Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

The kinetics of isthmal cells in mouse antrum were examined in three ways: the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; the duration of interphase and mitotic phases was determined from how frequently they occurred; and mice were killed at various intervals after an intravenous injection of 3H-thymidine to time the acquisition of label by the various phases of mitosis. The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G1 and G2 stages were 6.8 and 1.0 hr, respectively. From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurrence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr. Ten minutes after an intravenous injection of 3H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase. We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G2. Also, nuclei are in telophase during the early half of G1 but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G1 into early and late periods is practical.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. II. Duration of mitotic phases and cycle stages, and their relation to one another

Abstract. The kinetics of isthmal cells in mouse antrum were examined in three ways: (a) the duration of cell cycle and DNA-synthesizing (S) stage was measured by the 'fraction of labelled mitoses' method; (b) the duration of interphase and mitotic phases was determined from how frequently they occurred; and (c) mice were killed at various intervals after an intravenous injection of ³H-thymidine to time the acquisition of label by the various phases of mitosis.
The duration of the isthmal cell cycle was found to be 13.8 hr and that of the DNA-synthesizing (S) stage, 5.8 h. Estimates for the duration of the G₁ and G₂ stages were 6.8 and 1.0 hr, respectively.
From the frequency of mitotic phases, defined as indicated in the preceding article (El-Alfy & Leblond, 1987) and corrected for the probability of their occurence, it was estimated that prophase lasted 4.8 hr; metaphase, 0.2 hr; anaphase, 0.06 hr and telophase, 3.3 hr, while the interphase lasted 5.4 hr. In accordance with this, the duration of the whole mitotic process was 8.4 hr.
Ten minutes after an intravenous injection of ³H-thymidine, 38% of labelled isthmal cells were in interphase and 62% in early or mid prophase, while cells in late prophase and other mitotic phases were unlabelled. After 60 min, label was in late prophase, after 120 min, in mid telophase and after 180 min, in late telophase.
We conclude that there is overlap between some mitotic phases and cycle stages. Thus, while nuclei are at interphase during the early third of S, they are in prophase during the late two-thirds as well as during G₂. Also, nuclei are in telophase during the early half of G₁ but at interphase during the late half. Differences in nuclear diameter show that subdivision of both S and G₁ into early and late periods is practical.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. I. Identification of early and late steps of mitosis

In this series of two articles, the duration of mitosis and that of the cell cycle were examined in a group of proliferating cells located in the mouse pyloric antrum and known as isthmal cells. However, before measuring the duration of mitosis, as described in the second article, it is necessary to identify the early and late steps of the mitotic process. This is attempted in the present article, in which the four phases of mitosis and the interphase are described in semithin (0.5 micron thick) Epon serial sections stained with hemalun. The frequency of these phases is then estimated. The beginning of prophase is indicated by the appearance in the nucleus of numerous 0.2-0.3 micron thick basophilic threads. The threads gradually increase in thickness to become the typical chromosomes (about 0.7-micron thick) observed at the end of prophase. Metaphase and anaphase show no remarkable features. At telophase, chromosomes separate from one another, gradually acquire pale segments along their length eventually to look like rows of alternating dark and light patches, and finally vanish. When prophases and telophases are defined in this manner, the enumeration of isthmal cells yields a high proportion of prophases (28%) and telophases (31%), but a low proportion of metaphases (1%) and anaphases (0.3%). Forty per cent of the cells are in interphase.     

An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA-synthesizing (S) stage of the cycle

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.     

Long duration of mitosis and consequences for the cell cycle concept, as seen in the isthmal cells of the mouse pyloric antrum. I. Identification of early and late steps of mitosis

Abstract. In this series of two articles, the duration of mitosis and that of the cell cycle were examined in a group of proliferating cells located in the mouse pyloric antrum and known as isthmal cells. However, before measuring the duration of mitosis, as described in the second article, it is necessary to identify the early and late steps of the mitotic process. This is attempted in the present article, in which the four phases of mitosis and the interphase are described in semithin (0.5 μ m thick) Epon serial sections stained with hemalun. The frequency of these phases is then estimated.
The beginning of prophase is indicated by the appearance in the nucleus of numerous 0.2-0.3 μ m thick basophilic threads. The threads gradually increase in thickness to become the typical chromosomes (about 0.7- μ m thick) observed at the end of prophase. Metaphase and anaphase show no remarkable features. At telophase, chromosomes separate from one another, gradually acquire pale segments along their length eventually to look like rows of alternating dark and light patches, and finally vanish.
When prophases and telophases are defined in this manner, the enumeration of isthmal cells yields a high proportion of prophases (28%) and telophases (31%), but a low proportion of metaphases (1%) and anaphases (0.3%). Forty per cent of the cells are in interphase.     

