Free Tubulin Research Articles

Twist-Bending Elasticity and Energetics of the Plus-End Microtubule Tip Captured by Exascale Atomistic Simulations

Tubulin dimers associate longitudinally and laterally to form microtubules (MTs) that stochastically alternate between phases of growth and shrinkage. Assembly is favored upon GTP binding, whereas GTP hydrolysis within the MT encourages disassembly. Recent experiments with high-resolution cryoelectron microscopy, complemented by our molecular dynamics (MD) simulations of both free and MT-integrated tubulin [Igaev, M. and H. Grubmüller. eLife 2018;7:e34353, Igaev, M. and H. Grubmüller. PLoS Comp.

Microtubule Growth Is Slowed through Transient Exposure of GDP Tubulin

Microtubule plus-end growth dynamics are determined by a kinetic race dictated by tubulin on- and off-rates at the microtubule tip. The apparent tubulin on- and off-rates can be estimated by measuring the mean plus-end growth rate across a range of free tubulin concentrations. However, when growth phases are studied at higher temporal resolution, fluctuations around the average growth rate are observed, implying more complex dynamics. To understand growth dynamics at the microtubule plus-end, we used Interference Reflection Microscopy (IRM) to quantify time-dependent variations in microtubule growth using GMPCPP, a non-hydrolyzable GTP analog. We found an expected linear dependence of the average growth rate on tubulin concentration. However, there was also a linear relationship between growth variance and tubulin concentration. Utilizing these measured values, we were able to constrain a biochemical model of microtubule growth with an on-rate of 0.7/ μM/pf/s. To determine the degree to which growth in GMPCPP mimics the GTP cap, we repeated the measurements in GTP. We again found a linear dependence of the average growth rate on tubulin; but found a much higher tubulin-dependent growth variance than expected from the GMPCPP experiments. When the growth variance was normalized to growth rate for each condition, there was a 5-fold increase in the relative growth variance in GTP compared to GMPCPP. This result suggests that GTP hydrolysis impacts tubulin binding kinetics at the microtubule plus-end. Consistent with this hypothesis, we then artificially varied the hydrolysis rates from 0 to 0.15 s−1 in the biochemical model. Increasing the hydrolysis rate increased the growth variance, and transient suppression of growth coincided with increasing numbers of exposed GDP tubulin at the plus-end. This work suggests that during growth, exposure of terminal GDP tubulin slows growth until a terminal GTP is restored.

Crystal structure of human PACRG in complex with MEIG1 reveals roles in axoneme formation and tubulin binding

The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly.

Self-repair protects microtubules from destruction by molecular motors.

Microtubule instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Molecular motors use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the mechanical work produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. We also found that dimer removal by motors was compensated for the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of molecular motors and the renewal of the microtubule lattice.

Conjugate of podophyllotoxin with chlorambucil: synthesis, biological testing and molecular modeling

In the present work we have studied a novel conjugate of the DNA alkylating agent chlorambucil with podophyllotoxin, a ligand of the colchicine binding site in tubulin. The target compound was obtained by Steglich esterification of podophyllotoxin with the percentage yield of 41%. Results of biotesting carried out on the carcinoma A549 cell line revealed that at a concentration of 2 μM the conjugate caused full depolymerization of microtubules without any other effect on free tubulin. The conjugate inhibited proliferation (IC50=135±30 nM) and growth (EC50=240±30 nM) of A549 cells. The data of computer molecular docking of the novel compound into the 3D model of the colchicine binding site in α,β-tubulin and molecular dynamics modelling allowed to explain the observed difference in effects of chlorambucil-podophyllotoxin and chlorambucil-colchicine conjugates on microtubules.

Diffusion rather than intraflagellar transport likely provides most of the tubulin required for axonemal assembly in Chlamydomonas.

