Arrangement of spindle apparatus in mitoses of different ploidy.
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Animals↗
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Genetic damage induced by ethyl alcohol in Aspergillus nidulans.
Heterozygous diploid conidia of Aspergillus nidulans were treated during germination with ethyl alcohol in concentrations ranging from 0.25% to 20% (v/v). The diploid strain carried three recessive conidial color mutations, in addition to genetic markers on all eight pairs of linkage groups. It was thereby possible to detect events of crossing over, non-disjunction, and mutation. An increase in the dose of ethanol was associated with a decrease in conidial viability and an increase in the relative and absolute frequencies of formation of (a) normal colonies which produced colored sectors and (b) phenotypically abnormal colonies, the majority of which (83.1%) produced normal sectors. At a concentration of 5% (v/v) ethanol, the survivors included 17.59% of the former and 44.7% of the latter colonies. Genetic analysis of the various segregants suggested that the frequencies of both mitotic crossing over and non-disjunction or the misdistribution of chromosomes were increased by ethanol. Among 133 abnormal colonies which segregated normal clones, 79 (59.4%) were associated with one of these genetic events. A total of 297 haploids and 130 diploids arose as normal segregants from the abnormal colonies. There were 31 recognizable events of non-disjunction and 14 crossing over in linkage groups I and II, where these events could be distinguished. These data suggested that the predominant effect of ethanol was a disruption of chromosome distribution. A cytological examination of ethanol-treated, germinating conidia revealed an interference with the mitotic spindle apparatus. The frequency of detectable spindles decreased more than 3-fold after 8 h exposure to 5% (v/v) ethanol. This finding supported the conclusion that ethanol disrupted chromosome distribution, and suggested the mechanism by which it does so. Human clinical data on alcohol consumption were examined in light of these findings.
Aspergillus nidulans↗
What moves chromosomes, microtubules or microfilaments?
An investigation of the spindle apparatus of crane-fly (Nephrotoma suturalis) spermatocytes has been undertaken using methods that permit combined light and electron microscopy of selected cells. At the ultrastructural level, spindles contain microtubules in a granular matrix. Microtubules have been classified as kinetochore microtubules (which connect to kinetochores of chromosomes) and non-kinetochore microtubules (not attached to kinetochores). Kinetochore microtubules are distributed in densely packed bundles, which are the birefringent chromosomal fibers seen in living cells. Actin filaments were not observed in spindles of unglycerinated cells or in cells fixed in glutaraldehyde containing tannic acid, which negatively stains F-actin in situ and thus can be used to aid the localization of actin filaments in non-muscle cells. The absence of actin filaments in the spindle coupled with their presence in the "contractile ring" of spermatocytes fixed during cytokinesis is evidence against the hypothesis that chromosome movements are microfilament-based. The results are compatible with the hypothesis that microtubules are involved in the mechanism of chromosome transport. The details of that mechanism remain to be clarified.
Actins↗
Neurophysiological experiments on intention tremor (author's transl).
During a postural innervation, cerebellar intention tremor can be produced by applying direct stimulus to the muscle. The stimulus is followed by a silent period of 160 to 200 ms, which exceeds the normal variation. Simultaneously, a massive rebound in the antagonistic muscle is evoked due to the tension reflex in the agonistic muscle. Passive shortening or stretching of the voluntarily activated muscle has no effect: there is no silent period in the shortened muscle, no rebound in the stretched muscle, and, consequently, little or no oscillation follows. The spindle apparatus seems irrelevant to the development of intention tremor. The feedback mechanism of the Golgi-inhibition systems is discussed. These findings cannot be explained by assuming a central pacemaker.
Cerebellum↗
Chromosome distribution studies after inorganic lead exposure.
