Centrosome age breaks spindle size symmetry even in cells thought to divide symmetrically.

Centrosomes are the main microtubule-organizing centers in animal cells. Due to the semiconservative nature of centrosome duplication, the two centrosomes differ in age. In asymmetric stem cell divisions, centrosome age can induce an asymmetry in half-spindle lengths. However, whether centrosome age affects the symmetry of the two half-spindles in tissue culture cells thought to divide symmetrically is unknown. Here, we show that in human epithelial and fibroblastic cell lines centrosome age imposes a mild spindle asymmetry that leads to asymmetric cell daughter sizes. At the mechanistic level, we show that this asymmetry depends on a cenexin-bound pool of the mitotic kinase Plk1, which favors the preferential accumulation on old centrosomes of the microtubule nucleation-organizing proteins pericentrin, γ-tubulin, and Cdk5Rap2, and microtubule regulators TPX2 and ch-TOG. Consistently, we find that old centrosomes have a higher microtubule nucleation capacity. We postulate that centrosome age breaks spindle size symmetry via microtubule nucleation even in cells thought to divide symmetrically.

QSAR, ADMET, molecular docking, and dynamics studies of 1,2,4-triazine-3(2H)-one derivatives as tubulin inhibitors for breast cancer therapy.

Breast cancer remains a leading cause of cancer-related deaths among women globally, necessitating the development of more effective therapeutic agents with minimal side effects. This study explores novel 1,2,4-triazine-3(2H)-one derivatives as potential inhibitors of Tubulin, a pivotal protein in cancer cell division, highlighting a targeted approach in cancer therapy. Using an integrated computational approach, we combined quantitative structure-activity relationship (QSAR) modeling, ADMET profiling, molecular docking, and molecular dynamics simulations to evaluate and predict the efficacy and stability of these compounds. Our QSAR models, developed through rigorous statistical analysis, revealed that descriptors such as absolute electronegativity and water solubility significantly influence inhibitory activity, achieving a predictive accuracy (R2) of 0.849. Molecular docking studies identified compounds with high binding affinities, particularly Pred28, which exhibited the best docking score of - 9.6 kcal/mol. Molecular dynamics simulations conducted over 100 ns provided further insights into the stability of these interactions. Pred28 demonstrated notable stability, with the lowest root mean square deviation (RMSD) of 0.29 nm and root mean square fluctuation (RMSF) values indicative of a tightly bound conformation to Tubulin. The novelty of this work lies in its methodological rigor and the integration of multiple advanced computational techniques to pinpoint compounds with promising therapeutic potential. Our findings advance the current understanding of Tubulin inhibitors and open avenues for the synthesis and experimental validation of these compounds, aiming to offer new solutions for breast cancer treatment.

Vasohibin inhibition improves myocardial relaxation in a rat model of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a complex syndrome associated with increased myocardial stiffness and cardiac filling abnormalities. Prior studies implicated increased α-tubulin detyrosination, which is catalyzed by the vasohibin enzymes, as a contributor to increased stabilization of the cardiomyocyte microtubule network (MTN) and stiffness in failing human hearts. We explored whether increased MTN detyrosination contributed to impaired diastolic function in the ZSF1 obese rat model of HFpEF and designed a small-molecule vasohibin inhibitor to ablate MTN detyrosination in vivo. Compared with ZSF1 lean and Wistar Kyoto rats, obese rats exhibited increased tubulin detyrosination concomitant with diastolic dysfunction, left atrial enlargement, and cardiac hypertrophy with a preserved left ventricle ejection fraction, consistent with an HFpEF phenotype. Ex vivo myocardial phenotyping assessed cardiomyocyte mechanics and contractility. Vasohibin inhibitor treatment of isolated cardiomyocytes from obese rats resulted in reduced stiffness and faster relaxation. Acute in vivo treatment with vasohibin inhibitor improved diastolic relaxation in ZSF1 obese rats compared with ZSF1 lean and Wistar Kyoto rats. Vasohibin inhibition also improved relaxation in isolated human cardiomyocytes from both failing and nonfailing hearts. Our data suggest the therapeutic potential for vasohibin inhibition to reduce myocardial stiffness and improve relaxation in HFpEF.

