共查询到20条相似文献,搜索用时 15 毫秒
1.
《Cell cycle (Georgetown, Tex.)》2013,12(13):2162-2171
Taccalonolide A is a microtubule stabilizer that has cellular effects almost identical to paclitaxel. However, biochemical studies show that, unlike paclitaxel, taccalonolide A does not enhance purified tubulin polymerization or bind tubulin/microtubules. Mechanistic studies aimed at understanding the nature of the differences between taccalonolide A and paclitaxel were conducted. Our results show that taccalonolide A causes bundling of interphase microtubules at concentrations that cause antiproliferative effects. In contrast, the concentration of paclitaxel that initiates microtubule bundling is 31-fold higher than its IC50. Taccalonolide A’s effects are further differentiated from paclitaxel in that it is unable to enhance the polymerization of tubulin in cellular extracts. This finding extends previous biochemical results with purified brain tubulin to demonstrate that taccalonolide A requires more than tubulin and a full complement of cytosolic proteins to cause microtubule stabilization. Reversibility studies were conducted and show that the cellular effects of taccalonolide A persist after drug washout. In contrast, other microtubule stabilizers, including paclitaxel and laulimalide, demonstrate a much higher degree of cellular reversibility in both short-term proliferation and long-term clonogenic assays. The propensity of taccalonolide A to alter interphase microtubules at antiproliferative concentrations as well as its high degree of cellular persistence may explain why taccalonolide A is more potent in vivo than would be expected from cellular studies. The close linkage between the microtubule bundling and antiproliferative effects of taccalonolide A is of interest given the recent hypothesis that the effects of microtubule targeting agents on interphase microtubules might play a prominent role in their clinical anticancer efficacy. 相似文献
2.
Taccalonolide A is a microtubule stabilizer that has cellular effects almost identical to paclitaxel. However, biochemical studies show that, unlike paclitaxel, taccalonolide A does not enhance purified tubulin polymerization or bind tubulin/microtubules. Mechanistic studies aimed at understanding the nature of the differences between taccalonolide A and paclitaxel were conducted. Our results show that taccalonolide A causes bundling of interphase microtubules at concentrations that cause antiproliferative effects. In contrast, the concentration of paclitaxel that initiates microtubule bundling is 31-fold higher than its IC50. Taccalonolide A''s effects are further differentiated from paclitaxel in that it is unable to enhance the polymerization of tubulin in cellular extracts. This finding extends previous biochemical results with purified brain tubulin to demonstrate that taccalonolide A requires more than tubulin and a full complement of cytosolic proteins to cause microtubule stabilization. Reversibility studies were conducted and show that the cellular effects of taccalonolide A persist after drug washout. In contrast, other microtubule stabilizers, including paclitaxel and laulimalide, demonstrate a much higher degree of cellular reversibility in both short-term proliferation and long-term clonogenic assays. The propensity of taccalonolide A to alter interphase microtubules at antiproliferative concentrations as well as its high degree of cellular persistence may explain why taccalonolide A is more potent in vivo than would be expected from cellular studies. The close linkage between the microtubule bundling and antiproliferative effects of taccalonolide A is of interest given the recent hypothesis that the effects of microtubule targeting agents on interphase microtubules might play a prominent role in their clinical anticancer efficacy.Key words: taccalonolide, paclitaxel, microtubule stabilizer, microtubule targeted agent, tubulin, microtubule, laulimalide, antimitotic agent, drug persistence 相似文献
3.
Guillaume Laflamme Thierry Tremblay-Boudreault Marc-André Roy Parker Andersen éric Bonneil Kaleem Atchia Pierre Thibault Damien D'Amours Benjamin H. Kwok 《The Journal of biological chemistry》2014,289(40):27418-27431
Structural maintenance of chromosome (SMC) proteins are key organizers of chromosome architecture and are essential for genome integrity. They act by binding to chromatin and connecting distinct parts of chromosomes together. Interestingly, their potential role in providing connections between chromatin and the mitotic spindle has not been explored. Here, we show that yeast SMC proteins bind directly to microtubules and can provide a functional link between microtubules and DNA. We mapped the microtubule-binding region of Smc5 and generated a mutant with impaired microtubule binding activity. This mutant is viable in yeast but exhibited a cold-specific conditional lethality associated with mitotic arrest, aberrant spindle structures, and chromosome segregation defects. In an in vitro reconstitution assay, this Smc5 mutant also showed a compromised ability to protect microtubules from cold-induced depolymerization. Collectively, these findings demonstrate that SMC proteins can bind to and stabilize microtubules and that SMC-microtubule interactions are essential to establish a robust system to maintain genome integrity. 相似文献
4.
