Events Associated with Nuclear Assembly Following Metaphase in Mammalian Cells

Abstract

The process of nuclear ass~m~ly following metaphase in HeLa S3 has been shown to proceed in a serie~ ~f sequential st~ps (Welter et al., 1985). Each step is characterized -by a stage specific arrangement and orientation of chromatids to form anaphase and telophase chromatid configurations (Welter et al., 1985). To determine wh~ther the chromatid I • alignment and orientation observed in He La S3· is typical of mamm_alian cells in general, late mitotic configurations of HeLa S3 (a near triploid human cell line) were compared and contrasted to those of the Indian muntjac (a cell line with only seven chromosomes) and LN (a human primary cell line) with scanning electron microscopy (SEM). All three cell lines ~xhibit stage specific tnitoti,c ·configurations when visualized with SEM following acid isolation. While chromatid alignment and orientat~on within an anaphase or te_lophase configuration are consistent within a cell line, differences in chromatid alignment and orientation occur between cell lines. At metaphase, the chromatids of all three cell lines are arranged into a radial array around the· centromeric ring, with the larger chromatids located peripherally and the smaller chromatids in the center of the array. ·At anaphase, differences in chromatid alignment and orientation are obseryeci between the "cell lines. In early anaphase He La S3, many of the peripheral chromatids · are directed toward· the spindle pole, with the remaining telomeres of the peripheral chromatids directed toward the equatorial plane (Welter et al., 1985). In Indian muntjac anaphases, the centromeres, interconnected by a structural element, the centromeric ring, lead toward the spindle -pole while the telomeres of all ch,romatids except y 2 are directed toward the equatorial plane. The anaphase orientation of chromatids obser-ved - ·- in LN exhibited an orientation intermediate between that ·of Indian m-untjac and HeLa 83. HeLa S3 is a heteroploid (near triploid) cell line with > 65 chromosomes, several of which have been rearranged. The anaphase alignment and orientation of c.hromatids observed in the Indian muntjac and: LN. (both diploid) suggest that the alignment and orientation of He La 83: anaphase chromatids is the result of crowding the additional chromatids into the centromeric ring. Isolation of HeLa S3 mitotic chromatid configuration's with detergent followed by fixation in Karnovsky's solution and observation with scanning electron microscopy, indicates that the anaphase alignment and orientation is not an.artifact produced by the acid isolation procedure of Welter and Hodge ( 1985). However·. imm u~ofluorescence studies indicate that the acid, isolation does produce changes in the -molecular nature of the configurations. · A fibrous network of interconnecting fibers found on the· sur-face and betweenadjacent chromatids, appears to function toorganize the chromatids into the stage specific mitotic configurations and to stabilize the configurations during mitosis. High resolution ~EM of the fibrous network indicates that the inte-rconnecting fibers seen during late mitosis are actually chromatin. The f-ibrous network of the periphery of telophase and interphase nuclei is resistant to DNase· digestion. Non-histone chromosomal proteins may be complexed to the peripheral fibrous network, protectingthe fibrous network from DNase digestion. Since the lamin proteins repolymerize around telophase configurations and ate observed in acid isolated nuclei on the basis· of immunofluorescence and immunoblot ) . analysis, the lamin proteins are complexe'd with the peripheral chromatin although a morphologically distinct nuclear lamina is not observable , with SEM following acid isolation. A model is proposed in which the interconnecting fibers function to bind the chromatids together, maintaining chromatid orientation and alignment and, _in conjunction with the lam in proteins, provide the skeletal framework of the nuclear membrane and pore complexes.