Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis

1992 ◽  
Vol 102 (3) ◽  
pp. 401-416 ◽  
Author(s):  
M.A. Jordan ◽  
D. Thrower ◽  
L. Wilson
Keyword(s):  
Hela Cells ◽  
Mitotic Arrest ◽  
Mitotic Spindles ◽  
Microtubule Depolymerization ◽  
Metaphase Chromosomes ◽  
Low Concentrations ◽  
Subtle Effect ◽  
Full Complement ◽  
Spindle Microtubules

Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.

Mitotic block in HeLa cells by vinblastine: ultrastructural changes in kinetochore-microtubule attachment and in centrosomes

1993 ◽  
Vol 104 (2) ◽  
pp. 261-274 ◽  
Author(s):  
K.L. Wendell ◽  
L. Wilson ◽  
M.A. Jordan
Keyword(s):  
Cell Cycle ◽  
Hela Cells ◽  
Mitotic Spindle ◽  
Close Association ◽  
Ultrastructural Changes ◽  
Microtubule Depolymerization ◽  
Metaphase Chromosomes ◽  
Mitotic Block ◽  
Mother And Daughter ◽  
Spindle Microtubules

Previous work from this laboratory has indicated that very low concentrations of vinblastine block HeLa cells at mitosis in the presence of a full complement of microtubules and without major disruption of spindle organization. In the present study we analyzed the structural organization of mitotic spindle microtubules, chromosomes and centrosomes by electron microscopy after incubating HeLa cells for one cell cycle with 2 nM vinblastine. We found that mitotic block of HeLa cells by vinblastine was associated with alterations of the fine structure of the spindle that were subtle but profound in their apparent consequences. The cell cycle was blocked in a stage that resembled prometaphase or metaphase; chromosomes had not undergone anaphase segregation. Neither the structure of the microtubules nor the structure of the kinetochores was detectably altered by the drug. However, the number of microtubules attached to kinetochores was decreased significantly. In addition, the centrosomes were altered; the normal close association of mother and daughter centriole was lost, numerous membranous vesicles were found in the centrosomal region, and many centrioles exhibited abnormal ultrastructure and had microtubules coursing through their interiors. These findings are consistent with our previous results and indicate that inhibition of the polymerization dynamics of mitotic spindle microtubules and perhaps of centriole microtubules, rather than microtubule depolymerization, is responsible for the mitotic inhibition by vinblastine.

scholarly journals Mutations at the asp locus of Drosophila lead to multiple free centrosomes in syncytial embryos, but restrict centrosome duplication in larval neuroblasts

1990 ◽  
Vol 96 (4) ◽  
pp. 605-616 ◽  
Author(s):  
C. Gonzalez ◽  
R.D. Saunders ◽  
J. Casal ◽  
I. Molina ◽  
M. Carmena ◽  
...  
Keyword(s):  
Mitotic Index ◽  
Centrosome Duplication ◽  
Mitotic Spindles ◽  
Female Meiosis ◽  
Metaphase Chromosomes ◽  
Meiotic Spindles ◽  
Spindle Microtubules ◽  
As If

Mutations at abnormal spindle result in abnormally long and wavy microtubules in the meiotic spindles of males. Some of these spindles have a single pole and take the form of unopposed hemi-spindles. Unfertilised eggs produced by homozygous asp females may have either no nuclei, or a small number of large nuclei, consistent with there also being an effect upon female meiosis. Such eggs also display free centrosomes and independent arrays of microtubules. Embryos that have this phenotype are also present among the progeny of fertilised homozygous asp females, together with embryos that undergo varying degrees of aberrant morphogenesis, developing a variety of abnormal cuticle patterns. This latter category shows asynchronous mitoses prior to cellularisation, and has abnormal arrays of spindle microtubules. Such embryos can develop large areas that are either devoid of or have a reduced number of nuclei, in which there are centrosomes that have dissociated from the mitotic spindles. Neuroblasts in the brains of homozygous asp larvae display a high mitotic index, and have condensed chromosomes aligned as if blocked at metaphase. Immunostaining reveals that many cells contain a single centrosome connected to the metaphase chromosomes by microtubules in a hemi-spindle-like structure.

scholarly journals Physiological and ultrastructural analysis of elongating mitotic spindles reactivated in vitro.