Timing the phases of the microtubule cycles involved in cytoplasmic and nuclear divisions in cells of undisturbed onion root meristems

The duration of the different phases of the microtubule and chromosome cycles were estimated in the native diploid cell populations of Allium cepa L root meristems proliferating undisturbed, under steady state conditions, at the physiological temperature of 15°C. The cycles were coupled by considering their fitting in relation to the short process of nuclear envelope breakdown. In the cycle related to cytoplasmic division, the preprophase band which predicts the future position of the phragmoplast made its appearance, as a wide band, 16 mm before the G₂ to prophase transition, ie it was only present during the final 5% of the total G₂ timing (5 h 30 mm). The band became narrow only 6 mm after prophase had started and it was present in this form for the remaining prophase time (2 h 24 mm). Its disappearance occurred strictly coinciding with nuclear envelope breakdown, at the end of prophase. No microtubules related to cytoplasmic division were apparent until 9 mm after telophase had initiated. The two initial stages of phragmoplast formation which followed occupied, respectively, 27 mm and 54.5 mm of the 2-h long telophase. On the other hand, the third and last stage in phragmoplast formation covered both the final 35 mm of mitosis and the 6 initial mm of the G₁ of the next interphase. A very short (less than 4 mm) stage of microtubular nucleation around the nuclear envelope took place immediately afterwards, before the cortical array of microtubules appeared. The microtubule cycle related to nuclear division started with the apparent activation of the future spindle poles 7.4 mm before prophase was over. The mitotic spindle developed in the 5.6 mm long prometaphase. The spindle functioned in metaphase for the 42 mm it lasted, half spindles being separated for the 37 mm anaphase occupied in these cells.     

Nek2 localizes to multiple sites in mitotic cells, suggesting its involvement in multiple cellular functions during the cell cycle.

Nek2 is a mammalian protein kinase that is structurally homologous to NIMA, a mitotic regulator in Aspergillus nidulans. To understand the possible cellular processes in which Nek2 participates during the cell cycle, we investigated the expression and subcellular localization of Nek2 in mitotic cells. The Nek2 protein levels were observed to be regulated in a cell cycle stage-specific manner in cultured cells. The cell cycle stage specificity of Nek2 expression was also confirmed in cells undergoing mitosis in vivo. Nek2 proteins were localized in both the nucleus and cytoplasm throughout the cell cycle, but exhibited dynamic changes in distribution, depending on the cell cycle stage. Nek2 was associated with chromosomes from prophase to metaphase and then was dissociated upon entering into anaphase. Nek2 then appeared at the midbody of the cytoplasmic bridge at telophase. Nek2 was also associated with the centrosome throughout the cell cycle as observed previously by others. Additionally, the nuclear localization of Nek2 was increased during S phase. Such dynamic behavior of Nek2 suggests that Nek2 may be a mitotic regulator that is involved in diverse cell cycle events.     

Duration of the Mitotic Cycle in Cell Cultures of Haplopappus gracilis

The duration of the cell cycle and its component phases in cell cultures of Haplopappus gracilis was estimated by means of pulse labelling with tritiated thymidine and subsequent autoradiographic techniques. The total duration of the mitotic cycle was found to be 22.0 hours. The average durations of the following component phases were: the synthetic period (S) 6.4 hours, the postsynthetic period (G₂) 4.86 hours, prophase (P) 0.64 hours, metaphase (M) 0.40 hours, anaphase + early telophase (AT) 0.36 hours, the presynthetic period (G₁) 9.34 hours. The results indicate that G₁ and G₂ are the phases, which are most prolonged in populations of cultivated cells when compared to the same phases in root lip cells from the same species.     

