Tubulin Heterodimers Research Articles

Benzimidazole resistance-associated mutations improve the in silico dimerization of hookworm tubulin: An additional resistance mechanism

Background and Aim: Mutations in the β-tubulin genes of helminths confer benzimidazole (BZ) resistance by reducing the drug’s binding efficiency to the expressed protein. However, the effects of these resistance-associated mutations on tubulin dimer formation in soil-transmitted helminths remain unknown. Therefore, this study aimed to investigate the impact of these mutations on the in silico dimerization of hookworm α- and β-tubulins using open-source bioinformatics tools. Materials and Methods: Using AlphaFold 3, the α- and β-tubulin amino acid sequences of Ancylostoma ceylanicum were used to predict the structural fold of the hookworm tubulin heterodimer. The modeled complexes were subjected to several protein structure quality assurance checks. The binding free energies, overall binding affinity, dissociation constant, and interacting amino acids of the complex were determined. The dimer’s structural flexibility and motion were simulated through molecular dynamics. Results: BZ resistance-associated amino acid substitutions in the β-tubulin isotype 1 protein of hookworms altered tubulin dimerization. The E198K, E198V, and F200Y mutations conferred the strongest and most stable binding between the α and β subunits, surpassing that of the wild-type. In contrast, complexes with the Q134H and F200L mutations exhibited the opposite effect. Molecular dynamics simulations showed that wild-type and mutant tubulin dimers exhibited similar dynamic behavior, with slight deviations in those carrying the F200L and E198K mutations. Conclusion: Resistance-associated mutations in hookworms impair BZ binding to β-tubulin and enhance tubulin dimer interactions, thereby increasing the parasite’s ability to withstand treatment. Conversely, other mutations weaken these interactions, potentially compromising hookworm viability. These findings offer novel insights into helminth tubulin dimerization and provide a valuable foundation for developing anthelmintics targeting this crucial biological process. Keywords: Ancylostoma, anthelmintic resistance, microtubules, soil-transmitted helminths.

Visualizing the Submolecular Organization of αβ-Tubulin Subunits on the Microtubule Inner Surface Using Atomic Force Microscopy.

Microtubules (MTs) are dynamic cytoskeletal polymers essential for mediating fundamental cellular processes, including cell division, intracellular transport, and cell shape maintenance. Understanding the arrangement of tubulin heterodimers within MTs is key to their function. Using frequency modulation atomic force microscopy (FM-AFM) and simulations, we revealed the submolecular arrangement of α- and β-tubulin subunits on the inner MT surface. We observed an undulating molecular arrangement of protofilaments (PFs) with alternating height variations, attributed to different structural orientations and the confirmation of αβ-tubulin heterodimers in adjacent PFs, forming bimodal lateral contacts, as confirmed by AFM simulations. Structural defects resulting from missing tubulin units were directly identified. This detailed structural information provides critical insight into the MT functional properties. Our findings highlight the potential of FM-AFM in liquid as a powerful tool for elucidating the complex interactions among MTs, MT-associated proteins, and other molecules, which are essential for understanding MT dynamics in the cellular context.

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.

A tubulin-binding protein that preferentially binds to GDP-tubulin and promotes GTP exchange.

α- and β-tubulin form heterodimers, with GTPase activity, that assemble into microtubules. Like other GTPases, the nucleotide-bound state of tubulin heterodimers controls whether the molecules are in a biologically active or inactive state. While α-tubulin in the heterodimer is constitutively bound to GTP, β-tubulin can be bound to either GDP (GDP-tubulin) or GTP (GTP-tubulin). GTP-tubulin hydrolyzes its GTP to GDP following assembly into a microtubule and, upon disassembly, must exchange its bound GDP for GTP to participate in subsequent microtubule polymerization. Tubulin dimers have been shown to exhibit rapid intrinsic nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.

