Microtubule Lattice Research Articles

α-tubulin detyrosination fine-tunes kinetochore-microtubule attachments

Post-translational cycles of α-tubulin detyrosination and tyrosination generate microtubule diversity, the cellular functions of which remain largely unknown. Here we show that α-tubulin detyrosination regulates kinetochore-microtubule attachments to ensure normal chromosome oscillations and timely anaphase onset during mitosis. Remarkably, detyrosinated α-tubulin levels near kinetochore microtubule plus-ends depend on the direction of chromosome motion during metaphase. Proteomic analyses unveil that the KNL-1/MIS12/NDC80 (KMN) network that forms the core microtubule-binding site at kinetochores and the microtubule-rescue protein CLASP2 are enriched on tyrosinated and detyrosinated microtubules during mitosis, respectively. α-tubulin detyrosination enhances CLASP2 binding and NDC80 complex diffusion along the microtubule lattice in vitro. Rescue experiments overexpressing NDC80, including variants with slower microtubule diffusion, suggest a functional interplay with α-tubulin detyrosination for the establishment of a labile kinetochore-microtubule interface. These results offer a mechanistic explanation for how different detyrosinated α-tubulin levels near kinetochore microtubule plus-ends fine-tune load-bearing attachments to both growing and shrinking microtubules.

HURP facilitates spindle assembly by stabilizing microtubules and working synergistically with TPX2

In vertebrate spindles, most microtubules are formed via branching microtubule nucleation, whereby microtubules nucleate along the side of pre-existing microtubules. Hepatoma up-regulated protein (HURP) is a microtubule-associated protein that has been implicated in spindle assembly, but its mode of action is yet to be defined. In this study, we show that HURP is necessary for RanGTP-induced branching microtubule nucleation in Xenopus egg extract. Specifically, HURP stabilizes the microtubule lattice to promote microtubule formation from γ-TuRC. This function is shifted to promote branching microtubule nucleation through enhanced localization to TPX2 condensates, which form the core of the branch site on microtubules. Lastly, we provide a high-resolution cryo-EM structure of HURP on the microtubule, revealing how HURP binding stabilizes the microtubule lattice. We propose a model in which HURP stabilizes microtubules during their formation, and TPX2 preferentially enriches HURP to microtubules to promote branching microtubule nucleation and thus spindle assembly.

StableMARK-decorated microtubules in cells have expanded lattices.

Microtubules are crucial in cells and are regulated by various mechanisms like posttranslational modifications, microtubule-associated proteins, and tubulin isoforms. Recently, the conformation of the microtubule lattice has also emerged as a potential regulatory factor, but it has remained unclear to what extent different lattices co-exist within the cell. Using cryo-electron tomography, we find that, while most microtubules have a compacted lattice (∼41 Å monomer spacing), approximately a quarter of the microtubules displayed more expanded lattice spacings. The addition of the microtubule-stabilizing agent Taxol increased the lattice spacing of all microtubules, consistent with results on reconstituted microtubules. Furthermore, correlative cryo-light and electron microscopy revealed that the stable subset of microtubules labeled by StableMARK, a marker for stable microtubules, predominantly displayed a more expanded lattice spacing (∼41.9 Å), further suggesting a close connection between lattice expansion and microtubule stability. The coexistence of different lattices and their correlation with stability implicate lattice spacing as an important factor in establishing specific microtubule subsets.

Multiscale Computational Analysis of the Effect of Taxol on Microtubule Mechanics.

Microtubules (MTs) are widely recognized as targets for cancer therapies. They are directly related to unique mechanical properties, closely dependent on MT architecture and tubulin molecular features. Taxol is known to affect tubulin interactions resulting in the stabilization of the MT lattice, and thus the hierarchical organization stability, mechanics, and function. A deeper understanding of the molecular mechanisms through which taxol modulates intertubulin interactions in the MT lattice, and consequently, its stability and mechanical response is crucial to characterize how MT properties are regulated by environmental factors, such as interacting ligands. In this study, a computational analysis of the effect of taxol on the MT was performed at different scales, combining molecular dynamics simulation, dynamical network analysis, and elastic network modeling. The results show that the taxol-induced conformational differences at the M-loop region increase the stability of the lateral interactions and the amount of surface in contact between laterally coupled tubulins. Moreover, the conformational rearrangements in the taxane binding site result in a different structural communication pattern. Finally, the different conformation of the tubulin heterodimers and the stabilized lateral interactions resulted in a tendency toward higher deformation of the vibrating MT in the presence of taxol. Overall, this work provides additional insights into taxol-induced stabilization and relates the conformational changes at the tubulin level to the MT mechanics. Besides providing useful insights into taxol effect on MT mechanics, a methodological framework that could be used to characterize the effects of other MT stabilizing agents is presented.