Histone deacetylation is required for progression through mitosis in tobacco cells

Post-translational modifications of core histone proteins play a key role in chromatin structure and function. Here, we study histone post-translational modifications during reentry of protoplasts derived from tobacco mesophyll cells into the cell cycle and evaluate their significance for progression through mitosis. Methylation of histone H3 at lysine residues 4 and 9 persisted in chromosomes during all phases of the cell cycle. However, acetylation of H4 and H3 was dramatically reduced during mitosis in a stage-specific manner; while deacetylation of histone H4 commenced at prophase and persisted up to telophase, histone H3 remained acetylated up to metaphase but was deacetylated at anaphase and telophase. Phosphorylation of histone H3 at serine 10 was initiated at prophase, concomitantly with deacetylation of histone H4, and persisted up to telophase. Preventing histone deacetylation by the histone deacetylase inhibitor trichostatin A (TSA) led to accumulation of protoplasts at metaphase-anaphase, and reduced S10 phosphorylation during anaphase and telophase; in cultured tobacco cells, TSA significantly reduced the frequency of mitotic figures. Our results indicate that deacetylation of histone H4 and H3 in tobacco protoplasts occurs during mitosis in a phase-specific manner, and is important for progression through mitosis.     

Early replicating DNA in heterochromatin ofPlagiochila ovalifolia (Liverworts)

The timing of DNA replication of heterochromatin in malePlagiochila ovalifolia was investigated by the use of³H-thymidine autoradiography. The estimated duration of the mitotic cycle was as follows: S period, 19 hr: G₂+prophase, 10 hr; G₁+meta-, ana-, telophase, 5 hr; total mitotic cycle, 34 hr. The first appearance of silver grains over the chromosomes was observed at 8 hr after the beginning of pulse labelling at which time the silver grains were only over the euchromatic regions, not over the heterochromatic regions. This labelling pattern was also observed at 10 to 15 hr. The heterochromatic regions having more grains than the euchromatic regions were observed at 20 to 25 hr. These results show that the DNA of the heterochromatin of this species is replicated earlier than the euchromatin.     

Construction of three quadruple-fluorescent MDA435 cell lines that enable monitoring of the whole chromosome segregation process in the living state

Mitotic events from prophase to telophase are defined by morphology or movement of chromatin, nuclear envelope, centrosomes and spindles. Live-cell imaging is useful for characterizing the whole chromosome segregation process in the living state. In this study, we constructed three quadruple-fluorescent MDA435 cell lines in which chromatin, kinetochores, nuclear envelope and either inner centromere, microtubules or centrosomes/spindles were differentially visualized with cyan, green, orange and red fluorescent proteins (ECFP, EGFP, mKO and DsRed). Each mitotic stage of the individual cells could be identified by capturing live-cell images without the requirement of fixing or staining steps. In addition, we obtained four-color time-lapse images of one cell line, MDA-Auro/imp/H3/AF, from prophase to metaphase and from early anaphase to telophase. These quadruple-fluorescent cell lines will be useful for precisely analyzing the mitotic events from prophase through to telophase in single cells in the future.     

Entamoeba histolytica: cell cycle and nuclear division

The cell cycle of Entamoeba histolytica, the duration of its phases, and the details of the nuclear division stages are described in this paper. Trophozoites from clone L-6, strain HM1:IMSS, were synchronized by colchicine. Synchrony was observed immediately after treatment and cultures remained synchronous for at least three replicative cycles with synchrony indexes between 13 and 15 hr. The stages of nuclear division were studied by light and electron microscopy. Four stages of the nuclear division were defined: prophase, early anaphase, late anaphase, and telophase. No metaphase stage was observed by light or electron microscopy. One of the first events in the nuclear division was the presence of a bud close to the juxtanuclear body, which grew to a daughter nucleus. The karyosome and the nuclear membrane remained throughout the mitotic process. Bundles of intranuclear microtubules were observed forming a "V" from the center of the nucleus to one of the poles, and associated with them, 12 to 16 chromosomes-like structures appeared. The results of these studies strongly suggest that division of E. histolytica involved a pleuromitotic process which is carried out in about 120 min.     

Cell cycle dependent distribution of the proliferation-associated Ki-67 antigen in human embryonic lung cells

The cell cycle-dependent distribution of the proliferation-associated Ki-67 antigen has been evaluated immunocytochemically in L-132 human fetal lung cells. The cells were synchronized and cell cycle phases were determined: G1 = 6.7 h, S = 5.4 h, G2 = 8.5 h and mitosis = 1.3 h. The Ki-67 patterns were strictly correlated with the cell cycle phases. In late G1-phase, Ki-67 antigen was present only in the perinucleolar region. In the S-phase, Ki-67 staining was found homogeneously in the karyoplasm and in the perinucleolar region. G2-phase cells contained a finely granular Ki-67 staining in the karyoplasm with Ki-67-positive specks and perinucleolar staining. In early mitotic cells (pro- and metaphase) an intense perichromosomal Ki-67 staining was observed in addition to a homogeneously stained karyoplasm in prophase, and cytoplasm in metaphase. During ana- and telophase the Ki-67 antigen disappeared rapidly. In resting cells there was no Ki-67 staining.     