Tubulin enters the cilium by diffusion and motor-based intraflagellar transport (IFT). However, the respective contribution of each route in providing tubulin for axonemal assembly remains unknown. Using Chlamydomonas, we attenuated IFT-based tubulin transport of GFP-β-tubulin by altering the IFT74N-IFT81N tubulin-binding module and the C-terminal E-hook of tubulin. E-hook-deficient GFP-β-tubulin was incorporated into the axonemal microtubules, but its transport frequency by IFT was reduced by ∼90% in control cells and essentially abolished when the tubulin-binding site of IFT81 was incapacitated. Despite the strong reduction in IFT, the proportion of E-hook-deficient GFP-β-tubulin in the axoneme was only moderately reduced. In vivo imaging showed more GFP-β-tubulin particles entering cilia by diffusion than by IFT. Extrapolated to endogenous tubulin, the data indicate that diffusion provides most of the tubulin required for axonemal assembly. We propose that IFT of tubulin is nevertheless needed for ciliogenesis, because it augments the tubulin pool supplied to the ciliary tip by diffusion, thus ensuring that free tubulin there is maintained at the critical concentration for plus-end microtubule assembly during rapid ciliary growth.

Dynamic localization of αB-crystallin at the microtubule cytoskeleton network in beating heart cells.

αB-crystallin is highly expressed in the heart and slow skeletal muscle; however, the roles of αB-crystallin in the muscle are obscure. Previously, we showed that αB-crystallin localizes at the sarcomere Z-bands, corresponding to the focal adhesions of cultured cells. In myoblast cells, αB-crystallin completely colocalizes with microtubules and maintains cell shape and adhesion. In this study, we show that in beating cardiomyocytes α-tubulin and αB-crystallin colocalize at the I- and Z-bands of the myocardium, where it may function as a molecular chaperone for tubulin/microtubules. Fluorescence recovery after photobleaching (FRAP) analysis revealed that the striated patterns of GFP-αB-crystallin fluorescence recovered quickly at 37°C. FRAP mobility assay also showed αB-crystallin to be associated with nocodazole-treated free tubulin dimers but not with taxol-treated microtubules. The interaction of αB-crystallin and free tubulin was further confirmed by immunoprecipitation and microtubule sedimentation assay in the presence of 1-100 μM calcium, which destabilizes microtubules. Förster resonance energy transfer analysis showed that αB-crystallin and tubulin were at 1-10 nm apart from each other in the presence of colchicine. These results suggested that αB-crystallin may play an essential role in microtubule dynamics by maintaining free tubulin in striated muscles, such as the soleus or cardiac muscles.

Tubulin targeted antimitotic agents based on adamantane lead compound: synthesis, SAR and molecular modeling

5-Hydroxymethyl-2-methoxyphenyl adamantane-1-acetate inhibits cell proliferation and stimulates depolymerization of microtubules of cancer cells to free tubulin. Its analogues were synthesized via the Steglich or Mitsunobu reactions to determine the role of structural subunits of the molecule in tubulin binding. Based on the structure–activity relationship studies, metabolically stable 1-[2-(5-hydroxymethyl-2-methoxyphenyl)ethyl]adamantane was invented, which exhibits a dual-target profile and retains in vitro activity observed for the lead compound.

Combining optogenetics with sensitive FRET imaging to monitor local microtubule manipulations

Optogenetic methods for switching molecular states in cells are increasingly prominent tools in life sciences. Förster Resonance Energy Transfer (FRET)-based sensors can provide quantitative and sensitive readouts of altered cellular biochemistry, e.g. from optogenetics. However, most of the light-inducible domains respond to the same wavelength as is required for excitation of popular CFP/YFP-based FRET pairs, rendering the techniques incompatible with each other. In order to overcome this limitation, we red-shifted an existing CFP/YFP-based OP18 FRET sensor (COPY) by employing an sYFP2 donor and mScarlet-I acceptor. Their favorable quantum yield and brightness result in a red-shifted FRET pair with an optimized dynamic range, which could be further enhanced by an R125I point mutation that stimulates intramolecular interactions. The new sensor was named ROPY and it visualizes the interaction between the microtubule regulator stathmin/OP18 and free tubulin heterodimers. We show that through phosphorylation of the ROPY sensor, its tubulin sequestering ability can be locally regulated by photo-activatable Rac1 (PARac1), independent of the FRET readout. Together, ROPY and PARac1 provide spatiotemporal control over free tubulin levels. ROPY/PARac1-based optogenetic regulation of free tubulin levels allowed us to demonstrate that depletion of free tubulin prevents the formation of pioneer microtubules, while local upregulation of tubulin concentration allows localized microtubule extensions to support the lamellipodia.

