Quantitative analysis of a nuclear antigen in interphase and mitotic cells

The quantification of an interchromatin-associated antigen, designated p 105, during cellular passage through mitosis is described. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated a qualitative increase in p 105 within the mitotic cytoplasm. Multiparameter flow cytometric analysis was performed on fixed cells sequentially stained with anti-p 105 immunofluorescence and/or propidium iodide. This analysis demonstrated approximately a tenfold increase in intracellular p 105 content as a function of progression from the G2 to the M phase. This increase was corroborated by the quantitative immunoblot analysis of colchicine-treated cell cultures and of cells sorted on the basis of anti-p 105 immunofluorescence. The data reveal that the increased levels of anti-p 105 immunofluorescence in conjunction with flow cytometry may be used effectively to quantitate mitotic index and isolate mitotic cells. The function and modulation of p 105 throughout the cell cycle is discussed.     

Ultrastructural and immunocytological studies on the rhizoplast in the chrysophycean alga Ochromonas danica

The rhizoplast, a striated band elongating from the flagellar basal body to the nucleus, is conspicuous in cells of Ochromonas danica Prings. In interphase cells, it runs from the basal body of the anterior flagellum to the space between the nucleus and the Golgi body. In O. danica, the rhizoplast duplicates during mitosis and the two rhizoplasts serve as mitotic poles. In the present study, we reinvestigated mitosis of O. danica using transmission electron microscopy and immunofluorescence microscopy, especially focusing on the rhizoplast. The nuclear envelope became dispersed during metaphase, and the rhizoplasts from two sets of the flagellar basal bodies functioned as the mitotic poles. Immunofluorescence microscopy using anti‐α‐tubulin, anti‐centrin and anti‐γ‐tubulin antibodies showed that centrin molecules were localized at the flagellar basal bodies, whereas γ‐tubulin molecules were detected at the rhizoplast during the whole cell cycle.     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

By indirect immunofluorescence microscopy and electron microscopy, we studied the behavior of intermediate filaments during mitosis in three human epithelial cell lines, derived from normal epidermis (PcaSE-1, from a cancer patient), stratified epithelium (CNE, from nasopharyngeal carcinoma) and simple epithelium (SPC-A-1 from lung adenocarcinoma) respectively. CNE cells and SPC-A-1 cells express two different intermediate filament systems; keratin filaments and vimentin filaments, but PcaSE-1 cells only express keratin filaments. The keratin filament system in PcaSE-1 cells remained intact and encircled the developing mitotic spindle as the cells entered mitosis. In contrast, in CNE cells and SPC-A-1 cells, keratin filaments appeared to disassemble into amorphous cytoplasmic bodies during mitosis. However, their vimentin filaments remained morphologically intact throughout mitosis. We propose; (1) The disassembly of keratin filaments in mitotic epithelial cells is more or less associated with the degree of their cell malignancy rather than with the abundance of keratin filaments in interphase. (2) Intermediate filaments may be involved in the positioning and/or centering of the spindle during mitosis. (3) The possible function of vimentin filament system in CNE cells is positioning and orientation of chromosomes.     

中等纤维在几种人体上皮细胞有丝分裂过程中的行为

本文用间接免疫荧光法和电镜术观察了分别来自人表皮(PcaSE-1)、复层上皮(CNE)和单层上皮(SPC-A-1)的3个上皮细胞系的细胞在有丝分裂过程中中等纤维的行为。结果表明,CNE细胞和SPC-A-1细胞表达两种不同类型的中等纤维系统:角蛋白纤维和波形纤维,而PcaSE-1细胞仅表达角蛋白纤维。当细胞进入有丝分裂时,PcaSE-1细胞的角蛋白纤维维持完整的形态且将有丝分裂纺锤体围绕在细胞中央。相反,在CNE细胞和SPC-A-1细胞中,在细胞有丝分裂时,角蛋白纤维解聚成无定形的胞质小体,然而它们的波形纤维始终保持完整的形态。我们认为(1)在分裂上皮细胞中,角蛋白纤维的解聚与细胞的恶性程度有关,而与间期上皮细胞中是否含有丰富的角蛋白纤维无明显关系。(2)在上皮细胞有丝分裂时,中等纤维可能参于纺锤体的定位和趋中。(3)在分裂CNE细胞中,波形纤维的可能功能是染色体的定位和定向。     