THE EFFECT OF ACUTE RENAL FAILURE ON MITOTIC DURATION OF MOUSE ILEAL EPITHELIUM

Previous percentage labelled mitoses studies in acutely uraemic mice have demonstrated a lengthening of the cell cycle and the DNA synthetic phase of ileal epithelium. The mitotic index was unaltered. Further studies have been performed to obtain an estimate of mitotic duration. Acute renal failure was produced by urinary outflow obstruction in male mice. Controls were subjected to sham operation. The mean number of cells per crypt cell column, number of mitoses present per crypt section and differential mitotic stage count were assessed 18 hr after operation for uraemic and control mice. The mean number of metaphases accumulated per crypt section over a 2 hr interval following colchicine injection was obtained in other groups of mice and the mitotic duration calculated. The mean number of mitoses per crypt section was 1.30 ± 0.46 for the controls and 1.48 ± 0.66 for the uraemic group. No evidence for a block in mitosis was indicated by the differential mitotic stage count. After applying Tannock's correction factor the mitotic duration was estimated to be 0.91 ± 0.18 hr for the control group and 2.81 ± 0.89 hr for the uraemic group. The difference in duration between the groups, 1.90 ± 0.91 hr, was significant (P≤0.05). Reduction in cell proliferation may explain the development of uraemic lesions in the gastrointestinal tract.     

Trichomonas vaginalis: chromatin and mitotic spindle during mitosis

The mitotic phases and the changes that the chromatin and mitotic microtubules undergo during mitosis in the sexually transmitted parasite Trichomonas vaginalis are described. Parasites arrested in the gap 2 phase of the cell cycle by nutrient starvation were induced to mitosis by addition of fresh whole medium. [(3)H] Thymidine labeling of trichomonad parasites for 24 h showed that parasites have at least four synchronic duplications after mitosis induction. Fixed or live and acridine orange (AO)-stained trichomonads analyzed at different times during mitosis by epifluorescence microscopy showed that mitosis took about 45 min and is divided into five stages: prophase, metaphase, early and late anaphase, early and late telophase, and cytokinesis. The AO-stained nucleus of live trichomonads showed green (DNA) and orange (RNA) fluorescence, and the nucleic acid nature was confirmed by DNase and RNase treatment, respectively. The chromatin appeared partially condensed during interphase. At metaphase, it appeared as six condensed chromosomes, as recently reported, which decondensed at anaphase and migrated to the nuclear poles at telophase. In addition, small bundles of microtubules (as hemispindles) were detected only in metaphase with the polyclonal antibody anti-Entamoeba histolytica alpha-tubulin. This antibody showed that the hemispindle and an atractophore-like structure seem to duplicate and polarize during metaphase. In conclusion, T. vaginalis mitosis involves five mitotic phases in which the chromatin undergoes different degrees of condensation, from chromosomes to decondensed chromatin, and two hemispindles that are observed only in the metaphase stage.     

Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine.

Dividing epithelial cells in the mouse small intestine were examined by thin-section electron microscopy with special attention given to the mode of cytokinesis. As the columnar epithelial cells entered mitosis in the crypt, they became rounded, maintaining their junctional complexes with neighboring cells while detaching themselves from the basal lamina. In such rounded cells the mitotic apparatus was formed with its long axis parallel to the luminal surface. Replicated centrioles moved down from the apical region to locate themselves lateral to the nucleus, where they served as the poles of the mitotic spindle. During mitosis the cell retained microvilli on its luminal surface, though the terminal web became much thinner. At telophase the formation of a cleavage furrow proceeded asymmetrically from the basal side alone, and thus the contractile ring which was prominent at the base of the furrow, merged with the terminal web. Eventually, an intercellular bridge with a midbody was formed on the luminal surface. The space in the furrow was occupied by the flattened cytoplasmic processes of the neighboring cells. The tight junction was also seen on the basolateral surface of the intercellular bridge with the underlying neighboring cells. At very late telophase the intercellular bridge was disconnected from the neighboring cells and protruded into the lumen. These observations have led us to propose a mode by which the simple columnar epithelium maintain the tight junctional seal during cell division in the crypt of the small intestinal epithelium.     

