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PubMed1978

Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement.

Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus now permits continuous observation of mitosis in individual Barbulanympha cells. In Part I of this two-part paper, we report methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells, sequentially in differential interference contrast and rectified polarized light microscopy. We describe the following prophase events and structures: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays which were deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. The observations described here and in Part II have implications both for the evolution of mitosis and for understanding the mitotic process generally.

Anaerobiosis↗
PubMed1979

Role of spindle microtubules in the control of cell cycle timing.

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

Animals↗
PubMed1961

Nucleic acid and protein metabolism during the mitotic cycle in Vicia faba.

In order to investigate some of the cytochemical processes involved in interphase growth and culminating in cell division, a combined autoradiographic and microphotometric study of nucleic acids and proteins was undertaken on statistically seriated cells of Vicia faba root meristems. Adenine-8-C(14) and uridine-H(3) were used as ribonucleic acid (RNA) precursors, thymidine-H(3) as a deoxyribonucleic acid (DNA) precursor, and phenylalanine-3-C(14) as a protein precursor. Stains used in microphotometry were Feulgen (DNA), azure B (RNA), pH 2.0 fast green (total protein), and pH 8.1 fast green (histone). The autoradiographic data (representing rate of incorporation per organelle) and the microphotometric data (representing changes in amounts of the various components) indicate that the mitotic cycle may be divided into several metabolic phases, three predominantly anabolic (net increase), and a fourth phase predominantly catabolic (net decrease). The anabolic periods are: 1. Telophase to post-telophase during which there are high rates of accumulation of cytoplasmic and nucleolar RNA and nucleolar and chromosomal total protein. 2. Post-telophase to preprophase characterized by histone synthesis and a diphasic synthesis of DNA with the peak of synthesis at mid-interphase and a minor peak just preceding prophase. The minor peak is coincident with a relatively localized DNA synthesis in several chromosomal regions. This period is also characterized by minimal accumulations of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA. 3. Preprophase to prophase in which there are again high rates of accumulation of cytoplasmic RNA, and nucleolar and chromosomal total protein and RNA. The catabolic phase is: 4. The mitotic division during which there are marked losses of cytoplasmic RNA and chromosomal and nucleolar total protein and RNA.

Cell Division↗
PubMed2002

Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition.

We report a novel autosomal recessive disorder characterized by premature chromosome condensation in the early G2 phase. It was observed in two siblings, from consanguineous parents, affected with microcephaly, growth retardation, and severe mental retardation. Chromosome analysis showed a high frequency of prophase-like cells (>10%) in lymphocytes, fibroblasts, and lymphoblast cell lines with an otherwise normal karyotype. (3)H-thymidine-pulse labeling and autoradiography showed that, 2 h after the pulse, 28%-35% of the prophases were labeled, compared with 9%-11% in healthy control subjects, indicating that the phenomenon is due to premature chromosome condensation. Flow cytometry studies demonstrate that the entire cell cycle is not prolonged, compared with that in healthy control subjects, and compartment sizes did not differ from those in healthy control subjects. No increased reaction of the cells to X-irradiation or treatments with the clastogens bleomycin and mitomycin C was observed, in contrast to results in the cell-cycle mutants ataxia telangiectasia and Fanconi anemia. The rates of sister chromatid exchanges and the mitotic nondisjunction rates were inconspicuous. Premature entry of cells into mitosis suggests that a gene involved in cell-cycle regulation is mutated in these siblings.

Cell Cycle↗
PubMed2006

Meiotic recombination and spatial proximity in the etiology of the recurrent t(11;22).

Although balanced translocations are among the most common human chromosomal aberrations, the constitutional t(11;22)(q23;q11) is the only known recurrent non-Robertsonian translocation. Evidence indicates that de novo formation of the t(11;22) occurs during meiosis. To test the hypothesis that spatial proximity of chromosomes 11 and 22 in meiotic prophase oocytes and spermatocytes plays a role in the rearrangement, the positions of the 11q23 and 22q11 translocation breakpoints were examined. Fluorescence in situ hybridization with use of DNA probes for these sites demonstrates that 11q23 is closer to 22q11 in meiosis than to a control at 6q26. Although chromosome 21p11, another control, often lies as close to 11q23 as does 22q11 during meiosis, chromosome 21 rarely rearranges with 11q23, and the DNA sequence of chromosome 21 appears to be less susceptible than 22q11 to double-strand breaks (DSBs). It has been suggested that the rearrangement recurs as a result of the palindromic AT-rich repeats at both 11q23 and 22q11, which extrude hairpin structures that are susceptible to DSBs. To determine whether the DSBs at these sites coincide with normal hotspots of meiotic recombination, immunocytochemical mapping of MLH1, a protein involved in crossing over, was employed. The results indicate that the translocation breakpoints do not coincide with recombination hotspots and therefore are unlikely to be the result of meiotic programmed DSBs, although MRE11 is likely to be involved. Previous analysis indicated that the DSBs appear to be repaired by a mechanism similar to nonhomologous end joining (NHEJ), although NHEJ is normally suppressed during meiosis. Taken together, these studies support the hypothesis that physical proximity between 11q23 and 22q11--but not typical meiotic recombinational activity in meiotic prophase--plays an important role in the generation of the constitutional t(11;22) rearrangement.

