D. Hernandez-Verdun

Ultrastructural localization of Ag-NOR stained proteins in the nucleolus during the cell cycle and in other nucleolar structures

EM investigation of Ag-AS-NOR staining after short glutaraldehyde prefixation followed by Carnoy fixation maintained good ultrastructural preservation and reactive selectivity. This enables exact localization of silver deposits both in the fibrillar centers of typical or segregated nucleoli during interphase, and in chromosome NORs during mitosis. These results argue in favour of the possibility that fibrillar centers are the interphasic counterpart of chromosome NORs. Special structures such as nucleolar blobs and remnants usually considered to be of nucleolar origin, were also stained. — These findings seem to indicate a relationship between the distribution of the silver-stained proteins, the arrangement of the nucleolar structures and the degree of nucleolar activity resulting from the experimental conditions. These results are of interest at the time when the concept of the nucleolar matrix is gradually emerging.

Silver staining of NORs in electron microscopy

Abstract We adapted the Ag-As procedure for staining the nucleolus organizer regions (NORs) to the ultrastructural level, under conditions preserving the specificity of the method. The fibrillar centre and the dense fibrillar component appeared to constitute the interphasic counterpart of the NOR plus the RNA transcription sites.

Fine structure of nucleoli in micronucleated cells.

Abstract The correlation between the number of nucleolus organizing regions (NOR) on metaphase chromosomes and the number of nucleoli was studied in normal and micronucleate cells. Many micronuclei, but not all, were able to form complete nucleoli with fibrillar and granular RNP components and fibrillar centers. Micronuclei which failed to form complete nucleoli often contained multiple electron-dense bodies of fibrillar material. These structures, which were much smaller than nucleoli, reacted with nucleolus-specific antibodies and the Ag-As method in the same way as complete nucleoli, but lacked fibrillar centers and granular RNP components. The data suggest that these nucleolus-like ‘blobs’ contain nucleolar material which, following mitosis, has been enclosed in micronuclei which do not contain nucleolus organizing chromosomes. No evidence was found for the activation of latent NORs not expressed in mononucleate cells.

Nucleologenesis in chick erythrocyte nuclei reactivated by cell fusion

“Dormant” chick erythrocyte nuclei have been fused by Sendai virus with human TG cells. A quantitative analysis and a three-dimensional approach using staggered sections of flat embedded heterokaryons were used to analyze the various aspects of nucleolar structures differentiated during chick erythrocyte reactivation 12, 24, and 48 hr after fusion. Three types of nucleolar structures are described: (1) developed nucleoli , (2) prenucleoli , and (3) nucleolar organizer region. Reactivation of RNA synthesis was followed by electron microscope autoradiography after 10, 30, and 180 min [ 3 H]uridine pulse in the three types of nucleolar structures described. Developed nucleoli and prenucleoli were involved in RNA synthesis; the fibrillar component was first labeled in the periphery of the fibrillar center. To conclude, we propose a dynamic model of nucleologenesis which correlates ultrastructural features and RNA synthesis.

Three-dimensional organization of micronuclei induced by colchicine in PtK1 cells☆

In PtK1 cells micronucleated by colchicine, we previously demonstrated that some micronuclei contain a single chromosome. Here, we investigated interphase chromosome organization in micronucleated PtK1 cells using conventional electron microscopy and three-dimensional computer reconstruction. The distribution of micronuclei was not always polarized, but in some cells they formed a ring. When this occurred, centrioles and Golgi apparatus were located inside the ring. On freeze-fracture replicas, we observed that nuclear pore distribution among the micronuclei was heterogeneous, and on thin sections some micronuclei displayed an incomplete nuclear envelope, with gaps in the double membrane and areas without lamina or condensed chromatin. By autoradiography, we showed that the fibrillar dots were not sites of active transcription. We applied three-dimensional reconstruction to one micronucleated cell containing 22 micronuclei whose size indicated that each micronucleus probably contained one chromosome. In this cell we demonstrated that only the smallest micronuclei had an incomplete nuclear envelope. The presence in micronuclei of either nucleoli or fibrillar dots was found to be mutually exclusive. These dots might constitute stores of nucleolar proteins which migrate into micronuclei possessing no ribosomal genes. In NOR-bearing micronuclei, the structural organization was similar to that of diploid nuclei: the nucleoli were attached to the nuclear membrane and a nucleolar canal was seen, even in single-chromosome spherical micronuclei. Taken together, these findings indicate that in the diploid nuclei of PtK1 cells, the three-dimensional organization of the nucleolar domain seems to be directly controlled by the X-chromosome.