Editing of endogenous tubulins reveals varying effects of tubulin posttranslational modifications on axonal growth and regeneration.

Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or -disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans. We found that the status of ß-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.

Toxic Advanced Glycation End-Products Inhibit Axonal Elongation Mediated by ß-Tubulin Aggregation in Mice Optic Nerves.

Advanced glycation end-products (AGEs) form through non-enzymatic glycation of various proteins. Optic nerve degeneration is a frequent complication of diabetes, and retinal AGE accumulation is strongly linked to the development of diabetic retinopathy. Type 2 diabetes mellitus is a major risk factor for Alzheimer's disease (AD), with patients often exhibiting optic axon degeneration in the nerve fiber layer. Notably, a gap exists in our understanding of how AGEs contribute to neuronal degeneration in the optic nerve within the context of both diabetes and AD. Our previous work demonstrated that glyceraldehyde (GA)-derived toxic advanced glycation end-products (TAGE) disrupt neurite outgrowth through TAGE-ß-tubulin aggregation and tau phosphorylation in neural cultures. In this study, we further illustrated GA-induced suppression of optic nerve axonal elongation via abnormal ß-tubulin aggregation in mouse retinas. Elucidating this optic nerve degeneration mechanism holds promise for bridging the knowledge gap regarding vision loss associated with diabetes mellitus and AD.

Pathological Changes in the Gastrocnemius Muscle throughout Hind Limb Ischemia Modeling in Rats.

We present a two-stage model for the study of chronic hind limb ischemia in rats. In the area of ischemia, sclerotic changes with atrophic rhabdomyocytes and reduced vascularization were revealed. CD31 expression in the endothelium increased proportionally to the number of vessels in the ischemic zone, and at the same time, focal expression of ßIII-tubulin was detected in the newly formed nerve fibers. These histological features are equivalent to the development of peripheral arterial disease in humans, which allows using our model in the search for new therapeutic strategies.

Cryo-EM unveils kinesin KIF1A's processivity mechanism and the impact of its pathogenic variant P305L.

Mutations in the microtubule-associated motor protein KIF1A lead to severe neurological conditions known as KIF1A-associated neurological disorders (KAND). Despite insights into its molecular mechanism, high-resolution structures of KIF1A-microtubule complexes remain undefined. Here, we present 2.7-3.5 Å resolution structures of dimeric microtubule-bound KIF1A, including the pathogenic P305L mutant, across various nucleotide states. Our structures reveal that KIF1A binds microtubules in one- and two-heads-bound configurations, with both heads exhibiting distinct conformations with tight inter-head connection. Notably, KIF1A's class-specific loop 12 (K-loop) forms electrostatic interactions with the C-terminal tails of both α- and ß-tubulin. The P305L mutation does not disrupt these interactions but alters loop-12's conformation, impairing strong microtubule-binding. Structure-function analysis reveals the K-loop and head-head coordination as major determinants of KIF1A's superprocessive motility. Our findings advance the understanding of KIF1A's molecular mechanism and provide a basis for developing structure-guided therapeutics against KAND.

Roles of Tubulin Concentration during Prometaphase and Ran-GTP during Anaphase of Caenorhabditis elegans Meiosis.

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes.

Immunodetection of tubulin and centromeric histone H3 (CENH3) proteins in Glycine species.

