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.

New data on the in-situ position of the inactive X chromosome in the interphase nucleus of human fibroblasts.

The in situ spatial distribution of nucleolus-organizing-region (NOR) bearing chromosomes in relation to the inactive X chromosome was studied during interphase in human fibroblasts. The respective positions of these chromosomes were examined in 30 growing and 32 resting fibroblasts from reconstructed nuclei, using nucleoli and the Barr body as ultrastructural markers. Experimental values for the distance between the nucleoli and the Barr body were estimated by their coefficient of closeness and compared to the uniform distribution. The following results were obtained: the distribution patterns for the two populations of nuclei were similar, the distribution of the NOR-bearing chromosomes in relation to the inactive X chromosome varied and differed significantly from a uniform distribution, and in many cases the Barr body was observed to be in a juxta-nucleolar position. The internal distribution revealed by this study is compared with the data in the literature, especially with the conflicting data obtained by other methods used to determine the interphase arrangement of chromosomes. The relationship between interphase and metaphase arrangements such as can be deduced with these methods, is discussed in relation to the mechanisms of the formation of metaphase plates or chromatid translocations.

In situ arrangement of nor-bearing chromosomes in the interphase nucleus of Aotus trivirgatus.

The spatial organization of the two nucleolus-organizing region (NOR)-bearing chromosomes during interphase was studied in Aotus trivirgatus fibroblasts, using nucleoli as ultrastructural markers. Their distribution was examined by measuring the distances between them in 30 reconstructed nuclei, and comparing these experimental values with the theoretical ones obtained by simulation. Results were as follows: (1) the nucleoli are arranged in a polarized manner inside the nucleus; (2) the nucleoli are tightly bound to the nuclear envelope at two opposites sites; (3) the distance between the two nucleoli is variable, and is shorter than it would be if the two nucleoli were distributed at random. These findings indicate that the NOR-bearing chromosomes are fixed at the nuclear envelope in two opposite areas. They are also consistent with the hypothesis that each chromosome occupies a separate domain inside the nucleus. They can be interpreted according to the model in which chromosome arrangement within the interphase nucleus is based on the separation of the diploid complement into two independent haploid sets.