Study on the mesoscopic dynamic effects of tumor treating fields on cell tubulin

Tumor treatment fields (TTFields) can effectively inhibit the proliferation of tumor cells, but its mechanism remains exclusive. The destruction of cellular microtubule structure caused by TTFields through electric field force is considered to be the main reason for inhibiting tumor cell proliferation. However, the validity of this hypothesis still lacks exploration at the mesoscopic level. Therefore, in this study, we built force models for tubulins subjected to TTFields, based on the physical and electrical properties of tubulin molecules. We theoretically analyzed and simulated the dynamic effects of electric field force and torque on tubulin monomer polymerization, as well as the alignment and orientation of α/β tubulin heterodimer, respectively. Research results indicate that the interference of electric field force induced by TTFields on tubulin monomer is notably weaker than the inherent electrostatic binding force among tubulin monomers. Additionally, the electric field torque generated by the TTFileds on α/β tubulin dimers is also difficult to affect their random alignment. Therefore, at the mesoscale, our study affirms that TTFields are improbable to destabilize cellular microtubule structures via electric field dynamics effects. These results challenge the traditional view that TTFields destroy the microtubule structure of cells through TTFields electric field force, and proposes a new approach that should pay more attention to the "non-mechanical" effects of TTFields in the study of TTFields mechanism. This study can provide reliable theoretical basis and inspire new research directions for revealing the mesoscopic bioelectrical mechanism of TTFields.

Role of tubulin C-terminal tail on mechanical properties of microtubule

Tubulin C-terminal tail (CTT) is a disordered segment extended from each tubulin monomer of αβ tubulin heterodimers, the building blocks of microtubules. The tubulin CTT contributes to the cellular function of microtubules such as intracellular transportation by regulating their interaction with other proteins and cell shape regulation by controlling microtubule polymerization dynamics. Although the mechanical integrity of microtubules is crucial for their functions, the role of tubulin CTT on microtubule mechanical properties has remained elusive. In this work, we investigate the role of tubulin CTTs in regulating the mechanical properties of microtubules by estimating the persistence lengths and investigating the buckling behavior of microtubules with and without CTT. We find that microtubules with intact CTTs exhibit twice the rigidity of microtubules lacking tubulin CTTs. Our study will widen the scope of altering microtubule mechanical properties for its application in nano bio-devices and lead to novel therapeutic approaches for neurodegenerative diseases with altered microtubule properties.

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.

Quantitative Microtubule Fractionation Technique to Separate Stable Microtubules, Labile Microtubules, and Free Tubulin in Mouse Tissues.

Microtubules, composed of α/β-tubulin dimers, are a crucial component of the cytoskeleton in eukaryotic cells. These tube-like polymers exhibit dynamic instability as tubulin heterodimer subunits undergo repetitive polymerization and depolymerization. Precise control of microtubule stability and dynamics, achieved through tubulin post-translational modifications and microtubule-associated proteins, is essential for various cellular functions. Dysfunctions in microtubules are strongly implicated in pathogenesis, including neurodegenerative disorders. Ongoing research focuses on microtubule-targeting therapeutic agents that modulate stability, offering potential treatment options for these diseases and cancers. Consequently, understanding the dynamic state of microtubules is crucial for assessing disease progression and therapeutic effects. Traditionally, microtubule dynamics have been assessed in vitro or in cultured cells through rough fractionation or immunoassay, using antibodies targeting post-translational modifications of tubulin. However, accurately analyzing tubulin status in tissues using such procedures poses challenges. In this study, we developed a simple and innovative microtubule fractionation method to separate stable microtubules, labile microtubules, and free tubulin in mouse tissues. The procedure involved homogenizing dissected mouse tissues in a microtubule-stabilizing buffer at a 19:1 volume ratio. The homogenates were then fractionated through a two-step ultracentrifugation process following initial slow centrifugation (2,400 × g) to remove debris. The first ultracentrifugation step (100,000 × g) precipitated stable microtubules, while the resulting supernatant was subjected to a second ultracentrifugation step (500,000 × g) to fractionate labile microtubules and soluble tubulin dimers. This method determined the proportions of tubulin constituting stable or labile microtubules in the mouse brain. Additionally, distinct tissue variations in microtubule stability were observed that correlated with the proliferative capacity of constituent cells. These findings highlight the significant potential of this novel method for analyzing microtubule stability in physiological and pathological conditions.