Chromosomes, Human, Pair 11↗
PubMed1999

Unusual centrosome cycle in Dictyostelium: correlation of dynamic behavior and structural changes.

Centrosome duplication and separation are of central importance for cell division. Here we provide a detailed account of this dynamic process in Dictyostelium. Centrosome behavior was monitored in living cells using a gamma-tubulin-green fluorescent protein construct and correlated with morphological changes at the ultrastructural level. All aspects of the duplication and separation process of this centrosome are unusual when compared with, e.g., vertebrate cells. In interphase the Dictyostelium centrosome is a box-shaped structure comprised of three major layers, surrounded by an amorphous corona from which microtubules emerge. Structural duplication takes place during prophase, as opposed to G1/S in vertebrate cells. The three layers of the box-shaped core structure increase in size. The surrounding corona is lost, an event accompanied by a decrease in signal intensity of gamma-tubulin-green fluorescent protein at the centrosome and the breakdown of the interphase microtubule system. At the prophase/prometaphase transition the separation into two mitotic centrosomes takes place via an intriguing lengthwise splitting process where the two outer layers of the prophase centrosome peel away from each other and become the mitotic centrosomes. Spindle microtubules are now nucleated from surfaces that previously were buried inside the interphase centrosome. Finally, at the end of telophase, the mitotic centrosomes fold in such a way that the microtubule-nucleating surface remains on the outside of the organelle. Thus in each cell cycle the centrosome undergoes an apparent inside-out/outside-in reversal of its layered structure.

Animals↗
PubMed1999

Architecture of the nuclear periphery of rat pachytene spermatocytes: distribution of nuclear envelope proteins in relation to synaptonemal complex attachment sites.

The nucleus of spermatocytes provides during the first meiotic prophase an interesting model for investigating relationships of the nuclear envelope (NE) with components of the nuclear interior. During the pachytene stage, meiotic chromosomes are synapsed via synaptonemal complexes (SCs) and attached through both ends to the nuclear periphery. This association is dynamic because chromosomes move during the process of synapsis and desynapsis that takes place during meiotic prophase. The NE of spermatocytes possesses some peculiarities (e.g., lower stability than in somatic cells, expression of short meiosis-specific lamin isoforms called C2 and B3) that could be critically involved in this process. For better understanding of the association of chromosomes with the nuclear periphery, in the present study we have investigated the distribution of NE proteins in relation to SC attachment sites. A major outcome was the finding that lamin C2 is distributed in the form of discontinuous domains at the NE of spermatocytes and that SC attachment sites are embedded in these domains. Lamin C2 appears to form part of larger structures as suggested by cell fractionation experiments. According to these results, we propose that the C2-containing domains represent local reinforcements of the NE that are involved in the proper attachment of SCs.

Animals↗
PubMed1997

Protein-protein interactions in the synaptonemal complex.

In mammalian systems, an approximately M(r) 30,000 Cor1 protein has been identified as a major component of the meiotic prophase chromosome cores, and a M(r) 125,000 Syn1 protein is present between homologue cores where they are synapsed and form the synaptonemal complex (SC). Immunolocalization of these proteins during meiosis suggests possible homo- and heterotypic interactions between the two as well as possible interactions with yet unrecognized proteins. We used the two-hybrid system in the yeast Saccharomyces cerevisiae to detect possible protein-protein associations. Segments of hamsters Cor1 and Syn1 proteins were tested in various combinations for homo- and heterotypic interactions. In the cause of Cor1, homotypic interactions involve regions capable of coiled-coil formation, observation confirmed by in vitro affinity coprecipitation experiments. The two-hybrid assay detects no interaction of Cor1 protein with central and C-terminal fragments of Syn1 protein and no homotypic interactions involving these fragments of Syn1. Hamster Cor1 and Syn1 proteins both associate with the human ubiquitin-conjugation enzyme Hsubc9 as well as with the hamster Ubc9 homologue. The interactions between SC proteins and the Ubc9 protein may be significant for SC disassembly, which coincides with the repulsion of homologs by late prophase I, and also for the termination of sister centromere cohesiveness at anaphase II.

Animals↗
PubMed2002

Meiotic cohesion requires accumulation of ORD on chromosomes before condensation.

