Interphase Microtubules Research Articles

Unsaturated genistein disaccharide glycoside as a novel agent affecting microtubules

Genistein, due to its recognized chemopreventive and antitumor potential, is a molecule of interest as a lead compound in drug design. While multiple molecular targets for genistein have been identified, so far neither for this isoflavonoid nor for its natural or synthetic derivatives disruption of microtubules and mitotic spindles has been reported. Here we describe such properties of the synthetic glycosidic derivative of genistein significantly more cytotoxic than genistein, 7- O-(2,3,4,6-tetra- O-acetyl-β- d-galactopyranosyl)-(1→4)-(6- O-acetyl-hex-2-ene-α- d-erythro-pyranosyl)genistein, shortly named G21. We found that G21 causes significant mitotic delay, frequent appearance of multipolar spindles, and alteration of the interphase microtubule array.

Vitamin K3 Disrupts the Microtubule Networks by Binding to Tubulin: A Novel Mechanism of Its Antiproliferative Activity

Vitamin K3 (2-methyl-1,4-naphthoquinone), also known as menadione, is the synthetic precursor of all the naturally occurring vitamin K in the body. Vitamin K is necessary for the production of prothrombin and five other blood-clotting factors in humans. We have examined the effects of menadione on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human cervical epithelial cancer cells (HeLa) and human oral epithelial cancer cells (KB) indicated that the IC(50) values for menadione are 25.6 +/- 0.6 and 64.3 +/- 0.36 microM, respectively, in those cells. Mendione arrests HeLa cells in mitosis. Immunofluorescence studies using an anti-alpha-tubulin antibody showed a significant irreversible depolymeriztion of the interphase microtubule network and spindle microtubule in a dose-dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by menadione with an IC(50) value of 47 +/- 0.65 microM. The binding of menadione with tubulin was studied using menadione fluorescence and intrinsic tryptophan fluorescence of tubulin. Binding of menadione to tubulin is slow, taking 35 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 189.12 +/- 17 and 32.44 +/- 21 M(-1) s(-1) at 25 degrees C, respectively. The stoichiometry of menadione binding to tubulin is 1:1 (molar ratio) with a dissociation constant from 2.44 +/- 0.34 to 3.65 +/- 0.25 microM at 25 degrees C. Menadione competes for the colchicine binding site with a K(i) of 2.5 muM as determined from a modified Dixon plot. The obtained data suggested that menadione binds at the colchicine binding site to tubulin. Thus, we can conclude one novel mechanism of inhibition of cancer cell proliferation by menadione is through tubulin binding.

Embryogenesis of Romanomermis culicivorax: An alternative way to construct a nematode

The current picture of embryonic development in nematodes is essentially shaped by Caenorhabditis elegans and its close relatives. As their pattern of embryogenesis is rather similar, it is often considered to be representative for the taxon Nematoda as a whole. Here we give for the first time a comprehensive description of embryonic development in an ancestrally diverged nematode. Romanomermis culicivorax differs strikingly from C. elegans with respect to cell division pattern, spatial arrangement of blastomeres and tissue formation. Our study reveals a number of unexpected phenomena. These include (i) unique polar interphase microtubule caps forming in early blastomeres destined to undergo asymmetric cleavages, suggesting the presence of a so far undescribed MTOC; (ii) embryonic cell lineages of reduced complexity with predominantly monoclonal sublineages, generating just a single tissue type; (iii) construction of major parts of the body from duplicating building blocks consisting of rings of cells, a pattern showing some resemblance to segmentation; (iv) prominent differences in cell fate assignment which can be best explained with a global shift affecting all somatic founder cells. In summary, our data indicate that during nematode evolution massive alterations in the developmental program took place of how to generate a juvenile.

Pharmacological inhibition of Aurora-A but not Aurora-B impairs interphase microtubule dynamics

Aurora kinases are key cell cycle regulators and represent attractive new targets in cancer therapy. In this work we investigated the effect of specific inhibition of Aurora-A and Aurora-B on interphase microtubule dynamics using the GSK6000063A and AZD1152 HQPA compounds respectively. We show that Aurora-A inhibition results in microtubule network disorganization and bundling. Using video microscopy and laser-based photo ablation we demonstrate that Aurora-A inhibition decreases microtubule shrinkage, growth rate, frequency rescue and nucleation. These results open new perspectives on the role of Aurora-A in interphase and might be worth considering in a pharmacological perspective.

