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     

NuSAP,a novel microtubule-associated protein involved in mitotic spindle organization

Here, we report on the identification of nucleolar spindle-associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.     

Aurora-B regulates RNA methyltransferase NSUN2

Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G(1) phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis.     

Cell division and nucleolar ribonucleic acid synthesis in synchronous cultures

Summary Nucleolar RNA synthesis is inhibited and cell division delayed in synchronous cultures of mouse fibroblasts (strain L-929) treated with actinomycin D (0.04 μg per ml). The gradual loss of actinomycin D from the cells during a 2-hr period following incubation is accompanied by an increase in the rate of nucleolar RNA synthesis to the control level. Following this the rate of protein synthesis is decreased by 25% for approximately 9 hr. The length of time that nucleolar RNA and protein synthesis are inhibited accounts for the delay in mitosis 1 1/2 cell cycles later. These data support the contention that certain proteins produced during one interphase are prerequisite for division in a subsequent cycle.     

Cell division and nucleolar ribonucleic acid synthesis in synchronous cultures

Summary Nucleolar RNA synthesis is inhibited and cell division delayed in synchronous cultures of mouse fibroblasts (strain L-929) treated with actinomycin D(0.04 μ per ml). The gradual loss of actinomycin D from the cells during a 2-hr period following incubation is accompanied by an increased in the rate of nucleolar RNA synthesis to the control level. Following this the rate of protein synthesis is decreased by 25% for approximately 9 hr. The length of time that nucleolar RNA and protein synthesis are inhibited accounts for the delay in mitosis 1 1/2 cell cycles later. These data support the contention that certain proteins produced during one interphase are prerequisite for division in a subsequent cycle.     

Nucleologenesis: use of non-isotopic in situ hybridization and immunocytochemistry to compare the localization of rDNA and nucleolar proteins during mitosis

Using in situ hybridization and immunocytochemistry during interphase and mitosis, we have compared the distribution of ribosomal DNA (rDNA) to that of the nucleolar proteins fibrillarin and RNA polymerase I. During interphase, nucleolar proteins were localized at sites throughout the nucleolus while the bulk of rDNA was localized in a single restricted nucleolar area. During metaphase and anaphase, all six NORs were detected by in situ hybridization, Ag-staining, or by the immunolocalization of RNA polymerase I. During telophase, rDNA and RNA polymerase I were found in a distinct subset of the prenucleolar bodies (PNBs) which obviously must contain the nucleolar organizers. Other numerous PNBs are smaller in size and do not contain detectable amounts of rDNA or RNA polymerase I. Therefore, reconstruction of the nucleolus originates in telophase-specific domains which contain both rDNA and RNA polymerase I.     

A nucleolar protein RRS1 contributes to chromosome congression

We report here the functional analysis of human Regulator of Ribosome Synthesis 1 (RRS1) protein during mitosis. We demonstrate that RRS1 localizes in the nucleolus during interphase and is distributed at the chromosome periphery during mitosis. RNA interference experiments revealed that RRS1-depleted cells show abnormalities in chromosome alignment and spindle organization, which result in mitotic delay. RRS1 knockdown also perturbs the centromeric localization of Shugoshin 1 and results in premature separation of sister chromatids. Our results suggest that a nucleolar protein RRS1 contributes to chromosome congression.     

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).     

PROTEIN SYNTHESIS AND RNA SYNTHESIS DURING MITOSIS IN ANIMAL CELLS

Protein synthesis and RNA synthesis during mitosis were studied by autoradiography on mammalian tissue culture cells. Protein synthesis was followed by incubating hamster epithelial and human amnion cells for 10 or 15 minutes with phenylalanine-C¹⁴. To study RNA synthesis the hamster cells were incubated for 10 minutes with uridine-C¹⁴. Comparisons of the synthetic capacity of the interphase and mitotic cells were then made using whole cell grain counts. The rate of RNA synthesis decreased during prophase and reached a low of 13 to 16 per cent of the average interphase rate during metaphase-anaphase. Protein synthesis in the hamster cells showed a 42 per cent increase during prophase with a subsequent return to the average interphase value during metaphase-anaphase. The human amnion cells showed no significant change at prophase but there was a 52 to 56 per cent drop in phenylalanine incorporation at metaphase-anaphase as compared to the average interphase rate. Colcemide was used on the hamster cells to study the effect of a prolonged mitotic condition on protein and RNA synthesis. Under this condition, uridine incorporation was extremely low whereas phenylalanine incorporation was still relatively high. The drastic reduction of RNA synthesis observed under mitotic conditions is believed to be due to the coiled condition of the chromosomes. The lack of a comparable reduction in protein synthesis during mitosis is interpreted as evidence for the presence in these cells of a relatively stable messenger RNA.     

REPOPULATION OF POSTMITOTIC NUCLEOLI BY PREFORMED RNA : II. Ultrastructure

The reconstruction of the nucleolus after mitosis was analyzed by electron microscopy in cultured mammalian (L929) cells in which nucleolar RNA synthesis was inhibited for a 3 h period either after or before mitosis. When synchronized mitotic cells were plated into a concentration of actinomycin D sufficient to block nucleolar RNA synthesis preferentially, nucleoli were formed at telophase as usual. 3 h after mitosis, these nucleoli had fibrillar and particulate components and possessed the segregated appearance characteristic of nucleoli of actinomycin D-treated cells. Cells in which actinomycin D was present for the last 3 h preceding mitosis did not form nucleoli by 3 h after mitosis though small fibrillar prenucleolar bodies were detectable at this time. These bodies subsequently grew in size and eventually acquired a particulate component. It took about a full cell cycle before nucleoli of these cells were completely normal in appearance. Thus, nucleolar RNA synthesis after mitosis is not necessary for organization of nucleoli after mitosis. However, inhibition of nucleolar RNA synthesis before mitosis renders the cell incapable of forming nucleoli immediately after mitosis. If cells are permitted to resume RNA synthesis after mitosis, they eventually regain nucleoli of normal morphology.     