Mitotic drug targets

Mitosis is the key event of the cell cycle during which the sister chromatids are segregated onto two daughter cells. It is well established that abrogation of the normal mitotic progression is a highly efficient concept for anti‐cancer treatment. In fact, various drugs that target microtubules and thus interfere with the function of the mitotic spindle are in clinical use for the treatment of various human malignancies for many years. However, since microtubule inhibitors not only target proliferating cells severe side effects limit their use. Therefore, the identification of novel mitotic drug targets other than microtubules have gained recently much attention. This review will summarize the latest developments on the identification and clinical evaluation of novel mitotic drug targets and will introduce novel concepts for chemotherapy that are based on recent progress in our understanding how mitotic progression is regulated and how anti‐mitotic drugs induce tumor cell death. J. Cell. Biochem. 111: 258–265, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of cyclin B1 after induction of senescence and cell death in non-small cell lung carcinoma A549 cells

The purpose of this study was to evaluate the level of mitotic cyclin B1 in the context of senescence and cell death in A549 non-small cell lung carcinoma cells. This was performed through analysis of the cell cycle, the percentage of SA-β-galactosidase-positive, as well as TUNEL-positive cells. Morphological alterations were studied using a transmission electron microscope. Changes in the intracellular level and the presence of cyclin B1 in the nucleus and cytoplasm areas were detected by flow cytometry and confocal fluorescence microscopy, respectively. In the cells exposed to various concentrations of doxorubicin, different kinds of cell death and senescent phenotype were observed. Alterations in the cell cycle and increased polyploidy may be indicative of mitotic catastrophe execution. Changes in cyclin B1 may also be strictly related to its different regulation at mitotic catastrophe and senescence programs.     

Microtubule-organizing centers abnormal in number, structure, and nucleating activity in x-irradiated mammalian cells

Microtubule-organizing centers (MTOCs) in x-irradiated cells were visualized by immunofluorescence using antibody against tubulin. From two to ten reassembly sites of microtubules appeared after microtubule depolymerization at low temperature in an irradiated mitotic cell, in contrast to nonirradiated mitotic cells, which predominantly show 2 MTOCs. A time-course examination of MTOCs in synchronously cultured cells revealed that the multiple MTOCs appeared not immediately after irradiation but at the time of mitosis. Those multiple MTOCs formed at mitosis were inherited by the daughter cells in the next generation. The structure and capacity of the centrosomes to nucleate microtubules in vitro were then examined by electron microscopy of whole-mount preparations as well as by dark-field microscopy. About 70-80% of the centrosomes derived from nonirradiated cells were composed of a pair of centrioles and pericentriolar material, which initiated greater than 100 microtubules. The fraction of fully active complete centrosomes decreased with time of incubation after irradiation. These were replaced by disintegrated centrosomal components such as dissociated centrioles and pericentriolar cloud, a nucleating site with a single centriole, or only an amorphous structure of pericentriolar cloud. Assembly of less than 20 microtubules onto the amorphous cloud without centrioles was seen in 54% of the initiating sites in mitotic cells 2 d after irradiation. These results suggest that x-irradiation causes disintegration of centrosomes at mitosis when the structural and functional reorganization of centrosomes is believed to occur.     

The organizational fate of intermediate filament networks in two epithelial cell types during mitosis

Intermediate filaments (IF) appear to be attached to the nuclear envelope in various mammalian cell types. The nucleus of mouse keratinocytes is enveloped by a cagelike network of keratin-containing bundles of IF (IFB). This network appears to be continuous with the cytoplasmic IFB system that extends to the cell surface. Electron microscopy reveals that the IFB appear to terminate at the level of the nuclear envelope, frequently in association with nuclear pore complexes (Jones, J. C .R., A. E. Goldman, P. Steinert, S. Yuspa, and R. D. Goldman, 1982, Cell Motility, 2:197-213). Based on these observations of nuclear-IF associations, it is of interest to determine the fate and organizational states of IF during mitosis, a period in the cell cycle when the nuclear envelope disassembles. Immunofluorescence microscopy using a monoclonal keratin antibody and electron microscopy of thin and thick sections of mitotic mouse keratinocytes revealed that the IFB system remained intact as the cells entered mitosis and surrounded the developing mitotic spindle. IFB were close to chromosomes and often associated with chromosome arms. In contrast, in HeLa, a human epithelial cell, keratin-containing IFB appear to dissemble as cells enter mitosis (Franke, W. W., E. Schmid, C. Grund, and B. Geiger, 1982, Cell, 30:103-113). The keratin IFB in mitotic HeLa cells appeared to form amorphous nonfilamentous bodies as determined by electron microscopy. However, in HeLa, another IF system composed primarily of a 55,000-mol-wt protein (frequently termed vimentin) appears to remain morphologically intact throughout mitosis in close association with the mitotic apparatus (Celis, J.E., P.M. Larsen, S.J. Fey, and A. Celis, 1983, J. Cell Biol., 97:1429-34). We propose that the mitotic apparatus in both mouse epidermal cells and in HeLa cells is supported and centered within the cell by IFB networks.     

