microtubule dynamics
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Glioblastomas are aggressive brain tumors for which effective therapy is still lacking, resulting in dismal survival rates. These tumors display significant phenotypic plasticity, harboring diverse cell populations ranging from tumor core cells to dispersed, highly invasive cells. Neuron navigator 3 (NAV3), a microtubule-associated protein affecting microtubule growth and dynamics, is downregulated in various cancers, including glioblastoma, and has thus been considered a tumor suppressor. In this study, we challenge this designation and unveil distinct expression patterns of NAV3 across different invasion phenotypes. Using glioblastoma cell lines and patient-derived glioma stem-like cell cultures, we disclose an upregulation of NAV3 in invading glioblastoma cells, contrasting with its lower expression in cells residing in tumor spheroid cores. Furthermore, we establish an association between low and high NAV3 expression and the amoeboid and mesenchymal invasive phenotype, respectively, and demonstrate that overexpression of NAV3 directly stimulates glioblastoma invasive behavior in both 2D and 3D environments. Consistently, we observed increased NAV3 expression in cells migrating along blood vessels in mouse xenografts. Overall, our results shed light on the role of NAV3 in glioblastoma invasion, providing insights into this lethal aspect of glioblastoma behavior.
- MeSH
- fenotyp * MeSH
- glioblastom * patologie genetika metabolismus MeSH
- invazivní růst nádoru * genetika MeSH
- lidé MeSH
- membránové proteiny MeSH
- mikrotubuly metabolismus MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory mozku * patologie genetika metabolismus MeSH
- pohyb buněk genetika fyziologie MeSH
- proteiny nervové tkáně metabolismus genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Microtubule dynamic is exceptionally sensitive to modulation by small-molecule ligands. Our previous work presented the preparation of microtubule-targeting estradiol dimer (ED) with anticancer activity. In the present study, we explore the effect of selected linkers on the biological activity of the dimer. The linkers were designed as five-atom chains with carbon, nitrogen or oxygen in their centre. In addition, the central nitrogen was modified by a benzyl group with hydroxy or methoxy substituents and one derivative possessed an extended linker length. Thirteen new dimers were subjected to cytotoxicity assay and cell cycle profiling. Dimers containing linker with benzyl moiety substituted with one or more methoxy groups and longer branched ones were found inactive, whereas other structures had comparable efficacy as the original ED (e.g. D1 with IC50 = 1.53 μM). Cell cycle analysis and immunofluorescence proved the interference of dimers with microtubule assembly and mitosis. The proposed in silico model and calculated binding free energy by the MM-PBSA method were closely correlated with in vitro tubulin assembly assay.
- MeSH
- apoptóza MeSH
- ethinylestradiol * chemie farmakologie MeSH
- kontrolní body fáze G2 buněčného cyklu účinky léků MeSH
- mikrotubuly MeSH
- modulátory tubulinu * chemie farmakologie MeSH
- nádorové buněčné linie MeSH
- protinádorové látky * chemie farmakologie MeSH
- triazoly * chemie farmakologie MeSH
- tubulin * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.
- MeSH
- Bardetův-Biedlův syndrom * genetika metabolismus patologie MeSH
- cilie metabolismus MeSH
- lidé MeSH
- myši knockoutované MeSH
- myši MeSH
- proteiny asociované s mikrotubuly metabolismus MeSH
- proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.
