THE CYTONUCLEOPROTEINS OF AMEBAE : II. Some Aspects of Cytonucleoprotein Behavior and Synthesis

Radioactivity, apparently in cytonucleoproteins, from an amino acid-labeled nucleus implanted into a non-radioactive cell appeared in the host nucleus within 10 minutes, and the typical equilibrium ratio 70:30 donor nucleus radioactivity:host nucleus radioactivity was reached in 4 to 5 hours at 25°C. If such binucleates grew and divided, no localization of radioactivity was observable in cells fixed during mitosis, but the protein label remained concentrated in the daughter interphase nuclei for at least 4 generations. Continued migration of cytonucleoproteins was observed if these daughter nuclei were transplanted to other unlabeled cells. The Q₁₀ (19° to 29°C) of the migration rate of radioactive cytonucleoproteins was ca. 1.3, suggesting that passage through the cytoplasm occurred by diffusion. Both non-migratory nuclear proteins and cytonucleoproteins appear to be synthesized in the cytoplasm.     

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine     

THE EFFECT OF LIGHT ON THE PERMEABILITY OF PARAMECIUM

1. The permeability of Paramecium to NH₄OH is greater when the cells are exposed to light than it is when they are in darkness. 2. This change can be demonstrated in cells exposed to monochromatic red light, though it is small. It becomes greater as the wave lengths shorten, and is greatest in the near ultra-violet. 3. The permeability increases as the duration of exposure to light is prolonged. 4. These experiments demonstrate the necessity of controlling the illumination when using Paramecium in physiological tests.     

ULTRASTRUCTURE OF THE NUCLEOLUS DURING THE CHINESE HAMSTER CELL CYCLE

Changes in the structure of the nucleolus during the cell cycle of the Chinese hamster cell in vitro were studied. Quantitative electron microscopic techniques were used to establish the size and volume changes in nucleolar structures. In mitosis, nucleolar remnants, "persistent nucleoli," consisting predominantly of ribosome-like granular material, and a granular coating on the chromosomes were observed. Persistent nucleoli were also observed in some daughter nuclei as they were leaving telophase and entering G₁. During very early G₁, a dense, fibrous material characteristic of interphase nucleoli was noted in the nucleoplasm of the cells. As the cells progressed through G₁, a granular component appeared which was intimately associated with the fibrous material. By the middle of G₁, complete, mature nucleoli were present. The nucleolar volume enlarged by a factor of two from the beginning of G₁ to the middle of S primarily due to the accumulation of the granular component. During the G₂ period, there was a dissolution or breakdown of the nucleolus prior to the entry of the cells into mitosis. Correlations between the quantitative aspects of this study and biochemical and cytochemical data available in the literature suggest the following: nucleolar reformation following division results from the activation of the nucleolar organizer regions which transcribe for RNA first appearing in association with protein as a fibrous component (45S RNA) and then later as a granular component (28S and 32S RNA).     

Proteins in nucleocytoplasmic interactions. 3. Redistributions of nuclear proteins during and following mitosis in Amoeba proteus

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus.     

REPOPULATION OF THE POSTMITOTIC NUCLEOLUS BY PREFORMED RNA

This study is concerned with the fate of the nucleolar contents, particularly nucleolar RNA, during mitosis Mitotic cells harvested from monolayer cultures of Chinese hamster embryonal cells, KB6 (human) cells, or L929 (mouse) cells were allowed to proceed into interphase in the presence or absence (control) of 0.04–0 08 µg/ml of actinomycin D, a concentration which preferentially inhibits nucleolar (ribosomal) RNA synthesis 3 hr after mitosis, control cells had large, irregularly shaped nucleoli which stained intensely for RNA with azure B and for protein with fast green. In cells which had returned to interphase in the presence of actinomycin D, nucleoli were segregated into two components easily resolvable in the light microscope, and one of these components stained intensely for RNA with azure B. Both nucleolar components stained for protein with fast green In parallel experiments, cultures were incubated with 0.04–0 08 µg/ml actinomycin D for 3 hr before harvesting of mitotic cells, then mitotic cells were washed and allowed to return to interphase in the absence of actinomycin D. 3 hr after mitosis, nuclei of such cells were devoid of large RNA-containing structures, though small, refractile nucleolus-like bodies were observed by phase-contrast microscopy or in material stained for total protein. These experiments indicate that nucleolar RNA made several hours before mitosis persists in the mitotic cell and repopulates nucleoli when they reform after mitosis     

