scholarly journals NITROUS OXIDE: EFFECTS ON THE MITOTIC APPARATUS AND CHROMOSOME MOVEMENT IN HELA CELLS

1973 ◽  
Vol 58 (1) ◽  
pp. 96-106 ◽  
Author(s):  
B. R. Brinkley ◽  
Potu N. Rao
Keyword(s):  
Nitrous Oxide ◽  
Hela Cells ◽  
Metaphase Plate ◽  
Microtubule Assembly ◽  
Chromosome Movement ◽  
Spindle Microtubule ◽  
Mitotic Apparatus ◽  
Ultrastructural Studies ◽  
Multipolar Spindles ◽  
Spindle Microtubules

When HeLa cells were grown in the presence of nitrous oxide (N2O) under pressure (80 lb/in2) mitosis was inhibited and the chromosomes displayed a typical colchicine metaphase (c-metaphase) configuration when examined by light microscopy. When the cells were returned to a 37°C incubator, mitosis was resumed and the cells entered G1 synchronously. Ultrastructural studies of N2O-blocked cells revealed a bipolar spindle with centriole pairs at each pole. Both chromosomal and interpolar (pole-to-pole) microtubules were also present. Thus, N2O, unlike most c-mitotic agents, appeared to have little or no effect upon spindle microtubule assembly. However, the failure of chromo somes to become properly aligned onto the metaphase plate indicated an impairment in normal prometaphase movement. The alignment of spindle microtubules was frequently atypical with some chromosomal microtubules extending from kinetochores to the poles, while others extended out at acute angles from the spindle axis. These ultrastructural studies indicated that N2O blocked cells at a stage in mitosis more advanced than that produced by Colcemid or other c-mitotic agents. Like Colcemid, however, prolonged arrest in mitosis with N2O led to an increased incidence of multipolar spindles.

scholarly journals Hepatoma Up-Regulated Protein Is Required for Chromatin-induced Microtubule Assembly Independently of TPX2

2008 ◽  
Vol 19 (11) ◽  
pp. 4900-4908 ◽  
Author(s):  
Claudia M. Casanova ◽  
Sofia Rybina ◽  
Hideki Yokoyama ◽  
Eric Karsenti ◽  
Iain W. Mattaj
Keyword(s):  
Xenopus Laevis ◽  
Detailed Analysis ◽  
Spindle Assembly ◽  
Microtubule Assembly ◽  
Spindle Microtubule ◽  
Microtubule Stabilization ◽  
Egg Extracts ◽  
Spindle Midzone ◽  
Microtubule Growth ◽  
Spindle Microtubules

The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.

scholarly journals Kinetochore microtubules shorten by loss of subunits at the kinetochores of prometaphase chromosomes

1991 ◽  
Vol 98 (2) ◽  
pp. 151-158 ◽  
Author(s):  
L. Cassimeris ◽  
E.D. Salmon
Keyword(s):  
Metaphase Plate ◽  
Force Generation ◽  
Microtubule Assembly ◽  
Immunofluorescent Staining ◽  
Chromosome Movement ◽  
Mitotic Spindles ◽  
Subunit Dissociation ◽  
Spindle Poles ◽  
Tubulin Subunit ◽  
A Site

The site of tubulin subunit dissociation was determined during poleward chromosome movement in prometaphase newt lung cell mitotic spindles using fluorescence photobleaching techniques and nocodazole-induced spindle shortening. Synchronous shortening of all kinetochore microtubules was produced by incubating cells in 17 microM nocodazole to block microtubule assembly. Under these conditions the spindle poles moved towards the metaphase plate at a rate of 3.6 +/− 0.4 microns min-1 (n = 3). On the basis of anti-tubulin immunofluorescent staining of cells fixed after incubation in nocodazole, we found that nonkinetochore microtubules rapidly disappeared and only kinetochore fibers were present after 60–90 s in nocodazole. To localize the site of tubulin subunit dissociation, a narrow bar pattern was photobleached across one half-spindle in prometaphase-metaphase cells previously microinjected with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF)-labeled tubulin. Immediately after photobleaching, cells were perfused with 17 microM nocodazole to produce shortening of kinetochore microtubules. Shortening was accompanied by a decrease in the distance between the bleach bar and the kinetochores. In contrast, there was little or no decrease in the distance between the bleach bar and the pole. Compared to their initial lengths, the average kinetochore to pole distance shortened by 18%, the bleach bar to kinetochore distance shortened by 28% and the average bleached bar to pole distance shortened by 1.6%. The data provide evidence that tubulin subunits dissociate from kinetochore microtubules at a site near the kinetochore during poleward chromosome movement. These results are consistent with models of poleward force generation for chromosome movement in which prometaphase-metaphase poleward force is generated in association with the kinetochore.