Structural insights into the mechanism of GTP initiation of microtubule assembly

In eukaryotes, the dynamic assembly of microtubules (MT) plays an important role in numerous cellular processes. The underlying mechanism of GTP triggering MT assembly is still unknown. Here, we present cryo-EM structures of tubulin heterodimer at their GTP- and GDP-bound states, intermediate assembly states of GTP-tubulin, and final assembly stages of MT. Both GTP- and GDP-tubulin heterodimers adopt similar curved conformations with subtle flexibility differences. In head-to-tail oligomers of tubulin heterodimers, the inter-dimer interface of GDP-tubulin exhibits greater flexibility, particularly in tangential bending. Cryo-EM of the intermediate assembly states reveals two types of tubulin lateral contacts, “Tube-bond” and “MT-bond”. Further, molecular dynamics (MD) simulations show that GTP triggers lateral contact formation in MT assembly in multiple sequential steps, gradually straightening the curved tubulin heterodimers. Therefore, we propose a flexible model of GTP-initiated MT assembly, including the formation of longitudinal and lateral contacts, to explain the nucleation and assembly of MT.

α-tubulin regulation by 5' introns in Saccharomyces cerevisiae.

Across eukaryotic genomes, multiple α- and β-tubulin genes require regulation to ensure sufficient production of tubulin heterodimers. Features within these gene families that regulate expression remain underexplored. Here, we investigate the role of the 5' intron in regulating α-tubulin expression in Saccharomyces cerevisiae. We find that the intron in the α-tubulin, TUB1, promotes α-tubulin expression and cell fitness during microtubule stress. The role of the TUB1 intron depends on proximity to the TUB1 promoter and sequence features that are distinct from the intron in the alternative α-tubulin isotype, TUB3. These results lead us to perform a screen to identify genes that act with the TUB1 intron. We identified several genes involved in chromatin remodeling, α/β-tubulin heterodimer assembly, and the spindle assembly checkpoint. We propose a model where the TUB1 intron promotes expression from the chromosomal locus and that this may represent a conserved mechanism for tubulin regulation under conditions that require high levels of tubulin production.

Synthesis and biological evaluation of structurally diverse 6-aryl-3-aroyl-indole analogues as inhibitors of tubulin polymerization

The synthesis and evaluation of small-molecule inhibitors of tubulin polymerization remains a promising approach for the development of new therapeutic agents for cancer treatment. The natural products colchicine and combretastatin A-4 (CA4) inspired significant drug discovery campaigns targeting the colchicine site located on the beta-subunit of the tubulin heterodimer, but so far these efforts have not yielded an approved drug for cancer treatment in human patients. Interest in the colchicine site was enhanced by the discovery that a subset of colchicine site agents demonstrated dual functionality as both potent antiproliferative agents and effective vascular disrupting agents (VDAs). Our previous studies led to the discovery and development of a 2-aryl-3-aroyl-indole analogue (OXi8006) that inhibited tubulin polymerization and demonstrated low nM IC50 values against a variety of human cancer cell lines. A water-soluble phosphate prodrug salt (OXi8007), synthesized from OXi8006, displayed promising vascular disrupting activity in mouse models of cancer. To further extend structure-activity relationship correlations, a series of 6-aryl-3-aroyl-indole analogues was synthesized and evaluated for their inhibition of tubulin polymerization and cytotoxicity against human cancer cell lines. Several structurally diverse molecules in this small library were strong inhibitors of tubulin polymerization and of MCF-7 and MDA-MB-231 human breast cancer cells. One of the most promising analogues (KGP591) caused significant G2/M arrest of MDA-MB-231 cells, disrupted microtubule structure and cell morphology in MDA-MB-231 cells, and demonstrated significant inhibition of MDA-MB-231 cell migration in a wound healing (scratch) assay. A phosphate prodrug salt, KGP618, synthesized from its parent phenolic precursor, KGP591, demonstrated significant reduction in bioluminescence signal when evaluated in vivo against an orthotopic model of kidney cancer (RENCA-luc) in BALB/c mice, indicative of VDA efficacy. The most active compounds from this series offer promise as anticancer therapeutic agents.

Exploration of the Pharmacophore for Cytoskeletal Targeting Ruthenium Polypyridyl Complexes.