Jayant Asthana Anuradha Kuchibhatla Swadhin Chandra Jana Krishanu Ray Dulal Panda 《The Journal of biological chemistry》2012,287(48):40793-40805
Dynein light chain 1 (LC8), a highly conserved protein, is known to bind to a variety of different polypeptides. It functions as a dimer, which is inactivated through phosphorylation at the Ser-88 residue. A loss of LC8 function causes apoptosis in Drosophila embryos, and its overexpression induces malignant transformation of breast cancer cells. Here we show that LC8 binds to tubulin, promotes microtubule assembly, and induces the bundling of reconstituted microtubules in vitro. Furthermore, LC8 decorates microtubules both in Drosophila embryos and in HeLa cells, increases the microtubule stability, and promotes microtubule bundling in these cells. Microtubule stability influences a number of different cellular functions including mitosis and cell differentiation. The LC8 overexpression reduces the susceptibility of microtubules to cold and nocodazole-induced depolymerization in tissue-cultured cells and increases microtubule acetylation, suggesting that LC8 stabilizes microtubules. We also show that LC8 knockdown or transfection with inhibitory peptides destabilizes microtubules and inhibits bipolar spindle assembly in HeLa cells. In addition, LC8 knockdown leads to the mitotic block in HeLa cells. Furthermore, molecular docking analysis using the crystal structures of tubulin and LC8 dimer indicated that the latter may bind at α-β tubulin junction in a protofilament at sites distinct from the kinesin and dynein binding sites. Together, we provide the first evidence of a novel microtubule-associated protein-like function of LC8 that could explain its reported roles in cellular metastasis and differentiation. 相似文献
5.
6.
Plant morphogenesis is driven by a surprising number of microtubule arrays. The four arrays of vegetative tissues are hoop-like
cortical, preprophase band (PPB), spindle, and phragmoplast. When syncytia occur during the reproductive phase of the plant
life cycle, neither hoop-like corticals nor PPBs are present, and functional phragmoplasts fail to form following the proliferative
mitoses that give rise to the multinucleate cytoplasm. Instead, the interphase microtubules are radial microtubule systems
(RMSs) that emanate from the nuclei. These RMSs organize the cytoplasm into nascent cells and ultimately trigger phragmoplast
formation at their boundaries. During investigations of the syncytial stage that initiates development of the female gametophyte
in gymnosperms, we studied the large (3–4 mm) female gametophyte of Ginkgo biloba. Here we describe the microtubule cycle correlated with successive mitotic waves and discuss the importance of this system
in studying the acentrosomal nucleation and organization of cycling microtubule arrays.
Electronic Publication 相似文献
7.
Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells
Emmanuel Gallaud Renaud Caous Aude Pascal Franck Bazile Jean-Philippe Gagné Sébastien Huet Guy G. Poirier Denis Chrétien Laurent Richard-Parpaillon Régis Giet 《The Journal of cell biology》2014,204(7):1111-1121
The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1. 相似文献
8.
Summary Although there are numerous herbicides that disrupt mitosis as a mechanism of action, to date not one has compared the effects of these disrupters on a single species and over a range of concentrations. Oat seedlings, treated with a range of concentrations of nine different mitotic disrupter herbicides, were examined by immunofluorescence microscopy of tubulin in methacrylate sections. All herbicides caused the same kinds of microtubule disruption, although the concentrations required to cause the effects differed markedly between the herbicides. Effects on spindle and phragmoplast mitotic microtubule arrays were seen at the lowest concentrations and manifested as multipolar spindles and bifurcated phragmoplasts (which subsequently resulted in abnormal cell plate formation). At increasing concentrations, effects on mitotic microtubule arrays manifested as microtubule tufts at kinetochores and reduction of cortical microtubules resulting in arrested prometaphase figures and isodiametric cells. These data indicate that all mitotic disrupter herbicides have a common primary mechanism of action, inhibition of microtubule polymerization, and that marginal effects observed in the past were the result of incomplete inhibition and/or differential sensitivity of the microtubule arrays.Abbreviations DCPA
2,3,5,6-tetrachloroterephthalic acid dimethyl ester
- APM
amiprophosmethyl
- DAPI
4,6-diamidino-2-phenyl indole
- MTOC
microtubule organizing center 相似文献
9.