Polarised asymmetric inheritance of accumulated protein damage in higher eukaryotes

Disease-associated misfolded proteins or proteins damaged due to cellular stress are generally disposed via the cellular protein quality-control system. However, under saturating conditions, misfolded proteins will aggregate. In higher eukaryotes, these aggregates can be transported to accumulate in aggresomes at the microtubule organizing center. The fate of cells that contain aggresomes is currently unknown. Here we report that cells that have formed aggresomes can undergo normal mitosis. As a result, the aggregated proteins are asymmetrically distributed to one of the daughter cells, leaving the other daughter free of accumulated protein damage. Using both epithelial crypts of the small intestine of patients with a protein folding disease and Drosophila melanogaster neural precursor cells as models, we found that the inheritance of protein aggregates during mitosis occurs with a fixed polarity indicative of a mechanism to preserve the long-lived progeny.     

MCPH1 patient cells exhibit delayed release from DNA damage-induced G2/M checkpoint arrest

Mutations in the MCPH1 gene cause primary microcephaly associated with a unique cellular phenotype of misregulated chromosome condensation. The encoded protein contains three BRCT domains, and accumulating data show that MCPH1 is involved in the DNA damage response. However, most of this evidence has been generated by experiments using RNA interference (RNAi) and cells from non-human model organisms. Here, we demonstrate that patient-derived cell lines display a proficient G₂/M checkpoint following ionizing irradiation (IR) despite homozygous truncating mutations in MCPH1. Moreover, chromosomal breakage rates and the relocation to DNA repair foci of several proteins functioning putatively in an MCPH1-dependent manner are normal in these cells. However, the MCPH1-deficient cells exhibit a slight delay in re-entering mitosis and delayed resolution of γH2AX foci following IR. Analysis of chromosome condensation behavior following IR suggests that these latter observations may be related to hypercondensation of the chromatin in cells with MCPH1 mutations. Our results indicate that the DNA damage response in human cells with truncating MCPH1 mutations differs significantly from the damage responses in cells of certain model organisms and in cells depleted of MCPH1 by RNAi. These subtle effects of human MCPH1 deficiency on the cellular DNA damage response may explain the absence of cancer predisposition in patients with biallelic MCPH1 mutations.Key words: chromosome condensation, DNA damage, G₂/M checkpoint, ionizing radiation, PCC syndrome, primary microcephaly, repair foci     

The first mitosis of the mouse embryo is prolonged by transitional metaphase arrest

The first mitosis of the mouse embryo is almost twice as long as the second. The mechanism of the prolongation of the first mitosis remains unknown, and it is not clear whether prometaphase or metaphase or both are prolonged. Prometaphase is characterized by dynamic chromosome movements and spindle assembly checkpoint activity, which prevents anaphase until establishment of stable kinetochore-microtubule connections. The end of prometaphase is correlated with checkpoint inactivation and disappearance of MAD2L1 (MAD2) and RSN (CLIP-170) proteins from kinetochores. Spindle assembly checkpoint operates during the early mouse mitoses, but it is not clear whether it influences their duration. Here, we determine the length of prometaphases and metaphases during the first two embryonic mitoses by time-lapse video recording of chromosomes and by immunolocalization of MAD2L1 and RSN proteins. We show that the duration of the two prometaphases does not differ and that MAD2L1 and RSN disappear from kinetochores very early during each mitosis. The first metaphase is significantly longer than the second one. Therefore, the prolongation of the first embryonic mitosis is due to a prolonged metaphase, and the spindle assembly checkpoint cannot be involved in this process. We show also that MAD2L1 staining disappears gradually from kinetochores of oocytes arrested at metaphase of the second meiotic division. This shows a striking similarity between the first embryonic mitosis and metaphase arrest in oocytes. We postulate that the first embryonic mitosis is prolonged by a transient metaphase arrest that is independent of the spindle assembly checkpoint and is similar to metaphase II arrest. The molecular mechanism of this transient arrest remains to be elucidated.     

