Comprehensive analysis of two hotspot codons in the TUBB4B gene and associated phenotypes.

Our purpose was to elucidate the genotype and ophthalmological and audiological phenotype in TUBB4B-associated inherited retinal dystrophy (IRD) and sensorineural hearing loss (SNHL), and to model the effects of all possible amino acid substitutions at the hotspot codons Arg390 and Arg391. Six patients from five families with heterozygous missense variants in TUBB4B were included in this observational study. Ophthalmological testing included best-corrected visual acuity, fundus examination, optical coherence tomography, fundus autofluorescence imaging, and full-field electroretinography (ERG). Audiological examination included pure-tone and speech audiometry in adult patients and auditory brainstem response testing in a child. Genetic testing was performed by disease gene panel analysis based on genome sequencing. The molecular consequences of the substitutions of residues 390 and 391 on TUBB4B and its interaction with α-tubulin were predicted in silico on its three-dimensional structure obtained by homology modelling. Two independent patients had amino acid exchanges at position 391 (p.(Arg391His) or p.(Arg391Cys)) of the TUBB4B protein. Both had a distinct IRD phenotype with peripheral round yellowish lesions with pigmented spots and mild or moderate SNHL, respectively. Yet the phenotype was milder with a sectorial pattern of bone spicules in one patient, likely due to a genetically confirmed mosaicism for p.(Arg391His). Three patients were heterozygous for an amino acid exchange at position 390 (p.(Arg390Gln) or p.(Arg390Trp)) and presented with another distinct retinal phenotype with well demarcated pericentral retinitis pigmentosa. All showed SNHL ranging from mild to severe. One additional patient showed a variant distinct from codon 390 or 391 (p.(Tyr310His)), and presented with congenital profound hearing loss and reduced responses in ERG. Variants at codon positions 390 and 391 were predicted to decrease the structural stability of TUBB4B and its complex with α-tubulin, as well as the complex affinity. In conclusion, the twofold larger reduction in heterodimer affinity exhibited by Arg391 substitutions suggested an association with the more severe retinal phenotype, compared to the substitution at Arg390.

Ultraviolet Superradiance from Mega-Networks of Tryptophan in Biological Architectures.

Networks of tryptophan (Trp)─an aromatic amino acid with strong fluorescence response─are ubiquitous in biological systems, forming diverse architectures in transmembrane proteins, cytoskeletal filaments, subneuronal elements, photoreceptor complexes, virion capsids, and other cellular structures. We analyze the cooperative effects induced by ultraviolet (UV) excitation of several biologically relevant Trp mega-networks, thus giving insights into novel mechanisms for cellular signaling and control. Our theoretical analysis in the single-excitation manifold predicts the formation of strongly superradiant states due to collective interactions among organized arrangements of up to >105 Trp UV-excited transition dipoles in microtubule architectures, which leads to an enhancement of the fluorescence quantum yield (QY) that is confirmed by our experiments. We demonstrate the observed consequences of this superradiant behavior in the fluorescence QY for hierarchically organized tubulin structures, which increases in different geometric regimes at thermal equilibrium before saturation, highlighting the effect's persistence in the presence of disorder. Our work thus showcases the many orders of magnitude across which the brightest (hundreds of femtoseconds) and darkest (tens of seconds) states can coexist in these Trp lattices.

The α-tubulin acetyltransferase ATAT1: structure, cellular functions, and its emerging role in human diseases.

The acetylation of α-tubulin on lysine 40 is a well-studied post-translational modification which has been associated with the presence of long-lived stable microtubules that are more resistant to mechanical breakdown. The discovery of α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for lysine 40 acetylation on α-tubulin in a wide range of species, including protists, nematodes, and mammals, dates to about a decade ago. However, the role of ATAT1 in different cellular activities and molecular pathways has been only recently disclosed. This review comprehensively summarizes the most recent knowledge on ATAT1 structure and substrate binding and analyses the involvement of ATAT1 in a variety of cellular processes such as cell motility, mitosis, cytoskeletal organization, and intracellular trafficking. Finally, the review highlights ATAT1 emerging roles in human diseases and discusses ATAT1 potential enzymatic and non-enzymatic roles and the current efforts in developing ATAT1 inhibitors.