BACKGROUND: Cystathionine β-synthase (CBS), one of three enzymes that endogenously produce hydrogen sulfide, is extensively studied for its relevance in the cells of various tumors. In our previous work, we observed that the immunofluorescence pattern of CBS is very similar to that of tubulin and actin. Therefore, we focused on the potential interaction of CBS with cytoskeletal proteins β-actin and β-tubulin and the functional relevance of the potential interaction of these proteins in colorectal carcinoma cell lines. METHODS: To study the potential interaction of CBS with cytoskeletal proteins and its functional consequences, a CBS-knockout DLD1 (DLDx) cell line was established by using the CRISPR/Cas9 gene editing method. The interaction of the selected cytoskeletal protein with CBS was studied by immunoprecipitation, Western blot analysis, immunofluorescence, and proximity ligation assay. The functional consequences were studied by proliferation and migration assays and by generation of xenografts in SCID/bg mice. RESULTS: We have found that CBS, an enzyme that endogenously produces H2S, binds to cytoskeletal β-tubulin and, to a lesser extent, also to β-actin in colorectal carcinoma-derived cells. When CBS was knocked out by the CRISPR/Cas9 technique (DLDx), we observed a de-arranged cytoskeleton compared to the unmodified DLD1 cell line. Treatment of these cells with a slow sulfide donor GYY4137 resulted in normal organization of the cytoskeleton, thus pointing to the role of CBS in microtubule dynamics. To evaluate the physiological importance of this observation, both DLD1 and DLDx cells were injected into SCID/bg mice, and the size and mass of the developed xenografts were evaluated. Significantly larger tumors developed from DLDx compared to the DLD1 cells, which correlated with the increased proliferation of these cells. CONCLUSIONS: Taken together, in colorectal cancer DLD1 cells, CBS binds to the cytoskeleton, modulates microtubule dynamics, and thus affects the proliferation and migration in the colorectal carcinoma stable cell line.
- Publikační typ
- časopisecké články MeSH
Cortical projection neurons polarize and form an axon while migrating radially. Even though these dynamic processes are closely interwoven, they are regulated separately-the neurons terminate their migration when reaching their destination, the cortical plate, but continue to grow their axons. Here, we show that in rodents, the centrosome distinguishes these processes. Newly developed molecular tools modulating centrosomal microtubule nucleation combined with in vivo imaging uncovered that dysregulation of centrosomal microtubule nucleation abrogated radial migration without affecting axon formation. Tightly regulated centrosomal microtubule nucleation was required for periodic formation of the cytoplasmic dilation at the leading process, which is essential for radial migration. The microtubule nucleating factor γ-tubulin decreased at neuronal centrosomes during the migratory phase. As distinct microtubule networks drive neuronal polarization and radial migration, this provides insight into how neuronal migratory defects occur without largely affecting axonal tracts in human developmental cortical dysgeneses, caused by mutations in γ-tubulin.
- MeSH
- axony metabolismus MeSH
- centrozom MeSH
- lidé MeSH
- mikrotubuly metabolismus MeSH
- mozek metabolismus MeSH
- neurony * fyziologie MeSH
- tubulin * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Microtubules composed of αβ-tubulin dimers are dynamic cytoskeletal polymers that play key roles in essential cellular processes such as cell division, organelle positioning, intracellular transport, and cell migration. γ-Tubulin is a highly conserved member of the tubulin family that is required for microtubule nucleation. γ-Tubulin, together with its associated proteins, forms the γ-tubulin ring complex (γ-TuRC), that templates microtubules. Here we review recent advances in the structure of γ-TuRC, its activation, and centrosomal recruitment. This provides new mechanistic insights into the molecular mechanism of microtubule nucleation. Accumulating data suggest that γ-tubulin also has other, less well understood functions. We discuss emerging evidence that γ-tubulin can form oligomers and filaments, has specific nuclear functions, and might be involved in centrosomal cross-talk between microtubules and microfilaments.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Microtubules composed of tubulin heterodimers represent highly dynamic structures. These structures are essential for basic cellular functions, such as cell division. Microtubules can grow or shrink in response to environmental signals, principally chemical cues. Here, we provide an alternative-physical-strategy to modulate tubulin properties and its self-assembly process. The conformation and electrical properties of tubulin subunits are modulated by nanosecond electropulse signals. The formed structures of electrically treated tubulin are tightly linked to the degree of conformational and electrical properties changes induced by nanosecond electropulses. This strategy opens a new way for controlling the self-assembly process in biomolecules as well as in bioinspired materials.