A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase

A protein of 62 kD is a substrate of a calcium/calmodulin-dependent protein kinase, and both proteins copurify with isolated mitotic apparatuses (Dinsmore, J. H., and R. D. Sloboda. 1988. Cell. 53:769-780). Phosphorylation of the 62-kD protein increases after fertilization; maximum incorporation of phosphate occurs during late metaphase and anaphase and correlates directly with microtubule disassembly as determined by in vitro experiments with isolated mitotic apparatuses. Because 62-kD protein phosphorylation occurs in a pattern similar to the accumulation of the mitotic cyclin proteins, experiments were performed to determine the relationship between cyclin and the 62-kD protein. Continuous labeling of marine embryos with [35S]methionine, as well as immunoblots of marine embryo proteins using specific antibodies, were used to identify both cyclin and the 62-kD protein. These results clearly demonstrate that the 62-kD protein is distinct from cyclin and, unlike cyclin, is a constant member of the cellular protein pool during the first two cell cycles in sea urchin and surf clam embryos. Similar results were obtained using immunofluorescence microscopy of intact eggs and embryos. In addition, immunogold electron microscopy reveals that the 62-kD protein associates with the microtubules of the mitotic apparatus in dividing cells. Interestingly, the protein changes its subcellular distribution with respect to microtubules during the cell cycle. Specifically, during mitosis the 62-kD protein associates with the mitotic apparatus; before nuclear envelope breakdown, however, the 62-kD protein is confined to the nucleus. After anaphase, the 62-kD protein returns to the nucleus, where it resides until nuclear envelope disassembly of the next cell cycle.     

Virus Replication, Cytopathology, and Lysosomal Enzyme Response of Mitotic and Interphase Hep-2 Cells Infected with Poliovirus

Mitotic Hep-2 cells, selected by the PEL (colloidal silica) density gradient method and held in mitosis with Colcemid, are readily infected by poliovirus type I (Mahoney). They produce and release the same amount of virus as interphase, random-growing cells. In contrast to interphase cells, mitotic cells show no detectable virus-induced cytopathic effect at the light microscopy level and only slight alterations, consisting of small clusters of vacuoles, at the electron microscopy level. Mitotic cells contain the same total amount of lysosomal enzymes per cell as interphase cells, but they display no redistribution of lysosomal enzymes during the virus infection as interphase cells do. This supports the view that lysosomal enzyme redistribution is associated with the cytopathic effect in poliovirus infection but shows that virus synthesis and release is not dependent on either the cytopathic effect or lysosomal enzyme release. The possible reasons for the lack of cytopathic effect in mitotic cells are discussed.     

Effects of chemical manipulation of mitotic arrest and slippage on cancer cell survival and proliferation

Microtubule-targeting cancer therapies interfere with mitotic spindle dynamics and block cells in mitosis by activating the mitotic checkpoint. Cells arrested in mitosis may remain arrested for extended periods of time or undergo mitotic slippage and enter interphase without having separated their chromosomes. How extended mitotic arrest and mitotic slippage contribute to subsequent cell death or survival is incompletely understood. To address this question, automated fluorescence microscopy assays were designed and used to screen chemical libraries for modulators of mitotic slippage. Chlorpromazine and triflupromazine were identified as drugs that inhibit mitotic slippage and SU6656 and geraldol as chemicals that stimulate mitotic slippage. Using the drugs to extend mitotic arrest imposed by low concentrations of paclitaxel led to increased cell survival and proliferation after drug removal. Cells arrested at mitosis with paclitaxel or vinblastine and chemically induced to undergo mitotic slippage underwent several rounds of DNA replication without cell division and exhibited signs of senescence but eventually all died. By contrast, cells arrested at mitosis with the KSP/Eg5 inhibitor S-trityl-L-cysteine and induced to undergo mitotic slippage were able to successfully divide and continued to proliferate after drug removal. These results show that reinforcing mitotic arrest with drugs that inhibit mitotic slippage can lead to increased cell survival and proliferation, while inducing mitotic slippage in cells treated with microtubule-targeting drugs seems to invariably lead to protracted cell death.     

