The effects of diazepam on mitosis and the microtubule cytoskeleton. I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta

1994 ◽  
Vol 107 (9) ◽  
pp. 2643-2651
Author(s):  
T.P. Spurck ◽  
J.D. Pickett-Heaps
Keyword(s):  
Time Lapse ◽  
Detectable Effect ◽  
Metaphase Chromosomes ◽  
Central Spindle ◽  
Cell Cleavage ◽  
Spindle Poles ◽  
Transmission Electron ◽  
Live Diatoms ◽  
Interphase Cells ◽  
Chromosome Motion

The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy. DZP treatment (250 microM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle. Chromosomes remaining attached to one pole continued to display slight prometaphase oscillations; however, this activity was never observed in metaphase spindles. Following removal of DZP, some chromosomes still bipolarly attached, immediately released elastically from one pole. Within the first 2 minutes of recovery, all chromosomes recommenced spindle attachment, exhibiting normal prometaphase oscillations and proceeded through mitosis. DZP treatment during anaphase had no detectable effect on chromosome motion or cell cleavage. These results suggest that DZP acts as an anti-MT agent, selectively affecting polar MTs at prophase, prometaphase and metaphase, and thereby weakening kinetochore connection to the poles. From these and other results (unpublished), its mode of action is different to that of most anti-MT agents.

scholarly journals Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

2019 ◽  
Vol 30 (19) ◽  
pp. 2503-2514 ◽  
Author(s):  
Che-Hang Yu ◽  
Stefanie Redemann ◽  
Hai-Yin Wu ◽  
Robert Kiewisz ◽  
Tae Yeon Yoo ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Tomographic Reconstruction ◽  
Mitotic Spindles ◽  
Strongly Coupled ◽  
Central Spindle ◽  
Spindle Poles ◽  
Meiotic Spindles ◽  
Spindle Microtubules ◽  
Anaphase B ◽  
Chromosome Motion

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.

scholarly journals Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

2019 ◽  
Author(s):  
Che-Hang Yu ◽  
Stefanie Redemann ◽  
Hai-Yin Wu ◽  
Robert Kiewisz ◽  
Tae Yeon Yoo ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Tomographic Reconstruction ◽  
Mitotic Spindles ◽  
Strongly Coupled ◽  
Central Spindle ◽  
Spindle Poles ◽  
Meiotic Spindles ◽  
Spindle Microtubules ◽  
Anaphase B ◽  
Chromosome Motion

AbstractSpindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, i.e. on the region between chromosomes and poles. In comparison, microtubules in the central spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central spindle microtubules during chromosome segregation in human mitotic spindles, and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move towards spindle poles. In these systems, damaging central spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central spindle microtubules during chromosome segregation in diverse spindles, and suggest that central spindle microtubules and chromosomes are strongly coupled in anaphase.

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 Ablation of PRC1 by Small Interfering RNA Demonstrates that Cytokinetic Abscission Requires a Central Spindle Bundle in Mammalian Cells, whereas Completion of Furrowing Does Not

2005 ◽  
Vol 16 (3) ◽  
pp. 1043-1055 ◽  
Author(s):  
Cristiana Mollinari ◽  
Jean-Philippe Kleman ◽  
Yasmina Saoudi ◽  
Sandra A. Jablonski ◽  
Julien Perard ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Time Lapse ◽  
Cell Cortex ◽  
Central Spindle ◽  
Spatial Regulation ◽  
Daughter Cells ◽  
Cell Cleavage ◽  
Temporal And Spatial ◽  
Interfering Rna ◽  
Passenger Proteins

