Tubulin Stability Research Articles

SUMOylation of nuclear receptor Nor1/NR4A3 coordinates microtubule cytoskeletal dynamics and stability in neuronal cells

BackgroundNor1/NR4A3 is a member of the NR4A subfamily of nuclear receptors that play essential roles in regulating gene expression related to development, cell homeostasis and neurological functions. However, Nor1 is still considered an orphan receptor, as its natural ligand remains unclear for mediating transcriptional activation. Yet other activation signals may modulate Nor1 activity, although their precise role in the development and maintenance of the nervous system remains elusive.MethodsWe used transcriptional reporter assays, gene expression profiling, protein turnover measurement, and cell growth assays to assess the functional relevance of Nor1 and SUMO-defective variants in neuronal cells. SUMO1 and SUMO2 conjugation to Nor1 were assessed by immunoprecipitation. Tubulin stability was determined by acetylation and polymerization assays, and live-cell fluorescent microscopy.ResultsHere, we demonstrate that Nor1 undergoes SUMO1 conjugation at Lys-89 within a canonical ψKxE SUMOylation motif, contributing to the complex pattern of Nor1 SUMOylation, which also includes Lys-137. Disruption of Lys-89, thereby preventing SUMO1 conjugation, led to reduced Nor1 transcriptional competence and protein stability, as well as the downregulation of genes involved in cell growth and metabolism, such as ENO3, EN1, and CFLAR, and in microtubule cytoskeleton dynamics, including MAP2 and MAPT, which resulted in reduced survival of neuronal cells. Interestingly, Lys-89 SUMOylation was potentiated in response to nocodazole, a microtubule depolymerizing drug, although this was insufficient to rescue cells from microtubule disruption despite enhanced Nor1 gene expression. Instead, Lys-89 deSUMOylation reduced the expression of microtubule-severing genes like KATNA1, SPAST, and FIGN, and enhanced α-tubulin cellular levels, acetylation, and microfilament organization, promoting microtubule stability and resistance to nocodazole. These effects contrasted with Lys-137 SUMOylation, suggesting distinct regulatory mechanisms based on specific Nor1 input SUMOylation signals.ConclusionsOur study provides novel insights into Nor1 transcriptional signaling competence and identifies a hierarchical mechanism whereby selective Nor1 SUMOylation may govern neuronal cytoskeleton network dynamics and resistance against microtubule disturbances, a condition strongly associated with neurodegenerative diseases.

Abstract PO5-25-12: CDH1 mutations predict resistance to neoadjuvant taxane therapy