The kinesin-14 Klp2 organizes microtubules into parallel bundles by an ATP-dependent sorting mechanism.

The dynamic organization of microtubules into parallel arrays allows interphase cells to set up multi-lane highways for intracellular transport and M-phase cells to build the mitotic and meiotic spindles. Here we show that a minimally reconstituted system composed of Klp2, a kinesin-14 from the fission yeast Schizosaccharomyces pombe, together with microtubules assembled from purified S. pombe tubulin, autonomously assembles bundles of parallel microtubules. Bundles form by an ATP-dependent sorting mechanism that requires the full-length Klp2 motor. By this mechanism, antiparallel-overlapped microtubules slide over one another until they dissociate from the bundles, whereas parallel-overlapped microtubules are selectively trapped by an energy-dissipating force-balance mechanism. Klp2-driven microtubule sorting provides a robust pathway for the organization of microtubules into parallel arrays. In vivo evidence indicates that Klp2 is required for the proper organization of S. pombe interphase microtubules into bipolar arrays of parallel-overlapped microtubules, suggesting that kinesin-14-dependent microtubule sorting may have wide biological importance.

Ciliary Abnormalities Due to Defects in the Retrograde Transport Protein DYNC2H1 in Short-Rib Polydactyly Syndrome

The short-rib polydactyly (SRP) syndromes are a heterogeneous group of perinatal lethal skeletal disorders with polydactyly and multisystem organ abnormalities. Homozygosity by descent mapping in a consanguineous SRP family identified a genomic region that contained DYNC2H1, a cytoplasmic dynein involved in retrograde transport in the cilium. Affected individuals in the family were homozygous for an exon 12 missense mutation that predicted the amino acid substitution R587C. Compound heterozygosity for one missense and one null mutation was identified in two additional nonconsanguineous SRP families. Cultured chondrocytes from affected individuals showed morphologically abnormal, shortened cilia. In addition, the chondrocytes showed abnormal cytoskeletal microtubule architecture, implicating an altered microtubule network as part of the disease process. These findings establish SRP as a cilia disorder and demonstrate that DYNC2H1 is essential for skeletogenesis and growth.

Dynamic release of nuclear RanGTP triggers TPX2-dependent microtubule assembly during the apoptotic execution phase

During apoptosis, the interphase microtubule network is dismantled then later replaced by a novel, non-centrosomal microtubule array. These microtubules assist in the peripheral redistribution of nuclear fragments in the apoptotic cell; however, the regulation of apoptotic microtubule assembly is not understood. Here, we demonstrate that microtubule assembly depends upon the release of nuclear RanGTP into the apoptotic cytoplasm because this process is blocked in apoptotic cells overexpressing dominant-negative GDP-locked Ran (T24N). Actin-myosin-II contractility provides the impetus for Ran release and, consequently, microtubule assembly is blocked in blebbistatin- and Y27632-treated apoptotic cells. Importantly, the spindle-assembly factor TPX2 (targeting protein for Xklp2), colocalises with apoptotic microtubules, and siRNA silencing of TPX2, but not of the microtubule motors Mklp1 and Kid, abrogates apoptotic microtubule assembly. These data provide a molecular explanation for the assembly of the apoptotic microtubule network, and suggest important similarities with the process of RanGTP- and TPX2-mediated mitotic spindle formation.

Theoretical Description of Microtubule Dynamics in Fission Yeast During Interphase

Fission yeast (S. pombe) is a unicellular organism with a characteristic cylindrical shape. Cell growth during interphase is strongly influenced by microtubule self-organization - a process that has been experimentally well characterised. The microtubules are organized in 3 to 4 bundles, called “interphase microtubule assemblies” (IMAs). Each IMA is composed of several microtubules, arranged with their dynamic “plus” ends facing the cell tips and their “minus” ends overlapping at the cell middle. Although the main protein factors involved in interphase microtubule organization have been identified, an understanding of how their collective interaction with microtubules leads to the organization and structures observed in vivo is lacking. We present a physical model of microtubule dynamics that aims to provide a quantitative description of the self-organization process. First, we solve equations for the microtubule length distribution in steady-state, taking into account the way that a limited tubulin pool affects the nucleation, growth and shrinkage of microtubules. Then we incorporate passive and active crosslinkers (the bundling factor Ase1 and molecular motor Klp2) and investigate the formation of IMA structures. Analytical results are complemented by a 3D stochastic simulation.

