Prolonged depletion of profilin 1 or F-actin causes an adaptive response in microtubules.

In addition to its well-established role in actin assembly, profilin 1 (PFN1) has been shown to bind to tubulin and alter microtubule growth. However, whether PFN1's predominant control over microtubules in cells occurs through direct regulation of tubulin or indirectly through the polymerization of actin has yet to be determined. Here, we manipulated PFN1 expression, actin filament assembly, and actomyosin contractility and showed that reducing any of these parameters for extended periods of time caused an adaptive response in the microtubule cytoskeleton, with the effect being significantly more pronounced in neuronal processes. All the observed changes to microtubules were reversible if actomyosin was restored, arguing that PFN1's regulation of microtubules occurs principally through actin. Moreover, the cytoskeletal modifications resulting from PFN1 depletion in neuronal processes affected microtubule-based transport and mimicked phenotypes that are linked to neurodegenerative disease. This demonstrates how defects in actin can cause compensatory responses in other cytoskeleton components, which in turn significantly alter cellular function.

Stable GDP-tubulin islands rescue dynamic microtubules.

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics.

Transcription factor 4 promotes increased corneal endothelial cellular migration by altering microtubules in Fuchs endothelial corneal dystrophy.

Fuchs endothelial corneal dystrophy (FECD) is a complex corneal disease characterized by the progressive decline and morphological changes of corneal endothelial cells (CECs) that leads to corneal edema and vision loss. The most common mutation in FECD is an intronic CTG repeat expansion in transcription factor 4 (TCF4) that leads to its altered expression. Corneal endothelial wound healing occurs primarily through cell enlargement and migration, and FECD CECs have been shown to display increased migration speeds. In this study, we aim to determine whether TCF4 can promote cellular migration in FECD CECs. We generated stable CEC lines derived from FECD patients that overexpressed different TCF4 isoforms and investigated epithelial-to-mesenchymal (EMT) expression, morphological analysis and cellular migration speeds. We found that full length TCF4-B isoform overexpression promotes cellular migration in FECD CECs in an EMT-independent manner. RNA-sequencing identified several pathways including the negative regulation of microtubules, with TUBB4A (tubulin beta 4A class IVa) as the top upregulated gene. TUBB4A expression was increased in FECD ex vivo specimens, and there was altered expression of cytoskeleton proteins, tubulin and actin, compared to normal healthy donor ex vivo specimens. Additionally, there was increased acetylation and detyrosination of microtubules in FECD supporting that microtubule stability is altered in FECD and could promote cellular migration. Future studies could be aimed at investigating if targeting the cytoskeleton and microtubules would have therapeutic potential for FECD by promoting cellular migration and regeneration.

Design and synthesis of novel tetrabromophthalimide derivatives as potential tubulin inhibitors endowed with apoptotic induction for cancer treatment.

Although various approaches exist for treating cancer, chemotherapy continues to hold a prominent role in the management of this disease. Besides, microtubules serve as a vital component of the cellular skeleton, playing a pivotal role in the process of cell division making it an attractive target for cancer treatment. Hence, the scope of this work was adapted to design and synthesize new anti-tubulin tetrabromophthalimide hybrids (3-17) with colchicine binding site (CBS) inhibitory potential. The conducted in vitro studies showed that compound 16 displayed the lowest IC50 values (11.46 µM) at the FaDu cancer cell lines, whereas compound 17 exhibited the lowest IC50 value (13.62 µM) at the PC3 cancer cell line. However, compound 7b exhibited the lowest IC50 value (11.45 µM) at the MDA-MB-468 cancer cell line. Moreover, compound 17 was observed to be the superior antitumor candidate against all three tested cancer cell lines (MDA-MB-468, PC3, and FaDu) with IC50 values of 17.22, 13.15, and 13.62 µM, respectively. In addition, compound 17 showed a well-established upregulation of apoptotic markers (Caspases 3, 7, 8, and 9, Bax, and P53). Moreover, compound 17 induced downregulation of the antiapoptotic markers (MMP2, MMP9, and BCL-2). Furthermore, the colchicine binding site inhibition assay showed that compounds 15a and 17 exhibited particularly significant inhibitory potentials, with IC50 values of 23.07 and 4.25 µM, respectively, compared to colchicine, which had an IC50 value of 3.89 µM. Additionally, cell cycle analysis was conducted, showing that compound 17 could prompt cell cycle arrest at both the G0-G1 and G2-M phases. On the other hand, a molecular docking approach was applied to investigate the binding interactions of the examined candidates compared to colchicine towards CBS of the ß-tubulin subunit. Thus, the synthesized tetrabromophthalimide hybrids can be regarded as outstanding anticancer candidates with significant apoptotic activity.