Karcinom močového měchýře je celosvětově devátou nejčastější onkologickou diagnózou, s nejvyšší incidencí zaznamenanou u mužů v severní a západní Evropě, Severní Americe a také v některých zemích severní Afriky a západní Asie. Incidence je vyšší u mužů, ačkoli jejich podíl podle pohlaví se v jednotlivých zemích liší.1 V roce 2018 činila incidence zhoubných nádorů močového měchýře v České republice 19,93 na 100 000 obyvatel, mortalita 8,54 na 100 000 obyvatel, přičemž v 8 % případů je v době diagnózy zjištěno klinické stadium IV.2 Vinflunin je určen k monoterapii dospělých pacientů s pokročilým nebo metastazujícím karcinomem přechodného epitelu močového ústrojí po selhání předchozích režimů léčby obsahujících platinu.3 Protinádorový účinek vinfluninu lze významně přičíst jeho modulaci dynamiky mikrotubulů a je zprostředkován jeho schopností indukovat apoptózu v cílových buňkách. V necytotoxických koncentracích vinflunin též vykazuje antiangiogenní a antivaskulární aktivitu.4 V naší kazuistice popisujeme případ dlouhodobé léčebné odpovědi u pacienta s rozsáhlou diseminací uroteliálního karcinomu močového měchýře.
Bladder cancer ranks as the ninth most frequently diagnosed cancer worldwide, with the highest incidence rates observed in men in Southern and Western Europe, North America, as well in certain countries in Northern Africa or Western Asia. Incidence rates are consistently lower in women than men, although sex differences varied greatly between countries.1 In 2018 the incidence of bladder cancer in Czech Republic was 19.93 per 100 000 inhabitants and the mortality was 8.54 per 100 000 inhabitants and in 8 % of cases are diagnosed in clinical stage IV.2 The vinflunine is indicated in monotherapy for the treatment of adult patients with advanced or metastatic transitional cell carcinoma of the urothelial tract after failure of a prior platinum-containing regimen. The antitumor effect of vinflunine is largely attributable to its modulation of microtubule dynamics and is mediated by its ability to induce apoptosis in target cells. At non-cytotoxic concentrations, vinflunine also exerts antiangiogenic and antivascular activity.2 In our case report we are presenting the case of long-term response at the patient with large dissemination of bladder cancer.
- Klíčová slova
- vinflunin,
- MeSH
- karcinom z přechodných buněk farmakoterapie MeSH
- kvalita života MeSH
- lidé MeSH
- metastázy nádorů farmakoterapie MeSH
- nádory močového měchýře * farmakoterapie MeSH
- protinádorové látky terapeutické užití MeSH
- senioři MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- Publikační typ
- kazuistiky MeSH
Drug withdrawal is associated with abstinence symptoms including deficits in cognitive functions that may persist even after prolonged discontinuation of drug intake. Cognitive deficits are, at least partially, caused by alterations in synaptic plasticity but the precise molecular mechanisms have not yet been fully identified. In the present study, changes in proteomic and phosphoproteomic profiles of selected brain regions (cortex, hippocampus, striatum, and cerebellum) from rats abstaining for six months after cessation of chronic treatment with morphine were determined by label-free quantitative (LFQ) proteomic analysis. Interestingly, prolonged morphine withdrawal was found to be associated especially with alterations in protein phosphorylation and to a lesser extent in protein expression. Gene ontology (GO) term analysis revealed enrichment in biological processes related to synaptic plasticity, cytoskeleton organization, and GTPase activity. More specifically, significant changes were observed in proteins localized in synaptic vesicles (e.g., synapsin-1, SV2a, Rab3a), in the active zone of the presynaptic nerve terminal (e.g., Bassoon, Piccolo, Rims1), and in the postsynaptic density (e.g., cadherin 13, catenins, Arhgap35, Shank3, Arhgef7). Other differentially phosphorylated proteins were associated with microtubule dynamics (microtubule-associated proteins, Tppp, collapsin response mediator proteins) and the actin-spectrin network (e.g., spectrins, adducins, band 4.1-like protein 1). Taken together, a six-month morphine withdrawal was manifested by significant alterations in the phosphorylation of synaptic proteins. The altered phosphorylation patterns modulating the function of synaptic proteins may contribute to long-term neuroadaptations induced by drug use and withdrawal.
- Publikační typ
- časopisecké články MeSH