Investigation of the Electrical Properties of Microtubule Ensembles under Cell-Like Conditions

Microtubules are hollow cylindrical polymers composed of the highly negatively-charged (~23e), high dipole moment (1750 D) protein α, β- tubulin. While the roles of microtubules in chromosomal segregation, macromolecular transport, and cell migration are relatively well-understood, studies on the electrical properties of microtubules have only recently gained strong interest. Here, we show that while microtubules at physiological concentrations increase solution capacitance, free tubulin has no appreciable effect. Further, we observed a decrease in electrical resistance of solution, with charge transport peaking between 20–60 Hz in the presence of microtubules, consistent with recent findings that microtubules exhibit electric oscillations at such low frequencies. We were able to quantify the capacitance and resistance of the microtubules (MT) network at physiological tubulin concentrations to be 1.27 × 10−5 F and 9.74 × 104 Ω. Our results show that in addition to macromolecular transport, microtubules also act as charge storage devices through counterionic condensation across a broad frequency spectrum. We conclude with a hypothesis of an electrically tunable cytoskeleton where the dielectric properties of tubulin are polymerisation-state dependent.

The disordered N-terminus of HDAC6 is a microtubule-binding domain critical for efficient tubulin deacetylation

Histone deacetylase 6 (HDAC6) is a multidomain cytosolic enzyme having tubulin deacetylase activity that has been unequivocally assigned to the second of the tandem catalytic domains. However, virtually no information exists on the contribution of other HDAC6 domains on tubulin recognition. Here, using recombinant protein expression, site-directed mutagenesis, fluorimetric and biochemical assays, microscale thermophoresis, and total internal reflection fluorescence microscopy, we identified the N-terminal, disordered region of HDAC6 as a microtubule-binding domain and functionally characterized it to the single-molecule level. We show that the microtubule-binding motif spans two positively charged patches comprising residues Lys-32 to Lys-58. We found that HDAC6-microtubule interactions are entirely independent of the catalytic domains and are mediated by ionic interactions with the negatively charged microtubule surface. Importantly, a crosstalk between the microtubule-binding domain and the deacetylase domain was critical for recognition and efficient deacetylation of free tubulin dimers both in vitro and in vivo Overall, our results reveal that recognition of substrates by HDAC6 is more complex than previously appreciated and that domains outside the tandem catalytic core are essential for proficient substrate deacetylation.