The temporal and spatial regulation of cytokinesis requires an interaction between the anaphase mitotic spindle and the cell cortex. However, the relative roles of the spindle asters or the central spindle bundle are not clear in mammalian cells. The central spindle normally serves as a platform to localize key regulators of cell cleavage, including passenger proteins. Using time-lapse and immunofluorescence analysis, we have addressed the consequences of eliminating the central spindle by ablation of PRC1, a microtubule bundling protein that is critical to the formation of the central spindle. Without a central spindle, the asters guide the equatorial cortical accumulation of anillin and actin, and of the passenger proteins, which organize into a subcortical ring in anaphase. Furrowing goes to completion, but abscission to create two daughter cells fails. We conclude the central spindle bundle is required for abscission but not for furrowing in mammalian cells.

scholarly journals PRESENCE OF ABNORMAL CELL CLEAVAGE, CELL FUSION OR BOTH SEEN DURING EMBRYO DEVELOPMENT ON TIME-LAPSE MICROSCOPY IS NOT PREDICTIVE OF CHROMOSOMAL ANEUPLOIDY

2020 ◽  
Vol 114 (3) ◽  
pp. e44
Author(s):  
Alice J. Shapiro ◽  
Lindsay Kroener ◽  
Nicholas J. Jackson ◽  
Zachary Haimowitz ◽  
Alin Lina Akopians ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Embryo Development ◽  
Time Lapse ◽  
Abnormal Cell ◽  
Time Lapse Microscopy ◽  
Cell Cleavage ◽  
Chromosomal Aneuploidy

scholarly journals Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry.

1979 ◽  
Vol 27 (1) ◽  
pp. 478-485 ◽  
Author(s):  
Z Darzynkiewicz ◽  
F Traganos ◽  
M Andreeff ◽  
T Sharpless ◽  
M R Melamed
Keyword(s):  
Acridine Orange ◽  
Condensed Chromatin ◽  
Metaphase Chromosomes ◽  
Quiescent Cells ◽  
Acid Denaturation ◽  
Interphase Cells ◽  
The Difference ◽  
Cell Subpopulations ◽  
G1 Cells ◽  
Dna Complex

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.

scholarly journals Anastral meiotic spindle morphogenesis: role of the non-claret disjunctional kinesin-like protein.

1996 ◽  
Vol 134 (2) ◽  
pp. 455-464 ◽  
Author(s):  
H J Matthies ◽  
H B McDonald ◽  
L S Goldstein ◽  
W E Theurkauf
Keyword(s):  
Laser Scanning ◽  
Spindle Assembly ◽  
Time Lapse ◽  
Laser Scanning Confocal Microscopy ◽  
Meiotic Spindle ◽  
Bipolar Spindle ◽  
Scanning Confocal Microscopy ◽  
Spindle Poles ◽  
Assembly Kinetics

We have used time-lapse laser scanning confocal microscopy to directly examine microtubule reorganization during meiotic spindle assembly in living Drosophila oocytes. These studies indicate that the bipolarity of the meiosis I spindle is not the result of a duplication and separation of centrosomal microtubule organizing centers (MTOCs). Instead, microtubules first associate with a tight chromatin mass, and then bundle to form a bipolar spindle that lacks asters. Analysis of mutant oocytes indicates that the Non-Claret Disjunctional (NCD) kinesin-like protein is required for normal spindle assembly kinetics and stabilization of the spindle during metaphase arrest. Immunolocalization analyses demonstrate that NCD is associated with spindle microtubules, and that the centrosomal components gamma-tubulin, CP-190, and CP-60 are not concentrated at the meiotic spindle poles. Based on these observations, we propose that microtubule bundling by the NCD kinesin-like protein promotes assembly of a stable bipolar spindle in the absence of typical MTOCs.

scholarly journals Ordered Kinetochore Assembly in the Human-Pathogenic Basidiomycetous Yeast Cryptococcus neoformans

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Lukasz Kozubowski ◽  
Vikas Yadav ◽  
Gautam Chatterjee ◽  
Shreyas Sridhar ◽  
Masashi Yamaguchi ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Cryptococcus Neoformans ◽  
Budding Yeast ◽  
Genetic Material ◽  
Time Lapse ◽  
Content Type ◽  
Nuclear Envelope Breakdown ◽  
Kinetochore Assembly ◽  
Transmission Electron ◽  
Partial Opening