Induction of autophagy-dependent and caspase- and microtubule-acetylation-independent cell death by phytochemical-stabilized gold nanopolygons in colorectal adenocarcinoma cells.

Collective functionalization of the phytochemicals of medicinal herbs on nanoparticles is emerging as a potential cancer therapeutic strategy. This study presents the facile synthesis of surface-functionalized gold nanoparticles using Bacopa monnieri (Brahmi; Bm) phytochemicals and their therapeutically relevant mechanism of action in the colorectal cancer cell line, HT29. The nanoparticles were characterized using UV-visible spectroscopy, TEM-EDAX, zeta potential analysis, TGA, FTIR and 1H NMR spectroscopy, and HR-LC-MS. The particles (Bm-GNPs) were of polygonal shape and were stable against aggregation. They entered the target cells and inhibited the viability and clonogenicity of the cells with eight times more antiproliferative efficacy (25 ± 1.5 µg mL-1) than Bm extract (Bm-EX). In vitro studies revealed that Bm-GNPs bind tubulin (a protein crucial in cell division and a target of anticancer drugs) and disrupt its helical structure without grossly altering its tertiary conformation. Like other antitubulin agents, Bm-GNPs induced G2/M arrest and ultimately killed the cells, as confirmed using flow cytometry analyses. ZVAD-FMK-mediated global pan-caspase inhibition and the apparent absence of cleaved caspase-3 in treated cells indicated that the death did not involve the classic apoptosis pathway. Cellular ultrastructure analyses, western immunoblots, and in situ immunofluorescence visualization of cellular microtubules revealed microtubule-acetylation-independent induction of autophagy as the facilitator of cell death. Together, the data indicate strong antiproliferative efficacy and a possible mechanism of action for these designer nanoparticles. Bm-GNPs, therefore, merit further investigations, including preclinical evaluations, for their therapeutic potential as inducers of non-apoptotic cell death.

Synthesis of Estrone Heterodimers and Evaluation of Their In Vitro Antiproliferative Activity.

Directed structural modifications of natural products offer excellent opportunities to develop selectively acting drug candidates. Natural product hybrids represent a particular compound group. The components of hybrids constructed from different molecular entities may result in synergic action with diminished side effects. Steroidal homo- or heterodimers deserve special attention owing to their potentially high anticancer effect. Inspired by our recently described antiproliferative core-modified estrone derivatives, here, we combined them into heterodimers via Cu(I)-catalyzed azide-alkyne cycloaddition reactions. The two trans-16-azido-3-(O-benzyl)-17-hydroxy-13α-estrone derivatives were reacted with 3-O-propargyl-D-secoestrone alcohol or oxime. The antiproliferative activities of the four newly synthesized dimers were evaluated against a panel of human adherent gynecological cancer cell lines (cervical: Hela, SiHa, C33A; breast: MCF-7, T47D, MDA-MB-231, MDA-MB-361; ovarian: A2780). One heterodimer (12) exerted substantial antiproliferative activity against all investigated cell lines in the submicromolar or low micromolar range. A pronounced proapoptotic effect was observed by fluorescent double staining and flow cytometry on three cervical cell lines. Additionally, cell cycle blockade in the G2/M phase was detected, which might be a consequence of the effect of the dimer on tubulin polymerization. Computational calculations on the taxoid binding site of tubulin revealed potential binding of both steroidal building blocks, mainly with hydrophobic interactions and water bridges.

In-Cell Association of a Bioorthogonal Tubulin.