We studied the chromosome distribution in persons professionally exposed to inorganic lead. The degree of lead exposure was evaluated by biochemical measurements and cytogenetic analysis. The chromosome distribution was analyzed from trypsin banded karyotypes; in particular we studied centromere distances (delta2) and centromere-metaphase-center distances (d2) which were obtained by computer-aided mathematical transformation of the individual metaphase coordinates. Higher concentrations of blood lead and urine delta-ALA and a statistically significant increase in aneuploidy, hypoploidy, and type-B chromosome aberrations revealed appreciable exposure although none of the subjects showed signs of excessive lead absorption. However study of the chromosome distribution showed no major differences with that of the controls indicating that lead acts preferentially (directly or indirectly) on the chromosomes rather than on the spindle apparatus. A dissociation of the acrocentric chromosomes was observed in the lead group when compared with the controls. This is thought to reflect a secondary action of lead on the nucleolar organiser regions.
Adult↗
Ten-nanometer filaments and mitosis: maintenance of structural continuity in dividing endothelial cells.
By indirect immunofluorescence the behavior of the 10-nm filaments was studied at various stages of mitosis in guinea pig vascular endothelial cells. Interphase cells contain a ring of 10-nm filaments that encircles the nucleus and is maintained in a plane parallel to the substrate. During prophase and metaphase the cells round up and the 10-nm filament ring becomes wavy though still a closed structure. As anaphase progresses the ring then elongates into a rectangle that contains the spindle apparatus and chromosomes. In late telophase, cytokinesis cleaves the 10-nm filaments into crescents at the site of the contractile ring. These crescents then close into rings in the daughter cells. If cytokinesis is inhibited with 5 microgram of cytochalasin B per ml, then cleavage of the 10-nm filaments is blocked and the daughter nuclei remain surrounded by the parent ring. At no point during mitosis does the array of 10-nm filaments undergo major disassembly. These results indicate that, in contrast to the other major cytoplasmic structures, ventral microfilament bundles and cytoplasmic microtubules, which disassemble and reassemble during mitosis, 10-nm filaments remain intact throughout this process. The possibility is discussed that these filaments may function in transport of organelles and structural proteins, and provide the daughter cells with topological information about placement and assembly of these elements within the microtrabecular lattice.
Animals↗
The absence of centrioles from spindle poles of rat kangaroo (PtK2) cells undergoing meiotic-like reduction division in vitro.
Light and electron microscopy were used to study somatic cell reduction division occurring spontaneously in tetraploid populations of rat kangaroo Potorous tridactylis (PtK2) cells in vitro. Light microscopy coupled with time-lapse photography documented the pattern of reduction division which includes an anaphase-like movement of double chromatid chromosomes to opposite spindle poles followed by the organization of two separate metaphase plates and synchronous anaphase division to form four poles and four daughter nuclei. The resulting daughter cells were isolated and cloned, showing their viability, and karyotyped to determine their ploidy. Ultrastructural analysis of cells undergoing reduction consistently revealed two duplexes of centrioles (one at each of two spindle poles) and two spindle poles in each cell that lacked centrioles but with microtubules terminating in a pericentriolar-like cloud of material. These results suggest that the centriole is not essential for spindle pole formation and division and implicate the could region as a necessary component of the spindle apparatus.
Cell Line↗
Mitosis in milk secreting epithelial cells of mammary gland: an ultrastructural study.
In all stages of lactation mitotic configurations were observed in mammary gland epithelial cells of rats. An electron microscopic study is presented which shows that ultrastructure of such mitotic stages is normal and that mitotic cells contain typical products of milk secreting cells such as casein micelle-containing vesicles and milk fat droplets. Such secretory products can even be observed in the immediate vicinity of the chromosomes and microtubules of the spindle apparatus. The endoplasmic reticulum of mitotic cells appeared altered in that it did not show typical cisternal stacks characteristic of interphase cells. While the numbers of such mitotic cells were very low, especially from the second week of lactation on (always less than 0.1% of the milk secreting epithelial cells encountered), the observations clearly demonstrate that differentiation for milk secretory activity and cells division are not mutually exclusive. We conclude that postpartum growth of mammary gland epithelium and replacement of epithelial cells lost during desquamation into the milk liquids can occur by division of existing differentiated milk secreting cells and does not require mitotic activity of non-lactating 'stem cells' which are not observed in lactating alveoli.
Animals↗
Ultrastructural observations on renal schizogony of Leucocytozoon dubreuili in the American robin.
