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C L Rieder

Publications and source records attributed to C L Rieder.

At least 37 records · Page 2Linked to original sources
PubMed1998

Entry into mitosis in vertebrate somatic cells is guarded by a chromosome damage checkpoint that reverses the cell cycle when triggered during early but not late prophase.

When vertebrate somatic cells are selectively irradiated in the nucleus during late prophase (<30 min before nuclear envelope breakdown) they progress normally through mitosis even if they contain broken chromosomes. However, if early prophase nuclei are similarly irradiated, chromosome condensation is reversed and the cells return to interphase. Thus, the G2 checkpoint that prevents entry into mitosis in response to nuclear damage ceases to function in late prophase. If one nucleus in a cell containing two early prophase nuclei is selectively irradiated, both return to interphase, and prophase cells that have been induced to returned to interphase retain a normal cytoplasmic microtubule complex. Thus, damage to an early prophase nucleus is converted into a signal that not only reverses the nuclear events of prophase, but this signal also enters the cytoplasm where it inhibits e.g., centrosome maturation and the formation of asters. Immunofluorescent analyses reveal that the irradiation-induced reversion of prophase is correlated with the dephosphorylation of histone H1, histone H3, and the MPM2 epitopes. Together, these data reveal that a checkpoint control exists in early but not late prophase in vertebrate cells that, when triggered, reverses the cell cycle by apparently downregulating existing cyclin-dependent kinase (CDK1) activity.

Animals↗
PubMed1998

The disassembly and reassembly of functional centrosomes in vitro.

Animal cells contain a single centrosome that nucleates and organizes a polarized array of microtubules which functions in many cellular processes. In most cells the centrosome is composed of two centrioles surrounded by an ill-defined "cloud" of pericentriolar material. Recently, gamma-tubulin-containing 25-nm diameter ring structures have been identified as likely microtubule nucleation sites within the pericentriolar material of isolated centrosomes. Here we demonstrate that when Spisula centrosomes are extracted with 1.0 M KI they lose their microtubule nucleation potential and appear by three-dimensional electron microscopy as a complex lattice, built from 12- to 15-nm thick elementary fiber(s), that lack centrioles and 25-nm rings. Importantly, when these remnants are incubated in extracts prepared from Spisula oocytes they recover their 25-nm rings, gamma-tubulin, and microtubule nucleation potential. This recovery process occurs in the absence of microtubules, divalent cations, and nucleotides. Thus, in animals the centrosome is structurally organized around a KI-insoluble filament-based "centromatrix" that serves as a scaffold to which those proteins required for microtubule nucleation bind, either directly or indirectly, in a divalent cation and nucleotide independent manner.

Amino Acid Sequence↗
PubMed1998

The coordination of centrosome reproduction with nuclear events of the cell cycle in the sea urchin zygote.

Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1-cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Delta-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase-anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary "licensing" event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.

Anaphase↗
PubMed1998

A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution.

Three decades of structural analysis have produced the view that the kinetochore in vertebrate cells is a disk-shaped structure composed of three distinct structural domains. The most prominent of these consists of a conspicuous electron opaque outer plate that is separated by a light-staining electron-translucent middle plate from an inner plate associated with the surface of the pericentric heterochromatin. Spindle microtubules terminate in the outer plate and, in their absence, a conspicuous corona of fine filaments radiates from the cytoplasmic surface of this plate. Here we report for the first time the ultrastructure of kinetochores in untreated and Colcemid-treated vertebrate somatic (PtK1) cells prepared for optimal structural preservation using high-pressure freezing and freeze substitution. In serial thin sections, and electron tomographic reconstructions, the kinetochore appears as a 50-75 nm thick mat of light-staining fibrous material that is directly connected with the more electron-opaque surface of the centromeric heterochromatin. This mat corresponds to the outer plate in conventional preparations, and is surrounded on its cytoplasmic surface by a conspicuous 100-150 nm wide zone that excludes ribosomes and other cytoplasmic components. High magnification views of this zone reveal that it contains a loose network of light-staining, thin (<9 nm diameter) fibers that are analogous to the corona fibers in conventional preparations. Unlike the chromosome arms, which appear uniformly electron opaque, the chromatin in the primary constriction appears mottled. Since the middle plate is not visible in these kinetochore preparations this feature is likely an artifact produced by extraction and coagulation during conventional fixation and/or dehydration procedures.

Animals↗
PubMed1998

The vertebrate cell kinetochore and its roles during mitosis.