Studies of proteins from one organism in another organism's cells have shown that such exogenous proteins stick more, pointing toward coevolution of the cytoplasm and protein surface to minimize stickiness. Here we flip this question around by asking whether exogenous proteins can assemble efficiently into their target complexes in a non-native cytoplasm. We use as our model system the assembly of BtubA and BtubB from Prosthecobacter hosted in human U-2 OS cells. BtubA and B evolved from eukaryotic tubulins after horizontal gene transfer, but they have low surface sequence identity with the homologous human tubulins and do not respond to tubulin drugs such as nocodazole. In U-2 OS cells, BtubA and B assemble efficiently into dimers compared to in vitro, and the wild-type BtubA and B proteins subsequently are able to form microtubules as well. We find that generic crowding effects (Ficoll 70 in vitro) contribute significantly to efficient dimer assembly when compared to sticking interactions (U-2 OS cell lysate in vitro), consistent with the notion that a generic mechanism such as crowding can be effective at driving assembly of exogenous proteins, even when protein-cytoplasm quinary structure and sticking have been modified in a non-native cytoplasm. A simple Monte Carlo model of in vitro and in-cell interactions, treating BtubA and B as sticky dipoles in a matrix of sticky or nonsticky crowders, rationalizes all the experimental trends with two adjustable parameters and reveals nucleation as the likely mechanism for the time-scale separation between dimer- and tubule formation in-cell and in vitro.

Anthraquinones from the roots of Morinda scabrida Craib exhibit antiproliferative activity against A549 lung cancer cells and antitubulin polymerization.

Six anthraquinones were isolated from Morinda scabrida Craib, an unexplored species of Morinda found in the tropical forest of Thailand. All six anthraquinones showed cytotoxicity against A549 lung cancer cells, with the most active compound, nordamnacanthal (MS01), exhibiting the IC50 value of 16.3 ± 2.5 µM. The cytotoxic effect was dose-dependent and led to cell morphological changes characteristic of apoptosis. In addition, flow cytometric analysis showed dose-dependent apoptosis induction and the G2/M phase cell cycle arrest, which was in agreement with the tubulin polymerization inhibitory activity of MS01. Molecular docking analysis illustrated the binding between MS01 and the α/ß-tubulin heterodimer at the colchicine binding site, and UV-visible absorption spectroscopy revealed the DNA binding capacity of MS01.

Ivermectin affects Arabidopsis thaliana microtubules through predicted binding site of ß-tubulin.

The ivermectin is a potent nematocide and insecticide, which has low toxicity for humans and domestic animals, but due to low biotransformation, it can be dangerous for non-target organisms. The recent determination of ivermectin absorption and accumulation in tissues of higher plants and multiple shreds of evidence of its negative impact on plant physiology provide a basis for the search for ivermectin's molecular targets and mechanisms of action in plant cells. In this research, for the first time, the ivermectin effect on microtubules of Arabidopsis thaliana cells was studied. It was revealed that ivermectin (250 µg mL-1) disrupts the microtubule network, induces the loss of microtubule orientation, leads to microtubule curvature and shrinkage, and their longitudinal and cross-linked bundling in various cells of A. thaliana primary roots. Further, the previously proposed binding of ivermectin to the ß1-tubulin taxane site was developed and confirmed using molecular dynamics simulations of ivermectin complexes with Haemonchus contortus and A. thaliana ß1-tubulins. It was predicted that similar to other microtubule stabilizing agents ivermectin binding causes M-loop stabilization in both H. contortus and A. thaliana ß-tubulin, which leads to the enhancement of lateral contacts between subunits of adjacent protofilaments preventing microtubule depolymerization.

Beyond uniformity: Exploring the heterogeneous and dynamic nature of the microtubule lattice.

