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Microtubules are essential intracellular polymers, built from tubulin subunits, that establish cell shape, move organelles, and segregate chromosomes during cell division. Vemu et al. show that microtubule-severing enzymes extract tubulin subunits along the microtubule shaft. This nanoscale damage is repaired by the incorporation of free tubulin, which stabilizes the microtubule against depolymerization. When extraction outpaces repair, microtubules are severed, emerging with stabilized ends composed of fresh tubulin. The severed microtubules act as templates for new microtubule growth, leading to amplification of microtubule number and mass. Thus, seemingly paradoxically, severing enzymes can increase microtubule mass in processes such as neurogenesis and mitotic spindle assembly. Science , this issue p. eaau1504 Spastin and katanin extract tubulin dimers from microtubules and amplify microtubule arrays via lattice repair. Spastin and katanin sever and destabilize microtubules. Paradoxically, despite their destructive activity they increase microtubule mass in vivo. We combined single-molecule total internal reflection fluorescence microscopy and electron microscopy to show that the elemental step in microtubule severing is the generation of nanoscale damage throughout the microtubule by active extraction of tubulin heterodimers. These damage sites are repaired spontaneously by guanosine triphosphate (GTP)–tubulin incorporation, which rejuvenates and stabilizes the microtubule shaft. Consequently, spastin and katanin increase microtubule rescue rates. Furthermore, newly severed ends emerge with a high density of GTP-tubulin that protects them against depolymerization. The stabilization of the newly severed plus ends and the higher rescue frequency synergize to amplify microtubule number and mass. Thus, severing enzymes regulate microtubule architecture and dynamics by promoting GTP-tubulin incorporation within the microtubule shaft.

The AAA+ ATPase spastin remodels microtubule arrays through severing and its mutation is the most common cause of hereditary spastic paraplegias (HSP). Polyglutamylation of the tubulin C-terminal tail recruits spastin to microtubules and modulates severing activity. Here, we present a ~3.2 Å resolution cryo-EM structure of the Drosophila melanogaster spastin hexamer with a polyglutamate peptide bound in its central pore. Two electropositive loops arranged in a double-helical staircase coordinate the substrate sidechains. The structure reveals how concurrent nucleotide and substrate binding organizes the conserved spastin pore loops into an ordered network that is allosterically coupled to oligomerization, and suggests how tubulin tail engagement activates spastin for microtubule disassembly. This allosteric coupling may apply generally in organizing AAA+ protein translocases into their active conformations. We show that this allosteric network is essential for severing and is a hotspot for HSP mutations.AAA+ ATPase spastin recognizes tubulin polyglutamylated C-terminal tails and severs microtubules. A cryo-EM structure of fly spastin with polyGlu reveals how spastin engages with the substrate, an activity allosterically coupled to nucleotide binding and oligomerization.

Using a combination of crystallography, SAXS and cryo-EM, the katanin hexamer is observed in spiral or ring arrangements, suggesting a mechanism to generate the power stroke to severe microtubules. Microtubule-severing enzymes katanin, spastin and fidgetin are AAA ATPases important for the biogenesis and maintenance of complex microtubule arrays in axons, spindles and cilia. Because of a lack of known 3D structures for these enzymes, their mechanism of action has remained poorly understood. Here we report the X-ray crystal structure of the monomeric AAA katanin module from Caenorhabditis elegans and cryo-EM reconstructions of the hexamer in two conformations. The structures reveal an unexpected asymmetric arrangement of the AAA domains mediated by structural elements unique to microtubule-severing enzymes and critical for their function. The reconstructions show that katanin cycles between open spiral and closed ring conformations, depending on the ATP occupancy of a gating protomer that tenses or relaxes interprotomer interfaces. Cycling of the hexamer between these conformations would provide the power stroke for microtubule severing.

The central apparatus regulates the beating of motile cilia. High-resolution structures of the almost complete central apparatus are now reported in two separate studies, shedding light on the mechanism of ciliary beating and marking a new era in our molecular understanding of cilia architecture and function.

Microtubules have spatiotemporally complex posttranslational modification patterns. Tubulin tyrosine ligase-like (TTLL) enzymes introduce the most prevalent modifications on α-tubulin and β-tubulin. How TTLLs specialize for specific substrate recognition and ultimately modification-pattern generation is largely unknown. TTLL6, a glutamylase implicated in ciliopathies, preferentially modifies tubulin α-tails in microtubules. Cryo-electron microscopy, kinetic analysis and single-molecule biochemistry reveal an unprecedented quadrivalent recognition that ensures simultaneous readout of microtubule geometry and posttranslational modification status. By binding to a β-tubulin subunit, TTLL6 modifies the α-tail of the longitudinally adjacent tubulin dimer. Spanning two tubulin dimers along and across protofilaments (PFs) ensures fidelity of recognition of both the α-tail and the microtubule. Moreover, TTLL6 reads out and is stimulated by glutamylation of the β-tail of the laterally adjacent tubulin dimer, mediating crosstalk between α-tail and β-tail. This positive feedback loop can generate localized microtubule glutamylation patterns. Our work uncovers general principles that generate tubulin chemical and topographic complexity. Cryo-electron microscopy (cryo-EM), kinetic analysis and single-molecule biochemistry reveal how the tubulin tyrosine ligase-like 6 (TTLL6) glutamylase binds reads microtubule geometry and modification state of neighboring tubulins, enabling a spatial positive feedback loop for microtubule modification.

