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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.

Microtubules are dynamic polymers, which grow and shrink by addition and removal of tubulin dimers at their extremities. Within the microtubule shaft, dimers adopt a densely packed and highly ordered crystal-like lattice structure, which is generally not considered to be dynamic. Here we report that thermal forces are sufficient to remodel the microtubule shaft, despite its apparent stability. Our combined experimental data and numerical simulations on lattice dynamics and structure suggest that dimers can spontaneously leave and be incorporated into the lattice at structural defects. We propose a model mechanism, where the lattice dynamics is initiated via a passive breathing mechanism at dislocations, which are frequent in rapidly growing microtubules. These results show that we may need to extend the concept of dissipative dynamics, previously established for microtubule extremities, to the entire shaft, instead of considering it as a passive material.

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.

Background: Fat in wound blood observed in orthopedic or cardiac surgery might pose a risk for fat embolism during blood salvage. Fat removal was optimized in the washing process., Study Design and Methods: In an experimental study blood from fresh donations was adjusted to a hematocrit (Hct) of 25% and an admixture of 1.25% human tissue fat. This blood was processed with the cell salvage device XTRA in a modified program mode. Volumetric quantification of fat was performed after centrifugation of blood samples in Pasteur pipettes. From the volumes, the Hct levels and the concentrations of fat and other variables elimination rates and RBC recovery were calculated., Results: Pretests showed wash volume, wash flow, and process interruptions affecting fat elimination. With the new optimized fat elimination program Pfat removal rate of fat increased to 98.5 ± 0.9% for the 225-mL bowl. The product had a mean Hct of 48.7 ± 1.2% and a RBC recovery rate of 93.5 ± 2.3%. The program conserved the high elimination rates for albumin, heparin, potassium, and free plasma hemoglobin (98.8, 99.3, 95.3, and 94.9%, respectively). Similar high fat removal was also observed with bowls of smaller size, namely, 98.1% for the 175-mL bowl and 98.2% for the 125- and the 55-mL bowls. With test blood of Hct 10% a mean fat elimination of 99.6 ± 01% was observed., Conclusions: A special program modification Pfat involving extra washing and RBC concentration steps significantly improves fat removal by the Latham bowl-based autotransfusion device XTRA, thus yielding results equivalent to the continuous cell salvage system., (© 2015 AABB.)

Diatoms are heterokont unicellular algae with a widespread distribution throughout all aquatic habitats. Research on diatoms has advanced significantly over the last decade due to available genetic transformation methods and publicly available genome databases. Yet up to now, proteins involved in the regulation of the cytoskeleton in diatoms are largely unknown. Consequently, this work focuses on actin and actin-related proteins (ARPs) encoded in the diatom genomes of Thalassiosira pseudonana, Thalassiosira oceanica, Phaeodactylum tricornutum, Fragilariopsis cylindrus and Pseudo-nitzschia multiseries. Our comparative genomic study revealed that most diatoms possess only a single conventional actin and a small set of ARPs. Among these are the highly conserved cytoplasmic Arp1 protein and the nuclear Arp4 as well as Arp6. Diatom genomes contain genes coding for two structurally different homologues of Arp4 that might serve specific functions. All diatom species examined here lack ARP2 and ARP3 proteins, suggesting that diatoms are not capable of forming the Arp2/3 complex, which is essential in most eukaryotes for actin filament branching and plus-end dynamics. Interestingly, none of the sequenced representatives of the Bacillariophyta phylum code for profilin. Profilin is an essential actin-binding protein regulating the monomer actin pool and is involved in filament plus-end dynamics. This is the first report of organisms not containing profilin., (Copyright © 2015 Elsevier B.V. All rights reserved.)