P-080 Unveiling the molecular determinants of sperm endpiece stabilization

Abstract Study question How are microtubules at the endpiece of the mammalian sperm tail stabilized? Summary answer SAX01 and SPACA9 are microtubule inner proteins that binds at the endpieces. In addition, singlet microtubules are capped by a complex of proteins. What is known already At the core of the sperm flagellum is the axoneme, a supramolecular assembly of nine doublet microtubules around a central pair of singlet microtubules. At the endpiece, axoneme structure is lost and only singlet microtubules remain. Using cryo-electron microscopy (cryo-EM) our group showed that the singlet microtubules contain species-specific microtubule inner proteins in a helical arrangement. Furthermore, we observed a protein density capping the microtubules plus-ends in pig, horse, and mouse sperm. Study design, size, duration To elucidate the function of the sperm endpiece plug and its associated proteins, we apply single-particle analysis cryo-EM. Participants/materials, setting, methods - Main results and the role of chance We revealed that the microtubule inner proteins in the singlets are SAX01 and SPACA9. Our high-resolution structure shows that SPACA9 forms a discontinuously intraluminal spiral. It consists of a bundle of alpha helices that binds across the intradimer α/β-tubulin interface as well as between adjacent protofilaments. Specifically, SPACA9 interacts with both α- and β-tubulin within a dimer while also interacting with the M-loop of α-tubulin from the neighbouring protofilament. Our structure further shows that SAX01 binds longitudinally along individual protofilaments with its Mn motif, a 5- to 6-residue helix flanked by a Tyr/Phe residue on one end and by a Ser/Thr on the other that binds the shoulder of the α -tubulin S9/S10 loop. We further observed that microtubule termini are capped with yet to be identified protein complexes. Taken together, we show that microtubule inner proteins longitudinally and laterally reinforce the microtubule lattice, while capped in their termini. Limitations, reasons for caution Limitations include species-specific variations as we previously observed between mouse sperm endpieces and porcine or horse. Cryo-EM structures represent averages across singlet microtubules, so individual variation within particles may be lost. Wider implications of the findings Investigating the mechanism behind stabilization of the endpiece singlet microtubules using cryo-EM provides better understanding of sperm tail stabilization that holds potential for progress in understanding male infertility and can be utilized for a development of male contraception. Trial registration number not applicable

Chromosome segregation during spermatogenesis occurs through a unique center-kinetic mechanism in holocentric moth species.

Precise regulation of chromosome dynamics in the germline is essential for reproductive success across species. Yet, the mechanisms underlying meiotic chromosomal events such as homolog pairing and chromosome segregation are not fully understood in many species. Here, we employ Oligopaint DNA FISH to investigate mechanisms of meiotic homolog pairing and chromosome segregation in the holocentric pantry moth, Plodia interpunctella, and compare our findings to new and previous studies in the silkworm moth, Bombyx mori, which diverged from P. interpunctella over 100 million years ago. We find that pairing in both Bombyx and Plodia spermatogenesis is initiated at gene-rich chromosome ends. Additionally, both species form rod shaped cruciform-like bivalents at metaphase I. However, unlike the telomere-oriented chromosome segregation mechanism observed in Bombyx, Plodia can orient bivalents in multiple different ways at metaphase I. Surprisingly, in both species we find that kinetochores consistently assemble at non-telomeric loci toward the center of chromosomes regardless of where chromosome centers are located in the bivalent. Additionally, sister kinetochores do not seem to be paired in these species. Instead, four distinct kinetochores are easily observed at metaphase I. Despite this, we find clear end-on microtubule attachments and not lateral microtubule attachments co-orienting these separated kinetochores. These findings challenge the classical view of segregation where paired, poleward-facing kinetochores are required for accurate homolog separation in meiosis I. Our studies here highlight the importance of exploring fundamental processes in non-model systems, as employing novel organisms can lead to the discovery of novel biology.