Systematic profiling of Taxol resistance and sensitivity to tubulin missence mutations at molecular and cellular levels.

Taxol (paclitaxel) is the first approved microtubule-stabilizing agent (MSA) by binding stoichiometrically to tubulin, which is considered to be one of the most significant advances in first-line chemotherapy against diverse tumors. However, a large number of residue missence mutations harboring in the tubulin have been observed to cause acquired drug resistance, largely limiting the clinical application of Taxol and its analogs in chemotherapy. A systematic investigation of the intermolecular interactions between the Taxol and various tubulin mutants would help to establish a comprehensive picture of drug response to tubulin mutations in clinical treatment of cancer, and to design new MSA agents with high potency and selectivity to overcome drug resistance. In this study, we described an integration of in silico analysis and in vitro assay (iSiV) to profile Taxol against a panel of 149 clinically observed, cancer-associated missence mutations in ß-tubulin at molecular and cellular levels, aiming to a systematic understanding of molecular mechanism and biological implication underlying drug resistance and sensitivity conferring from tubulin mutations. It is revealed that the Taxol-resistant mutations can be classified into three types: (I) nonbonded interaction broken due to mutation, (II) steric hindrance caused by mutation, and (III) conformational change upon mutation. In addition, we identified three new Taxol-resistant mutations (C239Y, T274I, and R320P) that can largely reduce the binding affinity of Taxol to tubulin at molecular level, in which the T274I and R320P were observed to considerably impair the antitumor activity of Taxol at cellular level. Moreover, a novel drug-susceptible mutation (M363T) was also identified, which improves Taxol affinity by 2.6-fold and decreases Taxol antitumor EC50 values from 29.4 to 18.7 µM.

Crebinostat facilitates memory formation.

Protein modifications importantly contribute to memory formation. Protein acetylation is a post-translational modification of proteins that regulates memory formation. Acetylation level is determined by the relative activities of acetylases and deacetylases. Crebinostat is a histone deacetylase inhibitor. Here we show that in an object recognition task, crebinostat facilitates memory formation by a weak training. Further, this compound enhances acetylation of α-tubulin, and reduces the level of histone deacetylase 6, an α-tubulin deacetylase. The results suggest that enhanced acetylation of α-tubulin by crebinostat contributes to its facilitatory effect on memory formation.

STMND1 is a phylogenetically ancient stathmin which localizes to motile cilia and exhibits nuclear translocation that is inhibited when soluble tubulin concentration increases.

Stathmins are small, unstructured proteins that bind tubulin dimers and are implicated in several human diseases, but whose function remains unknown. We characterized a new stathmin, STMND1 (Stathmin Domain Containing 1) as the human representative of an ancient subfamily. STMND1 features a N-terminal myristoylated and palmitoylated motif which directs it to membranes and a tubulin-binding stathmin-like domain (SLD) that contains an internal nuclear localization signal. Biochemistry and proximity labeling showed that STMND1 binds tubulin, and live imaging showed that tubulin binding inhibits translocation from cellular membranes to the nucleus. STMND1 is highly expressed in multiciliated epithelial cells, where it localizes to motile cilia. Overexpression in a model system increased the length of primary cilia. Our study suggests that the most ancient stathmins have cilium-related functions that involve sensing soluble tubulin.

Damage-repair events increase the instability of cortical microtubules in Arabidopsis.

Microtubules rely on dynamic assembly and disassembly for their functions. Increasing evidences support that the damage-repair of microtubule lattices can affect microtubule dynamics in vitro and in animal cells. Here we successfully established a way for visualizing damage-repair sites on microtubule lattices in plant cells, via labeling the tubulin proteins with the photoconvertible fluorescent protein mEOS3.2. We observed that the crossovers of the microtubule lattice were more prone to be damaged and repaired, with the frequency of damage-repair events positively correlated with the crossing angle between microtubules. The microtubules with damage-repair events displayed shorter lifespans and significantly increased severing frequency compared with the undamaged microtubules. These observations suggested that the damage-repair events promoted instability of cortical microtubules in plant cells.

Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

γ-TuRCs and the augmin complex are required for the development of highly branched dendritic arbors in Drosophila.