Voltage Dependent Anion Channels Regulate Proliferation of Cancer Stem Cells

Cancer stem cells (CSCs) are associated with tumor progression and resistance to chemotherapy. Oxidative phosphorylation and aerobic glycolysis in CSCs are dynamically regulated. Mitochondrial metabolism and membrane potential (ΔΨ) are contributed by the ingress of respiratory substrates, ADP and Pi into mitochondria through voltage-dependent anion channels 1/2/3 (VDAC). We previously demonstrated that free tubulin closes VDAC and erastin antagonizes the tubulin inhibition of VDAC1/2 but not VDAC3. VDAC opening increases mitochondrial metabolism decreasing glycolysis and proliferation. Here, we hypothesized that different VDAC isoforms, by controlling oxidative metabolism, regulate proliferation of CSCs. CSCs from HepG2 and Huh7 human hepatocarcinoma and HCC4006 lung adenocarcinoma cells (spheres >50 µm diameter) were generated using ultra-low attachment plates (DMEM-F12 medium supplemented with B-27, EGF and bFGF). Markers of pluripotency were assessed by qPCR, western blotting and immunostaining. Spheres were counted using 50 µm side grid units. Tetramethylrhodamine methyl ester fluorescence to assess ΔΨ was determined both using a microplate reader and confocal fluorescence microscopy. The relative contribution of each VDAC isoform was similar in all CSCs and bulk cancer cells (CCs). By contrast, VDAC 2/3 mRNA levels were higher in CSCs compared to bulk HepG2, Huh7 and HCC4006 whereas VDAC1 was higher only in HepG2 and Huh7 CSCs. VDAC1 single knockdown in HepG2 and Huh7 CSCs decreased sphere formation by ∼30%. VDAC2 and VDAC3 single knockdown increased both the amount (∼18% and 102%, respectively) and the size of spheroids. VDAC1/2/3 single knockdown decreased ΔΨ. Erastin decreased sphere formation and promoted intra-sphere cell death in HepG2 and Huh7 CSCs. VDAC1/2 but not VDAC3 contribute to the maintenance and proliferation of CSCs. VDAC1/2 opening by erastin may decrease sphere formation and promote cell death by enhancing oxidative metabolism and decreasing glycolysis.

GTP-tubulin loves microtubule plus ends but marries the minus ends.

Microtubule minus ends are inherently more stable than plus ends despite the fact that free tubulin associates more avidly to the plus end. In this issue, Strothman et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201905019) measure, for the first time, the off-rate for GTP-tubulin and find that it is different for the two ends, suggesting that this parameter may control the transition to disassembly at microtubule ends.

Structures of TOG1 and TOG2 from the human microtubule dynamics regulator CLASP1.

Tubulin-binding TOG domains are found arrayed in a number of proteins that regulate microtubule dynamics. While much is known about the structure and function of TOG domains from the XMAP215 microtubule polymerase family, less in known about the TOG domain array found in animal CLASP family members. The animal CLASP TOG array promotes microtubule pause, potentiates rescue, and limits catastrophe. How structurally distinct the TOG domains of animal CLASP are from one another, from XMAP215 family TOG domains, and whether a specific order of structurally distinct TOG domains in the TOG array is conserved across animal CLASP family members is poorly understood. We present the x-ray crystal structures of Homo sapiens (H.s.) CLASP1 TOG1 and TOG2. The structures of H.s. CLASP1 TOG1 and TOG2 are distinct from each other and from the previously determined structure of Mus musculus (M.m.) CLASP2 TOG3. Comparative analyses of CLASP family TOG domain structures determined to date across species and paralogs supports a conserved CLASP TOG array paradigm in which structurally distinct TOG domains are arrayed in a specific order. H.s. CLASP1 TOG1 bears structural similarity to the free-tubulin binding TOG domains of the XMAP215 family but lacks many of the key tubulin-binding determinants found in XMAP215 family TOG domains. This aligns with studies that report that animal CLASP family TOG1 domains cannot bind free tubulin or microtubules. In contrast, animal CLASP family TOG2 and TOG3 domains have reported microtubule-binding activity but are structurally distinct from the free-tubulin binding TOG domains of the XMAP215 family. H.s. CLASP1 TOG2 has a convex architecture, predicted to engage a hyper-curved tubulin state that may underlie its ability to limit microtubule catastrophe and promote rescue. M.m. CLASP2 TOG3 has unique structural elements in the C-terminal half of its α-solenoid domain that our modeling studies implicate in binding to laterally-associated tubulin subunits in the microtubule lattice in a mode similar to, yet distinct from those predicted for the XMAP215 family TOG4 domain. The potential ability of the animal CLASP family TOG3 domain to engage lateral tubulin subunits may underlie the microtubule rescue activity ascribed to the domain. These findings highlight the structural diversity of TOG domains within the CLASP family TOG array and provide a molecular foundation for understanding CLASP-dependent effects on microtubule dynamics.