1986 ◽  
Vol 103 (2) ◽  
pp. 593-604 ◽  
Author(s):  
W Z Cande ◽  
K McDonald
Keyword(s):  
Cross Sections ◽  
Simple Procedure ◽  
Ultrastructural Level ◽  
Mitotic Spindles ◽  
Gap Formation ◽  
Microtubule Depolymerization ◽  
Central Spindle ◽  
Original Number ◽  
Spindle Elongation

We have developed a simple procedure for isolating mitotic spindles from the diatom Stephanopyxis turris and have shown that they undergo anaphase spindle elongation in vitro upon addition of ATP. The isolated central spindle is a barrel-shaped structure with a prominent zone of microtubule overlap. After ATP addition greater than 75% of the spindle population undergoes distinct structural rearrangements: the spindles on average are longer and the two half-spindles are separated by a distinct gap traversed by only a small number of microtubules, the phase-dense material in the overlap zone is gone, and the peripheral microtubule arrays have depolymerized. At the ultrastructural level, we examined serial cross-sections of spindles after 1-, 5-, and 10-min incubations in reactivation medium. Microtubule depolymerization distal to the poles is confirmed by the increased number of incomplete, i.e., c-microtubule profiles specifically located in the region of overlap. After 10 min we see areas of reduced microtubule number which correspond to the gaps seen in the light microscope and an overall reduction in the number of half-spindle microtubules to about one-third the original number. The changes in spindle structure are highly specific for ATP, are dose-dependent, and do not occur with nonhydrolyzable nucleotide analogues. Spindle elongation and gap formation are blocked by 10 microM vanadate, equimolar mixtures of ATP and AMPPNP, and by sulfhydryl reagents. This process is not affected by nocodazole, erythro-9-[3-(2-hydroxynonyl)]adenine, cytochalasin D, and phalloidin. In the presence of taxol, the extent of spindle elongation is increased; however, distinct gaps still form between the two half-spindles. These results show that the response of isolated spindles to ATP is a complex process consisting of several discrete steps including initiation events, spindle elongation mechanochemistry, controlled central spindle microtubule plus-end depolymerization, and loss of peripheral microtubules. They also show that the microtubule overlap zone is an important site of ATP action and suggest that spindle elongation in vitro is best explained by a mechanism of microtubule-microtubule sliding. Spindle elongation in vitro cannot be accounted for by cytoplasmic forces pulling on the poles or by microtubule polymerization.

scholarly journals Kinesin-8 from Fission Yeast: A Heterodimeric, Plus-End–directed Motor that Can Couple Microtubule Depolymerization to Cargo Movement

2009 ◽  
Vol 20 (3) ◽  
pp. 963-972 ◽  
Author(s):  
Paula M. Grissom ◽  
Thomas Fiedler ◽  
Ekaterina L. Grishchuk ◽  
Daniela Nicastro ◽  
Robert R. West ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Metaphase Plate ◽  
Sf9 Cells ◽  
Microtubule Depolymerization ◽  
Chromosome Congression ◽  
Motor Activities ◽  
Spindle Microtubules ◽  
Anaphase B

Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5+ and klp6+ genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end–directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A.

scholarly journals Kinetochore Fiber Maturation in PtK1 Cells and Its Implications for the Mechanisms of Chromosome Congression and Anaphase Onset

1997 ◽  
Vol 137 (7) ◽  
pp. 1567-1580 ◽  
Author(s):  
Bruce F. McEwen ◽  
Amy B. Heagle ◽  
Grisel O. Cassels ◽  
Karolyn F. Buttle ◽  
Conly L. Rieder
Keyword(s):  
Time Course ◽  
Spindle Pole ◽  
Metaphase Chromosomes ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Chromosome Congression ◽  
Early Anaphase ◽  
Full Complement ◽  
Spindle Microtubules ◽  
Chromosome Motion

