GTP- and GDP-tubulins are captured equivalently at the tips of microtubules, but then differentially retained, based on their differing abilities to form stable interprotofilament bonds. This biased retention mechanism allows mosaic lattices to be built and dynamic instability to be tuned. One sentence summary Microtubules grow not by biased capture of GTP tubulins, but by biased retention of tubulins that form stable interprotofilament bonds.

A genetic screen unveils key molecular steps in the polymerization cycle of the bacterial actin-like MreB

Key mechanistic steps coordinating ATP-driven MreB polymerization and turnover are established and provide a basis for a complete MreB assembly–disassembly cycle and for further elucidating how MreB dynamics contribute to cell-wall organization.