Meiosis-specific functions of kinetochore protein SPC105R required for chromosome segregation in Drosophila oocytes.

The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and coorientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that the SPC105R C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for lateral microtubule attachments and biorientation of homologues, which are critical for accurate chromosome segregation in meiosis I.

Mechanism of how the universal module XMAP215 γ-TuRC nucleates microtubules.

It has become increasingly evident in recent years that nucleation of microtubules from a diverse set of MTOCs requires both the γ-tubulin ring complex (γ-TuRC) and the microtubule polymerase XMAP215. Despite their essentiality, little is known about how these nucleation factors interact and work together to generate microtubules. Using biochemical domain analysis of XMAP215 and structural approaches, we find that a sixth TOG domain in XMAP215 binds γ-TuRC via γ-tubulin as part of a broader interaction involving the C-terminal region. Moreover, TOG6 is required for XMAP215 to promote nucleation from γ-TuRC to its full extent. Interestingly, we find that XMAP215 also depends strongly on TOG5 for microtubule lattice binding and nucleation. Accordingly, we report a cryo-EM structure of TOG5 bound to the microtubule lattice that reveals promotion of lateral interactions between tubulin dimers. Finally, we find that while XMAP215 constructs' effects on nucleation are generally proportional to their effects on polymerization, formation of a direct complex with γ-TuRC allows cooperative nucleation activity. Thus, we propose that XMAP215's C-terminal TOGs 5 and 6 play key roles in promoting nucleation by promoting formation of longitudinal and lateral bonds in γ-TuRC templated nascent microtubules at cellular MTOCs.

Stable GDP-tubulin islands rescue dynamic microtubules.

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

MEF2C contributes to axonal branching by regulating Kif2c transcription.

Neurons are post-mitotic cells, with microtubules playing crucial roles in axonal transport and growth. Kinesin family member 2c (KIF2C), a member of the Kinesin-13 family, possesses the ability to depolymerize microtubules and is involved in remodelling the microtubule lattice. Myocyte enhancer factor 2c (MEF2C) was initially identified as a regulator of muscle differentiation but has recently been associated with neurological abnormalities such as severe cognitive impairment, stereotyping, epilepsy and brain malformations when mutated or deleted. However, further investigation is required to determine which target genes MEF2C acts upon to influence neuronal function as a transcription regulator. Our data demonstrate that knockdown of both Mef2c and Kif2c significantly impacts spinal motor neuron development and behaviour in zebrafish. Luciferase reporter assays and chromosome immunoprecipitation assays, along with down/upregulated expression analysis, revealed that MFE2C functions as a novel transcription regulator for the Kif2c gene. Additionally, the knockdown of either Mef2c or Kif2c expression in E18 cortical neurons substantially reduces the number of primary neurites and axonal branches during neuronal development in vitro without affecting neurite length. Finally, depletion of Kif2c eliminated the effects of overexpression of Mef2c on the neurite branching. Based on these findings, we provided novel evidence demonstrating that MEF2C regulates the transcription of the Kif2c gene thereby influencing the axonal branching.

Damage-repair events increase the instability of cortical microtubules in Arabidopsis.

Microtubules rely on dynamic assembly and disassembly for their functions. Increasing evidences support that the damage-repair of microtubule lattices can affect microtubule dynamics in vitro and in animal cells. Here we successfully established a way for visualizing damage-repair sites on microtubule lattices in plant cells, via labeling the tubulin proteins with the photoconvertible fluorescent protein mEOS3.2. We observed that the crossovers of the microtubule lattice were more prone to be damaged and repaired, with the frequency of damage-repair events positively correlated with the crossing angle between microtubules. The microtubules with damage-repair events displayed shorter lifespans and significantly increased severing frequency compared with the undamaged microtubules. These observations suggested that the damage-repair events promoted instability of cortical microtubules in plant cells.

