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The properties of single microtubules within the microtubule network can be modulated through post-translational modifications (PTMs), including acetylation within the lumen of microtubules. To access the lumen, the enzymes could enter through the microtubule ends and at damage sites along the microtubule shaft. Here we show that the acetylation profile depends on damage sites, which can be caused by the motor protein kinesin-1. Indeed, the entry of the deacetylase HDAC6 into the microtubule lumen can be modulated by kinesin-1-induced damage sites. In contrast, activity of the microtubule acetylase αTAT1 is independent of kinesin-1-caused shaft damage. On a cellular level, our results show that microtubule acetylation distributes in an exponential gradient. This gradient results from tight regulation of microtubule (de)acetylation and scales with the size of the cells. The control of shaft damage represents a mechanism to regulate PTMs inside the microtubule by giving access to the lumen., (© 2024. The Author(s).)

Regulation of microtubule dynamics is essential for diverse cellular functions, and proteins that bind to dynamic microtubule ends can regulate network dynamics. Here, we show that two conserved microtubule end-binding proteins, CLIP-170 and EB3, undergo phase separation and form dense liquid networks. When CLIP-170 and EB3 act together, the multivalency of the network increases, which synergistically increases the amount of protein in the dense phase. In vitro and in cells, these liquid networks can concentrate tubulin. In vitro, in the presence of microtubules, phase separation of EB3/CLIP-170 can enrich tubulin all along the microtubule. In this condition, microtubule growth speed increases up to twofold and the frequency of depolymerization events are strongly reduced compared to conditions in which there is no phase separation. Our data show that phase separation of EB3/CLIP-170 adds an additional layer of regulation to the control of microtubule growth dynamics.

Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails-which are site-specifically glutamylated-to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Control of microtubule abundance, stability, and length is crucial to regulate intracellular transport as well as cell polarity and division. How microtubule stability depends on tubulin addition or removal at the dynamic ends is well studied. However, microtubule rescue, the event when a microtubule switches from shrinking to growing, occurs at tubulin exchange sites along the shaft. Molecular motors have recently been shown to promote such exchanges. Using a stochastic theoretical description, we study how microtubule stability and length depend on motor-induced tubulin exchange and thus rescue. Our theoretical description matches our in vitro experiments on microtubule dynamics in the presence of kinesin-1 molecular motors. Although the overall dynamics of a population of microtubules can be captured by an effective rescue rate, by assigning rescue to exchange sites, we reveal that the dynamics of individual microtubules within the population differ dramatically. Furthermore, we study in detail a transition from bounded to unbounded microtubule growth. Our results provide novel insights into how molecular motors imprint information of microtubule stability on the microtubule network., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Microtubules are dynamic polymers where tubulin exchanges not only at the ends but also all along the microtubule shaft. In vitro reconstitutions are a vital approach to study microtubule tip dynamics, while direct observation of shaft dynamics is challenging. Here, we describe a dual-color in vitro assay to visualize microtubule shaft dynamics using purified, labeled bovine brain tubulin. With this assay, we can quantitatively address how proteins or small molecules impact the dynamics at the microtubule shaft. For complete details on the use and execution of this protocol, please refer to Andreu-Carbó et al. (2022)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Tubulin dimers assemble into dynamic microtubules, which are used by molecular motors as tracks for intracellular transport. Organization and dynamics of the microtubule network are commonly thought to be regulated at the polymer ends, where tubulin dimers can be added or removed. Here, we show that molecular motors running on microtubules cause exchange of dimers along the shaft in vitro and in cells. These sites of dimer exchange act as rescue sites where depolymerizing microtubules stop shrinking and start re-growing. Consequently, the average length of microtubules increases depending on how frequently they are used as motor tracks. An increase of motor activity densifies the cellular microtubule network and enhances cell polarity. Running motors leave marks in the shaft, serving as traces of microtubule usage to organize the polarity landscape of the cell., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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 by 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.

Kinesin-1 is a processive motor protein that uses ATP-derived energy to transport a variety of intracellular cargoes toward the cell periphery. The ability to visualize and monitor kinesin transport in live cells is critical to study the myriad of functions associated with cargo trafficking. Herein we report the discovery of a fluorogenic small molecule substrate (QPD-OTf) for kinesin-1 that yields a precipitating dye along its walking path on microtubules (MTs). QPD-OTf enables to monitor native kinesin-1 transport activity in cellulo without external modifications. In vitro assays show that kinesin-1 and MTs are sufficient to yield fluorescent crystals; in cells, kinesin-1 specific transport of cargo from the Golgi appears as trails of fluorescence over time. These findings are further supported by docking studies, which suggest the binding of the activity-based substrate in the nucleotide binding site of kinesin-1.

The dynamic instability of microtubules is characterized by slow growth phases stochastically interrupted by rapid depolymerizations called catastrophes. Rescue events can arrest the depolymerization and restore microtubule elongation. However, the origin of these rescue events remains unexplained. Here we show that microtubule lattice self-repair, in structurally damaged sites, is responsible for the rescue of microtubule growth. Tubulin photo-conversion in cells revealed that free tubulin dimers can incorporate along the shafts of microtubules, especially in regions where microtubules cross each other, form bundles or become bent due to mechanical constraints. These incorporation sites appeared to act as effective rescue sites ensuring microtubule rejuvenation. By securing damaged microtubule growth, the self-repair process supports a mechanosensitive growth by specifically promoting microtubule assembly in regions where they are subjected to physical constraints., Competing Interests: Competing financial interests. The authors have no competing financial interests.