ABSTRACT Kinetochores facilitate interaction between chromosomes and the spindle apparatus. The formation of a metazoan trilayered kinetochore is an ordered event in which inner, middle, and outer layers assemble during disassembly of the nuclear envelope during mitosis. The existence of a similar strong correlation between kinetochore assembly and nuclear envelope breakdown in unicellular eukaryotes is unclear. Studies in the hemiascomycetous budding yeasts Saccharomyces cerevisiae and Candida albicans suggest that an ordered kinetochore assembly may not be evolutionarily conserved. Here, we utilized high-resolution time-lapse microscopy to analyze the localization patterns of a series of putative kinetochore proteins in the basidiomycetous budding yeast Cryptococcus neoformans, a human pathogen. Strikingly, similar to most metazoa but atypical of yeasts, the centromeres are not clustered but positioned adjacent to the nuclear envelope in premitotic C. neoformans cells. The centromeres gradually coalesce to a single cluster as cells progress toward mitosis. The mitotic clustering of centromeres seems to be dependent on the integrity of the mitotic spindle. To study the dynamics of the nuclear envelope, we followed the localization of two marker proteins, Ndc1 and Nup107. Fluorescence microscopy of the nuclear envelope and components of the kinetochore, along with ultrastructure analysis by transmission electron microscopy, reveal that in C. neoformans, the kinetochore assembles in an ordered manner prior to mitosis in concert with a partial opening of the nuclear envelope. Taken together, the results of this study demonstrate that kinetochore dynamics in C. neoformans is reminiscent of that of metazoans and shed new light on the evolution of mitosis in eukaryotes. IMPORTANCE Successful propagation of genetic material in progeny is essential for the survival of any organism. A proper kinetochore-microtubule interaction is crucial for high-fidelity chromosome segregation. An error in this process can lead to loss or gain of chromosomes, a common feature of most solid cancers. Several proteins assemble on centromere DNA to form a kinetochore. However, significant differences in the process of kinetochore assembly exist between unicellular yeasts and multicellular metaozoa. Here, we examined the key events that lead to formation of a proper kinetochore in a basidiomycetous budding yeast, Cryptococcus neoformans. We found that, during the progression of the cell cycle, nonclustered centromeres gradually clustered and kinetochores assembled in an ordered manner concomitant with partial opening of the nuclear envelope in this organism. These events have higher similarity to mitotic events of metazoans than to those previously described in other yeasts.

scholarly journals The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles

2001 ◽  
Vol 154 (6) ◽  
pp. 1135-1146 ◽  
Author(s):  
Aime A. Levesque ◽  
Duane A. Compton
Keyword(s):  
Cultured Cells ◽  
Time Lapse ◽  
Spindle Pole ◽  
Mitotic Spindles ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Poles ◽  
Ejection Force ◽  
Chromosome Congression ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy

Chromokinesins have been postulated to provide the polar ejection force needed for chromosome congression during mitosis. We have evaluated that possibility by monitoring chromosome movement in vertebrate-cultured cells using time-lapse differential interference contrast microscopy after microinjection with antibodies specific for the chromokinesin Kid. 17.5% of cells injected with Kid-specific antibodies have one or more chromosomes that remain closely opposed to a spindle pole and fail to enter anaphase. In contrast, 82.5% of injected cells align chromosomes in metaphase, progress to anaphase, and display chromosome velocities not significantly different from control cells. However, injected cells lack chromosome oscillations, and chromosome orientation is atypical because chromosome arms extend toward spindle poles during both congression and metaphase. Furthermore, chromosomes cluster into a mass and fail to oscillate when Kid is perturbed in cells containing monopolar spindles. These data indicate that Kid generates the polar ejection force that pushes chromosome arms away from spindle poles in vertebrate-cultured cells. This force increases the efficiency with which chromosomes make bipolar spindle attachments and regulates kinetochore activities necessary for chromosome oscillation, but is not essential for chromosome congression.

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