Orientation of spindle axis and distribution of plasma membrane proteins during cell division in polarized MDCKII cells

MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114- kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically. We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.     

Nuclear and microtubular cycles in heterophasic multinuclearTriticum root-tip cells induced by caffeine

Summary Nuclear and microtubular cycles were studied in large heterophasic multinuclear cells induced in root tips ofTriticum turgidum by caffeine treatment. Multinuclear cells and cells with polyploid nuclei exhibited various configurations of multiple and complex preprophase microtubule (Mt) bands (PPBs), including helical ones. The developmental stages of PPBs in some heterophasic cells did not comply with the cell cycle stages of the associated nuclei, a fact indicating that these events are not directly controlled by the associated nuclei. The heterophasic cells exhibited asynchronous nuclei at different stages of mitosis. In cells displaying prophase and interphase nuclei, the prophase spindle was either absent or developed around both of them or developed around the prophase nuclei earlier than around the interphase ones. During prometaphase-metaphase of the advanced nuclei the lagging interphase nuclei were induced to form prematurely condensed chromosomes (PCCs) along with spindle formation around them. These observations suggest that the mitotic transition in heterophasic cells is delayed but is ultimately achieved due to the effect of the advanced nuclei, which induces a premature mitotic entry of the lagging nuclei. Although kinetochore Mt bundles were found associated with PCCs, their metaphase and anaphase spindles were abnormal resulting in abnormal or abortive anaphases. In some heterophasic cells, metaphase-anaphase transition did not take place simultaneously in different chromosome groups, signifying that the cells do not exit from the mitotic state after anaphase initiation of the advanced nuclei. Asynchronous pace of mitosis of different chromosome groups was also observed during anaphase and telophase. Implications of these observations in understanding plant cell cycle regulation are discussed.Abbreviations cdk cyclin dependent kinase - Mt microtubule - PCC prematurely condensed chromosome - PPB preprophase band     

Enrichment of cell populations in metaphase, anaphase, and telophase by synchronization using nocodazole and blebbistatin: a novel method suitable for examining dynamic changes in proteins during mitotic progression

Mitosis is a continuous process to separate replicated chromosomes into two daughter cells through prophase, metaphase, anaphase, and telophase. Although a number of methods have been established to synchronize cells at different phases of the cell cycle, it is difficult to synchronize cells at the specific phases, anaphase and telophase, during mitosis because of the short duration of anaphase. Here, we show that HeLa S3 cells in anaphase and in telophase are successfully enriched by treatment with a combination of low concentrations of the microtubule-depolymerizing agent nocodazole and the myosin II inhibitor blebbistatin. After 9-h release from thymidine block at G1/S phase, addition of nocodazole at 20 ng/ml but not 40 ng/ml ensures rapid release from the nocodazole arrest. Subsequently, the cells are cultured in the presence of 50 μM blebbistatin for 20 and 50 min to enrich cells in anaphase and telophase, respectively. Western blot analysis verifies down-regulation of phospho-histone H3-Ser10, phospho-Aurora A/B/C, and cyclin B1 during M-phase progression. Furthermore, we show how the electrophoretic mobility shifts of the Src-family kinases c-Yes and c-Src can change in each phase of mitosis. These results provide a useful synchronization method for biochemically examining protein dynamics during M-phase progression.     

Expression of the calcium binding protein calretinin in WiDr cells and its correlation to their cell cycle

Ca2+ ions intervene during different phases of the progression of the cell cycle, but only one calcium-binding protein, calmodulin, has been shown to be associated with dividing cells. We therefore screened cancer cells for the presence of other related calcium-binding proteins. Using molecular biological and immunohistochemical techniques we show that human tumor cells of epithelial origin, express calretinin. Calretinin immunoreactivity can be demonstrated at precise moments of the cell cycle and, in particular, in phase G1 and during mitosis. During mitosis calretinin is localized both in the cytoplasm and in the mitotic spindle. In the cytoplasm we find calretinin after prophase and until telophase. In the spindle apparatus, calretinin is already present in cells in prometaphase and persists in all the succeeding mitotic phases. It is associated with the kinetochore microtubules but, in contrast to calmodulin, also with the polar microtubules. The role that calretinin plays in well-defined moments of the cell cycle of these cells is as yet unknown, but our results strongly suggest that, in collaboration with other molecules, calretinin intervenes in the dynamic phenomena regulating the separation of the chromosomes.