The kinetochore plays important roles in cell cycle progression. Interactions between chromosomes and spindle microtubules allow chromosomes to congress to the middle of the cell and to segregate the sister chromatids into daughter cells in mitosis. The chromosome passenger complex (CPC), composed of the Aurora B kinase and its regulatory subunits INCENP, Survivin, and Borealin, plays multiple roles in these chromosomal events. In the genome of the silkworm, Bombyx mori, which has holocentric chromosomes, the CPC components and their molecular interactions were highly conserved. In contrast to monocentric species, however, the silkworm CPC co-localized with the chromatin-driven spindles on the upper side of prometaphase chromosomes without forming bipolar mitotic spindles. Depletion of the CPC by RNAi arrested the cell cycle progression at prometaphase and disrupted the microtubule network of the chromatin-driven spindles. Interestingly, depletion of mitotic centromere-associated kinesin (MCAK) recovered formation of the microtubule network but did not overcome the cell cycle arrest at prometaphase. These results suggest that the CPC modulates the chromatin-induced spindle assembly and metaphase congression of silkworm holocentric chromosomes. 相似文献
10.
《Journal of structural biology》2020,209(1):107402
Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins. 相似文献
11.
Shubhandra Tripathi Akhil Kumar B. Sathish Kumar Arvind S. Negi 《Journal of biomolecular structure & dynamics》2016,34(6):1232-1240
Microtubule stabilizers provide an important mode of treatment via mitotic cell arrest of cancer cells. Recently, we reported two novel neolignans derivatives Cmp10 and Cmp19 showing anticancer activity and working as microtubule stabilizers at micromolar concentrations. In this study, we have explored the binding site, mode of binding, and stabilization by two novel microtubule stabilizers Cmp10 and Cmp19 using in silico molecular docking, molecular dynamics (MD) simulation, and binding free energy calculations. Molecular docking studies were performed to explore the β-tubulin binding site of Cmp10 and Cmp19. Further, MD simulations were used to probe the β-tubulin stabilization mechanism by Cmp10 and Cmp19. Binding affinity was also compared for Cmp10 and Cmp19 using binding free energy calculations. Our docking results revealed that both the compounds bind at Ptxl binding site in β-tubulin. MD simulation studies showed that Cmp10 and Cmp19 binding stabilizes M-loop (Phe272-Val288) residues of β-tubulin and prevent its dynamics, leading to a better packing between α and β subunits from adjacent tubulin dimers. In addition, His229, Ser280 and Gln281, and Arg278, Thr276, and Ser232 were found to be the key amino acid residues forming H-bonds with Cmp10 and Cmp19, respectively. Consequently, binding free energy calculations indicated that Cmp10 (?113.655 kJ/mol) had better binding compared to Cmp19 (?95.216 kJ/mol). This study provides useful insight for better understanding of the binding mechanism of Cmp10 and Cmp19 and will be helpful in designing novel microtubule stabilizers. 相似文献
12.
Cassimeris L 《Current opinion in cell biology》1999,11(1):134-141
A number of accessory proteins capable of stabilizing or destabilizing microtubule polymers in dividing cells have been identified recently. Many of these accessory proteins are modified and regulated by cell-cycle-dependent phosphorylation. Through this regulation, microtubule dynamics are modified to generate rapid microtubule turnover during mitosis. In general, although some microtubule-stabilizing proteins are inactivated at entry into mitosis, a critical balance between microtubule stabilizers and destabilizers is necessary for assembly of the mitotic spindle. 相似文献
13.
《Journal of molecular biology》2019,431(4):864-872
Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion. 相似文献
14.