Visualization of chromatin distribution in living PTO cells by Hoechst 33342 fluorescent staining

Chromatin distribution was visualized in living cells with the selective DNA fluorochrome Hoechst 33342. This dye was shown to be non-toxic on the rat kangaroo PTO cell line by measuring the labelled cell growth rate. The aim of this work was firstly to visualize chromatin distribution without fixation or dehydration and secondly to demonstrate that quantitative determination of DNA content was possible under these non-toxic labelling conditions. During interphase, condensed, decondensed and thin network chromatin configurations were visualized. In nucleolar regions the fluorochrome revealed well-defined chromocentres. During mitosis, fluorescent chromosome banding was observed in vital conditions and chromocentres on fixed chromosomes. Chromatin segregation was visualized after micronucleation, which induced chromosomal set distribution in individual micronuclei. By this means, we demonstrated that the chromocentres observed in interphase nuclei were part of nuclear organizer region (NOR)-bearing chromosomes. This vital staining of chromatin was shown to be compatible with the quantitative determination of DNA content, both in living PTO cells and in isolated nuclei.

A protein of Mr 80,000 is associated with the nucleolus organizer of human cell lines

A rabbit serum which had previously been reported to have an immunological affinity for centrosomes of human cell lines was shown also to be specific for the nucleus. Optical and ultrastructural immunolocalization in HeLa cells showed that this specificity is restricted to the fibrillar centre of nucleoli either in untreated or actinomycin D treated interphase cells. In mitotic cells discrete labelling was observed on chromosomes and shown to correspond, on spread metaphase plates, to the short arms of acrocentric chromosomes, i.e. to the nucleolar organizer regions (NORs). Using independent cell fractionation procedures in the human T-lymphoblastic KE 37 cell line and purification of immunoglobulins by affinity to antigens detected by electrophoresis and blotting, a strict correlation between immunoreactive proteins and cytological staining was established. The nucleolar specificity was shown to correspond to a protein with an Mr of 80,000 while the centrosomal specificity corresponded principally to a protein doublet of 60,000–65,000. These antigens share common epitopes as shown by the staining of both NOR and centrosome by immunoglobulins purified by affinity to either type of protein.

RNA polymerase activity in PtK1 micronuclei containing individual chromosomes: An in vitro and in situ study☆

Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.

Identification and sorting of micronuclei containing individual chromosomes

Micronuclei are diploid genome subfractions which can be induced by colchicine treatment. The purpose of the present study was to identify the chromosome content of micronuclei. Accordingly, PtK1 cells were micronucleated with colchicine and their DNA was labeled by the Hoechst 33342 fluorochrome. The fluorescence intensity of the micronuclei was measured by flow cytometry and compared to that of metaphase chromosomes of the same species. We observed histograms exhibiting five distinct peaks for both micronuclei and chromosomes. Each class of micronuclei corresponded to a peak in the flow karyotype with the same values. As the smallest micronuclei contained the same amount of DNA as one chromosome, it seemed possible to identify the DNA content of the micronuclei containing a single chromosome. This identification was confirmed by fluorescence microscope observations of the micronuclei and chromosomes sorted from their respective peaks. This was clear for peak number 3, which exhibited fluorescent markers both in micronuclei and in chromosomes. These markers, called chromocenters, were connected with the nucleoli in micronuclei and were specific for the nucleolar organizer regions of the X chromosomes. As we paid particular attention to maintaining the transcriptional activity of the micronuclei throughout the procedure used to sort and identify them, it is concluded that such an approach might be very suitable for investigating the expression of reduced nuclear domains corresponding to individual chromosomes.

Sorting of micronuclei from PtK1 cells

We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line.