Kinetochore microtubules (kMts) are a subset of spindle microtubules that bind directly to the kinetochore to form the kinetochore fiber (K-fiber). The K-fiber in turn interacts with the kinetochore to produce chromosome motion toward the attached spindle pole. We have examined K-fiber maturation in PtK1 cells using same-cell video light microscopy/serial section EM. During congression, the kinetochore moving away from its spindle pole (i.e., the trailing kinetochore) and its leading, poleward moving sister both have variable numbers of kMts, but the trailing kinetochore always has at least twice as many kMts as the leading kinetochore. A comparison of Mt numbers on sister kinetochores of congressing chromosomes with their direction of motion, as well as distance from their associated spindle poles, reveals that the direction of motion is not determined by kMt number or total kMt length. The same result was observed for oscillating metaphase chromosomes. These data demonstrate that the tendency of a kinetochore to move poleward is not positively correlated with the kMt number. At late prometaphase, the average number of Mts on fully congressed kinetochores is 19.7 ± 6.7 (n = 94), at late metaphase 24.3 ± 4.9 (n = 62), and at early anaphase 27.8 ± 6.3 (n = 65). Differences between these distributions are statistically significant. The increased kMt number during early anaphase, relative to late metaphase, reflects the increased kMt stability at anaphase onset. Treatment of late metaphase cells with 1 μM taxol inhibits anaphase onset, but produces the same kMt distribution as in early anaphase: 28.7 ± 7.4 (n = 54). Thus, a full complement of kMts is not sufficient to induce anaphase onset. We also measured the time course for kMt acquisition and determined an initial rate of 1.9 kMts/min. This rate accelerates up to 10-fold during the course of K-fiber maturation, suggesting an increased concentration of Mt plus ends in the vicinity of the kinetochore at late metaphase and/or cooperativity for kMt acquisition.

scholarly journals Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles

2004 ◽  
Vol 166 (4) ◽  
pp. 465-471 ◽  
Author(s):  
Jedidiah Gaetz ◽  
Tarun M. Kapoor
Keyword(s):  
Cell Division ◽  
Microtubule Depolymerization ◽  
Spindle Microtubule ◽  
Constant Length ◽  
Motor Complex ◽  
Spindle Poles ◽  
Microtubule Transport ◽  
Spindle Microtubules ◽  
Cross Linker

During cell division metaphase spindles maintain constant length, whereas spindle microtubules continuously flux polewards, requiring addition of tubulin subunits at microtubule plus-ends, polewards translocation of the microtubule lattice, and removal of tubulin subunits from microtubule minus-ends near spindle poles. How these processes are coordinated is unknown. Here, we show that dynein/dynactin, a multi-subunit microtubule minus-end–directed motor complex, and NuMA, a microtubule cross-linker, regulate spindle length. Fluorescent speckle microscopy reveals that dynactin or NuMA inhibition suppresses microtubule disassembly at spindle poles without affecting polewards microtubule sliding. The observed uncoupling of these two components of flux indicates that microtubule depolymerization is not required for the microtubule transport associated with polewards flux. Inhibition of Kif2a, a KinI kinesin known to depolymerize microtubules in vitro, results in increased spindle microtubule length. We find that dynein/dynactin contribute to the targeting of Kif2a to spindle poles, suggesting a model in which dynein/dynactin regulate spindle length and coordinate flux by maintaining microtubule depolymerizing activities at spindle poles.

scholarly journals Electron cryotomography analysis of Dam1C/DASH at the kinetochore–spindle interface in situ

2018 ◽  
Vol 218 (2) ◽  
pp. 455-473 ◽  
Author(s):  
Cai Tong Ng ◽  
Li Deng ◽  
Chen Chen ◽  
Hong Hwa Lim ◽  
Jian Shi ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Depolymerization ◽  
Yeast Chromosome ◽  
Dividing Cells ◽  
Spindle Microtubules ◽  
Electron Cryotomography ◽  
Binding Position

In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible “bridges”; there are no contacts with the protofilaments’ curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement.