Matesanz R Rodríguez-Salarichs J Pera B Canales A Andreu JM Jiménez-Barbero J Bras W Nogales A Fang WS Díaz JF 《Biophysical journal》2011,101(12):2970-2980
Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αβ-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 β-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loop. 相似文献
15.
Dong CF Shi S Wang XF An R Li P Chen JM Wang X Wang GR Shan B Zhang BY Han J Dong XP 《Archives of biochemistry and biophysics》2008,470(1):83-92
Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Some evidences demonstrate that PrP may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between PrP and tubulin was confirmed using pull-down assays, immunoprecipitation and ELISA. The interacting regions within PrP with tubulin were mapped in the N-terminus of PrP spanning residues 23-50 and 51-91. PrP octapeptide repeats are critical for the binding activity with tubulin, that the binding activity of PrP with tubulin became stronger along with the number of the octapeptide repeats increased. Microtubule assembly assays, sedimental tests and transmission electron microscopy demonstrated that the full-length PrP (aa 23-231) obviously inhibited the microtubule polymerization processes in vitro, whereas the N- (aa 23-91) and C- (aa 91-231) terminal peptides of PrP did not affect microtubule polymerization. Moreover, the familial Cruetzfeldt Jacob disease (fCJD) related PrP mutants with inserted or deleted octapeptide repeats showed much stronger inhibitive capacities on the microtubule dynamics in vitro than wild-type PrP. Our data highlight a potential role of PrP in regulating the microtubule dynamics in neurons. 相似文献
16.
Microtubule plus ends are dynamically regulated by a wide variety of proteins for performing diverse cellular functions. Here, we show that the fission yeast Schizosaccharomyces pombe uncharacterized protein mcp1p is a microtubule plus-end tracking protein which depends on the kinesin-8 klp6p for transporting along microtubules towards microtubule plus ends. In the absence of mcp1p, microtubule catastrophe and rescue frequencies decrease, leading to an increased dwell time of microtubule plus ends at cell tips. Thus, these findings suggest that mcp1p may synergize with klp6p at microtubule plus-ends to destabilize microtubules. 相似文献
17.
Lama Talje Khaled Ben El Kadhi Kaleem Atchia Thierry Tremblay-Boudreault Sébastien Carreno Benjamin H. Kwok 《FEBS letters》2014
The kinesin-13 family of microtubule depolymerases is a major regulator of microtubule dynamics. RNA interference-induced knockdown studies have highlighted their importance in many cell division processes including spindle assembly and chromosome segregation. Since microtubule turnovers and most mitotic events are relatively rapid (in minutes or seconds), developing tools that offer faster control over protein functions is therefore essential to more effectively interrogate kinesin-13 activities in living cells. Here, we report the identification and characterization of a selective allosteric kinesin-13 inhibitor, DHTP. Using high resolution microscopy, we show that DHTP is cell permeable and can modulate microtubule dynamics in cells. 相似文献
18.
Cornelia Tolg Sara R. Hamilton Lyndsey Morningstar Jing Zhang S. Zhang Kenneth V. Esguerra Patrick G. Telmer Len G. Luyt Rene Harrison James B. McCarthy Eva A. Turley 《The Journal of biological chemistry》2010,285(34):26461-26474
An oncogenic form of RHAMM (receptor for hyaluronan-mediated motility, mouse, amino acids 163–794 termed RHAMMΔ163) is a cell surface hyaluronan receptor and mitotic spindle protein that is highly expressed in aggressive human cancers. Its regulation of mitotic spindle integrity is thought to contribute to tumor progression, but the molecular mechanisms underlying this function have not previously been defined. Here, we report that intracellular RHAMMΔ163 modifies the stability of interphase and mitotic spindle microtubules through ERK1/2 activity. RHAMM−/− mouse embryonic fibroblasts exhibit strongly acetylated interphase microtubules, multi-pole mitotic spindles, aberrant chromosome segregation, and inappropriate cytokinesis during mitosis. These defects are rescued by either expression of RHAMM or mutant active MEK1. Mutational analyses show that RHAMMΔ163 binds to α- and β-tubulin protein via a carboxyl-terminal leucine zipper, but in vitro analyses indicate this interaction does not directly contribute to tubulin polymerization/stability. Co-immunoprecipitation and pulldown assays reveal complexes of RHAMMΔ163, ERK1/2-MEK1, and α- and β-tubulin and demonstrate direct binding of RHAMMΔ163 to ERK1 via a D-site motif. In vitro kinase analyses, expression of mutant RHAMMΔ163 defective in ERK1 binding in mouse embryonic fibroblasts, and blocking MEK1 activity collectively confirm that the effect of RHAMMΔ163 on interphase and mitotic spindle microtubules is mediated by ERK1/2 activity. Our results suggest a model wherein intracellular RHAMMΔ163 functions as an adaptor protein to control microtubule polymerization during interphase and mitosis as a result of localizing ERK1/2-MEK1 complexes to their tubulin-associated substrates. 相似文献
19.