BACKGROUND: The centromeres appear as primary constrictions on monocentric metaphase chromosomes; where sister chromatids are held together and assemble the proteinaceous kitechore complex at which microtubule proteins attach during nuclear divisions for pulling sister chromatids to opposite cell poles. The movement of chromosomes is usually governed by structural proteins that are either species-specific or highly conserved, such as the centromere-specific histone H3 (CENH3) and tubulin proteins, respectively. METHODS AND RESULTS: We aimed to detect these proteins across eight different Glycine species by an immunofluorescence assay using specific antibodies. Furthermore, with the α-tubulin antibody we traced the dynamics of microtubules during the mitotic cell cycle in Glycine max. With two-color immunofluorescence staining, we showed that both proteins interact during nuclear division. CONCLUSIONS: Finally, we proved that in different diploid and tetraploid Glycine species CENH3 can be detected in functional centromeres with spatial proximity of microtubule proteins.

Expanding the Structural Diversity of Tubulin-Targeting Agents: Development of Highly Potent Benzimidazoles for Treating Fungal Diseases.

Benzimidazoles, the representative pharmacophore of fungicides, have excellent antifungal potency, but their simple structure and single site of action have hindered their wider application in agriculture. In order to extend the structural diversity of tubulin-targeted benzimidazoles, novel benzimidazole derivatives were prepared by introducing the attractive pyrimidine pharmacophore. 2-((6-(4-(trifluoromethyl)phenoxy)pyrimidin-4-yl)thio)-1H-benzo[d]imidazole (A25) exhibited optimal antifungal activity against Sclerotinia sclerotiorum (S. s.), affording an excellent half-maximal effective concentration (EC50) of 0.158 µg/mL, which was higher than that of the reference agent carbendazim (EC50 = 0.594 µg/mL). Pot experiments revealed that compound A25 (200 µg/mL) had acceptable protective activity (84.7%) and curative activity (78.1%), which were comparable with that of carbendazim (protective activity: 90.8%; curative activity: 69.9%). Molecular docking displayed that multiple hydrogen bonds and π-π interactions could be formed between A25 and ß-tubulin, resulting in a stronger bonding effect than carbendazim. Fluorescence imaging revealed that the structure of intracellular microtubules can be changed significantly after A25 treatment. Overall, these remarkable antifungal profiles of constructed novel benzimidazole derivatives could facilitate the application of novel microtubule-targeting agents.

Impairment of α-tubulin and F-actin interactions of GJB3 induces aneuploidy in urothelial cells and promotes bladder cancer cell invasion.

BACKGROUND: We have previously identified an unsuspected role for GJB3 showing that the deficiency of this connexin protein induces aneuploidy in human and murine cells and accelerates cell transformation as well as tumor formation in xenograft models. The molecular mechanisms by which loss of GJB3 leads to aneuploidy and cancer initiation and progression remain unsolved. METHODS: GJB3 expression levels were determined by RT-qPCR and Western blot. The consequences of GJB3 knockdown on genome instability were assessed by metaphase chromosome counting, multinucleation of cells, by micronuclei formation and by the determination of spindle orientation. Interactions of GJB3 with α-tubulin and F-actin was analyzed by immunoprecipitation and immunocytochemistry. Consequences of GJB3 deficiency on microtubule and actin dynamics were measured by live cell imaging and fluorescence recovery after photobleaching experiments, respectively. Immunohistochemistry was used to determine GJB3 levels on human and murine bladder cancer tissue sections. Bladder cancer in mice was chemically induced by BBN-treatment. RESULTS: We find that GJB3 is highly expressed in the ureter and bladder epithelium, but it is downregulated in invasive bladder cancer cell lines and during tumor progression in both human and mouse bladder cancer. Downregulation of GJB3 expression leads to aneuploidy and genomic instability in karyotypically stable urothelial cells and experimental modulation of GJB3 levels alters the migration and invasive capacity of bladder cancer cell lines. Importantly, GJB3 interacts both with α-tubulin and F-actin. The impairment of these interactions alters the dynamics of these cytoskeletal components and leads to defective spindle orientation. CONCLUSION: We conclude that deregulated microtubule and actin dynamics have an impact on proper chromosome separation and tumor cell invasion and migration. Consequently, these observations indicate a possible role for GJB3 in the onset and spreading of bladder cancer and demonstrate a molecular link between enhanced aneuploidy and invasive capacity cancer cells during tumor cell dissemination.