Abstract Background: Despite recent advances in personalized medicine, conventional chemotherapy remains a backbone in breast cancer therapy. Thus, identifying markers predicting sensitivity or resistance to individual chemotherapeutics is of great importance. Methods: In the EpiTax neoadjuvant trial, enrolling patients between 1997-2003, patients with primary breast cancers (T2 >4cm, T3/T4 and/or N2/N3) were randomized to epirubicin 90mg/m2/3W or paclitaxel 200mg/m²/3W monotherapy, with cross-over in case of inferior response. Pre-treatment snap-frozen tumor biopsies from 223 patients were analyzed by targeted NGS of a 360 gene panel. Endpoint for comparison was clinical response to the first regimen, since pCR was rare due to the large tumor sizes at inclusion. For validation purposes we performed targeted sequencing of tumor samples from a total of 478 patients included in the Gepar Trio (n=132), Quattro (n=171) and Quinto (n=175) trials, in which patients with >2cm tumors received neoadjuvant anthracycline/taxane combination regimens. Here, the primary endpoint was clinical response to combined treatment, but since these tumors were smaller than in the EpiTax-trial, pCR was included as a secondary endpoint. In addition, experimental validations were performed -by CDH1 knock-down and CRISPR/Cas9 knock-out in cell line models. Results: In samples from the EpiTax-trial, CDH1 mutations predicted an inferior response (trend across response groups; cPD, cSD, cPR and cCR) in the paclitaxel arm (p=0.01) as well as the epirubicin arm (p=0.04). The predictive value was observed within the subgroup of ER-positive cases (both for paclitaxel (p=0.005) and epirubicin (p=0.003)) but not among ER-negative tumors. The majority of CDH1 mutations (24/34=71%) were observed in lobular cancers. While lobular histology predicted resistance to paclitaxel (but not epirubicin), CDH1 mutations predicted resistance also within the subgroup of lobular cases (p=0.002), demonstrating CDH1 mutations to be an independent predictor and not only a co-variate to lobular histology. As assessing functionally linked genes, mutations in GATA3, a transcriptional regulator of CDH1, were predominantly observed in ductal cancers, and were not predictive of resistance to any compound. Yet, combining GATA3 and CDH1 mutations into a composite biomarker predicted resistance to both paclitaxel (p=0.007) and epirubicin (p=0.01), especially in ER-positive cases (p=0.002 and p=0.0004, respectively). While EMT-signatures had predictive value, this effect was largely dominated by CDH1, while other EMT-related genes had limited impact on response. In the independent validation cohort from the Gepar trials, selected with enrichment for lobular cancers (34%), CDH1 mutations were not significantly associated with resistance to therapy (p=0.19) although predicted lack of pCR (p=0.01). Combining GATA3 and CDH1 mutations predicted lack of clinical response (p=0.05) and lack of pCR (p=0.0007) respectively in this cohort. In the in vitro analyses, resistance to paclitaxel was observed in three different breast cancer cell lines upon siRNA mediated knock-down of CDH1, as well as in a CRISPR/Cas9 mediated CDH1 knock-out model, as measured by growth rate, induction of apoptosis, G2 arrest, mitochondrial respiration and tubulin stability. For anthracyclines, similar effects were observed for mitochondrial respiration. Conclusions: In conclusion, mutations in CDH1 predicted resistance to paclitaxel and epirubicin. Our data suggest that CDH1 mutations should be explored further as a predictive biomarker for potential application. Citation Format: Stian Knappskog, Reham Helwa, Sivaramakrishna Rachakonda, Liv B. Gansmo, Carsten Denkert, Lucy R. Yates, Christine Solbach, Michael Untch, Bruno V. Sinn, Anne-Sophie Litmeyer, Beyhan Ataseven, Jens Huober, David C. Wedge, Thomas Karn, Oleksii Nikolaienko, Frederik Marmé, Peter A. Fasching, Hans Petter Eikesdal, Elmar Stickeler, Christian Schem, Paul Jank, Marion van Mackelenbergh, Volkmar Müller, Baerbel Felder, Johannes Holtschmidt, Peter J. Campbell, Sibylle Loibl, Per Lonning. CDH1 mutations predict resistance to neoadjuvant taxane therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-25-12.

Novel loss of function mutation in TUBA1A gene compromises tubulin stability and proteostasis causing spastic paraplegia and ataxia.

Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and β-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as "tubulinopathies" and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene.

A modified natural small molecule inhibits triple-negative breast cancer growth by interacting with Tubb3

BackgroundTriple-negative breast cancer (TNBC) is a malignant tumor without specific therapeutic targets and a poor prognosis. Chemotherapy is currently the first-line therapeutic option for TNBC. However, due to the heterogeneity of TNBC, not all of TNBC patients are responsive to chemotherapeutic agents. Therefore, the demand for new targeted agents is critical. β-tubulin isotype III (Tubb3) is a prognostic factor associated with cancer progression, including breast cancer, and targeting Tubb3 may lead to improve TNBC disease control. Shikonin, the active compound in the roots of Lithospermun erythrorhizon suppresses the growth of various types of tumors, and its efficacy can be improved by altering its chemical structure. PurposeIn this work, the anti-TNBC effect of a shikonin derivative (PMMB276) was investigated, and its mechanism was also investigated. Study Design/MethodsThis study combines flow cytometry, immunofluorescence staining, immunoblotting, immunoprecipitation, siRNA silencing, and the iTRAQ proteomics assay to analyze the inhibition potential of PMMB276 on TNBC. In vivo study was performed, Balb/c female murine models with or without the small molecule treatments. ResultsHerein, we screened 300 in-house synthesized analogs of shikonin against TNBC and identified a novel small molecule, PMMB276; it suppressed cell proliferation, induced apoptosis, and arrested the cell cycle at the G2/M phase, suggesting that it could have a tumor suppressive role in TNBC. Tubb3 was identified as the target of PMMB276 using proteomic and biological activity analyses. Meanwhile, PMMB276 regulated microtubule dynamics in vitro by inducing microtubule depolymerization and it could act as a tubulin stabilizer by a different process than that of paclitaxel. Moreover, suppressing or inhibiting Tubb3 with PMMB276 reduced the growth of breast cancer in an experimental mouse model, indicating that Tubb3 plays a significant role in TNBC progression. ConclusionThe findings support the therapeutic potential of PMMB276, a Tubb3 inhibitor, as a treatment for TNBC. Our findings might serve as a foundation for the utilization of shikonin and its derivatives in the development of anti-TNBC.

Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM

The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC. Our structural and XL-MS analyses suggest a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. In the closed TRiC-tubulin-S3 map, we capture a near-natively folded tubulin—with the tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions. Moreover, we also show the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated folding of tubulin along the ATPase cycle of TRiC, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.

Dual targeted 2-Benzylideneindanone pendant hydroxamic acid group exhibits selective HDAC6 inhibition along with tubulin stabilization effect

Abnormal epigenetics has been recognised as an early event in tumour progression and aberrant acetylation of lysine in particular has been understood in tumorigenesis. Therefore, it has become an attractive target for anticancer drug development. However, HDAC inhibitors have limited success due to toxicity and drug resistance concerns. Present study deals with design and synthesis of bivalent indanone based HDAC6 and antitubulin ligands as anticancer agents. Two of the analogues 9 and 21 exhibited potent antiproliferative activities (IC50, 0.36–3.27 µM) and high potency against HDAC 6 enzyme. Compound 21 showed high selectivity against HDAC 6 while 9 exhibited low selectivity. Both the compounds also showed microtubule stabilization effects and moderate anti-inflammatory effect. Dual targeted anticancer agents with concomitant anti-inflammatory effects will be more attractive clinical candidates in future.

Genome Analysis Using Whole-Exome Sequencing of Non-Syndromic Cleft Lip and/or Palate from Malagasy Trios Identifies Variants Associated with Cilium-Related Pathways and Asian Genetic Ancestry

Orofacial clefts (OFCs) are common congenital disabilities that can occur as isolated non-syndromic events or as part of Mendelian syndromes. OFC risk factors vary due to differences in regional environmental exposures, genetic variants, and ethnicities. In recent years, significant progress has been made in understanding OFCs, due to advances in sequencing and genotyping technologies. Despite these advances, very little is known about the genetic interplay in the Malagasy population. Here, we performed high-resolution whole-exome sequencing (WES) on non-syndromic cleft lip with or without palate (nCL/P) trios in the Malagasy population (78 individuals from 26 families (trios)). To integrate the impact of genetic ancestry admixture, we computed both global and local ancestries. Participants demonstrated a high percentage of both African and Asian admixture. We identified damaging variants in primary cilium-mediated pathway genes WNT5B (one family), GPC4 (one family), co-occurrence in MSX1 (five families), WDR11 (one family), and tubulin stabilizer SEPTIN9 (one family). Furthermore, we identified an autosomal homozygous damaging variant in PHGDH (one family) gene that may impact metabiotic activity. Lastly, all variants were predicted to reside on local Asian genetic ancestry admixed alleles. Our results from examining the Malagasy genome provide limited support for the hypothesis that germline variants in primary cilia may be risk factors for nCL/P, and outline the importance of integrating local ancestry components better to understand the multi-ethnic impact on nCL/P.