The ubiquitous Taxol binding site of microtubules also binds newly discovered ligands. We have designed a homogeneous assay for the high throughput detection of Taxol biomimetics, based on the displacement of 7-O-[N-(2,7-difluoro-4'-fluoresceincarbonyl)-L-alanyl]Taxol from its binding site in diluted solutions of preserved microtubules. The state of this reference ligand is measured by fluorescence anisotropy in a microplate reader, with varying concentrations of nonfluorescent competitors. The binding equilibrium constant of Taxol has a value K(b) = 3.7 x 10(7) M(-1). We have found that baccatin III, an analogue of Taxol without the C-13 side chain, binds with K(b) = 1.5 x 10(5) M(-1), whereas the side chain methyl ester is inactive. This was unexpected from the structure-activity relationship of taxoids but compatible with models of Taxol docked at the microtubule site. Baccatin III binding has been confirmed by displacement of [(3)H]Taxol and by direct HPLC measurements of its cosedimentation with microtubules, among other methods. Consequently, baccatin III induces microtubule bundles and multipolar spindles in PtK2 and U937 cells, and mitotic arrest and apoptotic death of the U937 cells, at concentrations 200-500-fold larger than Taxol. The simplest analysis of these results strongly suggests that the interaction of the C-2 C-4 substituted taxane ring system with the microtubule binding site provides most (ca. 75%) of the free energy change of Taxol binding and is sufficient to activate microtubule stabilization and transmit the antitumor effects of Taxol, whereas the C-13 side chain provides a weak specific anchor. 相似文献
20.
Amy E. Howard Jaime C. Fox Kevin C. Slep 《The Journal of biological chemistry》2015,290(16):10149-10162
Microtubule-associated proteins regulate microtubule (MT) dynamics spatially and temporally, which is essential for proper formation of the bipolar mitotic spindle. The XMAP215 family is comprised of conserved microtubule-associated proteins that use an array of tubulin-binding tumor overexpressed gene (TOG) domains, consisting of six (A–F) Huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin (HEAT) repeats, to robustly increase MT plus-end polymerization rates. Recent work showed that TOG domains have differentially conserved architectures across the array, with implications for position-dependent TOG domain tubulin binding activities and function within the XMAP215 MT polymerization mechanism. Although TOG domains 1, 2, and 4 are well described, structural and mechanistic information characterizing TOG domains 3 and 5 is outstanding. Here, we present the structure and characterization of Drosophila melanogaster Mini spindles (Msps) TOG3. Msps TOG3 has two unique features as follows: the first is a C-terminal tail that stabilizes the ultimate four HEAT repeats (HRs), and the second is a unique architecture in HR B. Structural alignments of TOG3 with other TOG domain structures show that the architecture of TOG3 is most similar to TOG domains 1 and 2 and diverges from TOG4. Docking TOG3 onto recently solved Stu2 TOG1· and TOG2·tubulin complex structures suggests that TOG3 uses similarly conserved tubulin-binding intra-HEAT loop residues to engage α- and β-tubulin. This indicates that TOG3 has maintained a TOG1- and TOG2-like TOG-tubulin binding mode despite structural divergence. The similarity of TOG domains 1–3 and the divergence of TOG4 suggest that a TOG domain array with polarized structural diversity may play a key mechanistic role in XMAP215-dependent MT polymerization activity. 相似文献