Kinesin-5 Promotes Microtubule Nucleation and Assembly by Stabilizing a Lattice-Competent Conformation of Tubulin

Besides sliding apart antiparallel microtubules during spindle elongation, the mitotic kinesin-5, Eg5, promotes microtubule polymerization, emphasizing its importance in mitotic spindle length control. Here, we characterize the Eg5 microtubule polymerase mechanism by assessing motor-induced changes in the longitudinal and lateral tubulin-tubulin bonds that form the microtubule lattice. Isolated Eg5 motor domains promote microtubule nucleation, growth, and stability; thus, crosslinking tubulin by pairs of motor heads is not necessary for polymerase activity. Eg5 binds preferentially to microtubules over free tubulin, which contrasts with microtubule-depolymerizing kinesins that preferentially bind free tubulin over microtubules. Colchicine-like inhibitors that stabilize the bent conformation of tubulin allosterically inhibit Eg5 binding, consistent with a model in which Eg5 induces a curved-to-straight transition in tubulin. Domain swap experiments establish that the family-specific loop11-helix 4 junction, which resides near the nucleotide-sensing switch-II domain, is necessary and sufficient for the polymerase activity of Eg5. Thus, we propose a microtubule polymerase mechanism in which Eg5 at the plus-end promotes a curved-to-straight transition in tubulin that enhances lateral bond formation and thereby promotes microtubule growth and stability. One implication is that regulation of Eg5 motile properties by regulatory proteins or small molecule inhibitors could also have effects on intracellular microtubule dynamics.

Identification and characterization of the land-plant-specific microtubule nucleation factor MACET4.

Here, we show that the embryophyte (land-plant)-specific protein MACERATOR4 (MACET4) binds microtubules in vitro and in vivo, promotes microtubule polymerization at sub-critical tubulin concentrations, decreases the lag phase in microtubule bulk polymerization assays, and colocalizes with microtubule nucleation sites. Furthermore, we find that MACET4 forms oligomers that induce aster formation in vitro in a manner that is similar to aster formation mediated by centrosomes and TPX2. MACET4 is expressed during cell division and accumulates at the microtubule nucleation regions of the plant-specific cytokinetic microtubule array, the phragmoplast. We found that MACET4 localizes to the preprophase band and the cortical division zone, but not the spindle. MACET4 appears as cytoplasmic foci in vivo and forms octamers in vitro Transient expression in tobacco leaf pavement cells results in labeling of shrinking plus- and minus-ends. MACET4 facilitates microtubule depolymerization by increasing the frequency of catastrophes in vivo and by suppressing rescues in vitro Microtubules formed in the presence of MACET4 in vitro are shorter, most likely due to the depletion of the free tubulin pool. Accordingly, MACET4 knockdown results in longer phragmoplasts. We conclude that the direct activity of MACET4 is in promoting microtubule nucleation.This article has an associated First Person interview with the first author of the paper.

Tubulin-VDAC Interaction: Molecular Basis for Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy.

Tubulin is a well-established target of microtubule-targeting agents (MTAs), a widely used class of chemotherapeutic drugs. Yet, aside from their powerful anti-cancer efficiency, MTAs induce a dose-limiting and debilitating peripheral neurotoxicity. Despite intensive efforts in the development of neuroprotective agents, there are currently no approved therapies to effectively manage chemotherapy-induced peripheral neuropathy (CIPN). Over the last decade, attempts to unravel the pathomechanisms underlying the development of CIPN led to the observation that mitochondrial dysfunctions stand as a common feature associated with axonal degeneration. Concomitantly, mitochondria emerged as crucial players in the anti-cancer efficiency of MTAs. The findings that free dimeric tubulin could be associated with mitochondrial membranes and interact directly with the voltage-dependent anion channels (VDACs) located in the mitochondrial outer membrane strongly suggested the existence of an interplay between both subcellular compartments. The biological relevance of the interaction between tubulin and VDAC came from subsequent in vitro studies, which found dimeric tubulin to be a potent modulator of VDAC and ultimately of mitochondrial membrane permeability to respiratory substrates. Therefore, one of the hypothetic mechanisms of CIPN implies that MTAs, by binding directly to the tubulin associated with VDAC, interferes with mitochondrial function in the peripheral nervous system. We review here the foundations of this hypothesis and discuss them in light of the current knowledge. A focus is set on the molecular mechanisms behind MTA interference with dimeric tubulin and VDAC interaction, the potential relevance of tubulin isotypes and availability as a free dimer in the specific context of MTA-induced CIPN. We further highlight the emerging interest for VDAC and its interacting partners as a promising therapeutic target in neurodegeneration.