Three-dimensional segmentation of nuclei and mitotic chromosomes for the study of cell divisions in live Drosophila embryos

Drosophila embryogenesis is an established model to investigate mechanisms and genes related to cell divisions in an intact multicellular organism. Progression through the cell cycle phases can be monitored in vivo using fluorescently labeled fusion proteins and time-lapse microscopy. To measure cellular properties in microscopic images, accurate and fast image segmentation methods are a critical prerequisite. To quantify static and dynamic features of interphase nuclei and mitotic chromosomes, we developed a three-dimensional (3D) segmentation method based on multiple level sets. We tested our method on 3D time-series images of live embryos expressing histone-2Av-green fluorescence protein. Our method is robust to low signal-to-noise ratios inherent to high-speed imaging, fluorescent signals in the cytoplasm, and dynamic changes of shape and texture. Comparisons with manual ground-truth segmentations showed that our method achieves more than 90% accuracy on the object as well as voxel levels and performs consistently throughout all cell cycle phases and developmental stages from syncytial blastoderm to postblastoderm mitotic domains.     

Preparation and characterization of nuclear matrix/chromosome scaffold in situ

A method of nuclear matrix and chromosomal scaffold preparation from cultured animal cells was developed. After the high-salt extraction, interphase and mitotic cells were not detached from the coverslips that enabled us to analyse the nuclear matrix and chromosomal scaffold in cells at all mitotic phases. Morphological methods (phase contrast microscopy and electron microscopy of ultrathin sections) did not reveal any structures that could be identified as a chromosomal scaffold. However, after staining with antibodies to XCAP-E and topoisomerase IIalpha some structures were revealed in metaphase cells having both localization and morphology of a chromosomal scaffold. The cell residuals were not stained with antibodies to XCAP-E and topoisomerase IIalpha, if the nuclear matrix and chromosomal scaffold were destabilized by addition of beta-mercaptoethanol.     

Growth characteristics of Heterosigma akashiwo virus and its possible use as a microbiological agent for red tide control

The growth characteristics of Heterosigma akashiwo virus clone 01 (HaV01) were examined by performing a one-step growth experiment. The virus had a latent period of 30 to 33 h and a burst size of 7.7 x 10(2) lysis-causing units in an infected cell. Transmission electron microscopy showed that the virus particles formed on the peripheries of viroplasms, as observed in a natural H. akashiwo cell. Inoculation of HaV01 into a mixed algal culture containing four phytoplankton species, H. akashiwo H93616, Chattonella antiqua (a member of the family Raphidophyceae), Heterocapsa triquetra (a member of the family Dinophyceae), and Ditylum brightwellii (a member of the family Bacillariophyceae), resulted in selective growth inhibition of H. akashiwo. Inoculation of HaV01 and H. akashiwo H93616 into a natural seawater sample produced similar results. However, a natural H. akashiwo red tide sample did not exhibit any conspicuous sensitivity to HaV01, presumably because of the great diversity of the host species with respect to virus infection. The growth characteristics of the lytic virus infecting the noxious harmful algal bloom-causing alga were considered, and the possibility of using this virus as a microbiological agent against H. akashiwo red tides is discussed.     

The organization of the mitotic apparatus poles in etoposide-treated CHO-K1 cells