Nanoparticle-assisted tubulin assembly is environment dependent.

Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.

Endosymbiosis in trypanosomatids: The bacterium division depends on microtubule dynamism.

Microtubules are components of the cytoskeleton that perform essential functions in eukaryotes, such as those related to shape change, motility and cell division. In this context some characteristics of these filaments are essential, such as polarity and dynamic instability. In trypanosomatids, microtubules are integral to ultrastructure organization, intracellular transport and mitotic processes. Some species of trypanosomatids co-evolve with a symbiotic bacterium in a mutualistic association that is marked by extensive metabolic exchanges and a coordinated division of the symbiont with other cellular structures, such as the nucleus and the kinetoplast. It is already established that the bacterium division is microtubule-dependent, so in this work, it was investigated whether the dynamism and remodeling of these filaments is capable of affecting the prokaryote division. To this purpose, Angomonas deanei was treated with Trichostatin A (TSA), a deacetylase inhibitor, and mutant cells for histone deacetylase 6 (HDAC6) were obtained by CRISPR-Cas9. A decrease in proliferation, an enhancement in tubulin acetylation, as well as morphological and ultrastructural changes, were observed in TSA-treated protozoa and mutant cells. In both cases, symbiont filamentation occurred, indicating that prokaryote cell division is dependent on microtubule dynamism.

Tubulin-binding region alters tau-lipid interactions and changes toxicity of tau fibrils formed in the presence of phosphatidylserine lipids.

Alzheimer's disease is the fastest-growing neurodegenerative disease that affects over six million Americans. The abnormal aggregation of amyloid ß peptide and Tau protein is the expected molecular cause of the loss of neurons in brains of AD patients. A growing body of evidence indicates that lipids can alter the aggregation rate of amyloid ß peptide and modify the toxicity of amyloid ß aggregates. However, the role of lipids in Tau aggregation remains unclear. In this study, we utilized a set of biophysical methods to determine the extent to which phospatidylserine (PS) altered the aggregation properties of Tau isoforms with one (1N4R) and two (2N4R) N terminal inserts that enhance the binding of Tau to tubulin. We found that the length and saturation of fatty acids (FAs) in PS altered the aggregation rate of 2N4R isoform, while no changes in the aggregation rate of 1N4R were observed. These results indicate that N terminal inserts play an important role in protein-lipid interactions. We also found that PS could change the toxicity of 1N4R and 2N4R Tau fibrils, as well as alter molecular mechanisms by which these aggregates exert cytotoxicity to neurons. Finally, we found that although Tau fibrils formed in the presence and absence of PS endocytosed by cells, only fibril species that were formed in the presence of PS exert strong impairment of the cell mitochondria.

Tissue-specific and endogenous protein labeling with split fluorescent proteins.

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

Improved Rigidin-Inspired Antiproliferative Agents with Modifications on the 7-Deazahypoxanthine C7/C8 Ring Systems.

To improve their aqueous solubility characteristics, water-solubilizing groups were added to some antiproliferative, rigidin-inspired 7-deazahypoxanthine frameworks after molecular modeling seemed to indicate that structural modifications on the C7 and/or C8 phenyl groups would be beneficial. To this end, two sets of 7-deazahypoxanthines were synthesized by way of a multicomponent reaction approach. It was subsequently determined that their antiproliferative activity against HeLa cells was retained for those derivatives with a glycol ether at the 4'-position of the C8 aryl ring system, while also significantly improving their solubility behavior. The best of these compounds were the equipotent 6-[4-(2-ethoxyethoxy)benzoyl]-2-(pent-4-yn-1-yl)-5-phenyl-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 33 and 6-[4-(2-ethoxyethoxy)benzoyl]-5-(3-fluorophenyl)-2-(pent-4-yn-1-yl)-1,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one 59. Similarly to the parent 1, the new derivatives were also potent inhibitors of tubulin assembly. In treated HeLa cells, live cell confocal microscopy demonstrated their impact on microtubulin dynamics and spindle morphology, which is the upstream trigger of mitotic delay and cell death.