Cohesion between sister chromatids is a prerequisite for accurate chromosome segregation during mitosis and meiosis. To allow chromosome condensation during prophase, the connections that hold sister chromatids together must be maintained but still permit extensive chromatin compaction. In Drosophila, null mutations in the orientation disruptor (ord) gene lead to meiotic nondisjunction in males and females because cohesion is absent by the time that sister kinetochores make stable microtubule attachments. We provide evidence that ORD is concentrated within the extrachromosomal domains of the nuclei of Drosophila primary spermatocytes during early G2, but accumulates on the meiotic chromosomes by mid to late G2. Moreover, using fluorescence in situ hybridization to monitor cohesion directly, we show that cohesion defects first become detectable in ord(null) spermatocytes shortly after the time when wild-type ORD associates with the chromosomes. After condensation, ORD remains bound at the centromeres of wild-type spermatocytes and persists there until centromeric cohesion is released during anaphase II. Our results suggest that association of ORD with meiotic chromosomes during mid to late G2 is required to maintain sister-chromatid cohesion during prophase condensation and that retention of ORD at the centromeres after condensation ensures the maintenance of centromeric cohesion until anaphase II.

Animals↗
PubMed2003

A kinesin mutant with an atypical bipolar spindle undergoes normal mitosis.

Motor proteins have been implicated in various aspects of mitosis, including spindle assembly and chromosome segregation. Here, we show that acentrosomal Arabidopsis cells that are mutant for the kinesin, ATK1, lack microtubule accumulation at the predicted spindle poles during prophase and have reduced spindle bipolarity during prometaphase. Nonetheless, all abnormalities are rectified by anaphase and chromosome segregation appears normal. We conclude that ATK1 is required for normal microtubule accumulation at the spindle poles during prophase and possibly functions in spindle assembly during prometaphase. Because aberrant spindle morphology in these mutants is resolved by anaphase, we postulate that mitotic plant cells contain an error-correcting mechanism. Moreover, ATK1 function seems to be dosage-dependent, because cells containing one wild-type allele take significantly longer to proceed to anaphase as compared with cells containing two wild-type alleles.

Alleles↗
PubMed2003

Organization and dynamics of growing microtubule plus ends during early mitosis.

A stable cell line expressing EB1-green fluorescent protein was used to image growing microtubule plus ends at the G(2)/M transition. By late prophase growing ends no longer extend to the cell periphery and were not uniformly distributed around each centrosome. Growing ends were much more abundant in the area surrounding the nuclear envelope, and microtubules growing around the nucleus were 1.5 fold longer than those growing in the opposite direction. The growth of longer ends toward the nucleus did not result from a localized faster growth rate, because this rate was approximately 11 microm/min in all directions from the centrosome. Rather, microtubule ends growing toward the nucleus seemed stabilized by dynein/dynactin associated with the nuclear envelope. Injection of p50 into late prophase cells removed dynein from the nuclear envelope, reduced the density of growing ends near the nuclear envelope and resulted in a uniform distribution of growing ends from each centrosome. We suggest that the cell cycle-dependent binding of dynein/dynactin to the nuclear envelope locally stabilizes growing microtubules. Both dynein and microtubules would then be in a position to participate in nuclear envelope breakdown, as described in recent studies.

Animals↗
PubMed2003

Depletion of centromeric MCAK leads to chromosome congression and segregation defects due to improper kinetochore attachments.

The complex behavior of chromosomes during mitosis is accomplished by precise binding and highly regulated polymerization dynamics of kinetochore microtubules. Previous studies have implicated Kin Is, unique kinesins that depolymerize microtubules, in regulating chromosome positioning. We have characterized the immunofluorescence localization of centromere-bound MCAK and found that MCAK localized to inner kinetochores during prophase but was predominantly centromeric by metaphase. Interestingly, MCAK accumulated at leading kinetochores during congression but not during segregation. We tested the consequences of MCAK disruption by injecting a centromere dominant-negative protein into prophase cells. Depletion of centromeric MCAK led to reduced centromere stretch, delayed chromosome congression, alignment defects, and severe missegregation of chromosomes. Rates of chromosome movement were unchanged, suggesting that the primary role of MCAK is not to move chromosomes. Furthermore, we found that disruption of MCAK leads to multiple kinetochore-microtubule attachment defects, including merotelic, syntelic, and combined merotelic-syntelic attachments. These findings reveal an essential role for Kin Is in prevention and/or correction of improper kinetochore-microtubule attachments.

Animals↗
PubMed2004

Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells.

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.

Adenosine Triphosphatases↗
PubMed2004

Plk1 regulates activation of the anaphase promoting complex by phosphorylating and triggering SCFbetaTrCP-dependent destruction of the APC Inhibitor Emi1.

