Ultrastructural localization of the nuclear protein mitotin during the cell cycle.

The work presents the results of the immunoelectron microscopical localization of mitotin in different phases of the cell cycle. The distribution of the protein was studied using its specific monoclonal antibody and immunogold labeling in synchronized WISH cells. In S phase the antigen was found in the nucleoplasm usually over the interchromatin granules. In G2 phase the amount of mitotin increases and it can be found also in the nucleolus. In mitosis the immunogold granules are always out of the condensed chromosomes.     

Dynamic localization of proteins and DNA during a bacterial cell cycle

A cellular differentiation programme that culminates in an asymmetric cell division is an integral part of the cell cycle in the bacterium Caulobacter crescentus. Recent work has uncovered mechanisms that ensure the execution of many events at different times during the cell cycle and at specific places in the cell. Surprisingly, in this one-micron bacterial cell, the dynamic spatial disposition of regulatory proteins, structural proteins and specific regions of the chromosome are important components of both cell-cycle progression and the generation of daughter cells with different cell fates.     

Ultrastructural cytochemistry of the mammalian cell nucleolus

In the present review on the organization of the mammalian cell nucleolus, we report and discuss data obtained during the past 10 years by means of cytochemical and immunocytochemical ultrastructural techniques. Particular emphasis is placed on the following topics: location of the nucleolus organizer regions in interphasic nucleolar components, structure of nucleolar chromatin in situ, and the structure-function relationship of the nucleolar components. The cytochemical and immunocytochemical results are compared and the concordant data are stressed for each topic.     

Change in phosphorylation of nucleolar proteins of Physarum polycephalum during the cell cycle in vivo and in vitro

We compared the phosphorylation of nucleolar proteins during the cell cycle of Physarum polycephalum labeled by pulse and continuous labeling methods in vivo with that obtained by in vitro labeling of isolated nucleoli. Both the phosphorylating activity of nucleoli and total incorporation of radioactive phosphate into nucleolar proteins increased and reached a maximum about 1.5-2.0 h before mitosis, confirming our previous observation. Analyses of labeled nucleolar proteins by SDS-polyacrylamide gel electrophoresis and by autoradiography indicated that most of the phosphoproteins labeled by in vitro labeling were labeled by in vivo pulse labeling. At least 10 nucleolar proteins underwent phosphorylation, which closely followed the cell cycle-dependent changes of the total phosphate incorporation into the nucleolar proteins. When mitosis was delayed by UV-irradiation, the maximal incorporation of radioactive phosphate into nucleolar proteins in vivo was not observed at the usual time, it shifted to about 2 h before the delayed mitosis, and the same set of nucleolar proteins that were phosphorylated without UV-irradiation were most heavily phosphorylated at this time. These results suggest the possibility that the increased phosphorylation of nucleolar proteins of Physarum just before mitosis is related to the onset of subsequent mitosis.     

Ultrastructural study of protein kinase C-betaII localization during the cell cycle of human glioma cells

Transmission electron microscopy and immunogold labeling were used to determine how PKC-betaII is localized at stages in the cell cycle of the human glioma cell line U-373MG. Results show that immunogold particles in both dimethylsulfoxide (DMSO) and calphostin C (0.5 microM)-treated cells were mainly located in the cytoplasm. The concentration of gold particles in the nucleus was relatively small and constant throughout the cell cycle of both DMSO and calphostin C treated cells. Micrographs revealed changes in PKC-betaII during the cell cycle. The concentration of gold particles in the DMSO-treated cells was constant until 8 h. Subsequently, cytoplasmic PKC-betaII oscillated with an increased at 10 h, a rapid decrease at 12 h, and a rise at 14 h. The concentration of the gold particles then gradually decreased. In contrast, immunogold labeling in calphostin C-treated cells increased gradually up to 10 h. Subsequently, the pattern of PKC-betaII labeling in calphostin C-treated cells recapitulated those of control cells as seen by the rapid decline of PKC-betaII labeling at 12 h and its re-accumulation at 14 h. Additionally, there was a rapid increase at 20 h. Western blots of PKC-betaII showed constant PKC-betaII immunoreactivity throughout the cell cycle. In comparison to Western blots, in-situ immunogold labeling revealed changes in PKC-II immunoreactivity at 10 h and 14 h. This technique may represent intracellular immunoreactivity of PKC-betaII. The results from the immunogold labeling technique suggest that binding of calphostin C to the regulatory domain of PKC-betaII provokes a conformation change in PKC-betaII, preventing its activation and degradation.     

Immunoelectron microscopic localization of the nucleolar protein B-36 (fibrillarin) during the cell cycle of Physarum polycephalum

Monoclonal antibodies raised against the 34-kD nucleolar protein, B-36, from the slime mold Physarum polycephalum have been used to examine the electron microscopic localization of B-36 during the cell cycle in Physarum. During interphase, B-36 is found primarily in regions corresponding to the dense fibrillar component. This is similar to what has been observed for the putative mammalian homologue of B-36, fibrillarin. During mitosis, B-36 remains associated with perichromosomal nucleolar remnants. With the Gautier DNA-specific staining procedure, the same nucleolar remnants are shown to contain short DNA segments, presumably rDNA molecules. These findings suggest that in Physarum, where the nucleolus is composed of several hundred extrachromosomal rDNA molecules, the dense fibrillar component and the "NOR" equivalents do not separate during mitosis as in mammalian cells. In addition, the B-36-enriched nucleolar remnants appear to be recycled from one cell cycle to the next.     

Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions

After extraction of Novikoff hepatoma nucleoli with 4 M urea/3 M LiCl, phosphoprotein C23 was isolated by DEAE-cellulose and Bio-Rad AG3-X4A column chromatography. Immunization of rabbits with the highly purified protein C23 resulted in the production of a specific antibody as determined by Ouchterlony diffusion analysis. When the immunoperoxidase method was used to localize protein C23 in cells, it was found in ‘fibrillar centers’ (nucleolonemas) in nucleoli. Protein C23 was also demonstrated to be present on the nucleolus organizer regions (NORs) of metaphase chromosomes.     

Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei

Summary Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver.In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes.In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules.In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes.In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes.In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components.The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.     

小麦细胞核仁中rRNA基因转录的超微定位（简报）

真核细胞核仁中rRNA基因转录位点是长期以来未能解决的问题。以小麦细胞为研究材料,应用常规电子显微镜技术,观察了小麦细胞核仁纤维中心（Fibrillar centers,FC)内染色质的超微结构;并通过DNA抗体阐明了核仁中DNA位于纤维中心、致密纤维组分（Dense fibrillar component,DFC）以及两者的过度区域;应用RNA聚合酶I相关转录因子UBF（Upstream binding factor) 抗体所做的分析显示,小麦细胞核仁中UBF位于FC与DFC的过渡区域以及DFC中,在FC中没有UBF的存在;进一步借助于RNA／DNA杂合体抗体选择性地直接标记核仁中rRNA基因的转录位点,结果表明了小麦细胞核仁rRNA基因的转录位点是在FC与DDFC的过渡区域及DFC中。     

Ultrastructural labeling of cell surface lectin receptors during the cell cycle.

The distribution of specific surface receptors in the course of the cell cycle has been studied on two transformed cell lines by means of ultrastructural labelling techniques employing Concanavalin A (ConA) and wheat germ agglutinin (WGA). Synchronized cultures of Cl₂TSV₅, an SV40-transformed hamster cell line and of CHO cells were labelled as monolayers or in suspension in the different phases of the cell cycle. In cells labelled in monolayers, a moderately discontinuous pattern of surface labelling was present during G 1, S, and G 2. On cells in mitosis, however, this pattern changes strikingly and becomes very discontinuous. These results indicate that the degree of receptor clustering is greater in mitosis than in interphase. In cells labelled in suspension, the differences in pattern between mitosis and interphase were absent. Colcemid treatment did not modify the distribution of the label, either in interphase or in mitosis. Moreover, cells in mitosis collected by Colcemid treatment and labelled at a moment in which parallel unblocked cultures had completed mitosis and passed into G 1 showed an interphase-type labelling pattern; this indicates that a certain dissociation exists between surface events and nuclear events during mitosis. These results are discussed in terms of several factors that may contribute to the production of receptor clustering, namely, direct lectin action, surface movement and membrane flow, participation of cytoplasmic structures and, finally, attachment of cells to a substratum.     

Ultrastructural and functional variations in the spermatid nucleolus during spermiogenesis in the mouse

The localization, structure, and activity of the nucleolus-organizers (NORs) were studied during spermiogenesis in the mouse by light and electron microscopy procedures including NOR-silver-staining and actinomycin D treatment. After the two meiotic divisions the NORs resume their activity during the Golgi phase of spermatid differentiation (steps 1-3), and the nucleolus displays a specific 'padlock' structure containing the fibrillar components of an active nucleolus. This activity drops during the cap phase (steps 4-7) during which the nucleolus undergoes a segregation process of its components. No nucleolar structure is visible during the acrosomal and maturation phases of spermatid differentiation.     

Fate of the nucleolar vacuole during resumption of cell cycle in pea cotyledonary buds

Summary Meristematic cells of pea cotyledonary buds blocked in G_0–1 state contain a small nucleolus with a large central clear area surrounded by a fibrillar rim. The nucleolar structure varies according to the cell cycle from the G_0–1-blocked state until the first mitoses occurring between 24 and 27h after removal of the main stem. In order to better identify and understand the role of the central area in the nucleolar function, its content was investigated by cytochemical and terminal deoxynucleotidyl transferase-immunogold methods. The central area showed the characteristics of a vacuole commonly constituted of the condensed chromatin, ribonucleoprotein granules, and lack of argyrophilic proteins. 3 h after decapitation, a thickening of the fibrillar rim occurred, accompanied by an increase of granules in the vacuole. After 6h, the unique vacuole broke up into two to four small vacuoles in which the granules are more abundant. After 12 h the nucleolus acquired compact structure with few minute vacuoles dispersed over the fibrillar component. During the whole cell cycle, the condensed chromatin is always observed in the vacuole. Our findings suggest that the appearance of the vacuoles is subsequent to the output of preribosomes from nucleolus. These vacuoles might play a role in condensation and decondensation of the chromatin.