The schizogonic development of Leucocytozoon dubreuili in the kidney proximal tubule cells of the American robin, Turdus migratorius, was studied by electron microscopy. Renal schizogony is initiated by the entry of certain hepatic merozoites into cells of the proximal tubules. Development of the schizont consists of coordinated sequence of events including extensive mitotic nuclear division, multiplication of mitochondria, increase in endoplasmic reticulum and ribosomes, differentiation of membranes, microtubules, micronemes and rhoptries, and cytoplasmic segmentation (cytomere formation). Merozoites form by budding around numerous centers in the schizont and, when mature, are bounded by a single plasma membrane subtended by microtubules. Each merozoite contains a large nucleus, a mitochondrion, and well developed apical complex consisting of 3 polar rings, paired rhoptries, and numerous micronemes. An atypical nuclear division observed in some maturing schizonts was characterized by the multiple fission of a nucleus within a persistent outer nuclear membrane and the absence of mitotic spindle apparatus. Alterations in infected renal cells consisted of disorganization and loss of cytoplasmic organelles and the accumulation of lipofuscin-like inclusions.
Animals↗
Chromatin behaviour during the mitotic cell cycle of Saccharomyces cerevisiae.
Chromatin behaviour during the cell division cycle of the yeast Saccharomyces cerevisiae has been investigated in cells which have been depleted of 90% of their RNA by digestion with ribonuclease. Removal of large amounts of RNA from the yeast nucleus before treatment of the cells with heavy metal fixatives and stains permits chromatin to be visualized with extreme clarity in thin sections of cells processed for electron microscopy by conventional procedures. Spindle pole bodies were also visualized by this treatment, although the associated microtubules were not. Chromatin is dispersed during interphase and occupies the non-nucleolar region of the nucleus which is known to be Feulgen-positive from light microscopy. Because spindle microtubules are not visualized, direct attachment of microtubules to chromatin fibrils could not be verified. However, chromatin was not attached directly to the spindle pole bodies and kinetochore differentiations were not observed in the nucleoplasm. During nuclear division chromatin remains dispersed and does not condense into discrete chromatids. As the nucleus expands into the bud, chromosomal distribution to the daughter cells is thought to result from the separation of the poles of the spindle apparatus with attached chromatin fibrils. However, that such distribution is occurring as the nucleus elongates is not obvious until an advanced stage of nuclear division is reached and partition of the nucleus is nearly complete. Thus, no aggregation of chromatin into metaphase or anaphase plates occurs and the appearance of chromatin during mitosis is essentially the same as in interphase. These observations indicate that the marked changes in the topological structure of chromatin which characterize mitosis in the higher eukaryotes do not occur in S. cerevisiae.
Cell Division↗
Aspects of the sporogonic development of Leucocytozoon tawaki of the Fiordland crested penguin in its primary vector, Austrosimulium ungulatum: an ultrastructural study.
Early oocysts of Leucocytozoon tawaki Fallis, Bisset and Allison were located between the basal lamina and the midgut epithelium of the vector, Austrosimulium ungulatum. The spherical oocysts were surrounded by an amorphous, electron-dense wall and contained a large, central core of closely spaced dense particles, the crystalloid inclusion. Around the latter were many concentrically arranged cisternae of granular endoplasmic reticulum. A few large, poorly defined nuclei, some of which contained spindle apparatus, were seen. Sporozoite formation occurred around the peripheral cytoplasm of maturing oocysts. The sporozoite pellicle was subtended by 30 microtubules which appeared to originate from the most posterior of 3 dense, polar rings. Each forming sporozoite contained a central nucleus, mitochondrion, and a crystalloid inclusion both anterior and posterior to the nucleus. The nature and significance of the crystalloid in Leucocytozoan species and other apicomplexans is discussed with special reference to similar viruslike inclusions in the sporogonic stages of certain species of Plasmodium.
Animals↗
Zea mays Meiotic Spindle Ultrastructure Reveals Kinetochore-Microtubule Interface and Embedded Membrane Components.