A fair amount of research on microtubules since their discovery in 1963 has focused on their dynamic tips. In contrast, the microtubule lattice was long believed to be highly regular and static, and consequently received far less attention. Yet, as it turned out, the microtubule lattice is neither as regular, nor as static as previously believed: structural studies uncovered the remarkable wealth of different conformations the lattice can accommodate. In the last decade, the microtubule lattice was shown to be labile and to spontaneously undergo renovation, a phenomenon that is intimately linked to structural defects and was called "microtubule self-repair". Following this breakthrough discovery, further recent research provided a deeper understanding of the lattice self-repair mechanism, which we review here. Instrumental to these discoveries were in vitro microtubule reconstitution assays, in which microtubules are grown from the minimal components required for their dynamics. In this review, we propose a shift from the term "lattice self-repair" to "lattice dynamics", since this phenomenon is an inherent property of microtubules and can happen without microtubule damage. We focus on how in vitro microtubule reconstitution assays helped us learn (1) which types of structural variations microtubules display, (2) how these structural variations influence lattice dynamics and microtubule damage caused by mechanical stress, (3) how lattice dynamics impact tip dynamics, and (4) how microtubule-associated proteins (MAPs) can play a role in structuring the lattice. Finally, we discuss the unanswered questions about lattice dynamics and how technical advances will help us tackle these questions.

Controlling Supramolecular Fiber Formation of Nucleopeptide by Guanosine Triphosphate.

Cells use dynamic self-assembly to construct functional structures for maintaining cellular homeostasis. However, using a natural biological small molecule to mimic this phenomenon remains challenging. This work reports the dynamic microfiber formation of nucleopeptide driven by guanosine triphosphate, the small molecule that controls microtubule polymerization in living cells. Deactivation of GTP by enzyme dissociates the fibers, which could be reactivated by adding GTP. Molecular dynamic simulation unveils the mystery of microfiber formation of GBM-1 and GTP. Moreover, the microfiber formation can also be controlled by diffusion-driven GTP gradients across a semipermeable membrane in bulk conditions and the microfluidic method in the defined droplets. This study provides a new platform to construct dynamic self-assembly materials of molecular building blocks driven by GTP.

The Tubulin Code, from Molecules to Health and Disease.

Microtubules are essential dynamic polymers composed of α/ß-tubulin heterodimers. They support intracellular trafficking, cell division, cellular motility, and other essential cellular processes. In many species, both α-tubulin and ß-tubulin are encoded by multiple genes with distinct expression profiles and functionality. Microtubules are further diversified through abundant posttranslational modifications, which are added and removed by a suite of enzymes to form complex, stereotyped cellular arrays. The genetic and chemical diversity of tubulin constitute a tubulin code that regulates intrinsic microtubule properties and is read by cellular effectors, such as molecular motors and microtubule-associated proteins, to provide spatial and temporal specificity to microtubules in cells. In this review, we synthesize the rapidly expanding tubulin code literature and highlight limitations and opportunities for the field. As complex microtubule arrays underlie essential physiological processes, a better understanding of how cells employ the tubulin code has important implications for human disease ranging from cancer to neurological disorders.

Microtubule rescue control by drugs and MAPs examined with in vitro pedestal assay.

Microtubules are essential cytoskeletal polymers, which exhibit stochastic transitions between assembly and disassembly, known as catastrophes and rescues. Understanding of catastrophes, rescues, and their control by drugs and microtubule associated proteins (MAPs) has been informed by in vitro reconstitutions of microtubule dynamics. In such experiments microtubules are typically observed on a flat surface of the coverslip. In contrast, we have recently proposed a modified setup in which microtubules assemble from stabilized seeds, overhanging from microfabricated pedestals, so that their dynamic extensions are fully isolated from contact with the coverslip. This assay allows to eliminate potential artifacts, which may substantially affect the frequency of microtubule rescues in vitro. Here we use the pedestal assay to study the sensitivity of microtubules to paclitaxel, one of the best-known inhibitors of microtubule dynamics. By comparing observations in the conventional and the pedestal assays, we find that microtubule dynamics are substantially more sensitive to paclitaxel when the polymers can contact the coverslip. We interpret this as a consequence of the coverslip-induced microtubule assembly perturbation, leading to formation of lattice with defects, and thereby enhancing the efficiency of paclitaxel binding to microtubules in the conventional assay. To test this idea, we use vinblastine, another small-molecule inhibitor, which had been previously shown to cause microtubule growth perturbations. We find that in the pedestal assay vinblastine sensitizes microtubules to paclitaxel to the level, observed in the conventional assay. Interestingly, a minimal fragment of MAP called CLASP2, a previously characterized rescue factor, has a strong effect on microtubule rescues, regardless of the type of assay. Overall, our study underscores the role of microtubule damage in promoting rescues and highlights the utility of the in vitro pedestal assay to study microtubule dynamics modulation by tubulin inhibitors and MAPs.

