Ion microprobe imaging of 44Ca-labeled mammalian chromosomes

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 (freezedrying 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 Ca2þ, in particular, with structural proteins. In addition, both Ca2þ and Mg2þ 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 Ca2þ throughout the different cell cycle stages, e.g. G1, G2 and mitosis.We have chosen the stable isotope 44Ca as a tracer to follow Ca2þ throughout the cell cycle. This nuclide occurs naturally in the ratio 44Ca=40Ca þ 44Ca 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 44Ca, or by replacing the cell culture medium with the 44Ca medium for a gated time span (pulsing), after appropriate cell cycle stage synchronization. In this paper, we describe our experimental approach to the 44Ca labeling of Indian muntjac deer and human fibroblast cells, which involve: (1) the preparation of culture medium where 40Ca has been replaced by 44Ca; (2) the development of appropriate cell culture labeling protocols; (3) the methodology that enabled us to determine accurate isotopic SIMS measurements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% 44Ca. We are presently developing cell culture synchronization protocols, which will allow us to analyze the binding dynamics of Ca throughout the cell cycle. # 2004 Elsevier B.V. All rights reserved.