Force‐ and kinesin‐8‐dependent effects in the spatial regulation of fission yeast microtubule dynamics

Microtubules (MTs) are central to the organisation of the eukaryotic intracellular space and are involved in the control of cell morphology. For these purposes, MT polymerisation dynamics are tightly regulated. Using automated image analysis software, we investigate the spatial dependence of MT dynamics in interphase fission yeast cells with unprecedented statistical accuracy. We find that MT catastrophe frequencies (switches from polymerisation to depolymerisation) strongly depend on intracellular position. We provide evidence that compressive forces generated by MTs growing against the cell pole locally reduce MT growth velocities and enhance catastrophe frequencies. Furthermore, we find evidence for an MT length-dependent increase in the catastrophe frequency that is mediated by kinesin-8 proteins (Klp5/6). Given the intrinsic susceptibility of MT dynamics to compressive forces and the widespread importance of kinesin-8 proteins, we propose that similar spatial regulation of MT dynamics plays a role in other cell types as well. In addition, our systematic and quantitative data should provide valuable input for (mathematical) models of MT organisation in living cells.

Force‐ and length‐dependent catastrophe activities explain interphase microtubule organization in fission yeast

The cytoskeleton is essential for the maintenance of cell morphology in eukaryotes. In fission yeast, for example, polarized growth sites are organized by actin, whereas microtubules (MTs) acting upstream control where growth occurs. Growth is limited to the cell poles when MTs undergo catastrophes there and not elsewhere on the cortex. Here, we report that the modulation of MT dynamics by forces as observed in vitro can quantitatively explain the localization of MT catastrophes in Schizosaccharomyces pombe. However, we found that it is necessary to add length-dependent catastrophe rates to make the model fully consistent with other previously measured traits of MTs. We explain the measured statistical distribution of MT–cortex contact times and re-examine the curling behavior of MTs in unbranched straight tea1Δ cells. Importantly, the model demonstrates that MTs together with associated proteins such as depolymerizing kinesins are, in principle, sufficient to mark the cell poles.

Disturbance of the radial system of interphase microtubules in the presence of excess serum in cell culture medium

The role of the orderliness of microtubules in the cell has been studied. For this purpose, a population of Vero cells with chaotically arranged microtubules was used, which was obtained after the addition of excess serum to cell culture medium. An increase in the total area and a slight dispersion of the Golgi apparatus were found; however, the rate of culture growth as a whole remained normal. Thus, the radial arrangement of microtubules is not vital even for cells where it is usually well pronounced.

Stress-regulated kinase pathways in the recovery of tip growth and microtubule dynamics following osmotic stress inS. pombe

The cell-integrity and stress-response MAP kinase pathways (CIP and SRP, respectively) are stimulated by various environmental stresses. Ssp1 kinase modulates actin dynamics and is rapidly recruited to the plasma membrane following osmotic stress. Here, we show that osmotic stress arrested tip growth, induced the deposition of abnormal cell-wall deposits at tips and led to disassociation of F-actin foci from cell tips together with a reduction in the amount of F-actin in these foci. Osmotic stress also ;froze' the dynamics of interphase microtubule bundles, with microtubules remaining static for approximately 38 minutes (at 30 degrees C) before fragmenting upon return to dynamic behaviour. The timing with which microtubules resumed dynamic behaviour relied upon SRP activation of Atf1-mediated transcription, but not on either CIP or Ssp1 signalling. Analysis of the recovery of tip growth showed that: (1) the timing of recovery was controlled by SRP-stimulated Atf1 transcription; (2) re-establishment of polarized tip growth was absolutely dependent upon SRP and partially dependent upon Ssp1 signalling; and (3) selection of the site for polarized tip extension required Ssp1 and the SRP-associated polarity factor Wsh3 (also known as Tea4). CIP signalling did not impact upon any aspect of recovery. The normal kinetics of tip growth following osmotic stress of plo1.S402A/E mutants established that SRP control over the resumption of tip growth after osmotic stress is distinct from its control of tip growth following heat or gravitational stresses.