Ruthenium(II) trisdiimine complexes of the formula, [Ru(dip)n (L-L)3-n ]2+ , where n=0-3; dip=4,7-diphenyl-1,10-phenanthroline; L-L=2,2'-bipyridine (bpy) or 1,10-phenanthroline (phen) were prepared and tested for cytotoxicity in two cell lines (H358, MCF7). Cellular uptake and subcellular localization were determined by harvesting treated cells and determining the ruthenium concentration in whole or fractionated cells (cytosolic, nuclear, mitochondrial/ ER/Golgi, and cytoskeletal proteins) by Ru ICP-MS. The logP values for the chloride salts of these complexes were measured and the data were analyzed to determine the role of lipophilicity versus structure in the various biological assays. Cellular uptake increased with lipophilicity but shows the biggest jump when the complex contains two or more dip ligands. Significantly, preferential cytoskeletal localization is also correlated with increased cytotoxicity. All of the RPCs promote tubulin polymerization in vitro, but [Ru(dip)2 phen]2+ and [Ru(dip)3 ]2+ show the strongest activity. Analysis of the pellet formed by centrifugation of MTs formed in the presence of [Ru(dip)2 phen]2+ establish a binding stoichiometry of one RPC per tubulin heterodimer. Complexes of the general formula [Ru(dip)2 (L-L)]2+ possess the necessary characteristics to target the cytoskeleton in live cells and increase cytotoxicity, however the nature of the L-L ligand does influence the extent of the effect.

Effect of Colchicine Binding Site Inhibitors on the Tubulin Intersubunit Interaction.

Microtubules are dynamic, non-covalent polymers consisting of α- and β-tubulin subunits that are involved in a wide range of intracellular processes. The polymerization and dynamics of microtubules are regulated by many factors, including small molecules that interact with different sites on the tubulin dimer. Colchicine binding site inhibitors (CBSIs) destabilize microtubules and inhibit tubulin polymerization, leading to cell cycle arrest. Because of their therapeutic potential, the molecular mechanism of CBSI function is an area of active research. Nevertheless, important details of this mechanism have yet to be resolved. In this study, we use atomistic molecular dynamics simulations to show that the binding of CBSIs to the tubulin heterodimer leads to the weakening of tubulin intersubunit interaction. Using atomistic molecular dynamics simulations and binding free energy calculations, we show that CBSIs act as protein-protein interaction inhibitors and destabilize interlinkage between α and β subunits, which is crucial for longitudinal contacts in the microtubule lattice. Our results offer new insight into the mechanisms of microtubule polymerization inhibition by colchicine and its analogs.

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.

Structure Guided Design, Synthesis, and Biological Evaluation of Oxetane-Containing Indole Analogues

The oxetane functional group offers a variety of potential advantages when incorporated within appropriate therapeutic agents as a ketone surrogate. OXi8006, a 2-aryl-3-aroyl-indole analogue, functions as a small-molecule inhibitor of tubulin polymerization that has a dual mechanism of action as both an antiproliferative agent and a tumor-selective vascular disrupting agent. Replacement of the bridging ketone moiety in OXi8006 with an oxetane functional group has expanded structure activity relationship (SAR) knowledge and provided insights regarding oxetane incorporation within this class of molecules. A new synthetic method using an oxetane-containing tertiary alcohol subjected to Lewis acid catalyzed conditions led to successful Friedel–Crafts alkylation and yielded fourteen new oxetane-containing indole-based molecules. This synthetic approach represents the first method to successfully install an oxetane ring at the 3-position of a 2-aryl-indole system. Several analogues showed potent cytotoxicity (micromolar GI50 values) against human breast cancer cell lines (MCF-7 and MDA-MB-231) and a pancreatic cancer cell line (PANC-1), although they proved to be ineffective as inhibitors of tubulin polymerization. Molecular docking studies comparing colchicine with the OXi8006-oxetane analogue 5m provided a rationale for the differential interaction of these molecules with the colchicine site on the tubulin heterodimer.

Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination.

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.

Structural basis of protein condensation on microtubules underlying branching microtubule nucleation

Targeting protein for Xklp2 (TPX2) is a key factor that stimulates branching microtubule nucleation during cell division. Upon binding to microtubules (MTs), TPX2 forms condensates via liquid-liquid phase separation, which facilitates recruitment of microtubule nucleation factors and tubulin. We report the structure of the TPX2 C-terminal minimal active domain (TPX2α5-α7) on the microtubule lattice determined by magic-angle-spinning NMR. We demonstrate that TPX2α5-α7 forms a co-condensate with soluble tubulin on microtubules and binds to MTs between two adjacent protofilaments and at the intersection of four tubulin heterodimers. These interactions stabilize the microtubules and promote the recruitment of tubulin. Our results reveal that TPX2α5-α7 is disordered in solution and adopts a folded structure on MTs, indicating that TPX2α5-α7 undergoes structural changes from unfolded to folded states upon binding to microtubules. The aromatic residues form dense interactions in the core, which stabilize folding of TPX2α5-α7 on microtubules. This work informs on how the phase-separated TPX2α5-α7 behaves on microtubules and represents an atomic-level structural characterization of a protein that is involved in a condensate on cytoskeletal filaments.