Systematic profiling of taxol and its analogues (taxalogues) binding to β‐tubulin and molecular analysis of their effects on microtubule stabilization

AbstractMicrotubules are highly dynamic polymers of α/β‐tubulin that represent major components of the cytoskeleton and have been established as an attractive druggable target of tumors. Taxol, also known as Paclitaxel, is a microtubule‐stabilizing agent that binds stoichiometrically to a specific site in β‐tubulin, where is out of but nearby the interacting interface of α/β‐tubulin complex, to improve the complex stability through an allosterically regulatory mechanism. In this study, the systematic binding profile of taxol and its 22 structurally diverse, medicinally relevant analogues (termed as taxalogues) to the specific site of β‐tubulin as well as their effects on the α/β‐tubulin complex stability were created and investigated by using structural modeling, dynamics simulation, and energetics analysis. Two helices H1 and H2 of β‐tubulin were identified as key hotspots at the α/β‐tubulin interface to mediate the binding event of β‐tubulin to α‐tubulin. Taxalogue binding can trigger a conformational displacement in the H1 and H2 to elicit the stabilization (or destabilization) of α/β‐tubulin. However, strong taxalogue binding potency to β‐tubulin does not mean effective α/β‐tubulin stabilization; there is only an indirect, moderate correlation between them. Cell viability assay revealed that the taxalogue‐induced α/β‐tubulin stabilization, but not the taxalogue binding, directly influences the antitumor activity of taxalogues. The activity increases in the order: SB‐T‐1214 < docetaxel < taxol < larotaxel < cisplatin < cabazitaxel.

In silico analysis of TUBA4A mutations in Amyotrophic Lateral Sclerosis to define mechanisms of microtubule disintegration

Amyotrophic lateral sclerosis (ALS) is an inexorably progressive and degenerative disorder of motor neurons with no currently-known cure. Studies to determine the mechanism of neurotoxicity and the impact of ALS-linked mutations (SOD1, FUS, TARDP, C9ORF72, PFN1, TUBA4A and others) have greatly expanded our knowledge of ALS disease mechanisms and have helped to identify potential targets for ALS therapy. Cellular pathologies (e.g., aggregation of mutant forms of SOD1, TDP43, FUS, Ubiqulin2, PFN1, and C9ORF72), mitochondrial dysfunction, neuroinflammation, and oxidative damage are major pathways implicated in ALS. Nevertheless, the selective vulnerability of motor neurons remains unexplained. The importance of tubulins for long-axon infrastructure, and the special morphology and function of motor neurons, underscore the central role of the cytoskeleton. The recent linkage of mutations to the tubulin α chain, TUBA4A, to familial and sporadic cases of ALS provides a new investigative opportunity to shed light on both mechanisms of ALS and the vulnerability of motor neurons. In the current study we investigate TUBA4A, a structural microtubule protein with mutations causal to familial ALS, using molecular-dynamic (MD) modeling of protein structure to predict the effects of each mutation and its overall impact on GTP binding, chain stability, tubulin assembly, and aggregation propensity. These studies predict that each of the reported mutations will cause notable structural changes to the TUBA4A (α chain) tertiary protein structure, adversely affecting its physical properties and functions. Molecular docking and MD simulations indicate certain α chain mutations (e.g. K430N, R215C, and W407X) may cause structural deviations that impair GTP binding, and plausibly prevent or destabilize tubulin polymerization. Furthermore, several mutations (including R320C and K430N) confer a significant increase in predicted aggregation propensity of TUBA4A mutants relative to wild-type. Taken together, these in silico modeling studies predict structural perturbations and disruption of GTP binding, culminating in failure to form a stable tubulin heterocomplex, which may furnish an important pathogenic mechanism to trigger motor neuron degeneration in ALS.

Triptolide exposure induces oxidative stress and decreases oocyte quality in mouse

Triptolide is a major active ingredient isolated from the traditional Chinese medicine Tripterygium wilfordii, which has anti-inflammatory, anti-cancer, and immunomodulatory effects. However, in clinical studies, triptolide has toxic side effects on the heart, kidney, liver and reproductive organs. With respect to female reproductive toxicity, damaging effects of triptolide on the ovary have been reported, but it has remained unknown whether oocytes are affected by triptolide. Therefore, this study established a concentration gradient of triptolide exposure in mice using 0 (control), 30, 60, and 90 μg triptolide/kg body weight/day administered by gavage. Triptolide administration for 28 d reduced body weight and ovarian weight and affected the developmental potential of oocytes. The triptolide-treated group exhibited meiotic failure of oocytes due to impaired spindle assembly, chromosome alignment, and tubulin stability. Triptolide was also found to induce mitochondrial dysfunction, autophagy and early apoptosis, iron homeostasis, and abnormal histone modifications. These adverse effects could be associated with oxidative stress induced by triptolide. In conclusion, our findings suggest detrimental effects of triptolide on mouse oocytes and, thus, on female reproduction.