Microtubule function is modulated by the tubulin code, diverse posttranslational modifications that are altered dynamically by writer and eraser enzymes 1 . Glutamylation—the addition of branched (isopeptide-linked) glutamate chains—is the most evolutionarily widespread tubulin modification 2 . It is introduced by tubulin tyrosine ligase-like enzymes and erased by carboxypeptidases of the cytosolic carboxypeptidase (CCP) family 1 . Glutamylation homeostasis, achieved through the balance of writers and erasers, is critical for normal cell function 3 – 9 , and mutations in CCPs lead to human disease 10 – 13 . Here we report cryo-electron microscopy structures of the glutamylation eraser CCP5 in complex with the microtubule, and X-ray structures in complex with transition-state analogues. Combined with NMR analysis, these analyses show that CCP5 deforms the tubulin main chain into a unique turn that enables lock-and-key recognition of the branch glutamate in a cationic pocket that is unique to CCP family proteins. CCP5 binding of the sequences flanking the branch point primarily through peptide backbone atoms enables processing of diverse tubulin isotypes and non-tubulin substrates. Unexpectedly, CCP5 exhibits inefficient processing of an abundant β-tubulin isotype in the brain. This work provides an atomistic view into glutamate branch recognition and resolution, and sheds light on homeostasis of the tubulin glutamylation syntax. Cryo-electron microscopy and X-ray structures of the glutamylation eraser CCP5 in complexes with glutamylated microtubules and tubulin tails show that the substrate backbone adopts a bent conformation that presents the branch glutamate to a substrate-binding pocket on CCP5.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microtubules are dynamic polymers assembled from αβ-tubulin dimers. Mammals have multiple α and β-tubulin isoforms. Despite a high degree of conservation, microtubules assembled from different tubulin isoforms have unique dynamic properties. How isoform sequence variation affects polymerization interfaces and nucleotide dependent conformational changes in microtubules is still not well understood. Here we report 2.9Å resolution cryo-EM structures of human α1B/βI+βIVb microtubules in the GDP state, and a GTP-like state, bound to GMPCPP. Our structures show that, similar to microtubules assembled from other mammalian isoforms, transition from the GTP to the GDP states in α1B/βI+βIVb microtubules results in strengthening of the longitudinal interface and an overall compaction of the axial dimer repeat distance in the lattice. Interestingly, we find that α-tail residues link longitudinally adjacent tubulin dimers through interactions with two conserved arginine residues in β-tubulin that are mutated in human disease. Comparative analysis of tubulin isoforms shows minimal isoform-specific effects at the longitudinal interface or the α-tubulin lateral interface, but a high concentration of sequence variability in the second shell of residues away from the β-tubulin lateral interface which can modulate polymerization interfaces and thus impact microtubule dynamics.Competing Interest StatementThe authors have declared no competing interest.

Tubulin is a universally conserved molecule, found in all three domains of life. Tubulin filaments use energy derived from GTP binding and hydrolysis to organize cytoplasm. Although tubulin homologues share the same fold to their monomers and similar protofilament architecture, they assemble filaments with unique geometric designs. Moreover, despite sharing the mechanism of self-assembly, wherein tubulin subunits require GTP binding to polymerize, and their polymerization stimulates GTP hydrolysis, tubulins exhibit distinctive dynamic properties. Together, unique filament architectures and dynamic properties determine a specific set of biological functions each tubulin family performs. The origins of tubulin filament architectural diversity and distinctive dynamic behavior are not well understood. We have been studying PhuZ tubulins, which are encoded by a few very large Pseudomonas ΦKZ-like bacteriophages. Our studies have shown that PhuZ assembles dynamically unstable spindle-like arrays that organize bacteriophage DNA at the cell midpoint, which somehow facilitates phage infectivity. Moreover, PhuZ monomer structure has the canonical tubulin fold, with a unique, highly conserved and extended C-terminus. The C-terminus mediates protofilament contacts and is critical for polymerization both in vitro and in vivo. The main focus of this manuscript is the structural investigation of the molecular mechanisms underlying PhuZ dynamic behavior. In an attempt to explain how GTP binding and hydrolysis drives PhuZ filament turnover, we have conducted a number of electron cryomicroscopy studies (cryo-EM) on PhuZ filaments in both pre- and post- hydrolysis states. PhuZ forms a polar three-stranded polymer with an unusual subunit orientation. Its C-terminus guides a cooperative assembly of the three-stranded filament by mediating both longitudinal and lateral interactions. Upon assembly, the longitudinal interface, C-terminus and tubulin fold undergo polymerization-competent rearrangements. We propose that the energy of GTP binding is stored in the displacement of these structural elements. Also, our structural studies have revealed how the energy of GTP hydrolysis is stored in the PhuZ filament lattice. In particular, GTP hydrolysis, initially sensed by the T3 loop and C-terminus, is accompanied by unwinding and supercoiling of the filament lattice. Based on these observations, we propose an assembly/disassembly pathway of PhuZ filament.