Abstract 2009: Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance

Abstract Taxanes are microtubule (MT)-stabilizing drugs widely used for the treatment of solid tumors, including the highly prevalent cancers of the breast, the prostate and gastric cancer. However, despite their initial activity, taxane resistance invariably occurs, leaving patients with limited therapeutic options. Currently, the molecular determinants of clinical taxane resistance remain poorly understood. We recently identified CLIP-170S, a novel truncated variant of the MT plus-end binding protein CLIP-170, that confers taxane resistance in vitro and is associated with clinical taxane resistance in patients with gastric cancer. We further showed that CLIP-170S is aberrantly localized along the MT lattice, thus impairing taxane access to its binding site in the MT lumen. However, the mechanism underlying the resistance-inducing extensive lattice localization of CLIP-170S is unknown. The activity and MT binding properties of canonical CLIP-170 are regulated by conformational changes mediated by the interaction of its N- and C-terminal domains. In its inactive, “closed” conformation, CLIP-170 is soluble as the N- and C-terminal domains interact with each other. In the active “open” conformation, CLIP-170’s N- and C-terminal domains are apart, thus exposing its MT-binding domain and allowing MT plus-end binding. As CLIP-170S is missing part of its N-terminal domain, we hypothesized that CLIP-170S is constitutively displayed in an open conformation that promotes its extensive and aberrant localization to the MT lattice. To prove this hypothesis, we used the FKBP-rapalog-FRB system to “force” CLIP-170S to assume a “closed” conformation. We generated a fusion CLIP-170S protein tagged with FKBP and FRB domains in its N- and C-terminal, respectively. The addition of rapalog induced FKBP (N-ter) and FRB (C-ter) domains to interact, promoting CLIP-170S to adopt a “closed” conformation. CLIP-170S in this “closed” conformation resulted in its release from the MT-lattice and restored taxane binding to MTs. We next extended our search to other CLIP-170 clinically prevalent variants with sequence alteration predicted to bind to the MT-lattice and potentially interfere with taxane sensitivity. This search identified CLIP-170IR (an intron retention variant) and several other truncation mutants prevalent in gastric, breast and lung cancer cell lines. Experimentally, we showed that these variants not only phenocopy CLIP-170S binding to the MT- lattice but also confer taxane-resistance. Together, these data demonstrate a novel mechanism of taxane resistance mediated by CLIP-170 mutations that induce CLIP-170 to adopt a constitutively “open” MT-lattice binding conformation, which renders them susceptible to causing taxane resistance. Citation Format: Urko del Castillo, Kiran Kumari Sahu, William G. Stone, Manish A. Shah, Paraskevi Giannakakou. Novel clinically relevant CLIP-170 variants display an extended conformation microtubule-lattice bound conformation associated with taxane resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2009.

Complexes of tubulin oligomers and tau form a viscoelastic intervening network cross-bridging microtubules into bundles

The axon-initial-segment (AIS) of mature neurons contains microtubule (MT) fascicles (linear bundles) implicated as retrograde diffusion barriers in the retention of MT-associated protein (MAP) tau inside axons. Tau dysfunction and leakage outside of the axon is associated with neurodegeneration. We report on the structure of steady-state MT bundles in varying concentrations of Mg2+ or Ca2+ divalent cations in mixtures containing αβ-tubulin, full-length tau, and GTP at 37 °C in a physiological buffer. A concentration-time kinetic phase diagram generated by synchrotron SAXS reveals a wide-spacing MT bundle phase (Bws), a transient intermediate MT bundle phase (Bint), and a tubulin ring phase. SAXS with TEM of plastic-embedded samples provides evidence of a viscoelastic intervening network (IN) of complexes of tubulin oligomers and tau stabilizing MT bundles. In this model, αβ-tubulin oligomers in the IN are crosslinked by tau’s MT binding repeats, which also link αβ-tubulin oligomers to αβ-tubulin within the MT lattice. The model challenges whether the cross-bridging of MTs is attributed entirely to MAPs. Tubulin-tau complexes in the IN or bound to isolated MTs are potential sites for enzymatic modification of tau, promoting nucleation and growth of tau fibrils in tauopathies.

Transport memory effects on coupled nonlinear waves in microtubule dynamics

The paper examines how microtubules (MTs) function in their biological environment, focusing on soliton-breather solutions. These solutions are nonlinear localized excitations that propagate in an angular continuous model of microtubule dynamics. Our model considers competitive effects from transport memory and nonlinearity, which arise due to collisions between tubulin dimers and particulate entities during energy processing in eukaryotic cells due to the hydrolysis of guanosine triphosphate (GTP). Our mathematical approach determines that the angular displacement of a dimer is governed by a modified system of coupled complex nonlinear Schrödinger equations. Analytical solutions are obtained and propagated in the MT lattice through direct numerical simulations, and small variations in coefficients significantly impact energy processing in cytoplasmic MTs.