UNLABELLED: Introduction: Spindles are microtubules-based machines whose primary function is to accurately segregate chromosomes in both mitotic and meiotic cell division. The structure of spindles is critical for their function; errors in morphology or attachment to chromosomes lead to aneuploidy, potentially resulting in disease, infertility, and lethality. Electron microscopy studies have yielded fine-detail spindle ultrastructures in many plant and animal species, but no studies have investigated the spindle of Zea mays, a critical crop, and cytogenetic model system. METHODS: Here we use electron tomography (ET), reconstruction, and modeling to obtain three-dimensional, nanometer-resolution of the Z. mays meiotic spindle. Structures such as microtubules, kinetochores, vesicles, membrane channels, and nuclear envelope were modeled through a partial spindle reconstruction, and confirmed using immunostaining and live fluorescence microscopy. RESULTS: ET revealed that maize spindles contain 8-18 kinetochore microtubules (kMTs) per kinetochore, which are approximately 776 nm in diameter and 316 nm in depth. Small ∼37 nm vesicles were identified, as well as larger (∼5 µm long, 800 nm wide) membrane structures with channels that allow spindle microtubules to pass through. These membrane channels stain positively for the ER-marker protein disulfide isomerase. Imaging of prophase meiotic cells revealed a cross-hatch microtubule arrangement in the perinuclear ring on the external surface of the nuclear envelope, which also contained type II nuclear grooves with transnuclear microtubules passing from the nucleus to the cytoplasm. CONCLUSIONS: Z. mays meiotic spindles are similar to animal counterparts with a comparable number of kMTs and pre-spindle transnuclear microtubules but also plant-specific features such as Golgi-derived vesicles to assist cell plate formation, internal ER membrane channels, and a perinuclear microtubule ring that aids spindle assembly. Maize kinetochores have an electron-diffuse ball in cup morphology that is comparable in size to Drosophila kinetochores and larger than mammalian kinetochores. .
Zea mays↗
Overexpression of GFP Fusions of Regulators of the SAC from Arabidopsis thaliana.
Cell division is a fundamental biological process, essential for sustaining life on Earth. Accurate replication followed by uniform segregation of the genome is required to ensure cell division is sustainable and reduces the likelihood of aneuploidy. The cell cycle has various checkpoints to safeguard proper replication, for example, the spindle assembly checkpoint (SAC) which ensures that all chromosomes are correctly aligned and attached to the spindle, before the transition to anaphase. The precise function of the SAC and SAC components in plants is so far unclear. First, the high level of polyploidy in plants raises concerns about the efficacy of the SAC. Second, many plant SAC components are implicated in other cellular processes, such as MAD1, which has been implicated in the reproductive transition of Arabidopsis thaliana. Overexpression of GFP fusions of core SAC components provides a key route to establish the functions of the different SAC components in plants. Here we describe two methods for agrobacterium-mediated transformation of plants.
Arabidopsis↗
Comparative Pharmacological Analysis of Mitotic Inhibitors Using Isogenic Ploidy Series of HAP1 Cells.
Drastic changes in chromosome number and cellular contents upon ploidy alterations profoundly affect the stability of mitotic regulation in different biological and pathological processes. Isogenic ploidy series of somatic cell lines are useful for studying the effects of ploidy differences on mitotic regulation at cellular and molecular levels. This chapter describes experimental procedures using isogenic human HAP1 cell lines that cover haploid, diploid, and tetraploid states. We first describe methods to establish and maintain these isogenic HAP1 ploidy series using a flow cytometer. We then describe a procedure of comparative pharmacological assay for analyzing ploidy-dependent changes in the functionality of the mitotic spindle components.
Humans↗
4D Microscopy and Tracking of Chromosomes and the Spindle in C. elegans Early Embryos.
Maintaining genomic integrity throughout successive cell divisions is essential for the proper development and functioning of organisms. Chromosome alignment and segregation occur on a microtubule-based spindle originating from centrosomes. The molecular and cellular mechanisms involved in accurate chromosome segregation during early embryonic divisions are highly conserved between worms and humans. Therefore, C. elegans serves as a robust model for investigating mitotic cell divisions within a metazoan system. Throughout early embryonic development, filming and tracking successive cell divisions becomes progressively more challenging as the number of cells increases and cell size decreases. To address this challenge, we describe a method for preparing live samples, performing 4D time-lapse imaging, and semi-automated tracking of chromosomes and spindle poles during early mitotic divisions in C. elegans embryos.