Benomyl and Colchicine Synergistically Inhibit Cell Proliferation and Mitosis: Evidence of Distinct Binding Sites for These Agents in Tubulin

Benomyl, a tubulin-targeted antimitotic antifungal agent, belongs to the benzimidazole group of compounds, which are known to inhibit the binding of colchicine to tubulin. Therefore, benomyl was thought to bind at or near the colchicine-binding site on tubulin. However, recent mutational studies in yeast and fluorescence studies involving competitive binding of benomyl and colchicine on goat brain tubulin suggested that benomyl may bind to tubulin at a site distinct from the colchicine-binding site. We set out to examine whether colchicine and benomyl bind to tubulin at distinct sites using a human cervical cancer (HeLa) cell line with the thinking that these agents should exert either additive or synergistic activity on cell proliferation if their binding sites on tubulin are different. We found that benomyl and colchicine synergistically inhibited the proliferation of HeLa cells and blocked their cell cycle progression at mitosis. The synergistic activity of benomyl and colchicine was also apparent from their strong depolymerizing effects on both the spindle and interphase microtubules when used in combinations, providing further evidence that these agents bind to tubulin at different sites. Using NMR spectroscopy, we finally demonstrated that benomyl and colchicine bind to tubulin at different sites and that the binding of colchicine seems to positively influence the binding of benomyl to tubulin and vice versa. Further, an analysis of the saturation transfer difference NMR data yielded an interesting insight into the colchicine-tubulin interaction. The data presented in this study provided a mechanistic understanding of the synergistic effects of benomyl and colchicine on HeLa cell proliferation.

Dynein, Lis1 and CLIP-170 counteract Eg5-dependent centrosome separation during bipolar spindle assembly

Bipolar spindle assembly critically depends on the microtubule plus-end-directed motor Eg5 that binds antiparallel microtubules and slides them in opposite directions. As such, Eg5 can produce the necessary outward force within the spindle that drives centrosome separation and inhibition of this antiparallel sliding activity results in the formation of monopolar spindles. Here, we show that upon depletion of the minus-end-directed motor dynein, or the dynein-binding protein Lis1, bipolar spindles can form in human cells with substantially less Eg5 activity, suggesting that dynein and Lis1 produce an inward force that counteracts the Eg5-dependent outward force. Interestingly, we also observe restoration of spindle bipolarity upon depletion of the microtubule plus-end-tracking protein CLIP-170. This function of CLIP-170 in spindle bipolarity seems to be mediated through its interaction with dynein, as loss of CLIP-115, a highly homologous protein that lacks the dynein-dynactin interaction domain, does not restore spindle bipolarity. Taken together, these results suggest that complexes of dynein, Lis1 and CLIP-170 crosslink and slide microtubules within the spindle, thereby producing an inward force that pulls centrosomes together.

Suppression of microtubule dynamic instability and turnover in MCF7 breast cancer cells by sulforaphane

Sulforaphane (SFN), a prominent isothiocyanate present in cruciferous vegetables, is believed to be responsible along with other isothiocyanates for the cancer preventive activity of such vegetables. SFN arrests mitosis, possibly by affecting spindle microtubule function. A critical property of microtubules is their rapid and time-sensitive growth and shortening dynamics (dynamic instability), and suppression of dynamics by antimitotic anticancer drugs (e.g. taxanes and the vinca alkaloids) is central to the anticancer mechanisms of such drugs. We found that at concentrations that inhibited proliferation and mitosis of MCF7-green fluorescent protein-alpha-tubulin breast tumor cells by approximately 50% (~15 microM), SFN significantly modified microtubule organization in arrested spindles without modulating the spindle microtubule mass, in a manner similar to that of much more powerful antimitotic drugs. By using quantitative fluorescence video microscopy, we determined that at its mitotic concentration required to inhibit mitosis by 50%, SFN suppressed the dynamic instability of the interphase microtubules in these cells, strongly reducing the rate and extent of growth and shortening and decreasing microtubule turnover, without affecting the polymer mass. SFN suppressed the dynamics of purified microtubules in a similar fashion at concentrations well below those required to depolymerize microtubules, indicating that the suppression of dynamic instability by SFN in cells is due to a direct effect on the microtubules. The results indicate that SFN arrests proliferation and mitosis by stabilizing microtubules in a manner weaker than but similar to more powerful clinically used antimitotic anticancer drugs and strongly support the hypothesis that inhibition of mitosis by microtubule stabilization is important for SFN's chemopreventive activity.

