Recombinant Tubulin Research Articles

The Impact of a Catalytic Site Mutation on the Shape and Mechanics of Tubulin Protofilaments

Alpha-beta heterodimers of tubulin proteins serve as the building blocks of microtubules, which are key biopolymers forming one of the principal systems of the cellular cytoskeleton. A detailed study of these building blocks, as well as their alterations caused by point mutations, contributes to a deeper understanding of physiological and pathological processes related to the cytoskeleton. This study presents an analysis of the impact of the E254N point mutation in the catalytic site of α-tubulin on the bending conformations of human recombinant tubulin tetramers using molecular dynamics methods. The models were constructed based on high-resolution cryo-electron microscopy data, allowing the reconstruction of three-dimensional structures of both wild-type and mutant tetramers. The simulations revealed that the primary difference between wild-type and mutant tubulin lies in the equilibrium bending direction of the protofilaments, while the bending amplitude, twisting, and associated stiffness remain largely unchanged. We propose that the observed differences in bending directions may be related to variations in protofilament tilts within microtubules, which aligns with previously published cryo-electron microscopy data. These findings provide valuable insights into the principles underlying the formation of the polymeric structure of microtubules based on the properties of their individual building blocks.

Design, synthesis and biological evaluation of novel tubulin-targeting agents with a dual-mechanism for polymerization inhibition and protein degradation

Microtubules are recognized as one of the most vital and attractive targets in anticancer therapy. The development of novel tubulin-targeting agents with a new action mechanism is imperative. Based on the hydrophobic tagging strategy, the molecular scaffold of tirbanibulin was selected as tubulin target-binding moiety, subsequent to which a series of target compounds were rationally designed by selecting various combinations of linkers and hydrophobic tags. A set of novel molecules were synthesized and most of them exhibited potent antiproliferative activity against tumor cells in vitro. The most active compound 14b inhibited polymerization of purified recombinant tubulin and induced degradation of α- and β-tubulin in MCF-7 cells. Notably, following treatment with compound 14b, an unexpected phenomenon of “microtubules fragmentation” was observed via immunofluorescence staining. Furthermore, compound 14b possessed antitumor activity in the 4T1 allograft models with TGI of 74.27 % without significant toxicity. In this work, we report the discovery of novel dual-mechanism tubulin-targeting agents.

Cryo-EM of α-tubulin isotype-containing microtubules revealed a contracted structure of α4A/β2A microtubules.

Microtubules are hollow α/β-tubulin heterodimeric polymers that play critical roles in cells. In vertebrates, both α- and β-tubulins have multiple isotypes encoded by different genes, which are intrinsic factors in regulating microtubule functions. However, the structures of microtubules composed of different tubulin isotypes, especially α-tubulin isotypes, remain largely unknown. Here, we purify recombinant tubulin heterodimers composed of different mouse α-tubulin isotypes, including α1A, α1C and α4A, with the β-tubulin isotype β2A. We further assemble and determine the cryo-electron microscopy (cryo-EM) structures of α1A/β2A, α1C/β2A, and α4A/β2A microtubules. Our structural analysis demonstrates that α4A/β2A microtubules exhibit longitudinal contraction between tubulin interdimers compared with α1A/β2A and α1C/β2A microtubules. Collectively, our findings reveal that α-tubulin isotype composition can tune microtubule structures, and also provide evidence for the "tubulin code" hypothesis.

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the β-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than β-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and β2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct rolein vivo.

α1A and α1C form microtubules to display distinct properties mainly mediated by their C-terminal tails.

Microtubules consisting of α/β-tubulin dimers play critical roles in cells. More than seven genes encode α-tubulin in vertebrates. However, the property of microtubules composed of different α-tubulin isotypes is largely unknown. Here, we purified recombinant tubulin heterodimers of mouse α-tubulin isotypes including α1A and α1C with β-tubulin isotype β2A. In vitro microtubule reconstitution assay detected that α1C/β2A microtubules grew faster and underwent catastrophe less frequently than α1A/β2A microtubules. Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal (C-terminal) tails of α-tubulin isotypes largely accounted for the differences in polymerization dynamics of α1A/β2A and α1C/β2A microtubules. Kinetics analysis showed that in comparison to α1A/β2A microtubules, α1C/β2A microtubules displayed higher on-rate, lower off-rate, and similar GTP hydrolysis rate at the plus-end, suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of α1C/β2A microtubules. Furthermore, EB1 had a higher binding ability to α1C/β2A microtubules than to α1A/β2A ones, which could also be attributed to the difference in the C-terminal tails of these two α-tubulin isotypes. Thus, α-tubulin isotypes diversify microtubule properties, which, to a great extent, could be accounted by their C-terminal tails.