An adhesive peptide specifically induces microtubule condensation.

Cell function-associated biomolecular condensation has great potential in modulation of molecular activities. We develop a microtubule-trapping peptide that first self-assembles into nanoparticles and then in situ transforms into nanofibers via ligand-receptor interactions when targeted to tubulin. The nanofibers support the increased exposed targets for further adhering to microtubules and induce the self-assembly of microtubules into networks due to multivalent effects. Microtubule condensation with prolonged retention in cells for up to 24 h, which is 6 times longer than that of the non-transformable nanoparticle group, efficiently induces in vitro cell apoptosis and inhibits in vivo tumour growth. These smart transformable peptide materials for targeted protein condensation have the potential for improving retention and inducing cell apoptosis in tumour therapy.

New insights into the mechanochemical coupling mechanism of kinesin-microtubule complexes from their high-resolution structures.

Kinesin motor proteins couple mechanical movements in their motor domain to the binding and hydrolysis of ATP in their nucleotide-binding pocket. Forces produced through this 'mechanochemical' coupling are typically used to mobilize kinesin-mediated transport of cargos along microtubules or microtubule cytoskeleton remodeling. This review discusses the recent high-resolution structures (<4 Å) of kinesins bound to microtubules or tubulin complexes that have resolved outstanding questions about the basis of mechanochemical coupling, and how family-specific modifications of the motor domain can enable its use for motility and/or microtubule depolymerization.

Identification of Novel Dual-Target Estrogen Receptor α Degraders with Tubulin Inhibitory Activity for the Treatment of Endocrine-Resistant Breast Cancer.

Endocrine resistance remains a significant problem in the clinical treatment of estrogen receptor α-positive (ERα+) breast cancer (BC). In this study, we developed a series of novel dual-functional ERα degraders based on a bridged bicyclic scaffold with selenocyano (SeCN) side chains. These compounds displayed potent ERα degradation and tubulin depolymerization activity. Among them, compounds 35s and 35t exhibited the most promising antiproliferative and ERα degradation activity in multiple ERα+ BC cell lines bearing either wild-type or mutant ERα. Meanwhile, compounds 35s and 35t disrupted the microtubule network by restraining tubulin polymerization, evidenced by 35t inducing cell cycle arrest in the G2/M phase. In MCF-7 and LCC2 xenograft models, compounds 35s and 35t remarkably suppressed tumor growth without noticeable poisonousness. Finally, this study provided guidance for developing new dual-target antitumor drug candidates for the ERα+ BC therapy, especially for the resistant variant.

Evolution of a minimal cell.

Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.

Microtubules and Cell Division: Potential Pharmacological Targets in Cancer Therapy.

Microtubules are a well-known target in cancer chemotherapy because of their critical role in cell division. Chromosome segregation during mitosis depends on the establishment of the mitotic spindle apparatus through microtubule dynamics. The disruption of microtubule dynamics through the stabilization or destabilization of microtubules results in the mitotic arrest of the cells. Microtubule-targeted drugs, which interfere with microtubule dynamics, inhibit the growth of cells at the mitotic phase and induce apoptotic cell death. The principle of microtubule-targeted drugs is to arrest the cells at mitosis and reduce their growth because cancer is a disease of unchecked cell proliferation. Many anti-microtubule agents produce significant inhibition of cancer cell growth and are widely used as chemotherapeutic drugs for the treatment of cancer. The drugs that interact with microtubules generally bind at one of the three sites vinblastine site, taxol site, or colchicine site. Colchicine binds to the interface of tubulin heterodimer and induces the depolymerization of microtubules. The colchicine binding site on microtubules is a much sought-after target in the history of anti-microtubule drug discovery. Many colchicine-binding site inhibitors have been discovered, but their use in the treatment of cancer is limited due to their dose-limiting toxicity and resistance in humans. Combination therapy can be a new treatment strategy to overcome these drawbacks of currently available microtubule-targeted anticancer drugs. This review discusses the significance of microtubules as a potential pharmacological target for cancer and stresses the necessity of finding new microtubule inhibitors to fight the disease.