[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/ß 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/ß 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.

A Platform for Medium-Throughput Cell-Free Analyses of Microtubule-Interacting Proteins Using Mammalian Cell Lysates.

The microtubule (MT) cytoskeleton performs a variety of functions in cell division, cell architecture, neuronal differentiation, and ciliary beating. These functions are controlled by proteins that directly interact with MTs, commonly referred to as microtubule-associated proteins (MAPs). Out of the many proteins reported interact with MTs, only a some have been biochemically and functionally characterized so far. One of the limitations of classical in vitro assays and single-MT reconstitution approaches is that they are typically performed with purified proteins. As purification of proteins can be difficult and time-consuming, many previous studies have only focused on a few proteins, while systematic analyses of many different proteins by in vitro reconstitution assays were not possible. Here we present a detailed protocol using lysates of mammalian cells instead of purified proteins that overcomes this limitation. Those lysates contain all molecular components required for in vitro MT reconstitution including the endogenous tubulin and the recombinant MAPs, which form MT assemblies upon the injection of the lysates into a microscopy chamber. This allows to directly observe the dynamic behavior of growing MTs, as well as the fluorescently labeled associated proteins by total internal reflection fluorescence (TIRF) microscopy. Strikingly, all proteins tested so far were functional in our approach, thus providing the possibility to test virtually any protein of interest. This also opens the possibility to screen the impact of patient mutations on the MT binding behavior of MAPs in a medium-throughput manner. In addition, the lysate approach can easily be adapted to other applications that have predominantly been performed with purified proteins so far, such as investigating other cytoskeletal systems and cytoskeletal crosstalk, or to study structures of MAPs bound to MTs by cryo-electron microscopy. Our approach is thus a versatile, expandable, and easy-to-use method to characterize the impact of a broad spectrum of proteins on cytoskeletal behavior and function. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of lysates of human cells for TIRF reconstitution assays Basic Protocol 2: Quantification of GFP-tagged MAP concentration in cell lysates Support Protocol 1: Purification of KIF5B(N555/T92A) (dead kinesin) protein for TIRF reconstitution assays Support Protocol 2: Preparation of GMPCPP MT seeds for TIRF reconstitution assays Basic Protocol 3: TIRF-based MT-MAP reconstitution assays using cell lysates.

Anti-tumor effects of tirbanibulin in squamous cell carcinoma cells are mediated via disruption of tubulin-polymerization.

Topical tirbanibulin is a highly effective and well tolerated novel treatment option for actinic keratoses (AKs). This study aimed to characterize the mode of action of tirbanibulin in keratinocytes (NHEK) and cutaneous squamous cell carcinoma (cSCC) cell lines (A431, SCC-12) in vitro. Tirbanibulin significantly reduced proliferation in a dose-dependent manner in all investigated cell lines, inhibited migration, and induced G2/M-cell cycle arrest only in the cSCC cell lines analyzed, and induced apoptosis solely in A431, which showed the highest sensitivity to tirbanibulin. In general, we detected low basal expression of phosphorylated SRC in all cell lines analyzed, therefore, interference with SRC signaling does not appear to be the driving force regarding the observed effects of tirbanibulin. The most prominent tirbanibulin-mediated effect was on ß-tubulin-polymerization, which was especially impaired in A431. Additionally, tirbanibulin induced an increase of the proinflammatory cytokines IL-1α, bFGF and VEGF in A431. In conclusion, tirbanibulin mediated anti-tumor effects predominantly in A431, while healthy keratinocytes and more dedifferentiated SCC-12 were less influenced. These effects of tirbanibulin are most likely mediated via dysregulation of ß-tubulin-polymerization and may be supported by proinflammatory aspects.