In vitro human cell-based aneugen molecular mechanism assay.

This laboratory previously described an in vitro human cell-based assay and data analysis scheme that discriminates common molecular targets responsible for chemical-induced in vitro aneugenicity: tubulin destabilization, tubulin stabilization, and inhibition of Aurora kinases (Bernacki et al., Toxicol. Sci. 170 [2019] 382-393). The current report describes updated procedures that simplify benchtop processing and data analysis methods. For these experiments, human lymphoblastoid TK6 cells were exposed to each of 25 aneugens over a range of concentrations in the presence of fluorescent paclitaxel (488 Taxol). After a 4h treatment period, cells were lysed and nuclei were stained with a nucleic acid dye and labeled with fluorescent antibodies against phospho-histone H3 (p-H3). Flow cytometric analyses revealed several unique signatures: tubulin stabilizers caused increased frequencies of p-H3-positive events with concentration-dependent increases in 488 Taxol-associated fluorescence; tubulin destabilizers caused increased frequencies of p-H3-positive events with concomitant decreases in 488 Taxol-associated fluorescence; and Aurora kinase B inhibitors caused reduced frequencies of p-H3-positive events and lower median fluorescent intensities of p-H3-positive events. These results demonstrate a simple rubric based on 488 Taxol- and p-H3-associated metrics can reliably discriminate between several commonly encountered aneugenic molecular mechanisms.

TubulinTracker, a Novel In Vitro Reporter Assay to Study Intracellular Microtubule Dynamics, Cell Cycle Progression, and Aneugenicity.

Aneuploidy is characterized by the presence of an abnormal number of chromosomes and is a common hallmark of cancer. However, exposure to aneugenic compounds does not necessarily lead to cancer. Aneugenic compounds are mainly identified using the in vitro micronucleus assay but this assay cannot standardly discriminate between aneugens and clastogens and cannot be used to identify the exact mode-of-action (MOA) of aneugens; tubulin stabilization, tubulin destabilization, or inhibition of mitotic kinases. To improve the classification of aneugenic substances and determine their MOA, we developed and validated the TubulinTracker assay that uses a green fluorescent protein-tagged tubulin reporter cell line to study microtubule stability using flow cytometry. Combining the assay with a DNA stain also enables cell cycle analysis. Substances whose exposure resulted in an accumulation of cells in G2/M phase, combined with increased or decreased tubulin levels, were classified as tubulin poisons. All known tubulin poisons included were classified correctly. Moreover, we correctly classified compounds, including aneugens that did not affect microtubule levels. However, the MOA of aneugens not affecting tubulin stability, such as Aurora kinase inhibitors, could not be identified. Here, we show that the TubulinTracker assay can be used to classify microtubule stabilizing and destabilizing compounds in living cells. This insight into the MOA of aneugenic agents is important, eg, to support a weight-of-evidence approach for risk assessment, and the classification as an aneugen as opposed to a clastogen or mutagen, has a big impact on the assessment.

Continued androgen signalling inhibition improves cabazitaxel efficacy in prostate cancer.

Background: The androgen receptor (AR) pathway is a key driver of neoplastic behaviour in the different stages of metastatic prostate cancer (mPCa). Targeting the AR therefore remains the cornerstone for mPCa treatment. We have previously reported that activation of AR signalling affects taxane chemo-sensitivity in preclinical models of castration resistant PCa (CRPC). Here, we explored the anti-tumour efficacy of the AR targeted inhibitor enzalutamide combined with cabazitaxel.Methods: We used the AR positive CRPC model PC346C-DCC-K to assess the in vitro and in vivo activity of combining enzalutamide with cabazitaxel. Subsequent validation studies were performed using an enzalutamide resistant VCaP model. To investigate the impact of AR signalling on cabazitaxel activity we used quantitative live-cell imaging of tubulin stabilization and apoptosis related nuclear fragmentation.Findings: Enzalutamide strongly amplified cabazitaxel anti-tumour activity in the patient-derived xenograft models PC346C-DCC-K (median time to humane endpoint 77 versus 48 days, P<0.0001) and VCaP-Enza-B (median time to humane endpoint 80 versus 53 days, P<0.001). Although enzalutamide treatment by itself was ineffective in reducing tumour growth, it significantly suppressed AR signalling in PC346C-DCC-K tumours as shown by AR target gene expression. The addition of enzalutamide enhanced cabazitaxel induced apoptosis as shown by live-cell imaging (P<0.001).Interpretation: Our study demonstrates that cabazitaxel efficacy can be improved by simultaneous blocking of AR signalling by enzalutamide, even if AR targeted treatment no longer affects tumour growth. These findings support clinical studies that combine AR targeted inhibitors with cabazitaxel in CRPC.