scholarly journals Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

1985 ◽  
Vol 101 (5) ◽  
pp. 1966-1976 ◽  
Author(s):  
J B Tucker ◽  
S A Mathews ◽  
K A Hendry ◽  
J B Mackie ◽  
D L Roche
Keyword(s):  
Nuclear Envelope ◽  
Early Stage ◽  
Microtubule Assembly ◽  
Membrane Association ◽  
Paramecium Tetraurelia ◽  
Spindle Microtubule ◽  
Spatial Control ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Anaphase B

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

scholarly journals THE DISTRIBUTION OF SPINDLE MICROTUBULES DURING MITOSIS IN CULTURED HUMAN CELLS

1971 ◽  
Vol 49 (2) ◽  
pp. 468-497 ◽  
Author(s):  
J. Richard McIntosh ◽  
Story C. Landis
Keyword(s):  
Electron Microscopy ◽  
Hela Cells ◽  
Metaphase Plate ◽  
Human Cells ◽  
Late Anaphase ◽  
Monolayer Cultures ◽  
Spindle Axis ◽  
Early Anaphase ◽  
Cultured Human Cells ◽  
Spindle Microtubules

WI-38 and HeLa cells in mitosis have been selected from fixed monolayer cultures and serially sectioned for electron microscopy. Sections perpendicular to the spindle axis permit counting of the number of microtubules at each position on the spindle axis and hence the preparation of tubule distribution profiles. Errors intrinsic to this method are discussed. The changes in the tubule distributions from one mitotic stage to another provide evidence concerning the behavior of the spindle tubules during mitosis. The ratio of the number of tubules passing the chromosomes on the metaphase plate to the maximum number in each half spindle is about 1/2. This ratio changes little in early anaphase, and then decreases in late anaphase at about the same time that a zone of increased tubule number develops at the middle of the interzone. The region where the stem bodies form contains about 3/2 the number of tubules seen elsewhere in the interzone. This ratio is almost constant as the mid-body forms in telophase and then increases to 2/1 in early interphase before the final stages of cytokinesis occur.

scholarly journals Pressure-induced depolymerization of spindle microtubules. III. Differential stability in HeLa cells.

1976 ◽  
Vol 69 (2) ◽  
pp. 443-454 ◽  
Author(s):  
E D Salmon ◽  
D Goode ◽  
T K Maugel ◽  
D B Bonar
Keyword(s):  
Hydrostatic Pressure ◽  
Light Microscopy ◽  
Hela Cells ◽  
Atmospheric Pressure ◽  
Differential Effect ◽  
Polarization Microscopy ◽  
Spindle Fiber ◽  
Ultrastructural Studies ◽  
Differential Stability ◽  
Spindle Microtubules

Evidence from light microscopy (principally polarization microscopy) has demonstrated that hydrostatic pressure can reversibly inhibit mitosis by rapidly depolymerizing the spindle fiber microtubules. We have confirmed this finding in ultrastructural studies of mitotic HeLa cells incubated at 37 degrees C and pressurized at 680 atm (10,000 psi). Althouth there are many spindle microtubules in the cells at atmospheric pressure, electron micographs of cells pressurized for 10 min (and fixed while under pressure in a Landau-Thibodeau chamber) show few microtubules. Pressure has a differential effect on the various types of spindle microtubules. Astral and interpolar MTs appear to be completely depolymerized in pressurized cells, but occasional groups of kinetochore fiber microtubules are seen. Surprisingly, the length and density of microtubules of the stem bodies and midbody of telophase cells appear unchanged by pressurization. In cells fixed 10 min after pressure was released, microtubules were again abundant, the density often appearing to be higher than in control cells. Reorganization seems incomplete, however, since many of the microtubules are randomly oriented. Unexpectedly, kinetochores appeared diffuse and were difficult to identify in sections of pressurized cells. Even after 10 min of recovery at atmospheric pressure, their structure was less distinct than in unpressurized cells.

scholarly journals Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells.

