Stabilization of macromolecular chromatin complexes in mitotic chromosomes by light irradiation in the presence of ethidium bromide Eugene V. Sheval a,b, * , Igor I. Kireev a , Vladimir Yu. Polyakov a,b a A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, GSP-2, Moscow 119992, Russia b Institute of Agricultural Biotechnology of the Russian Academy of Agricultural Sciences, Timiryazevskaya Street 42, Moscow 127550, Russia Received 10 June 2004; accepted 30 July 2004 Abstract A method was developed for stabilizing mitotic chromosomes. Light irradiation of permeabilized cells in a low concentration of ethidium bromide made chromatin resistant to high salt concentrations and decondensing buffer. This resistance was abolished by proteinase treatment, but not by DNase or RNase treatment. In photostabilized and extracted chromosomes, chromatin appeared as thick fibers with discrete high electron density regions. These stabilized structures might correspond to the higher-level structures (chromonemata) observed in native chromatin. Moreover, the electron density was higher in the centromeric regions than the chromosome arm material. Thus, the method allows chromatin substructures (chromonemata and centromeric heterochromatin) to be stabilized inside mitotic chromosomes. Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Chromosome; Chromonema; Scaffold; Chromatin photostabilization 1. Introduction Chromosome structure can be described as a hierar- chy of levels of DNA compaction. Histones are necessary for packaging the DNA into 10-nm nucleo- somic fibers and for compacting these into 30-nm fibers. The histones are not only structural proteins; histone H3 phosphorylation is required for cell cycle progression and for the changes in chromatin structure during chromosome condensation (Van Hooser et al., 1998; De la Barre et al., 2000). Little is known about the higher levels of chromo- some organization and several different models have been proposed. According to the radial loop model, the structural non-histone proteins (so-called ‘scaffold- ing proteins’) form a protein skeleton (chromosome scaffold) organizing the 30-nm fibers into topolo- gically constrained loops during mitosis (Marsden and Laemmli, 1979; Stack and Anderson, 2001; Swedlow and Hirano, 2003). The main proteins of the chromo- some scaffold are DNA topoisomerase IIa and compo- nents of the 13S condensin complex (De, 2002; Swedlow and Hirano, 2003; Gassman et al., 2004). However, according to published data, the formation of 30-nm fibers is not the final step in DNA folding. Thick fibers (chromonemata) have been described in mitotic chro- mosomes fixed in situ (Sparvoli et al., 1965; Chentsov et al., 1984; Zatsepina et al., 1983; Belmont et al., 1989; Hao et al., 1990, 1994; Iwano et al., 1997). Thus, the higher-order organization of mitotic chro- mosomes still remains unclear. To study chromosome * Corresponding author. Department of Electron Microscopy, A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Leninskie Gory GSP-2, Moscow 119992, Russia. Tel.: C7 095 9395528; fax: C7 095 9393181. E-mail address: sheval_e@genebee.msu.su (E.V. Sheval). 1065-6995/$ - see front matter Ó 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2004.07.013 www.elsevier.com/locate/cellbi Cell Biology International 28 (2004) 835e843