Sequestration of Polo kinase to microtubules by phosphopriming-independent binding to Map205 is relieved by phosphorylation at a CDK site in mitosis

The conserved Polo kinase controls multiple events in mitosis and cytokinesis. Although Polo-like kinases are regulated by phosphorylation and proteolysis, control of subcellular localization plays a major role in coordinating their mitotic functions. This is achieved largely by the Polo-Box Domain, which binds prephosphorylated targets. However, it remains unclear whether and how Polo might interact with partner proteins when priming mitotic kinases are inactive. Here we show that Polo associates with microtubules in interphase and cytokinesis, through a strong interaction with the microtubule-associated protein Map205. Surprisingly, this interaction does not require priming phosphorylation of Map205, and the Polo-Box Domain of Polo is required but not sufficient for this interaction. Moreover, phosphorylation of Map205 at a CDK site relieves this interaction. Map205 can stabilize Polo and inhibit its cellular activity in vivo. In syncytial embryos, the centrosome defects observed in polo hypomorphs are enhanced by overexpression of Map205 and suppressed by its deletion. We propose that Map205-dependent targeting of Polo to microtubules provides a stable reservoir of Polo that can be rapidly mobilized by the activity of Cdk1 at mitotic entry.

Fission Yeast Kinesin-8 Klp5 and Klp6 Are Interdependent for Mitotic Nuclear Retention and Required for Proper Microtubule Dynamics

Fission yeast has two kinesin-8s, Klp5 and Klp6, which associate to form a heterocomplex. Here, we show that Klp5 and Klp6 are mutually dependent on each other for nuclear mitotic localization. During interphase, they are exported to the cytoplasm. In sharp contrast, during mitosis, Klp5 and Klp6 remain in the nucleus, which requires the existence of each counterpart. Canonical nuclear localization signal (NLS) is identified in the nonkinesin C-terminal regions. Intriguingly individual NLS mutants (NLSmut) exhibit loss-of-function phenotypes, suggesting that Klp5 and Klp6 enter the nucleus separately. Indeed, although neither Klp5-NLSmut nor Klp6-NLSmut enters the nucleus, wild-type Klp6 or Klp5, respectively, does so with different kinetics. In the absence of Klp5/6, microtubule catastrophe/rescue frequency and dynamicity are suppressed, whereas growth and shrinkage rates are least affected. Remarkably, chimera strains containing only the N-terminal Klp5 kinesin domains cannot disassemble interphase microtubules during mitosis, leading to the coexistence of cytoplasmic microtubules and nuclear spindles with massive chromosome missegregation. In this strain, a marked reduction of microtubule dynamism, even higher than in klp5/6 deletions, is evident. We propose that Klp5 and Klp6 play a vital role in promoting microtubule dynamics, which is essential for the spatiotemporal control of microtubule morphogenesis.

The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L.

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.

The Natural Naphthoquinone Plumbagin Exhibits Antiproliferative Activity and Disrupts the Microtubule Network through Tubulin Binding

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.

Antiangiogenic vinflunine affects EB1 localization and microtubule targeting to adhesion sites

The motile behavior of endothelial cells is a crucial event for neoangiogenesis. We previously showed that noncytotoxic concentrations of vinflunine inhibit capillary-like tube formation on Matrigel and endothelial cell migration with a concomitant increase in interphase microtubule dynamic instability. In this article, we further investigated the effects of vinflunine on migration and cytoskeleton interaction dynamics in HMEC-1 endothelial cells. We confirmed that vinflunine, at low and noncytotoxic concentrations (0.01-1 nmol/L), inhibited endothelial cell random motility by 54%. This effect was associated with a decrease in the percentage of stable microtubules and in the mean duration of pauses for dynamic ones. Moreover, we found that vinflunine altered adhesion site targeting by microtubules and suppressed the microtubule (+) end pause that occurs at adhesion sites during cell migration (from 151 +/- 20 seconds in control cells to 38 +/- 7 seconds in vinflunine-treated cells, P < 0.001). This effect was associated with the inhibition of adhesion site dynamics and the formation of long-lived stress fibers. Importantly, we found that vinflunine altered EB1 localization at microtubule (+) ends. These results highlight a new mechanism of action of vinflunine, which act by disrupting the mutual control between microtubule and adhesion site dynamics and strengthen the role of +TIPs proteins such as EB1 as key regulators of endothelial cell motility.