Microtubules are nucleated by γ-tubulin ring complexes (γ-TuRCs) and are essential for neuronal development. Nevertheless, γ-TuRC depletion has been reported to perturb only higher-order branching in elaborated Drosophila larval class IV dendritic arborization (da) neurons. This relatively mild phenotype has been attributed to defects in microtubule nucleation from Golgi outposts, yet most Golgi outposts lack associated γ-TuRCs. By analyzing dendritic arbor regrowth in pupae, we show that γ-TuRCs are also required for the growth and branching of primary and secondary dendrites, as well as for higher-order branching. Moreover, we identify the augmin complex (hereafter augmin), which recruits γ-TuRCs to the sides of pre-existing microtubules, as being required predominantly for higher-order branching. Augmin strongly promotes the anterograde growth of microtubules in terminal dendrites and thus terminal dendrite stability. Consistent with a specific role in higher-order branching, we find that augmin is expressed less strongly and is largely dispensable in larval class I da neurons, which exhibit few higher-order dendrites. Thus, γ-TuRCs are essential for various aspects of complex dendritic arbor development, and they appear to function in higher-order branching via the augmin pathway, which promotes the elaboration of dendritic arbors to help define neuronal morphology.

Circulating miR-30e-3p induces disruption of neurite development in SH-SY5Y cells by targeting ABI1, a novel biomarker for schizophrenia.

Schizophrenia (SCZ) represents a set of enduring mental illnesses whose underlying etiology remains elusive, posing a significant challenge to public health. Previous studies have shown that the neurodevelopmental process involving small molecules such as miRNA and mRNA is one of the etiological hypotheses of SCZ. We identified and verified that miR-30e-3p and ABI1 can be used as biomarkers in peripheral blood transcriptome sequencing data of patients with SCZ, and confirmed the regulatory relationship between them. To further explore their involvement, we employed retinoic acid (RA)-treated SH-SY5Y differentiated cells as a model system. Our findings indicate that in RA-induced SH-SY5Y cells, ABI1 expression is up-regulated, while miR-30e-3p expression is down-regulated. Functionally, both miR-30e-3p down-regulation and ABI1 up-regulation promote apoptosis and inhibit the proliferation of SH-SY5Y cells. Subsequently, the immunofluorescence assay detected the expression location and abundance of the neuron-specific protein ß-tubulinIII. The expression levels of neuronal marker genes MAPT, TUBB3 and SYP were detected by RT-qPCR. We observed that these changes of miR-30e-3p and ABI1 inhibit the neurite growth of SH-SY5Y cells. Rescue experiments further support that ABI1 silencing can correct miR-30e-3p down-regulation-induced SH-SY5Y neurodevelopmental defects. Collectively, our results establish that miR-30e-3p's regulation of neurite development in SH-SY5Y cells is mediated through ABI1, highlighting a potential mechanism in SCZ pathogenesis.

Design, synthesis, and biological evaluation of novel 2,3-Di-O-Aryl/Alkyl sulfonate derivatives of l-ascorbic acid: Efficient access to novel anticancer agents via in vitro screening, tubulin polymerization inhibition, molecular docking study and ADME predictions.

A series of novel l-ascorbic acid derivatives bearing aryl and alkyl sulfonate substituents were synthesized and characterized. In vitro anticancer evaluation against MCF-7 (breast) and A-549 (lung) cancer cell lines revealed potent activity for most of the compounds, with 2b being equipotent to the standard drug colchicine against MCF-7 (IC50 = 0.04 µM). Notably, compound 2b displayed 89-fold selectivity for MCF-7 breast cancer over MCF-10A normal breast cells. Derivatives with two sulfonate groups (2a-g, 3a-g) exhibited superior potency over those with one sulfonate (4a-c,5g, 6b). Compounds 2b and 2c potently inhibited tubulin polymerization in A-549 cancer cells (73.12 % and 62.09 % inhibition, respectively), substantiating their anticancer potential through microtubule disruption. Molecular docking studies showed higher binding scores and affinities for these compounds at the colchicine-binding site of α, ß-tubulin compared to colchicine itself. In-silico ADMET predictions indicated favourable drug-like properties, with 2b exhibiting the highest binding affinity. These sulfonate derivatives of l-ascorbic acid represents promising lead scaffolds for anticancer drug development.

Design, synthesis, and biological evaluation of novel benzimidazole derivatives as anti-cervical cancer agents through PI3K/Akt/mTOR pathway and tubulin inhibition.

Phosphatidylinositol 3-kinase (PI3K) is one of the most attractive therapeutic targets for cervical cancer treatment. In this study, we designed and synthesized a series of benzimidazole derivatives and evaluated their anti-cervical cancer activity. Compound 4r exhibited strong antiproliferative activity in different cervical cancer cell lines HeLa, SiHa and Ca Ski, and relative lower cytotoxicity to normal hepatic and renal cell lines LO2 and HEK-293t (IC50 values were at 21.08 µM and 23.96 µM respectively). Its IC50 value was at 3.38 µM to the SiHa cells. Further mechanistic studies revealed that 4r induced apoptosis, arrested cell cycle in G2/M phase, suppressed PI3K/Akt/mTOR pathway and inhibit the polymerization of tubulin. Molecular docking study suggested that 4r formed key H-bonds action with PI3Kα (PDB ID:8EXU) and tubulin (PDB ID:1SA0). Zebrafish acute toxicity experiments showed that high concentrations of 4r did not cause death or malformation of zebrafish embryos. All these results demonstrated that 4r would be a promising lead candidate for further development of novel PI3K and tubulin dual inhibitors in cervical cancer treatment.

IFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans.

Intraflagellar transport (IFT) orchestrates entry of proteins into primary cilia. At the ciliary base, assembled IFT trains, driven by kinesin-2 motors, can transport cargo proteins into the cilium, across the crowded transition zone. How trains assemble at the base and how proteins associate with them is far from understood. Here, we use single-molecule imaging in the cilia of C. elegans chemosensory neurons to directly visualize the entry of kinesin-2 motors, kinesin-II and OSM-3, as well as anterograde cargo proteins, IFT dynein and tubulin. Single-particle tracking shows that IFT components associate with trains sequentially, both in time and space. Super-resolution maps of IFT components in wild-type and mutant worms reveal ciliary ultrastructure and show that kinesin-II is essential for axonemal organization. Finally, imaging cilia lacking kinesin-II and/or transition zone function uncovers the interplay of kinesin-II and OSM-3 in driving efficient transport of IFT trains across the transition zone.

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.

Antiproliferative efficacy and mechanism of action of garlic phytochemicals-functionalized gold nanoparticles in triple-negative breast cancer cells.

Fabrication of gold nanoparticles (GNPs) with phytochemicals is an emerging green nanotechnology approach with therapeutic implications. Garlic, known for its culinary and medicinal properties, has been extensively investigated for its anticancer properties. Here, we report a method to substantially enhance the antiproliferative potency of garlic by functionalizing its phytochemicals to GNPs and demonstrate a possible mechanism of action of these nanoparticles in the triple-negative breast cancer cell line, MDA-MB-231. Garlic gold nanoparticles (As-GNPs) were synthesized using garlic extract (As-EX) and gold chloride and characterized using a variety of spectroscopy techniques, and transmission electron microscopy (TEM). Compared to As-EX, which has a negligible effect on the viability of the cells, As-GNPs inhibited cell viability with an IC50of 0.310 ± 0.04 mg ml-1and strongly inhibited the clonogenic and migratory propensities of these cells. As indicated by TEM, the As-GNPs entered the cells via endocytosis and dispersed in the cellular milieu. Since tubulin, the protein involved in cell division, is a verified target for several antiproliferative drugs, we next examined whether the As-GNPs interact with this protein. The As-GNPs showed concentration-dependent binding to purified tubulin, slightly but consistently perturbing its secondary helical integritywithout grossly damaging the tertiary structure of the protein or the net polymer mass of the microtubules, as indicated by a tryptophan-quenching assay, far UV-circular dichroism spectroscopy, anilinonaphthalene sulfonate-binding assay, and polymer mass analysis, respectively. In cells, As-GNPs killed the cancer cells without cell cycle arrest, as evidenced by flow cytometry.

Novel PIKfyve/Tubulin Dual-target Inhibitor as a Promising Therapeutic Strategy for B-cell Acute Lymphoblastic Leukemia.

OBJECTIVE: In B-cell acute lymphoblastic leukemia (B-ALL), current intensive chemotherapies for adult patients fail to achieve durable responses in more than 50% of cases, underscoring the urgent need for new therapeutic regimens for this patient population. The present study aimed to determine whether HZX-02-059, a novel dual-target inhibitor targeting both phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) and tubulin, is lethal to B-ALL cells and is a potential therapeutic for B-ALL patients. METHODS: Cell proliferation, vacuolization, apoptosis, cell cycle, and in-vivo tumor growth were evaluated. In addition, Genome-wide RNA-sequencing studies were conducted to elucidate the mechanisms of action underlying the anti-leukemia activity of HZX-02-059 in B-ALL. RESULTS: HZX-02-059 was found to inhibit cell proliferation, induce vacuolization, promote apoptosis, block the cell cycle, and reduce in-vivo tumor growth. Downregulation of the p53 pathway and suppression of the phosphoinositide 3-kinase (PI3K)/AKT pathway and the downstream transcription factors c-Myc and NF-κB were responsible for these observations. CONCLUSION: Overall, these findings suggest that HZX-02-059 is a promising agent for the treatment of B-ALL patients resistant to conventional therapies.

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.

γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/ß-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.