The tail wags the tubulin.

The C-terminal tail of tubulin influences microtubule assembly and stability. In this issue of Developmental Cell, Chen etal. combine invitro experiments using recombinant tubulin with molecular dynamics simulations to provide molecular-level insights into the importance of α-tubulin tail and its post-translational modifications in microtubule assembly and stability.

Direct observation of individual tubulin dimers binding to growing microtubules

The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.

Structural Basis for the Lipid-Mediated Interaction of Tubulin with VDAC Revealed by Neutron Reflectometry

A preponderance of evidence suggests that the mitochondrial outer membrane (MOM) binds dimeric tubulin, which blocks the flux of metabolites through VDAC, the major MOM channel, by inserting its negatively charged, disordered C-terminal tail into the VDAC pore. The discovery of this novel regulatory mechanism of mitochondrial respiration has raised several fundamental questions, including how tubulin binds to the MOM surface, whether its membrane-bound conformation is consistent with the accessibility of its C-termini to the membrane surface, and what role mitochondrial lipids assume in mediating the tubulin-VDAC interaction. Here we report neutron reflectivity (NR) studies on tubulin-coated and VDAC-containing biomimetic membranes. In combination with molecular dynamics simulations, these reveal, first, that dimeric tubulin is anchored to the membrane surface by an amphipathic a-helix of the a-tubulin subunit in an orientation that presents the C-termini of both tubulin subunits to the membrane surface. The structural model is supported by electrophysiological evidence using recombinant tubulin constructs in which C-terminal tails bound to either subunit are individually observed in a VDAC nanopore. Second, we demonstrate using NR that surface-immobilized VDAC can be reconstituted into a complete lipid membrane, thus creating a “protein-tethered bilayer lipid membrane” (ptBLM) over a large sample area. The ptBLM architecture allows dynamic changes of the solution environment, while NR reveals the corresponding structural response of the protein and its lipid environment. This allows us to experimentally observe the effects of pH-induced changes in VDAC's structure on the local lipid environment. Molecular dynamics simulations are in good agreement with NR experiments. We speculate that local curvature or lipid packing stress induced by membrane-embedded VDAC and its structural dynamics serves to localize cytosolic regulators such as tubulin to the vicinity of the channel.

Direct Observation of Individual Tubulin Dimers Binding to Growing Microtubules

The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.

How Microtubules Activate Kinesin & Dynein Atpase Activity

Understanding the role of cytoskeletal filaments in motility could be essential to the complete elucidation of the motility mechanism, because the movements of motors involve an intimate and concerted partnership between the motor and the cytoskeletal filaments. However, with microtubules, the technical difficulty involved in generating recombinant tubulin has impeded the application of molecular biological methods to the study of the structure-function relationship in microtubules in vitro. We developed experiment systems to purify recombinant tubulins (yeast and human tubulin) and analyzed how microtubule binding activates the ATPase activity of kinesin and dynein motors. The charged-to-alanine scanning mutagenesis analyses of tubulin showed that the H11’-H12 loop and H12 in alpha-tubulin, consisting of only four amino acids (415EEGE), accelerates the rate of ATP hydrolysis in both motors, despite the distinct molecular mechanisms in each kinesin and dynein motor. In both motors, the H12 of beta-tubulin contributes to the stability of the motor-microtubule interactions. Our results indicate that the two motors have independently evolved to use the same loop in alpha-tubulin for ATPase activation, probably because the loop plays, either architecturally or mechanically, an essential role in the motility mechanism. (The talk will also include a practical guide to the use of recombinant tubulin.)

Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips.

Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the 'conformational wave' model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry.

Toward Discovery of Novel Microtubule Targeting Agents: A SNAP-tag–Based High-Content Screening Assay for the Analysis of Microtubule Dynamics and Cell Cycle Progression

Microtubule targeting agents (MTAs) are used for the treatment of cancer. Novel MTAs could provide additional and beneficial therapeutic options. To improve the sensitivity and throughput of standard immunofluorescence assays for the characterization of MTAs, we used SNAP-tag technology to produce recombinant tubulin monomers. To visualize microtubule filaments, A549 cells transfected with SNAP-tubulin were stained with a membrane-permeable, SNAP-reactive dye. The treatment of SNAP-tubulin cells with stabilizing MTAs such as paclitaxel resulted in the formation of coarsely structured microtubule filaments, whereas depolymerizing MTAs such as nocodazole resulted in diffuse staining patterns in which the tubulin filaments were no longer distinguishable. By combining these components with automated microscopy and image analysis algorithms, we established a robust high-content screening assay for MTAs with a Z′ factor of 0.7. Proof of principle was achieved by testing a panel of 10 substances, allowing us to identify MTAs and to distinguish between stabilizing and destabilizing modes of action. By extending the treatment of the cells from 2 to 20 h, our assay also detected abnormalities in cell cycle progression and in the formation of microtubule spindles, providing additional readouts for the discovery of new MTAs and facilitating their early identification during drug-screening campaigns.