The Golgi protein GM130 regulates centrosome morphology and function

The Golgi apparatus (GA) of mammalian cells is positioned in the vicinity of the centrosome, the major microtubule organizing center of the cell. The significance of this physical proximity for organelle function and cell cycle progression is only beginning to being understood. We have identified a novel function for the GA protein, GM130, in the regulation of centrosome morphology, position and function during interphase. RNA interference-mediated depletion of GM130 from five human cell lines revealed abnormal interphase centrosomes that were mispositioned and defective with respect to microtubule organization and cell migration. When GM130-depleted cells entered mitosis, they formed multipolar spindles, arrested in metaphase, and died. We also detected aberrant centrosomes during interphase and multipolar spindles during mitosis in ldlG cells, which do not contain detectable GM130. Although GA proteins have been described to regulate mitotic centrosomes and spindle formation, this is the first report of a role for a GA protein in the regulation of centrosomes during interphase.     

Erythrocyte membrane sulfhydryl groups and the active transport of cations

RNA synthesis was studied by autoradiographic analysis using tritiated uridine incorporation in the Chinese hamster cell line Dede after a one-minute pulse labeling period. RNA synthesis continues during all stages of interphase and mitosis except during metaphase and anaphase. Cytoplasmic RNA was apparently synthesized in the nucleus, since no grains were observed above the background level in the sample immediately following the labeling. Nucleoli synthesize their own RNA and are not reservoirs for RNA synthesized elsewhere. Both actinomycin D and nogalamycin inhibited the RNA synthetic activity of chromatin and nucleoli. However, the nucleolar synthetic activity was more susceptible to these agents than that of chromatin. Furthermore, actinomycin D was a stronger inhibitor than nogalamycin.     

Migration of newly synthesized RNA during mitosis : III. Nuclear RNA in the cytoplasm of metaphase cells

It was shown by autoradiography in previous papers that RNA which is synthesized before mitosis and located in the nuclei, enters the cytoplasm at the onset of mitosis and returns to the nuclei of the daughter cells after mitosis. In order to study thenature of this migrating RNA we performed a sedimentation analysis of RNA isolated from the cytoplasm and chromosomes (nuclei) of metaphase and interphase cells in the synchronized culture of the Chinese hamster. Whereas the cytoplasm of interphase cells is found to contain RNA with sedimentation constants not higher than 28S, the cytoplasm of metaphase cells includes precursors of ribosomal and messenger RNA with sedimentation constants 32S, 45S and even higher. This means that RNA migrating from nuclei to cytoplasm during cell division retains its nuclear character. It is suggested that this property provides for the return of RNA synthesized before mitosis to the nuclei of the daughter cells.     

Effects of protein synthesis inhibition during plant mitosis

The role of protein synthesis in onion root tips during mitosis has been studied, by using synchronous cell populations. Incubation in cycloheximide (CHM) or anisomycin during early or middle prophase induces the return of these cells to interphase. Therefore, it is suggested that essential proteins are synthesized, which determine the continuation of the cells in mitosis. In late prophase these treatments caused a certain delay in the entry into further stages, suggesting that a protein synthesis probably occurs which determines the duration of the transition from metaphase to anaphase. Mitotic processes which develop after metaphase do not seem morphologically dependent on protein synthesis, in spite of the fact that one of them, the nucleolar reconstruction, is markedly dependent on RNA synthesis. Unexpectedly this reorganization increases its rate in the absence of protein synthesis.     

Phosphorylation of keratin and vimentin polypeptides in normal and transformed mitotic human epithelial amnion cells: behavior of keratin and vimentin filaments during mitosis

Analysis by means of two-dimensional gel electrophoresis (IEF) of [32P]orthophosphate-labeled proteins from mitotic and interphase transformed amnion cells (AMA) has shown that keratins IEF 31 (Mr = 50,000; Hela protein catalogue number), 36 (Mr = 48,500), 44 (Mr = 44,000), 46 (Mr = 43,500), as well as vimentin (IEF 26; Mr = 54,000) are phosphorylated above their interphase level during mitosis. Similar studies of normal human amnion epithelial cells (AF type) confirmed the above observations except in the case of keratin IEF 44 whose relative proportion was too low to be analyzed. Immunofluorescent staining of methanol/acetone-treated mitotic transformed amnion cells with a mouse polyclonal antibody elicited against human keratin IEF 31 showed a dotted staining (with a fibrillar background) in all of the cells in late anaphase/early telophase (characteristic "domino" pattern) and in a sizeable proportion of the cells in other stages of mitosis. Normal mitotic amnion cells on the other hand showed a fine fibrillar staining of keratins at all stages of mitosis. Similar immunofluorescent staining of normal and transformed mitotic cells with vimentin antibodies revealed a fibrillar distribution of vimentin in both cell types. Taken together the results indicate that the transformed amnion cells may contain a factor(s) that modulates the organization of keratin filaments during mitosis. This putative factor(s), however, is most likely not a protein kinase as transformed amnion cells and amnion keratins are modified to similar extents. It is suggested that in general the preferential phosphorylation of intermediate-sized filament proteins during mitosis may play a role in modulating the various proposed associations of these filaments with organelles and other cellular structures.