A replicated chromosome possesses two discrete, complex, dynamic, macromolecular assemblies, known as kinetochores, that are positioned on opposite sides of the primary constriction of the chromosome. Here, the authors review how kinetochores control chromosome segregation during mitosis in vertebrates. They attach the chromosome to the opposing spindle poles by trapping the dynamic plus-ends of microtubules growing from the poles. They then produce much of the force for chromosome poleward motion, regulate when this force is applied, and act as a site for microtubule assembly and disassembly. Finally, they control the metaphase-anaphase transition by inhibiting chromatid separation until the chromatids are properly attached.

Animals↗
PubMed1997

Kinetochore fiber maturation in PtK1 cells and its implications for the mechanisms of chromosome congression and anaphase onset.

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 microM 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.

Anaphase↗
PubMed1997

Mitosis in vertebrate somatic cells with two spindles: implications for the metaphase/anaphase transition checkpoint and cleavage.

During mitosis an inhibitory activity associated with unattached kinetochores prevents PtK1 cells from entering anaphase until all kinetochores become attached to the spindle. To gain a better understanding of how unattached kinetochores block the metaphase/anaphase transition we followed mitosis in PtK1 cells containing two independent spindles in a common cytoplasm. We found that unattached kinetochores on one spindle did not block anaphase onset in a neighboring mature metaphase spindle 20 microm away that lacked unattached kinetochores. As in cells containing a single spindle, anaphase onset occurred in the mature spindles x = 24 min after the last kinetochore attached regardless of whether the adjacent immature spindle contained one or more unattached kinetochores. These findings reveal that the inhibitory activity associated with an unattached kinetochore is functionally limited to the vicinity of the spindle containing the unattached kinetochore. We also found that once a mature spindle entered anaphase the neighboring spindle also entered anaphase x = 9 min later regardless of whether it contained monooriented chromosomes. Thus, anaphase onset in the mature spindle catalyzes a "start anaphase" reaction that spreads globally throughout the cytoplasm and overrides the inhibitory signal produced by unattached kinetochores in an adjacent spindle. Finally, we found that cleavage furrows often formed between the two independent spindles. This reveals that the presence of chromosomes and/or a spindle between two centrosomes is not a prerequisite for cleavage in vertebrate somatic cells.

Animals↗
PubMed1997

Centrosomes isolated from Spisula solidissima oocytes contain rings and an unusual stoichiometric ratio of alpha/beta tubulin.

Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with pericentriolar material. To study the biochemical and structural basis of centrosome-dependent microtubule nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed that each centrosome was composed of a single centriole surrounded by pericentriolar material that was studded with ring-shaped structures approximately 25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b) alpha-, beta-, and gamma-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD. gamma-Tubulin was not an MPM-2 phosphoprotein and was the most abundant form of tubulin in centrosomes. Relatively little alpha- or beta-tubulin copurified with centrosomes, and the ratio of alpha- to beta-tubulin in centrosomes was not 1:1 as expected, but rather 1:4.6, suggesting that centrosomes contain beta-tubulin that is not dimerized with alpha-tubulin.

Animals↗
PubMed1997

Chromosome fragments possessing only one kinetochore can congress to the spindle equator.

We used laser microsurgery to cut between the two sister kinetochores on bioriented prometaphase chromosomes to produce two chromosome fragments containing one kinetochore (CF1K). Each of these CF1Ks then always moved toward the spindle pole to which their kinetochores were attached before initiating the poleward and away-from-the-pole oscillatory motions characteristic of monooriented chromosomes. CF1Ks then either: (a) remained closely associated with this pole until anaphase (50%), (b) moved (i.e., congressed) to the spindle equator (38%), where they usually (13/19 cells) remained stably positioned throughout the ensuing anaphase, or (c) reoriented and moved to the other pole (12%). Behavior of congressing CF1Ks was indistinguishable from that of congressing chromosomes containing two sister kinetochores. Three-dimensional electron microscopic tomographic reconstructions of CF1Ks stably positioned on the spindle equator during anaphase revealed that the single kinetochore was highly stretched and/or fragmented and that numerous microtubules derived from the opposing spindle poles terminated in its structure. These observations reveal that a single kinetochore is capable of simultaneously supporting the function of two sister kinetochores during chromosome congression and imply that vertebrate kinetochores consist of multiple domains whose motility states can be regulated independently.

Anaphase↗
PubMed1997

A synergy of technologies: combining laser microsurgery with green fluorescent protein tagging.