In vitro study on cytotoxicity and intracellular formaldehyde concentration changes after exposure to formaldehyde and its derivatives

2013 ◽  
Vol 33 (8) ◽  
pp. 822-830 ◽  
Author(s):  
YJ Ke ◽  
XD Qin ◽  
YC Zhang ◽  
H Li ◽  
R Li ◽  
...  
Keyword(s):  
Cell Growth ◽  
Formic Acid ◽  
Hela Cells ◽  
Cell Activity ◽  
In Vitro Study ◽  
Formaldehyde Concentration ◽  
Low Concentrations ◽  
Formaldehyde Degradation ◽  
Concentration Changes

HeLa cells were exposed to formaldehyde and its metabolic derivatives, methanol, formic acid, and acetaldehyde, to investigate that the toxicity of formaldehyde is not caused by the chemical group. After 1 h of treatment with formaldehyde, mitochondrial assays showed that low concentrations (e.g. 10 μmol/L) of formaldehyde promoted growth of the HeLa cells, while higher concentrations (e.g. ≥62.5 μmol/L) inhibited cell growth; while all four chemicals at a concentration of 125 μmol/L affected cell growth, formaldehyde affected the largest. Reactive oxygen species concentration increased with the concentration of the exposure chemical. The endogenous formaldehyde content increased the most in the formaldehyde group, but in other three groups, it did not increase as the exposure concentration increased. Expression of dehydrogenase (formaldehyde dehydrogenase (FDH)) in the formaldehyde (10.40) and methanol (10.60) groups increased significantly compared with the control (1), while it was similar to the control in formic acid (0.90) and acetaldehyde (1.10) groups. Our results suggest that formaldehyde could affect cell activity and even enter cells. Exposure to formaldehyde changes the endogenous formaldehyde concentration in cells within 24 h, and this induces expression of FDH for formaldehyde degradation to maintain the formaldehyde balance. The toxicity of formaldehyde is not caused by the carbon atoms in the aldehyde, hydroxyl, or carboxyl groups. Formaldehyde is hypothesized to be an important signaling molecule in the regulation of cell growth and maintenance of the endogenous formaldehyde level.

scholarly journals Bod1, a novel kinetochore protein required for chromosome biorientation

2007 ◽  
Vol 179 (2) ◽  
pp. 187-197 ◽  
Author(s):  
Iain M. Porter ◽  
Sarah E. McClelland ◽  
Guennadi A. Khoudoli ◽  
Christopher J. Hunter ◽  
Jens S. Andersen ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Hela Cells ◽  
Aurora B ◽  
Macromolecular Complex ◽  
Mitotic Spindles ◽  
Kinetochore Protein ◽  
Spindle Poles ◽  
Interfering Rna ◽  
Rna Depletion

We have combined the proteomic analysis of Xenopus laevis in vitro–assembled chromosomes with RNA interference and live cell imaging in HeLa cells to identify novel factors required for proper chromosome segregation. The first of these is Bod1, a protein conserved throughout metazoans that associates with a large macromolecular complex and localizes with kinetochores and spindle poles during mitosis. Small interfering RNA depletion of Bod1 in HeLa cells produces elongated mitotic spindles with severe biorientation defects. Bod1-depleted cells form syntelic attachments that can oscillate and generate enough force to separate sister kinetochores, suggesting that microtubule–kinetochore interactions were intact. Releasing Bod1-depleted cells from a monastrol block increases the frequency of syntelic attachments and the number of cells displaying biorientation defects. Bod1 depletion does not affect the activity or localization of Aurora B but does cause mislocalization of the microtubule depolymerase mitotic centromere- associated kinesin and prevents its efficient phosphorylation by Aurora B. Therefore, Bod1 is a novel kinetochore protein that is required for the detection or resolution of syntelic attachments in mitotic spindles.

scholarly journals Vinblastine suppresses dynamics of individual microtubules in living interphase cells.

1995 ◽  
Vol 6 (9) ◽  
pp. 1215-1229 ◽  
Author(s):  
R Dhamodharan ◽  
M A Jordan ◽  
D Thrower ◽  
L Wilson ◽  
P Wadsworth
Keyword(s):  
Dynamic Instability ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Microtubule Depolymerization ◽  
Dynamic Activity ◽  
Low Concentrations ◽  
Interphase Cells ◽  
Endogenous Regulators ◽  
Microtubule Growth

We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells.

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