In this study, we have examined the organization of the mitotic spindle poles in CHO-K1 cells dividing after treatment with the etoposide (1 h, 25 microM). We studied at various periods after the treatment: 1) the distribution of gamma-tubulin in mitotic cells by immunofluorescent staining; 2) the level of posttranslational modification of a-tubulin in the spindle microtubules by immunoelectron microscopy; 3) the ultrastructure of the mitotic apparatus poles by standard electron microscopy. In 48 h after the addition of the agent we identified considerable changes in the ultrastructure of poles in etoposide-treated CHO-K1 cells with bipolar and multipolar spindles. The number of centrioles increased. The centrioles were unevenly distributed among the poles, and some centrioles were not explicitly involved in the organization of mitotic spindle, furthermore they can differ in the number of outgrowing microtubules. Most centrioles were without fibrillar halo. In 48 h after the addition of etoposide, electron microscopy of cells after immunoperoxidase staining with antibodies to acetylated and tyrosinated alpha-tubulin has shown that different poles of a multipolar spindle within the same cell are stained differently for tyr-tubulin but not for acet-tubulin. Immunofluorescence staining for gamma-tubulin also points to different organization of poles in the same spindle. Our findings provide the first evidence that the pattern of immunostaning and the ultrastructure of mitotic apparatus poles differ in the cells dividing at various periods after etoposide treatment.     

Active participation of apoptosis and mitosis in sublingual gland regeneration of the rat following release from duct ligation

Summary This study was designed to establish how mitotic cell proliferation and apoptotic cell death participate in the regeneration of atrophied rat sublingual glands. To induce atrophy to the sublingual gland of rats, the excretory duct was ligated unilaterally near the hilum, and after 1 week of ligation (day 0) the duct ligation was released to enable gland regeneration. The regenerating glands were examined with routine histology, immunohistochemistry for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) as a marker of apoptotic cells, and transmission electron microscopy. At day 0, a few acini and many ducts remained in the atrophic sublingual glands, and newly formed immature acini were observed at day 3. Thereafter acinar cells progressively matured and increased in number, although the number of ducts decreased. Many PCNA- and some TUNEL-positive cells were seen in acini and ducts during regeneration. The labeling indices for both cell types were statistically significantly different from that of the control at several time points of the regeneration. Apoptotic and mitotic cells were also confirmed to be present in the experimental sublingual glands by electron microscopy. These observations suggest that apoptosis as well as mitosis of duct and acinar cells actively participate in and play important roles in sublingual gland regeneration.     

Evolution and function of the mitotic checkpoint

The mitotic checkpoint evolved to prevent cell division when chromosomes have not established connections with the chromosome segregation machinery. Many of the fundamental molecular principles that underlie the checkpoint, its spatiotemporal activation, and its timely inactivation have been uncovered. Most of these are conserved in eukaryotes, but important differences between species exist. Here we review current concepts of mitotic checkpoint activation and silencing. Guided by studies in model organisms and our phylogenomics analysis of checkpoint constituents and their functional domains and motifs, we highlight ancient and taxa-specific aspects of the core checkpoint modules in the context of mitotic checkpoint function.     

Erythrocyte ghost-mediated microinjection into cells in monolayer cultures: a highly efficient and low toxic technique

We describe a technique by which macromolecules can be microinjected into mammalian cells in monolayer cultures. This technique employs erythrocyte ghosts as the vehicle for microinjection, calcium as attachment agent and polyethylene glycol as fusogen. The use of calcium allows a reduction of the time of exposure to polyethylene glycol, and results in a high injection efficiency and a high cell viability when followed by incubation in a buffer free of divalent cations. Injecting over 90% of the cells, a reduction of cell viability is not observed and the mitotic index is never lower than 2.3%. Light and electron microscopy suggest that erythrocyte ghost-cell fusion is only a short event.     

Inhibition of the Proliferative Cycle and Apoptotic Events in WiDr Cells after Down-Regulation of the Calcium-Binding Protein Calretinin Using Antisense Oligodeoxynucleotides

The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [³H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca²⁺]_iwas not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.     

Progressive sheet-to-tubule transformation is a general mechanism for endoplasmic reticulum partitioning in dividing mammalian cells

The endoplasmic reticulum (ER) is both structurally and functionally complex, consisting of a dynamic network of interconnected sheets and tubules. To achieve a more comprehensive view of ER organization in interphase and mitotic cells and to address a discrepancy in the field (i.e., whether ER sheets persist, or are transformed to tubules, during mitosis), we analyzed the ER in four different mammalian cell lines using live-cell imaging, high-resolution electron microscopy, and three dimensional electron microscopy. In interphase cells, we found great variation in network organization and sheet structures among different cell lines. In mitotic cells, we show that the ER undergoes both spatial reorganization and structural transformation of sheets toward more fenestrated and tubular forms. However, the extent of spatial reorganization and sheet-to-tubule transformation varies among cell lines. Fenestration and tubulation of the ER correlates with a reduced number of membrane-bound ribosomes.