5-AMINOURACIL TREATMENT : A Method for Estimating G2

Treatment of Vicia faba lateral roots with a range of concentrations of 5-aminouracil (5-AU) indicate that cells are stopped at a particular point in interphase. The timing of the fall in mitotic index suggests that cells are held at the S - G₂ transition. When cells are held at this point, treatments with 5-AU can be used to estimate the duration of G₂ + mitosis/2 of proliferating cells. Treatment with 5-AU can also be used to demonstrate the presence of subpopulations of dividing cells that differ in their G₂ duration. Using this method, 5-AU-induced inhibition, we have confirmed that in V. faba lateral roots there are two populations of dividing cells: (a) a fast-dividing population, which makes up ~85% of the proliferating cell population and has a G₂ + mitosis/2 duration of 3.3 hr, and (b) a slow-dividing population, which makes up ~15% of dividing cells and has a G₂ duration in excess of 12 hr. These estimates are similar to those obtained from percentage labeled mitosis (PLM) curves after incorporation of thymidine-³H.     

THE FINE STRUCTURE OF THE NUCLEOLUS IN MITOTIC DIVISIONS OF CHINESE HAMSTER CELLS IN VITRO

The nucleolus of Chinese hamster tissue culture cells (strain Dede) was studied in each stage of mitosis with the electron microscope. Mitotic cells were selectively removed from the cultures with 0.2 per cent trypsin and fixed in either osmium tetroxide or glutaraldehyde followed by osmium tetroxide. The cells were embedded in both prepolymerized methacrylate and Epon 812. Thin sections of interphase nucleoli revealed two consistent components; dense 150-A granules and fine fibrils which measured 50 A or less in diameter. During prophase, distinct zones which were observed in some interphase nucleoli (i.e. nucleolonema and pars amorpha) were lost and the nucleoli were observed to disperse into smaller masses. By late prophase or prometaphase, the nucleoli appeared as loosely wound, predominantly fibrous structures with widely dispersed granules. Such structures persisted throughout mitosis either free in the cytoplasm or associated with the chromosomes. At telophase, those nucleolar bodies associated with the chromosomes became included in the daughter nuclei, resumed their compact granular appearance, and reorganized into an interphase-type structure.     

Immobility,inheritance and plasticity of shape of the yeast nucleus

Background  

Since S. cerevisiae undergoes closed mitosis, the nuclear envelope of the daughter nucleus is continuous with that of the maternal nucleus at anaphase. Nevertheless, several constitutents of the maternal nucleus are not present in the daughter nucleus. The present study aims to identify proteins which impact the shape of the yeast nucleus and to learn whether modifications of shape are passed on to the next mitotic generation. The Esc1p protein of S. cerevisiae localizes to the periphery of the nucleoplasm, can anchor chromatin, and has been implicated in targeted silencing both at telomeres and at HMR.     

THE EFFECT OF VINCRISTINE ON MOUSE JEJUNAL CRYPT CELLS OF DIFFERING CELL AGE: DOUBLE LABELLING AUTORADIOGRAPHIC STUDIES USING 3H- AND 14C-TdR

The mechanism of action of the alkaloid vincristine (VCR) has been investigated in vitro on HeLa cells in culture and in vivo on jejunal crypt cells of the mouse. The in vitro experiments with HeLa cells show that VCR affects not only mitotic but also interphase cells. The VCR-affected cells first continue their passage through the cell cycle undisturbed but after reaching mitosis they are arrested in metaphase. This agrees well with the results obtained by Madoc-Jones & Mauro (1968) and Madoc-Jones (1973) on synchronized cell cultures. Until now there has been no investigation of the mechanism of action of VCR in vivo. This is due to the absence of a suitable technique for synchronization in vivo. The present study is based on a method which permits the assessment of the VCR sensitivity as a function of the cell age without synchronization in the usual sense. The jejunal crypt epithelium of the normal mouse was double labelled with ³H- and ¹⁴C-thymidine (TdR) in such a way as to produce a narrow subpopulation of crypt cells with a maximum age difference of 1 hr. On autoradiographs these cells can be distinguished by their characteristic labelling from other cells. As this ‘pseudo’-synchronized subpopulation passes through the cycle the effect of VCR can be studied, i.e. one can analyse the effect in well-defined time intervals of the cycle. The results show that the effect of VCR is the same in vivo as in vitro. The crypt cells which are affected by VCR in interphase continue their passage through the cycle, but upon entering mitosis they are arrested in metaphase. VCR has, at the concentration used in the present study, no effect on the duration of the S and G₂ phases. The necrotic cells seen after VCR application are formed from arrested metaphases.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