When focused through an objective lens with a high numerical aperture, nanosecond pulses of high-intensity green (532-nm) laser light can be used to selectively destroy any cellular component whose boundaries can be defined by light microscopy. These components include, for example, chromosomes, spindle fibers, bundles of keratin, or actin filaments, mitochondria, vacuoles, and so forth. In addition, the definition of poorly resolved components can be enhanced for selective destruction by tagging one or more of their constituent proteins with green fluorescence protein (GFP). As a example we show that the centrosome in living PtK1 cells can be clearly defined, and then destroyed by green laser light, after transforming the cells with gamma-tubulin/GFP fusion protein. In some transformed cells it is even possible to target and selectively destroy just one of the centrioles.

Centrosome↗
PubMed1997

Mitosis and checkpoints that control progression through mitosis in vertebrate somatic cells.

During mitosis in vertebrates the sister kinetochores on each replicated chromosome interact with two separating arrays of astral microtubules to form a bipolar spindle that produces and/or directs the forces for chromosome motion. In order to ensure faithful chromosome segregation cells have evolved mechanisms that delay progress into and out of mitosis until certain events are completed. At least two of these mitotic "checkpoint controls" can be identified in vertebrates. The first prevents nuclear envelope breakdown, and thus spindle formation, when the integrity of some nuclear component(s) is compromised. The second prevents chromosome disjunction and exit from mitosis until all of the kinetochores are attached to the spindle.

Animals↗
PubMed1997

The checkpoint control for anaphase onset does not monitor excess numbers of spindle poles or bipolar spindle symmetry.

Exit from mitosis in animal cells is substantially delayed when spindle assembly is inhibited, spindle bipolarity is disrupted, or when a monopolar spindle is formed. These observations have led to the proposal that animal cells have a 'spindle assembly' checkpoint for the metaphase-anaphase transition that monitors bipolar spindle organization. However, the existence of such a checkpoint is uncertain because perturbations in spindle organization can produce unattached kinetochores, which by themselves are known to delay anaphase onset. In this study we have tested if cells monitor bipolar spindle organization, independent of kinetochore attachment, by analyzing the duration of mitosis in sea urchin zygotes and vertebrate somatic cells containing multipolar spindles in which all kinetochores are attached to spindle poles. We found that sea urchin zygotes containing tripolar or tetrapolar spindles progressed from nuclear envelope breakdown to anaphase onset with normal timing. We also found that the presence of supernumerary, unpaired spindle poles did not greatly prolong mitosis. Observation of untreated PtK1 cells that formed tripolar or tetrapolar spindles revealed that they progressed through mitosis, on average, at the normal rate. More importantly, the interval between the bipolar attachment of the last monooriented chromosome and anaphase onset was normal. Thus, neither of these cell types can detect the presence of gross aberrations in spindle architecture that inevitably lead to aneuploidy. We conclude that animal cells do not have a checkpoint for the metaphase-anaphase transition that monitors defects in spindle architecture independent of the checkpoint that monitors kinetochore attachment to the spindle. For dividing cells in which spindle microtubule assembly is not experimentally compromised, we propose that the completion of kinetochore attachment is the event which limits the time of the metaphase-anaphase transition.

Anaphase↗
PubMed1996

The force for poleward chromosome motion in Haemanthus cells acts along the length of the chromosome during metaphase but only at the kinetochore during anaphase.

The force for poleward chromosome motion during mitosis is thought to act, in all higher organisms, exclusively through the kinetochore. We have used time-lapse. video-enhanced, differential interference contrast light microscopy to determine the behavior of kinetochore-free "acentric" chromosome fragments and "monocentric" chromosomes containing one kinetochore, created at various stages of mitosis in living higher plant (Haemanthus) cells by laser microsurgery. Acentric fragments and monocentric chromosomes generated during spindle formation and metaphase both moved towards the closest spindle pole at a rate (approximately 1.0 microm/min) similar to the poleward motion of anaphase chromosomes. This poleward transport of chromosome fragments ceased near the onset of anaphase and was replaced. near midanaphase, by another force that now transported the fragments to the spindle equator at 1.5-2.0 microm/min. These fragments then remained near the spindle midzone until phragmoplast development, at which time they were again transported randomly poleward but now at approximately 3 microm/min. This behavior of acentric chromosome fragments on anastral plant spindles differs from that reported for the astral spindles of vertebrate cells, and demonstrates that in forming plant spindles, a force for poleward chromosome motion is generated independent of the kinetochore. The data further suggest that the three stages of non-kinetochore chromosome transport we observed are all mediated by the spindle microtubules. Finally, our findings reveal that there are fundamental differences between the transport properties of forming mitotic spindles in plants and vertebrates.