Caenorhabditis elegans↗
Cumulus cells enhance oocyte genomic quality control by promoting DNA damage-induced meiotic arrest.
Cumulus cells are known to maintain oocyte arrest at prophase I through gap junction-mediated cAMP signalling, but their role after meiotic resumption remains unclear. Here, we show that cumulus cells enhance oocyte genomic quality control by sensitizing mouse oocytes to DNA damage-induced meiotic arrest. Time-lapse imaging of SiR-tubulin-labelled spindles revealed that oocytes from cumulus-oocyte complexes (COCs) matured faster than denuded oocytes (DOs). Upon mild DNA damage induced by low-dose etoposide, COC oocytes arrested at metaphase I, whereas DOs completed maturation despite similar levels of DNA lesions. This arrest required spindle assembly checkpoint (SAC) activity, as reversine rescued polar body extrusion and BubR1 and Mad2 were elevated in COCs but not DOs. Disruption of gap junctions or inhibition of mTOR signalling abolished the checkpoint response. Notably, cumulus cells did not enhance oocyte response to minor spindle perturbations. These findings reveal a previously unrecognized role of cumulus cells in mediating DNA damage-induced SAC activation, providing post-GVBD genomic surveillance beyond prophase I arrest.
Animals↗
Cyclin A/Cdk1 promotes chromosome alignment and timely mitotic progression.
To ensure genomic fidelity, a series of spatially and temporally coordinated events is executed during prometaphase of mitosis, including bipolar spindle formation, chromosome attachment to spindle microtubules at kinetochores, the correction of erroneous kinetochore-microtubule (k-MT) attachments, and chromosome congression to the spindle equator. Cyclin A/Cdk1 kinase plays a key role in destabilizing k-MT attachments during prometaphase to promote correction of erroneous k-MT attachments. However, it is unknown whether Cyclin A/Cdk1 kinase regulates other events during prometaphase. Here, we investigate additional roles of Cyclin A/Cdk1 in prometaphase by using an siRNA knockdown strategy to deplete endogenous Cyclin A from human cells. We find that depleting Cyclin A significantly extends mitotic duration, specifically prometaphase, because chromosome alignment is delayed. Unaligned chromosomes display erroneous monotelic, syntelic, or lateral k-MT attachments suggesting that bioriented k-MT attachment formation is delayed in the absence of Cyclin A. Mechanistically, chromosome alignment is likely impaired because the localization of the kinetochore proteins BUB1 kinase, KNL1, and MPS1 kinase are reduced in Cyclin A-depleted cells. Moreover, we find that Cyclin A promotes BUB1 kinetochore localization independently of its role in destabilizing k-MT attachments. Thus, Cyclin A/Cdk1 facilitates chromosome alignment during prometaphase to support timely mitotic progression.
Humans↗
Kinetochore targeting of fission yeast Mad and Bub proteins is essential for spindle checkpoint function but not for all chromosome segregation roles of Bub1p.
Several lines of evidence suggest that kinetochores are organizing centers for the spindle checkpoint response and the synthesis of a "wait anaphase" signal in cases of incomplete or improper kinetochore-microtubule attachment. Here we characterize Schizosaccharomyces pombe Bub3p and study the recruitment of spindle checkpoint components to kinetochores. We demonstrate by chromatin immunoprecipitation that they all interact with the central domain of centromeres, consistent with their role in monitoring kinetochore-microtubule interactions. Bub1p and Bub3p are dependent upon one another, but independent of the Mad proteins, for their kinetochore localization. We demonstrate a clear role for the highly conserved N-terminal domain of Bub1p in the robust targeting of Bub1p, Bub3p, and Mad3p to kinetochores and show that this is crucial for an efficient checkpoint response. Surprisingly, neither this domain nor kinetochore localization is required for other functions of Bub1p in chromosome segregation.
Chromosomes, Fungal↗