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The University of Chicago high resolution scanning ion microprobe (UC-SIM) was used to image, by Secondary Ion Mass Spectrometry (SIMS), the distribution of Ca 2+ and Mg 2+ in the chromosomes of Indian muntjac (IM) deer mitotic fibroblasts. This is part of a systematic study of the cation composition of mammalian cells and chromosomes throughout the cell cycle, after having shown that Ca 2+ and Mg 2+ appear to be important for chromosome condensation and structure at metaphase. We focus here on a detailed description of the metaphase-anaphase transition at narrow time intervals beyond the G2/M border, made possible by controlled cell synchronization procedures. High-density distributions of chromosome spreads showed progressive stages of mitosis, identified by their morphology, within the same UC-SIM field of view. Subtle differences in cation contents between successive mitotic stages could thus be quantified in identical experimental conditions. Preliminary results indicate maximal chromosomal concentrations of Ca 2+ and Mg 2+ at metaphase, and a progressive decrease of the same with advancing stages of anaphase. Ca 2+ and Mg 2+ distributions were also imaged in the polytene chromosomes of Drosophila melanogaster, whose DNA distribution had been previously studied by BrdU labeling. These cations may play a common role in mitosis from lower eukaryotes to mammals.

At SIMS XIV, we reported SIMS evidence of specific Mg 2+ binding to the AT-rich regions of human metaphase chromosomes represented by G-bands. Subsequent Mg 2+ -depletion experiments supported a direct role for Mg 2+ in promoting and maintaining the higher order chromatin structure originating G-bands, possibly due to both Mg 2+ -DNA and Mg 2+ -protein interactions. An in-depth study, reported elsewhere, implicated also Ca 2+ in the maintenance of chromatin ultrastructure in the scaffold of mammalian chromosomes, in association with topoisomerase II. We examine here the association of Mg 2+ with AT-rich regions of chromatin in the chromosomes of the Indian muntjac deer (IMD), leading to conclusions similar to the above. To answer the question whether the presumed divalent cation role in the chromosomes of advanced eukaryotes had an evolutionary history to be traced back to earlier evolutionary stages, we have SIMS-mapped Ca 2+ and Mg 2+ in BrdU-labeled polytene chromosomes from the salivary gland of the Dipteran Drosophila melanogaster. Striking Ca 2+ and Mg 2+ SIMS banding patterns correlating with those of the Br label (a thymidine analogue) implicate unequivocally a close association of both these cations with the AT-rich regions of DNA for these primitive eukaryotes.

Our main research interests have focused on cation–DNA and cation–protein interactions in terms of their roles in higher order chromosomal structure. Our previous study directly analyzed the cation composition of cryo-preserved mammalian interphase and mitotic cells, as well as fractionated untreated and cation-depleted chromosomes at a resolution of 50 nm using the University of Chicago high resolution scanning ion microprobe (UC-SIM). Quantitative direct UC-SIMS signals and subsequent imaging of cryo-preserved cells demonstrated that Na+ and K+ were associated with chromatin throughout the cell cycle, whereas in contrast Ca2+ and Mg2+ exhibited a localization change during interphase and mitosis. Interphase Ca2+ and Mg2+ were found mainly throughout the cytoplasm, but at mitosis associated with chromatin. Our findings of chromatin association of Na+ and K+ support a role of these cations in both interphase and mitotic chromatin compaction, whereas Ca2+ and Mg2+ binding points to an essential function in mitotic chromatin compaction. In our present investigation using SIMS images obtained from both human and Indian muntjac metaphase chromosomes, we reveal specific binding of Mg2+ to chromosomal “p” and “q” arm heterochromatic regions correlating with conventional Giemsa (G-) bands. In addition, detailed Mg2+ image density peaks along the chromosomes indicate that Mg2+ peaks correspond to the location of G-bands. Our results support a direct role for Mg2+ in promoting and maintaining the higher order chromatin structure of heterochromatic regions on chromosome arms represented by G-bands, possibly due to both Mg2+–DNA and Mg2+–protein interactions.

In our previous investigation, we showed for the first time high-resolution analytical images of the cation composition of mammalian interphase and mitotic cells as well as of isolated metaphase chromosomes using the University of Chicago scanning ion microprobe (UC-SIM). In order to preserve the ionic integrity of the analyzed cells and prevent the well known occurrence of analytical artifacts due to the high diffusivity of cations in biological samples we used fast cryo-preservation methods (freeze-drying and freeze fracture), without any pre-fixations or washes. We identified the role of the cations in chromosome structure and maintenance using SIMS imaging and immunfluorescence methodologies. Importantly, we determined that the above cations are essential participants in chromosome condensation and maintenance of chromatin higher order structure, through their presumed function in DNA electrostatic neutralization and the direct interaction of Ca2+, in particular, with structural proteins. In addition, both Ca2+ and Mg2+ showed the same cell cycle regulation where during interphase both cations were enriched in the cytosol, particularly in organelles then at mitosis became specifically bound to chromatin. Our present research interest focuses on a more detailed analysis of the distribution of Ca2+ throughout the different cell cycle stages, e.g. G1, G2 and mitosis. We have chosen the stable isotope 44 Ca as a tracer to follow Ca2+ throughout the cell cycle. This nuclide occurs naturally in the ratio 44 Ca / 40 Ca + 44 Ca of 2.06%, so that incorporation at higher concentrations into chromatin or other cellular components should be easily detected by SIMS. Such incorporation can be obtained either by growing cells in a medium where ordinary Ca is replaced entirely by 44 Ca , or by replacing the cell culture medium with the 44 Ca medium for a gated time span (pulsing), after appropriate cell cycle stage synchronization. In this paper, we describe our experimental approach to the 44 Ca labeling of Indian muntjac deer and human fibroblast cells, which involve: (1) the preparation of culture medium where 40 Ca has been replaced by 44 Ca ; (2) the development of appropriate cell culture labeling protocols; (3) the methodology that enabled us to determine accurate isotopic SIMS measurements on chromosomes; (4) the first results where metaphase chromosomes obtained and imaged by SIMS bind up to 60–70% 44 Ca . We are presently developing cell culture synchronization protocols, which will allow us to analyze the binding dynamics of Ca throughout the cell cycle.