Modeling Prostate Cancer Treatment Responses in the Organoid Era: 3D Environment Impacts Drug Testing.

Organoid-based studies have revolutionized in vitro preclinical research and hold great promise for the cancer research field, including prostate cancer (PCa). However, experimental variability in organoid drug testing complicates reproducibility. For example, we observed PCa organoids to be less affected by cabazitaxel, abiraterone and enzalutamide as compared to corresponding single cells prior to organoid assembly. We hypothesized that three-dimensional (3D) organoid organization and the use of various 3D scaffolds impact treatment efficacy. Live-cell imaging of androgen-induced androgen receptor (AR) nuclear translocation and taxane-induced tubulin stabilization was used to investigate the impact of 3D scaffolds, spatial organoid distribution and organoid size on treatment effect. Scaffolds delayed AR translocation and tubulin stabilization, with Matrigel causing a more pronounced delay than synthetic hydrogel as well as incomplete tubulin stabilization. Drug effect was further attenuated the more centrally organoids were located in the scaffold dome. Moreover, cells in the organoid core revealed a delayed treatment effect compared to cells in the organoid periphery, underscoring the impact of organoid size. These findings indicate that analysis of organoid drug responses needs careful interpretation and requires dedicated read-outs with consideration of underlying technical aspects.

Structure and Energetics of GTP- and GDP-Tubulin Isodesmic Self-Association.

Tubulin self-association is a critical process in microtubule dynamics. The early intermediate structures, energetics, and their regulation by fluxes of chemical energy, associated with guanosine triphosphate (GTP) hydrolysis, are poorly understood. We reconstituted an in vitro minimal model system, mimicking the key elements of the nontemplated tubulin assembly. To resolve the distribution of GTP- and guanosine diphosphate (GDP)-tubulin structures, at low temperatures (∼10 °C) and below the critical concentration for the microtubule assembly, we analyzed in-line size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS) chromatograms of GTP- and GDP-tubulin solutions. Both solutions rapidly attained steady state. The SEC-SAXS data were consistent with an isodesmic thermodynamic model of longitudinal tubulin self-association into 1D oligomers, terminated by the formation of tubulin single rings. The analysis showed that free dimers coexisted with tetramers and hexamers. Tubulin monomers and lateral association between dimers were not detected. The dimer-dimer longitudinal self-association standard Helmholtz free energies were -14.2 ± 0.4 kBT (-8.0 ± 0.2 kcal mol-1) and -13.1 ± 0.5 kBT (-7.4 ± 0.3 kcal mol-1) for GDP- and GTP-tubulin, respectively. We then determined the mass fractions of dimers, tetramers, and hexamers as a function of the total tubulin concentration. A small fraction of stable tubulin single rings, with a radius of 19.2 ± 0.2 nm, was detected in the GDP-tubulin solution. In the GTP-tubulin solution, this fraction was significantly lower. Cryo-TEM images and SEC-multiangle light-scattering analysis corroborated these findings. Our analyses provide an accurate structure-stability description of cold tubulin solutions.