Systematic Studies on Anti-Cancer Evaluation of Stilbene and Dibenzo[b,f]oxepine Derivatives.

Cancer is one of the most common causes of human death worldwide; thus, numerous therapies, including chemotherapy, have been and are being continuously developed. In cancer cells, an aberrant mitotic spindle-a microtubule-based structure necessary for the equal splitting of genetic material between daughter cells-leads to genetic instability, one of the hallmarks of cancer. Thus, the building block of microtubules, tubulin, which is a heterodimer formed from α- and β-tubulin proteins, is a useful target in anti-cancer research. The surface of tubulin forms several pockets, i.e., sites that can bind factors that affect microtubules' stability. Colchicine pockets accommodate agents that induce microtubule depolymerization and, in contrast to factors that bind to other tubulin pockets, overcome multi-drug resistance. Therefore, colchicine-pocket-binding agents are of interest as anti-cancer drugs. Among the various colchicine-site-binding compounds, stilbenoids and their derivatives have been extensively studied. Herein, we report systematic studies on the antiproliferative activity of selected stilbenes and oxepine derivatives against two cancer cell lines-HCT116 and MCF-7-and two normal cell lines-HEK293 and HDF-A. The results of molecular modeling, antiproliferative activity, and immunofluorescence analyses revealed that compounds 1a, 1c, 1d, 1i, 2i, 2j, and 3h were the most cytotoxic and acted by interacting with tubulin heterodimers, leading to the disruption of the microtubular cytoskeleton.

Maize tubulin folding cofactor B is required for cell division and cell growth through modulating microtubule homeostasis.

Tubulin folding cofactors (TFCs) are required for tubulin folding, α/β tubulin heterodimer formation, and microtubule (MT) dynamics in yeast and mammals. However, the functions of their plant counterparts remain to be characterized. We identified a natural maize crumpled kernel mutant, crk2, which exhibits reductions in endosperm cell number and size, as well as embryo/seedling lethality. Map-based cloning and functional complementation confirmed that ZmTFCB is causal for the mutation. ZmTFCB is targeted mainly to the cytosol. It facilitates α-tubulin folding and heterodimer formation through sequential interactions with the cytosolic chaperonin-containing TCP-1 ε subunit ZmCCT5 and ZmTFCE, thus affecting the organization of both the spindle and phragmoplast MT array and the cortical MT polymerization and array formation, which consequently mediated cell division and cell growth. We detected a physical association between ZmTFCB and the maize MT plus-end binding protein END-BINDING1 (ZmEB1), indicating that ZmTFCB1 may modulate MT dynamics by sequestering ZmEB1. Our data demonstrate that ZmTFCB is required for cell division and cell growth through modulating MT homeostasis, an evolutionarily conserved machinery with some species-specific divergence.

Structure-Guided Prediction of the Functional Impact of DCLK1 Mutations on Tumorigenesis.

Doublecortin-like kinase 1 (DCLK1) is a functional serine/threonine (S/T)-kinase and a member of the doublecortin family of proteins which are characterized by their ability to bind to microtubules (MTs). DCLK1 is a proposed cancer driver gene, and its upregulation is associated with poor overall survival in several solid cancer types. However, how DCLK1 associates with MTs and how its kinase function contributes to pro-tumorigenic processes is poorly understood. This review builds on structural models to propose not only the specific functions of the domains but also attempts to predict the impact of individual somatic missense mutations on DCLK1 functions. Somatic missense mutations in DCLK1 are most frequently located within the N-terminal MT binding region and likely impact on the ability of DCLK1 to bind to αβ-tubulin and to polymerize and stabilize MTs. Moreover, the MT binding affinity of DCLK1 is negatively regulated by its auto-phosphorylation, and therefore mutations that affect kinase activity are predicted to indirectly alter MT dynamics. The emerging picture portrays DCLK1 as an MT-associated protein whose interactions with tubulin heterodimers and MTs are tightly controlled processes which, when disrupted, may confer pro-tumorigenic properties.