1979 ◽  
Vol 81 (3) ◽  
pp. 484-497 ◽  
Author(s):  
B R Telzer ◽  
J L Rosenbaum
Keyword(s):  
Hela Cells ◽  
Dark Field ◽  
Microtubule Assembly ◽  
Supernatant Fraction ◽  
Microtubule Organizing Center ◽  
Mitotic Apparatus ◽  
Discontinuous Sucrose Gradient ◽  
In Vitro Assembly ◽  
Pericentriolar Material

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.

Confocal analysis of chromosome behavior in wheat × maize zygotes

Genome ◽  
2004 ◽  
Vol 47 (1) ◽  
pp. 199-205 ◽  
Author(s):  
Keiichi Mochida ◽  
Hisashi Tsujimoto ◽  
Tetsuo Sasakuma
Keyword(s):  
Metaphase Plate ◽  
Chromosome Elimination ◽  
Chromosome Movement ◽  
Hybridization Method ◽  
Chromosome Behavior ◽  
Whole Mount ◽  
Hybrid Embryo ◽  
Spindle Microtubules ◽  
Spindle Structure

Herein, we profile the first embryonic mitosis in a hybrid of wheat and maize by using a whole-mount genomic in situ hybridization method and immunofluorescence staining with a tubulin-specific antibody. We have successfully captured the dynamics of each set of parental chromosomes in the first zygotic division of the hybrid embryo 24-28 h after crossing. During the first zygotic metaphase, although both sets of parental chromosomes congressed into the equatorial plate of the zygote, the maize chromosomes tended to lag in comparison with the wheat chromosomes. During anaphase, each parental chromosome separated into its sister chromosomes; however, some of the maize chromosomes lagged around the metaphase plate as segregants. The maize sister chromosomes that did move toward the pole showed delayed and asymmetric movement as compared with the wheat ones. Immunological staining of tubulin revealed a bipolar spindle structure in the first zygotic metaphase. The kinetochores of the maize chromosomes that lagged around the metaphase plate did not attach to the spindle microtubules. These results suggest that factors on the kinetochores of maize chromosomes that are required to control chromosome movement are deficient in the zygotic cell cycle.Key words: whole-mount, GISH, chromosome elimination, hybrid embryogenesis.

scholarly journals GTSE1 regulates spindle microtubule dynamics to control Aurora B kinase and Kif4A chromokinesin on chromosome arms

2017 ◽  
Vol 216 (10) ◽  
pp. 3117-3132 ◽  
Author(s):  
Aaron R. Tipton ◽  
Jonathan D. Wren ◽  
John R. Daum ◽  
Joseph C. Siefert ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Meta Analysis ◽  
Metaphase Plate ◽  
Spindle Pole ◽  
Aurora B ◽  
Chromosome Movement ◽  
Aurora B Kinase ◽  
Nuclear Envelope Breakdown ◽  
M Phase ◽  
Spindle Microtubules

In mitosis, the dynamic assembly and disassembly of microtubules are critical for normal chromosome movement and segregation. Microtubule turnover varies among different mitotic spindle microtubules, dictated by their spatial distribution within the spindle. How turnover among the various classes of spindle microtubules is differentially regulated and the resulting significance of differential turnover for chromosome movement remains a mystery. As a new tactic, we used global microarray meta-analysis (GAMMA), a bioinformatic method, to identify novel regulators of mitosis, and in this study, we describe G2- and S phase–expressed protein 1 (GTSE1). GTSE1 is expressed exclusively in late G2 and M phase. From nuclear envelope breakdown until anaphase onset, GTSE1 binds preferentially to the most stable mitotic spindle microtubules and promotes their turnover. Cells depleted of GTSE1 show defects in chromosome alignment at the metaphase plate and in spindle pole integrity. These defects are coupled with an increase in the proportion of stable mitotic spindle microtubules. A consequence of this reduced microtubule turnover is diminished recruitment and activity of Aurora B kinase on chromosome arms. This decrease in Aurora B results in diminished binding of the chromokinesin Kif4A to chromosome arms.

scholarly journals Isolation and partial characterization of a cage of filaments that surrounds the mammalian mitotic spindle.