Studies on the Involvement of Integrin and the Tubulin Cytoskeleton in the Mechanism Responsible for TRPV1‐dependent NKCC1 Activation in Mouse Lens

Previously we showed shrinkage of the mouse lens by hyperosmotic solution causes TRPV1-dependent activation of NKCC1. In various tissues the cytoskeleton has been shown to play a role in responses to osmotic stress. Here we present evidence for the involvement of integrin and tubulin in the activation of NKCC1 in mouse lens epithelium. Studies were carried out on lenses from wild-type (WT) and TRPV1 knockout (KO) mice. Rubidium (Rb) uptake, measured using an atomic absorption spectrophotometer, was used as a measure of NKCC activity. ERK1/2 phosphorylation was measured by quantitative Western blot analysis. Intracellular calcium was measured in Fura-2 loaded cultured lens epithelium by a ratiometric imaging technique. In WT mice, the integrin agonist leukadherin 1 (LA-1, 25 μM) increased Rb uptake by 33% in intact lenses and increased intracellular calcium concentration in cultured lens epithelium from 197±18 to 341±25 nM in (n=5, P<0.001). In contrast, LA-1 failed to produce any significant change of Rb uptake in TRPV1 KO lenses and did not increase calcium in cultured lens epithelium from TRPV1 KO animals. The increase of Rb uptake elicited by LA-1 in WT lenses was abolished by the putative tubulin stabilizer, paclitaxel (100 nm). The TRPV1 antagonist, A889425 (1.0 μM) also abolished the Rb uptake response to LA-1. LA-1, the TRPV1 agonist capsaicin, and hyperosmotic solution (350 mOsm) all caused ERK1/2 activation in WT cultured lens epithelium. In each case, the ERK1/2 response was transient with a peak response at ~5 min. The ERK1/2 activation responses to hyperosmotic stress and LA-1 induced were markedly suppressed by A889425 and by paclitaxel. Taken together, the results point to a link between integrin, the tubulin cytoskeleton, and TRPV1-dependent activation of NKCC1 in the mouse lens. It is noteworthy that the integrin agonist LA-1 as well as hyperosmotic stress both were found to activate ERK1/2, the upstream kinase that was shown previously associated with activation of NKCC1 in the lens.

Furan-2-carboxamide derivative, a novel microtubule stabilizing agent induces mitotic arrest and potentiates apoptosis in cancer cells

The vital role played by microtubules in the cell division process, marks them as a potential druggable target to decimate cancer. A novel furan-2-carboxamide based small molecule, is a selective microtubule stabilizing agent (MSA) with IC50 ranging from 4 µM to 8 µM in different cancer cell lines. Inhibition of tubulin polymerization or stabilization of tubulin polymers abrogates chromosomal segregation during cell division, results in cell cycle arrest and leads to cell death due to the delayed repair mechanism. A novel furan-2-carboxamide based small molecule exhibited potent anti-proliferative and anti-metastatic property In-Vitro against the panel of cancer cells. Annexin V-FITC/PI, double staining reveals potent cytotoxic effect of SH09 against HeLa cells. FACS analysis displays induction of G2/M arrest and accumulation of subG1 population of cells upon treatment with SH09. Molecular docking study unveils SH09 binding affinity to the Taxol binding pocket of tubulin proteins and MM-GBSA also confirms strong binding energies of SH09 with tubulin proteins.

Development of Taccalonolide AJ-Hydroxypropyl-β-Cyclodextrin Inclusion Complexes for Treatment of Clear Cell Renal-Cell Carcinoma.

Background: Microtubule-targeted drugs are the most effective drugs for adult patients with certain solid tumors. Taccalonolide AJ (AJ) can stabilize tubulin polymerization by covalently binding to β-tubulin, which enables it to play a role in the treatment of tumors. However, its clinical applications are largely limited by low water solubility, chemical instability in water, and a narrow therapeutic window. Clear-cell renal-cell carcinoma (cc RCC) accounts for approximately 70% of RCC cases and is prone to resistance to particularly targeted therapy drugs. Methods: we prepared a water-soluble cyclodextrin-based carrier to serve as an effective treatment for cc RCC. Results: Compared with AJ, taccalonolide AJ-hydroxypropyl-β-cyclodextrin (AJ-HP-β-CD) exhibited superior selectivity and activity toward the cc RCC cell line 786-O vs. normal kidney cells by inducing apoptosis and cell cycle arrest and inhibiting migration and invasion of tumor cells in vitro. According to acute toxicity testing, the maximum tolerated dose (MTD) of AJ-HP-β-CD was 10.71 mg/kg, which was 20 times greater than that of AJ. Assessment of weight changes showed that mouse body weight recovered over 7–8 days, and the toxicity could be greatly reduced by adjusting the injections from once every three days to once per week. In addition, we inoculated 786-O cells to generate xenografted mice to evaluate the anti-tumor activity of AJ-HP-β-CD in vivo and found that AJ-HP-β-CD had a better tumor inhibitory effect than that of docetaxel and sunitinib in terms of tumor growth and endpoint tumor weight. These results indicated that cyclodextrin inclusion greatly increased the anti-tumor therapeutic window of AJ. Conclusions: the AJ-HP-β-CD complex developed in this study may prove to be a novel tubulin stabilizer for the treatment of cc RCC. In addition, this drug delivery system may broaden the horizon in the translational study of other chemotherapeutic drugs.