A structural and dynamic visualization of the interaction between MAP7 and microtubules

Microtubules (MTs) are key components of the eukaryotic cytoskeleton and are essential for intracellular organization, organelle trafficking and mitosis. MT tasks depend on binding and interactions with MT-associated proteins (MAPs). MT-associated protein 7 (MAP7) has the unusual ability of both MT binding and activating kinesin-1-mediated cargo transport along MTs. Additionally, the protein is reported to stabilize MTs with its 112 amino-acid long MT-binding domain (MTBD). Here we investigate the structural basis of the interaction of MAP7 MTBD with the MT lattice. Using a combination of solid and solution-state nuclear magnetic resonance (NMR) spectroscopy with electron microscopy, fluorescence anisotropy and isothermal titration calorimetry, we shed light on the binding mode of MAP7 to MTs at an atomic level. Our results show that a combination of interactions between MAP7 and MT lattice extending beyond a single tubulin dimer and including tubulin C-terminal tails contribute to formation of the MAP7-MT complex.

CKAP5 enables formation of persistent actin bundles templated by dynamically instable microtubules

Cytoskeletal rearrangements and crosstalk between microtubules and actin filaments are vital for living organisms. Recently, an abundantly present microtubule polymerase, CKAP5 (XMAP215 homolog), has been reported to play a role in mediating crosstalk between microtubules and actin filaments in the neuronal growth cones. However, the molecular mechanism of this process is unknown. Here, we demonstrate, in a reconstituted system, that CKAP5 enables the formation of persistent actin bundles templated by dynamically instable microtubules. We explain the templating by the difference in CKAP5 binding to microtubules and actin filaments. Binding to the microtubule lattice with higher affinity, CKAP5 enables the formation of actin bundles exclusively on the microtubule lattice, at CKAP5 concentrations insufficient to support any actin bundling in the absence of microtubules. Strikingly, when the microtubules depolymerize, actin bundles prevail at the positions predetermined by the microtubules. We propose that the local abundance of available CKAP5-binding sites in actin bundles allows the retention of CKAP5, resulting in persisting actin bundles. In line with our observations, we found that reducing CKAP5 levels invivo results in a decrease in actin-microtubule co-localization in growth cones and specifically decreases actin intensity at microtubule plus ends. This readily suggests a mechanism explaining how exploratory microtubules set the positions of actin bundles, for example, in cytoskeleton-rich neuronal growth cones.

The tubulin database: Linking mutations, modifications, ligands and local interactions.

Microtubules are polymeric filaments, constructed of α-β tubulin heterodimers that underlie critical subcellular structures in eukaryotic organisms. Four homologous proteins (γ-, δ-, ε- and ζ-tubulin) additionally contribute to specialized microtubule functions. Although there is an immense volume of publicly available data pertaining to tubulins, it is difficult to assimilate all potentially relevant information across diverse organisms, isotypes, and categories of data. We previously assembled an extensive web-based catalogue of published missense mutations to tubulins with >1,500 entries that each document a specific substitution to a discrete tubulin, the species where the mutation was described and the associated phenotype with hyperlinks to the amino acid sequence and citation(s) for research. This report describes a significant update and expansion of our online resource (TubulinDB.bio.uci.edu) to nearly 18,000 entries. It now encompasses a cross-referenced catalog of post-translational modifications (PTMs) to tubulin drawn from public datasets, primary literature, and predictive algorithms. In addition, tubulin protein structures were used to define local interactions with bound ligands (GTP, GDP and diverse microtubule-targeting agents) and amino acids at the intradimer interface, within the microtubule lattice and with associated proteins. To effectively cross-reference these datasets, we established a universal tubulin numbering system to map entries into a common framework that accommodates specific insertions and deletions to tubulins. Indexing and cross-referencing permitted us to discern previously unappreciated patterns. We describe previously unlinked observations of loss of PTM sites in the context of cancer cells and tubulinopathies. Similarly, we expanded the set of clinical substitutions that may compromise MAP or microtubule-motor interactions by collecting tubulin missense mutations that alter amino acids at the interface with dynein and doublecortin. By expanding the database as a curated resource, we hope to relate model organism data to clinical findings of pathogenic tubulin variants. Ultimately, we aim to aid researchers in hypothesis generation and design of studies to dissect tubulin function.