1980 ◽  
Vol 87 (1) ◽  
pp. 160-169 ◽  
Author(s):  
G W Zieve ◽  
S R Heidemann ◽  
J R McIntosh
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Intermediate Filaments ◽  
Hela Cells ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Microtubule Assembly ◽  
Polypeptide Composition ◽  
Mitotic Apparatus ◽  
Filamentous Material

Mitotic cells have been detergent extracted under conditions that support microtubule assembly. When HeLa cells are lysed in the presence of brain tubulin, mitotic-arrested cells nucleate large asters and true metaphase cells yield spindles that remain enclosed within a roughly spherical cage of filamentous material. Detergent-extracted mitotic Chinese hamster ovary (CHO) cells show a similar, insoluble cage but the mitotic apparatus is only occasionally stabilized. In later stages of mitosis, HeLa cages are observed in elongated and furrowed configurations. In the terminal stages of cell division, two daughter filamentous networks are connected by the intercellular bridge. When observed in the electron microscope the cages include fibers 7-11 nm in diameter. The polypeptide composition of cages isolated from mitotic HeLa cells is complex, but the major polypeptides are a group with mol wt ranging from 43,000-60,000 daltons and a high molecular weight polypeptide. CHO cells contain a subset of these proteins which includes a major 58,000-dalton and a high molecular weight polypeptide. Two different antisera directed against the vimentin-containing intermediate filaments bind to polypeptides in the electrophoretic profiles of isolated HeLa and CHO cages and stain the cages, as visualized by indirect immunofluorescence. These results suggest that the HeLa and CHO cages include intermediate filaments of the vimentin type. The polypeptide composition of HeLa cages suggests that they also contain tonofilaments. The cages apparently form as the cells enter mitosis. We propose that these filamentous cages maintain the structural continuity of the cytoplasm while the cell is in mitosis.

Functional midbody assembly in the absence of a central spindle

2022 ◽  
Vol 221 (3) ◽  
Author(s):  
Sophia M. Hirsch ◽  
Frances Edwards ◽  
Mimi Shirasu-Hiza ◽  
Julien Dumont ◽  
Julie C. Canman
Keyword(s):  
Microtubule Assembly ◽  
Primary Role ◽  
Intercellular Bridge ◽  
Contractile Ring ◽  
Spindle Microtubule ◽  
Central Spindle ◽  
Family Protein ◽  
Spindle Midzone ◽  
Ring Constriction ◽  
Spindle Microtubules

Contractile ring constriction during cytokinesis is thought to compact central spindle microtubules to form the midbody, an antiparallel microtubule bundle at the intercellular bridge. In Caenorhabditis elegans, central spindle microtubule assembly requires targeting of the CLASP family protein CLS-2 to the kinetochores in metaphase and spindle midzone in anaphase. CLS-2 targeting is mediated by the CENP-F–like HCP-1/2, but their roles in cytokinesis and midbody assembly are not known. We found that although HCP-1 and HCP-2 mostly function cooperatively, HCP-1 plays a more primary role in promoting CLS-2–dependent central spindle microtubule assembly. HCP-1/2 codisrupted embryos did not form central spindles but completed cytokinesis and formed functional midbodies capable of supporting abscission. These central spindle–independent midbodies appeared to form via contractile ring constriction–driven bundling of astral microtubules at the furrow tip. This work suggests that, in the absence of a central spindle, astral microtubules can support midbody assembly and that midbody assembly is more predictive of successful cytokinesis than central spindle assembly.

Sign in / Sign up

Export Citation Format

Share Document