An Efficient Screen for Cell-Intrinsic Factors Identifies the Chaperonin CCT and Multiple Conserved Mechanisms as Mediating Dendrite Morphogenesis

Dendritic morphology is inextricably linked to neuronal function. Systematic large-scale screens combined with genetic mapping have uncovered several mechanisms underlying dendrite morphogenesis. However, a comprehensive overview of participating molecular mechanisms is still lacking. Here, we conducted an efficient clonal screen using a collection of mapped P-element insertions that were previously shown to cause lethality and eye defects in Drosophila melanogaster. Of 280 mutants, 52 exhibited dendritic defects. Further database analyses, complementation tests, and RNA interference validations verified 40 P-element insertion genes as being responsible for the dendritic defects. Twenty-eight mutants presented severe arbor reduction, and the remainder displayed other abnormalities. The intrinsic regulators encoded by the identified genes participate in multiple conserved mechanisms and pathways, including the protein folding machinery and the chaperonin-containing TCP-1 (CCT) complex that facilitates tubulin folding. Mutant neurons in which expression of CCT4 or CCT5 was depleted exhibited severely retarded dendrite growth. We show that CCT localizes in dendrites and is required for dendritic microtubule organization and tubulin stability, suggesting that CCT-mediated tubulin folding occurs locally within dendrites. Our study also reveals novel mechanisms underlying dendrite morphogenesis. For example, we show that Drosophila Nogo signaling is required for dendrite development and that Mummy and Wech also regulate dendrite morphogenesis, potentially via Dpp- and integrin-independent pathways. Our methodology represents an efficient strategy for identifying intrinsic dendrite regulators, and provides insights into the plethora of molecular mechanisms underlying dendrite morphogenesis.

Dynamic information of the time-dependent tobullian biomolecular structure using a high-accuracy size-dependent theory

As the most rigid cytoskeletal filaments, tubulin–labeled microtubules bear compressive forces in living cells, balancing the tensile forces within the cytoskeleton to maintain the cell shape. The current structure is often under several environmental conditions as well as various dynamic or static loads that can decrease the stability of the viscoelastic tubulin–labeled microtubules. For this issue, the dynamic stability analysis of size-dependent viscoelastic tubulin–labeled microtubules using modified strain gradient theory by considering the exact three-length scale parameter. Viscoelastic properties are modeled using Kelvin-Voight model to study the time-dependent tubulin–labeled microtubules structure. By applying energy methods (known as Hamilton’s principle), the motion equations of the tubulin–labeled microtubules are developed. The dynamic equations are based on first-order shear deformation theory (FSDT), and generalized differential quadrature and fourth-order Runge-Kutta methods are employed to find the model for the natural frequencies. The novelty of the current study is to consider the effects of viscoelastic properties, and exact values of size-dependent parameters on dynamic behaviors of the tubulin–labeled microtubules. Considering three-length scale parameters (l0 = h, l1 = h, l2 = h) in this size-dependent theory leads to a better agreement with molecular dynamic (MD) simulation in comparison with other theories. The results show that when the rigidity of the edges is improved by changing the simply supported to clamped supported boundary conditions, the maximum deflection and stability of the living part would be damped much more quickly.Communicated by Ramaswamy H. Sarma