A divergent tumor overexpressed gene domain and oligomerization contribute to SPIRAL2 function in stabilizing microtubule minus ends.

The acentrosomal cortical microtubules (MTs) of higher plants dynamically assemble into specific array patterns that determine the axis of cell expansion. Recently, the Arabidopsis (Arabidopsis thaliana) SPIRAL2 (SPR2) protein was shown to regulate cortical MT length and light-induced array reorientation by stabilizing MT minus ends. SPR2 autonomously localizes to both the MT lattice and MT minus ends, where it decreases the minus end depolymerization rate. However, the structural determinants that contribute to the ability of SPR2 to target and stabilize MT minus ends remain unknown. Here, we present the crystal structure of the SPR2 N-terminal domain, which reveals a unique tumor overexpressed gene (TOG) domain architecture with 7 HEAT repeats. We demonstrate that a coiled-coil domain mediates the multimerization of SPR2, which provides avidity for MT binding, and is essential to bind soluble tubulin. In addition, we found that an SPR2 construct spanning the TOG domain, basic region, and coiled-coil domain targets and stabilizes MT minus ends similar to full-length SPR2 in plants. These results reveal how a TOG domain, which is typically found in microtubule plus-end regulators, has been appropriated in plants to regulate MT minus ends.

Beyond uniformity: Exploring the heterogeneous and dynamic nature of the microtubule lattice

A fair amount of research on microtubules since their discovery in 1963 has focused on their dynamic tips. In contrast, the microtubule lattice was long believed to be highly regular and static, and consequently received far less attention. Yet, as it turned out, the microtubule lattice is neither as regular, nor as static as previously believed: structural studies uncovered the remarkable wealth of different conformations the lattice can accommodate. In the last decade, the microtubule lattice was shown to be labile and to spontaneously undergo renovation, a phenomenon that is intimately linked to structural defects and was called “microtubule self-repair". Following this breakthrough discovery, further recent research provided a deeper understanding of the lattice self-repair mechanism, which we review here. Instrumental to these discoveries were in vitro microtubule reconstitution assays, in which microtubules are grown from the minimal components required for their dynamics. In this review, we propose a shift from the term “lattice self-repair” to “lattice dynamics”, since this phenomenon is an inherent property of microtubules and can happen without microtubule damage. We focus on how in vitro microtubule reconstitution assays helped us learn (1) which types of structural variations microtubules display, (2) how these structural variations influence lattice dynamics and microtubule damage caused by mechanical stress, (3) how lattice dynamics impact tip dynamics, and (4) how microtubule-associated proteins (MAPs) can play a role in structuring the lattice. Finally, we discuss the unanswered questions about lattice dynamics and how technical advances will help us tackle these questions.

Abl2 repairs microtubules and phase separates with tubulin to promote microtubule nucleation

Abl family kinases are evolutionarily conserved regulators of cell migration and morphogenesis. Genetic experiments in Drosophila suggest that Abl family kinases interact functionally with microtubules to regulate axon guidance and neuronal morphogenesis. Vertebrate Abl2 binds to microtubules and promotes their plus-end elongation, both invitro and in cells, but the molecular mechanisms by which Abl2 regulates microtubule (MT) dynamics are unclear. We report here that Abl2 regulates MT assembly via condensation and direct interactions with both the MT lattice and tubulin dimers. We find that Abl2 promotes MT nucleation, which is further facilitated by the ability of the Abl2 C-terminal half to undergo liquid-liquid phase separation (LLPS) and form co-condensates with tubulin. Abl2 binds to regions adjacent to MT damage, facilitates MT repair via fresh tubulin recruitment, and increases MT rescue frequency and lifetime. Cryo-EM analyses strongly support a model in which Abl2 engages tubulin C-terminal tails along an extended MT lattice conformation at damage sites to facilitate repair via fresh tubulin recruitment. Abl2Δ688-790, which closely mimics a naturally occurring splice isoform, retains binding to the MT lattice but does not bind tubulin, promote MT nucleation, or increase rescue frequency. In COS-7 cells, MT reassembly after nocodazole treatment is greatly slowed in Abl2 knockout COS-7 cells compared with wild-type cells, and these defects are rescued by re-expression of Abl2, but not Abl2Δ688-790. We propose that Abl2 locally concentrates tubulin to promote MT nucleation and recruits it to defects in the MT lattice to enable repair and rescue.