Anaphase↗
PubMed1996

Kinetochores moving away from their associated pole do not exert a significant pushing force on the chromosome.

We used video-light microscopy and laser microsurgery to test the hypothesis that as a bioriented prometaphase chromosome changes position in PtK1 cells, the kinetochore moving away from its associated pole (AP) exerts a pushing force on the centromere. When we rapidly severed congressing chromosomes near the spindle equator between the sister kinetochores, the kinetochore that was originally "leading" the motion towards a pole (P) always (17/17 cells) continued moving P whereas the "trailing" kinetochore moving AP always stopped moving as soon as the operation was completed. This trailing kinetochore then initiated motion towards the pole it was originally moving away from up to 50 s later. The same result was observed (15/15 cells) when we selectively destroyed the leading (P moving) kinetochore on a congressing chromosome positioned > or = 3 microns from the pole it was moving away from. When we conducted this experiment on congressing chromosomes positioned within 3 microns of the pole, the centromere region either stopped moving, before switching into motion towards the near pole (2/4 cells), or it continued to move AP for 30-44 s (2/4 cells) before switching into P motion. Finally, kinetochore-free chromosome fragments, generated in the polar regions of PtK1 spindles, were ejected AP and often towards the spindle equator at approximately 2 microns/min. From these data we conclude that the kinetochore moving AP on a moving chromosome does not exert a significant pushing force on the chromosome. Instead, our results reveal that, when not generating a P force, kinetochores are in a "neutral" state that allows them to remain stationary or to coast AP in response to external forces sufficient to allow their K-fiber to elongate.

Animals↗
PubMed1996

The kinetochore microtubule minus-end disassembly associated with poleward flux produces a force that can do work.

During metaphase and anaphase in newt lung cells, tubulin subunits within the kinetochore microtubule (kMT) lattice flux slowly poleward as kMTs depolymerize at their minus-ends within in the pole. Very little is known about how and where the force that moves the tubulin subunits poleward is generated and what function it serves during mitosis. We found that treatment with the drug taxol (10 microM) caused separated centrosomes in metaphase newt lung cells to move toward one another with an average velocity of 0.89 microns/min, until the interpolar distance was reduced by 22-62%. This taxol-induced spindle shortening occurred as kMTs between the chromosomes and the poles shortened. Photoactivation of fluorescent marks on kMTs revealed that taxol inhibited kinetochore microtubule assembly/disassembly at kinetochores, whereas minus-end MT disassembly continued at a rate typical of poleward flux in untreated metaphase cells. This poleward flux was strong enough to stretch the centromeric chromatin between sister kinetochores as much as it is stretched in control metaphase cells. In anaphase, taxol blocked kMT disassembly/assembly at the kinetochore whereas minus-end disassembly continued at a rate similar to flux in control cells (approximately 0.2 microns/min). These results reveal that the mechanism for kMT poleward flux 1) is not dependent on kMT plus-end dynamics and 2) produces pulling forces capable of generating tension across the centromeres of bioriented chromosomes.

Anaphase↗
PubMed1996

Long crocidolite asbestos fibers cause polyploidy by sterically blocking cytokinesis.

Asbestos has been described as a physical carcinogen in that its carcinogenic effects appear to be related primarily to fiber dimensions. It has been hypothesized that long asbestos fibers may interfere with chromosome distribution during cell division, causing genomic changes that lead to cell transformation and neoplastic progression. Using high-resolution time-lapse light microscopy and serial-section electron microscopy, we have followed individual crocidolite asbestos fibers through the later stages of cell division in LLC-MK2 epithelial cells, and have detailed for the first time their effect on cytokinesis. We found that long fibers (15-55 microgram), trapped by the cleavage furrow, sterically blocked cytokinesis, sometimes resulting in the formation of a binucleated cell. The ends of blocking fibers were usually found within invaginations of the newly formed nuclei. Nuclear envelope-fiber attachment was evident when a chromatin strand ran with the fiber into the intercellular bridge. Such strands may break, causing chromosome structural rearrangements. Our data are the first to show that individual crocidolite fibers can cause genomic changes by sterically blocking cytokinesis and that fiber length and affinity for the nuclear envelope are important factors. Such genomic changes may be among the initial events leading to asbestos-induced cancers.

Animals↗