Progression through mitosis requires activation of cyclin B/Cdk1 and its downstream targets, including Polo-like kinase and the anaphase-promoting complex (APC), the ubiquitin ligase directing degradation of cyclins A and B. Recent evidence shows that APC activation requires destruction of the APC inhibitor Emi1. In prophase, phosphorylation of Emi1 generates a D-pS-G-X-X-pS degron to recruit the SCF(betaTrCP) ubiquitin ligase, causing Emi1 destruction and allowing progression beyond prometaphase, but the kinases directing this phosphorylation remain undefined. We show here that the polo-like kinase Plk1 is strictly required for Emi1 destruction and that overexpression of Plk1 is sufficient to trigger Emi1 destruction. Plk1 stimulates Emi1 phosphorylation, betaTrCP binding, and ubiquitination in vitro and cyclin B/Cdk1 enhances these effects. Plk1 binds to Emi1 in mitosis and the two proteins colocalize on the mitotic spindle poles, suggesting that Plk1 may spatially control Emi1 destruction. These data support the hypothesis that Plk1 activates the APC by directing the SCF-dependent destruction of Emi1 in prophase.

Anaphase↗
PubMed2002

Chfr expression is downregulated by CpG island hypermethylation in esophageal cancer.

Cell cycle progression is monitored by checkpoint mechanisms to ensure the integrity of the genome and the fidelity of sister chromatid separation. Failure of such checkpoint functions results in genomic instability, a condition that predisposes cells to neoplastic transformation and tumor progression. Recently, Scolnick and Halazonetis defined a new mitotic checkpoint that acts at prophase and delays chromosome condensation in response to mitotic stress, and identified a gene, named checkpoint with FHA and ring finger (Chfr), that seems to be required for delaying prophase in human cells. In the present study, we examined human Chfr mRNA expression in 15 human esophageal cancer cell lines and 43 primary esophageal cancers to investigate the potential involvement of Chfr in the pathogenesis of esophageal cancers. We report here that a significant proportion of human esophageal cancer has loss of expression of Chfr gene. Furthermore, we found aberrant hypermethylation of the promoter region of this checkpoint gene in four of 15 (26.7%) esophageal cancer cell lines and in seven of 43 (16.3%) primary cancers.

Adenocarcinoma↗
PubMed2003

Frequent hypermethylation of the 5' CpG island of the mitotic stress checkpoint gene Chfr in colorectal and non-small cell lung cancer.

Chfr, a mitotic stress checkpoint gene, regulates a prophase delay in cells exposed to agents that disrupt microtubules, such as nocodazole and taxol. In the present study, we report that Chfr is frequently methylated in cell lines derived from tumors of the colon (80%), brain (100%) and bone (100%). In addition, Chfr was methylated in 37% of primary colon adenocarcinomas and in 10% of primary non-small cell lung carcinomas. In normal colon tissue, but not lung, there was evidence for age-related methylation of Chfr, suggesting that in some cases the tumor may have arisen from a methylated clonal precursor. Methylation was associated with loss of Chfr mRNA and protein expression in cancer cell lines. In cells with methylated Chfr, treatment with the demethylating agent 5-aza-2'-deoxycytidine resulted in re-expression of Chfr, and partial restoration of the prophase checkpoint. These results suggest that epigenetic inactivation of Chfr may be responsible for many of the checkpoint defects observed in human cancers.

Adult↗
PubMed2003

Mitotic regulation of the human anaphase-promoting complex by phosphorylation.

The anaphase-promoting complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and Cdc20 binding. We have identified 43 phospho-sites on APC of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro, at least 15 of the mitotic phospho-sites can be generated by cyclin-dependent kinase 1 (Cdk1), and 3 by Polo-like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased Cdc20 binding and APC activation. Immunofluorescence microscopy using phospho-antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase phospho-APC accumulates on centrosomes where cyclin B ubiquitination is initiated, appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo. These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1995

gld-1, a tumor suppressor gene required for oocyte development in Caenorhabditis elegans.

We have characterized 31 mutations in the gld-1 (defective in germline development) gene of Caenorhabditis elegans. In gld-1 (null) hermaphrodites, oogenesis is abolished and a germline tumor forms where oocyte development would normally occur. By contrast, gld-1 (null) males are unaffected. The hermaphrodite germline tumor appears to derive from germ cells that enter the meiotic pathway normally but then exit pachytene and return to the mitotic cycle. Certain gld-1 partial loss-of-function mutations also abolish oogenesis, but germ cells arrest in pachytene rather than returning to mitosis. Our results indicate that gld-1 is a tumor suppressor gene required for oocyte development. The tumorous phenotype suggests that gld-1(+) may function to negatively regulate proliferation during meiotic prophase and/or act to direct progression through meiotic prophase. We also show that gld-1(+) has an additional nonessential role in germline sex determination: promotion of hermaphrodite spermatogenesis. This function of gld-1 is inferred from a haplo-insufficient phenotype and from the properties of gain-of-function gld-1 mutations that cause alterations in the sexual identity of germ cells.

Animals↗