EFFECT OF CYCLOHEXIMIDE ON THE CELL CYCLE OF THE CRYPTS OF THE SMALL INTESTINE OF THE RAT

A single injection of 1.5 mg/kg of cycloheximide induces a complete disappearance of mitotic activity in rat intestinal crypts within 1.5–2 hr. No significant necrosis of crypt cells is observed even though this phenomenon is accompanied by a marked decrease in uptake of labeled precursors into protein and DNA. Mitoses reappear 6 hr after injection and recovery then follows a cyclic pattern over a period equivalent to one cell cycle, thereby reflecting at least a partial synchronization of cell division. Concurrent use of colchicine, an agent known to induce metaphase arrest, has demonstrated that cycloheximide, while having no apparent effect on cells already in division, prevents the entrance of new cells into visible mitosis. Analysis of the cell cycle suggests that one block initiated by cycloheximide occurs in G₂, presumably as the result of an interference with the formation of protein(s) required for the normal progression of cells from this phase of the cycle into mitosis.     

AN IN VIVO DOUBLE LABELLING STUDY OF THE SUBSEQUENT FATE OF CELLS ARRESTED IN METAPHASE BY VINCRISTINE IN THE JB-1 MOUSE ASCITES TUMOUR

The fate of cells arrested by Vincristine (VCR) in metaphase is of interest because of the wide use of this substance in cancer chemotherapy and, particularly, in relation to its use in so-called ‘synchronization’ therapy. The present study was designed to answer the question of whether cells blocked in metaphase by VCR subsequently proliferate further or whether they become infertile and die. By means of a double labelling technique with [³H] and [¹⁴C]thymidine (TdR) it was shown that all VCR-arrested metaphases in the JB-1 ascites tumour subsequently became necrotic. These cells did not re-enter a viable G₂ phase following arrest and thus could not take part in a wave of synchronous proliferation. In agreement with earlier studies, VCR was found to lead to arrest in metaphase, not only of cells in or shortly prior to mitosis at the time of VCR administration, but also of the majority of cells which had at this time been in the S and G₂ phase.     

THE EFFECTS OF CARBON MONOXIDE INHIBITION ON ATP LEVEL AND THE RATE OF MITOSIS IN THE SEA URCHIN EGG

The requirements for ATP synthesis during the various phases of mitosis were investigated in the oxygen-requiring eggs of the sea urchin, Strongylocentrotus purpuratus. CO in the dark, a specific inhibitor of respiration, was used to inhibit ATP synthesis. The kinetics of respiratory inhibition were determined by analyzing ATP levels with the luciferin-luciferase assay. The kinetics of mitotic inhibition were determined by analysis of the rate of mitosis. It was found that CO inhibition resulted in a decrease in the normal ATP level. Coincident with this decrease was a decrease in the rate of mitosis which stops completely when the ATP drops below 50 per cent of the normal level. With the use of various degrees of CO inhibition, the rate of mitosis is shown to be related to the resultant ATP level. These results contradict the basic premise of the energy reservoir hypothesis, and also disagree with other reports that cells in mitosis are insensitive to inhibitors of energy metabolism. Data are presented which demonstrate that these conflicting reports result from insufficient inhibition of ATP synthesis. The above findings all indicate that mitosis depends on the continuous synthesis and utilization of ATP.     

THE RELATIONSHIP BETWEEN DIURNAL VARIATION OF THE NUMBER OF CELLS IN MITOSIS AND OF THE NUMBER OF CELLS SYNTHESIZING DNA IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH

1. The numbers of cells in mitosis and in DNA synthesis in the epithelium of the hamster cheek pouch have been studied at different times of the day and night. 2. By accumulation of mitotic cells using colcemid, both the rate of entry of cells into mitosis and the duration of mitosis have been estimated at two different times of day. 3. A diurnal variation has been demonstrated in both the mitotic index and in the tritiated thymidine labelling index. Although these variations are of different amplitude and timing, the experimental data fit closely to the hypothesis that the diurnal mitotic variation is the result of a partially synchronous population moving through the DNA synthetic period. No direct action on the mitotic process need be postulated. 4. From the results of mitotic accumulation, it is clear that the rate of entry of cells into mitosis depends on the time of day at which this is studied. There is also the possibility that the duration of mitosis is slightly longer when the mitotic index is high. 5. It is concluded that, at least in the epithelium of the hamster cheek pouch, the diurnal rhythm in the number of mitoses present is a reflection of the diurnal variation in the number of cells synthesizing DNA at some time earlier. Small fluctuations in the mitotic pattern imposed by this partially synchronous population moving from S into mitosis, could be caused by slight variations in the duration of mitosis.