Developing Hybrid All-Atom and Ultra-Coarse-Grained Models to Investigate Taxol-Binding and Dynein Interactions on Microtubules.

Simulating the conformations and functions of biological macromolecules by using all-atom (AA) models is a challenging task due to expensive computational costs. One possible strategy to solve this problem is to develop hybrid all-atom and ultra-coarse-grained (AA/UCG) models of the biological macromolecules. In the AA/UCG scheme, the interest regions are described by AA models, while the other regions are described in the UCG representation. In this study, we develop the hybrid AA/UCG models and apply them to investigate the conformational changes of microtubule-bound tubulins. The simulation results of the hybrid models elucidated the mechanism of why the taxol molecules selectively bound microtubules but not tubulin dimers. In addition, we also explore the interactions of the microtubules and dyneins. Our study shows that the hybrid AA/UCG model has great application potential in studying the function of complex biological systems.

Cryo-EM of α-tubulin isotype-containing microtubules revealed a contracted structure of α4A/ß2A microtubules.

Microtubules are hollow α/ß-tubulin heterodimeric polymers that play critical roles in cells. In vertebrates, both α- and ß-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 ß-tubulin isotype ß2A. We further assemble and determine the cryo-electron microscopy (cryo-EM) structures of α1A/ß2A, α1C/ß2A, and α4A/ß2A microtubules. Our structural analysis demonstrates that α4A/ß2A microtubules exhibit longitudinal contraction between tubulin interdimers compared with α1A/ß2A and α1C/ß2A microtubules. Collectively, our findings reveal that α-tubulin isotype composition can tune microtubule structures, and also provide evidence for the "tubulin code" hypothesis.

Unveiling the catalytic mechanism of GTP hydrolysis in microtubules.

Microtubules (MTs) are large cytoskeletal polymers, composed of αß-tubulin heterodimers, capable of stochastically converting from polymerizing to depolymerizing states and vice versa. Depolymerization is coupled with hydrolysis of guanosine triphosphate (GTP) within ß-tubulin. Hydrolysis is favored in the MT lattice compared to a free heterodimer with an experimentally observed rate increase of 500- to 700-fold, corresponding to an energetic barrier lowering of 3.8 to 4.0 kcal/mol. Mutagenesis studies have implicated α-tubulin residues, α:E254 and α:D251, as catalytic residues completing the ß-tubulin active site of the lower heterodimer in the MT lattice. The mechanism for GTP hydrolysis in the free heterodimer, however, is not understood. Additionally, there has been debate concerning whether the GTP-state lattice is expanded or compacted relative to the GDP state and whether a "compacted" GDP-state lattice is required for hydrolysis. In this work, extensive quantum mechanics/molecular mechanics simulations with transition-tempered metadynamics free-energy sampling of compacted and expanded interdimer complexes, as well as a free heterodimer, have been carried out to provide clear insight into the GTP hydrolysis mechanism. α:E254 was found to be the catalytic residue in a compacted lattice, while in the expanded lattice, disruption of a key salt bridge interaction renders α:E254 less effective. The simulations reveal a barrier decrease of 3.8 ± 0.5 kcal/mol for the compacted lattice compared to a free heterodimer, in good agreement with experimental kinetic measurements. Additionally, the expanded lattice barrier was found to be 6.3 ± 0.5 kcal/mol higher than compacted, demonstrating that GTP hydrolysis is variable with lattice state and slower at the MT tip.