Using Engineered Recombinant Tubulin Sotypes to Study VDAC Regulation

Highly variable anionic C-terminal tails (CTTs) of tubulin had been implicated in regulation of permeability of the mitochondrial voltage-dependent anion channel (VDAC). Tubulin, a structural protein of microtubules, is a heterodimer comprising α/β subunits. In cells tubulin possesses significant variability due to the expression of several tubulin genes. Seven α- and eight β-tubulin isoforms in the human tubulin gene family vary mostly in the length and primary sequence of the CTTs. This inherent non-homogeneity of the endogenous tubulin CTTs hindered the analysis of their interaction with VDAC reconstituted into planar lipid bilayers. A recent study (Sheldon et al., 2015) with peptide-protein chimeras evaluated the frequency of the VDAC blockage events to demonstrate that the on-rate is highly dependent on the tail chemical composition. In that study CTT-like peptides were grafted to albumin, a protein that does not bind to the membrane surface, though it is now conclusively established that membrane binding of the tubulin body is an essential step in tubulin/VDAC interaction. Here we took an advantage of recently generated recombinant yeast α/β-tubulin constructs with CTTs from various human tubulin isotypes (Sirajuddin et al., 2014). The effects of each of the engineered constructs on the reconstituted VDAC were compared to those of w.t. yeast tubulin and recombinant tubulin without CTTs. Single-molecule studies of the tubulin-VDAC interaction showed that length and total anionic charge of the individual CTTs correlate strongly with the average duration of the blockage. In particular, at 30-mV applied voltage the characteristic time of VDAC blockage by the longer and more highly charged β3-CTT is up to hundredfold greater than that observed for the α1-CTT, with the apparent gating charge doubled. These results suggest novel implications in VDAC regulation by the diversity of tubulin isotypes found in vertebrates.

The structured core of human β tubulin confers isotype-specific polymerization properties.

Diversity in cytoskeleton organization and function may be achieved through variations in primary sequence of tubulin isotypes. Recently, isotype functional diversity has been linked to a "tubulin code" in which the C-terminal tail, a region of substantial sequence divergence between isotypes, specifies interactions with microtubule-associated proteins. However, it is not known whether residue changes in this region alter microtubule dynamic instability. Here, we examine recombinant tubulin with human β isotype IIB and characterize polymerization dynamics. Microtubules with βIIB have catastrophe frequencies approximately threefold lower than those with isotype βIII, a suppression similar to that achieved by regulatory proteins. Further, we generate chimeric β tubulins with native tail sequences swapped between isotypes. These chimeras have catastrophe frequencies similar to that of the corresponding full-length construct with the same core sequence. Together, our data indicate that residue changes within the conserved β tubulin core are largely responsible for the observed isotype-specific changes in dynamic instability parameters and tune tubulin's polymerization properties across a wide range.

On the Force-Generating Capacity of Disassembling Microtubules

Microtubules can generate force independently of motor enzymes, especially at kinetochores where disassembling microtubules drive movement of mitotic chromosomes. A popular explanation for this microtubule-powered motility is the conformational wave model, where individual rows of tubulin subunits, the protofilaments, push or pull on a kinetochore as they curl outward from a disassembling microtubule tip. One pioneering study has demonstrated that curling protofilaments are capable of generating force, but the measured forces were far weaker (∼0.24 pN) than forces generated at kinetochores in vivo (4 - 8 pN) or in vitro (∼9 pN). Thus it has remained unclear whether the wave mechanism can contribute significantly to kinetochore force production. Here, by developing a wave assay using recombinant tubulin and a feedback-controlled laser trap, we show that the conformational wave can generate forces 50-fold higher than previously recorded. Measuring its full capacity for work output reveals that the wave carries at least three times the energy harnessed by kinetochores in vitro, and enables an estimate of the mechanical strain energy trapped per tubulin dimer in the microtubule lattice. Surprisingly, a β-tubulin mutation that allosterically enhances microtubule stability has little effect on wave energy. Our work indicates that the conformational wave mechanism can make a major contribution to kinetochore motility and it provides a new, direct way to examine tubulin mechanochemistry.