Tubulin Acetylation Mediates Bisphenol A Effects on the Microtubule Arrays of Allium cepa and Triticum turgidum.

The effects of bisphenol A (BPA), a prevalent endocrine disruptor, on both interphase and mitotic microtubule array organization was examined by immunofluorescence microscopy in meristematic root cells of Triticum turgidum (durum wheat) and Allium cepa (onion). In interphase cells of A. cepa, BPA treatment resulted in substitution of cortical microtubules by annular/spiral tubulin structures, while in T. turgidum BPA induced cortical microtubule fragmentation. Immunolocalization of acetylated α-tubulin revealed that cortical microtubules of T. turgidum were highly acetylated, unlike those of A. cepa. In addition, elevation of tubulin acetylation by trichostatin A in A. cepa resulted in microtubule disruption similar to that observed in T. turgidum. BPA also disrupted all mitotic microtubule arrays in both species. It is also worth noting that mitotic microtubule arrays were acetylated in both plants. As assessed by BPA removal, its effects are reversible. Furthermore, taxol-stabilized microtubules were resistant to BPA, while recovery from oryzalin treatment in BPA solution resulted in the formation of ring-like tubulin conformations. Overall, these findings indicate the following: (1) BPA affects plant mitosis/cytokinesis by disrupting microtubule organization. (2) Microtubule disassembly probably results from impairment of free tubulin subunit polymerization. (3) The differences in cortical microtubule responses to BPA among the species studied are correlated to the degree of tubulin acetylation.

Oxidative Stress Restructures the Cellular Microtubule Cytoskeleton via Repair-Mediated Rescue Events

Microtubules are cylindrical cytoskeletal polymers composed of a/β-tubulin heterodimers that make up an ordered tubulin lattice. Microtubules display dynamic length changes, termed “dynamic instability”, in which slow elongation phases are followed by rapid shortening. In cells, microtubules form a network that is a key component of the cellular cytoskeleton. Under pathological conditions of oxidative stress, we and others have found that cardiomyocytes display a denser microtubule cytoskeleton, which may lead to the progressive structural and functional cellular changes associated with myocardial ischemia and systolic dysfunction. This reorganization of the microtubule network occurs despite only small increases in tubulin expression, suggesting that alterations to microtubule length regulation are involved. Using biophysical reconstitution experiments and live-cell imaging, we found that oxidative stress directly provokes microtubule ‘rescue,’ the transition from rapid microtubule shortening to slow elongation, while it has little effect on other microtubule dynamic instability parameters or the microtubule nucleation rate. To explore a mechanism for this observation, we used electron microscopy, and observed structural damage, consisting of holes and sheet-like structures, under conditions of moderate oxidative stress. Further, using a quantitative two-color tubulin “repair” assay (Reid et al, 2017), we found that repair of structural defects within the microtubule lattice via the incorporation of free tubulin was 133% higher under conditions of oxidative stress compared to controls. Such repaired regions have been termed ‘rescue islands’ because they facilitate rescue events, and thus promote net microtubule elongation (Dimitrov et al., 2008). We conclude that microtubule structural damage may explain our observations of oxidative stress-mediated increases in microtubule density in cardiomyocytes, potentially providing insight into the progressive myocardial changes that accompany Ischemic Heart Disease.

Severing to build microtubules

Cytoskeleton![Figure][1] New tubulin (pink) patches small holes in microtubule lattices made by severing enzymes (purple). CREDIT: JANET IWASA 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][2] [1]: pending:yes [2]: /lookup/doi/10.1126/science.aau1504