Preparation of Immobilized Recombinant Tubulin Beta(TuBb) on Chitosan Nanoparticles by Covalent Binding Method

Objective In order to screen TuBb inhibitors, this paper describes the preparation of immobilized TuBb on chitosan nanoparticles. Methods TuBb was immobilized onto chitosan nanoparticles by covalent binding method. Results The results of the univariate test indicated that the highest immobilized yield can be obtained when the optimal immobilization condition was 1 mg of TuBb, 0.5 mol/L of buffer solution with pH 6.5, immobilization 30 min and immobilization at 0-4 °C. Conclusions The authors conclude that the immobilized TuBb maintain the catalysis properties and can be used as the screening of TuBb inhibitors.

Versatile and Efficient Site-Specific Protein Functionalization by Tubulin Tyrosine Ligase.

A novel chemoenzymatic approach for simple and fast site-specific protein labeling is reported. Recombinant tubulin tyrosine ligase (TTL) was repurposed to attach various unnatural tyrosine derivatives as small bioorthogonal handles to proteins containing a short tubulin-derived recognition sequence (Tub-tag). This novel strategy enables a broad range of high-yielding and fast chemoselective C-terminal protein modifications on isolated proteins or in cell lysates for applications in biochemistry, cell biology, and beyond, as demonstrated by the site-specific labeling of nanobodies, GFP, and ubiquitin.

Overexpression, purification, and functional analysis of recombinant human tubulin dimer

Microtubules consisting of tubulin dimers play essential roles in various cellular functions. Investigating the structure–function relationship of tubulin dimers requires a method to prepare sufficient quantities of recombinant tubulin. To this end, we simultaneously expressed human α1- and β3-tubulin using a baculovirus-insect cell expression system that enabled the purification of 5mg recombinant tubulin per litre of cell culture. The purified recombinant human tubulin could be polymerized into microtubules that glide on a kinesin-coated glass surface. The method provides a powerful tool for in vitro functional analyses of microtubules.

Engineering tubulin: microtubule functionalization approaches for nanoscale device applications

With the emergences of engineered devices at microscale and nanoscale dimensions, there is a growing need for controlled actuation and transport at these length scales. The kinesin-microtubule system provides a highly evolved biological transport system well suited for these tasks. Accordingly, there is an ongoing effort to create hybrid nanodevices that integrate biological components with engineered materials for applications such as biological separations, nanoscale assembly, and sensing. Adopting microtubules for these applications generally requires covalent attachment of biotin, fluorophores, or other biomolecules to tubulin enable surface or cargo attachment, or visualization. This review summarizes different strategies for functionalizing microtubules for application-focused as well as basic biological research. These functionalization strategies must maintain the integrity of microtubule proteins so that they do not depolymerize and can be transported by kinesin motors, while adding utility such as the ability to reversibly bind cargo. The relevant biochemical and electrical properties of microtubules are discussed, as well as strategies for microtubule stabilization and long-term storage. Next, attachment strategies, such as antibodies and DNA hybridization that have proven useful to date, are discussed in the context of ongoing hybrid nanodevice research. The review concludes with a discussion of less explored opportunities, such as harnessing the utility of tubulin posttranslational modifications and the use of recombinant tubulin that may enable future progress in nanodevice development.

Microtubules as antifungal and antiparasitic drug targets

Introduction: Diseases caused by fungi and parasites are major illnesses in humans as well as in animals. Microtubule-targeted drugs are highly effective for the treatment of fungal and parasitic infections; however, several human parasitic infections such as malaria, trypanosomiasis and leishmaniasis do not have effective remedial drugs. In addition, the emergence of drug-resistant fungi and parasites makes the discovery of new drugs imperative.Areas covered: This article describes similarities and dissimilarities between parasitic, fungal and mammalian tubulins and focuses on microtubule-targeting agents and therapeutic approaches for the treatment of fungal and parasitic diseases. New microtubule-targeted antileishmanial, antimalarial and antifungal drugs, with structures, biological activities and related patents, are described. The potential of dsRNA against tubulin to inhibit proliferation of protozoan and helminthic parasites is also discussed. Patent documents up to 2010 have been searched on USPTO, Patentscope, and Espacenet resources.Expert opinion: The article suggests that vaccination with tubulin may offer novel opportunities for the antiparasitic treatment. Native or recombinant tubulin used as antigen has been shown to elicit immune response and cure infection partially or fully in animals upon challenge by protozoan parasites and helminths, thus indicating the suitability of tubulin as a vaccine against parasitic diseases.