Structure of Metaphase Chromosomes: A Role for Effects of Macromolecular Crowding

Abstract

In metaphase chromosomes, chromatin is compacted to a concentration of several hundred mg/ml by mechanisms which remain elusive. Effects mediated by the ionic environment are considered most frequently because mono- and di-valent cations cause polynucleosome chains to form compact ∼30-nm diameter fibres in vitro, but this conformation is not detected in chromosomes in situ. A further unconsidered factor is predicted to influence the compaction of chromosomes, namely the forces which arise from crowding by macromolecules in the surrounding cytoplasm whose measured concentration is 100–200 mg/ml. To mimic these conditions, chromosomes were released from mitotic CHO cells in solutions containing an inert volume-occupying macromolecule (8 kDa polyethylene glycol, 10.5 kDa dextran, or 70 kDa Ficoll) in 100 µM K-Hepes buffer, with contaminating cations at only low micromolar concentrations. Optical and electron microscopy showed that these chromosomes conserved their characteristic structure and compaction, and their volume varied inversely with the concentration of a crowding macromolecule. They showed a canonical nucleosomal structure and contained the characteristic proteins topoisomerase IIα and the condensin subunit SMC2. These observations, together with evidence that the cytoplasm is crowded in vivo, suggest that macromolecular crowding effects should be considered a significant and perhaps major factor in compacting chromosomes. This model may explain why ∼30-nm fibres characteristic of cation-mediated compaction are not seen in chromosomes in situ. Considering that crowding by cytoplasmic macromolecules maintains the compaction of bacterial chromosomes and has been proposed to form the liquid crystalline chromosomes of dinoflagellates, a crowded environment may be an essential characteristic of all genomes.

Crowding-Induced Formation and Structural Alteration of Nuclear Compartments

“…4.4. Our simulation results were consistent with experimental findings (Albiez et al, 2006;Richter et al, 2007;Hancock, 2007Hancock, , 2012, showing a clear condensation of chromosome subcompartments with increasing degree of crowding. Quantitative analysis in terms of the polymer size and volume fraction (Fig.…”

“…When the crowder density is increased, macromolecular associations are expected to increase because of the increase in the equilibrium constant or equivalently because of the amplified effective attraction. Based on this understanding, we can expect that the volume exclusion effect caused by macromolecular crowding induces compaction of chromosome subcompartments (Hancock, 2007(Hancock, , 2012 and a stronger association between the components of NBs. A more detailed discussion of the effects of crowding, which are more complex than indicated by the simple statement above, is presented throughout the rest of this chapter.…”

HEAT repeats – versatile arrays of amphiphilic helices working in crowded environments?

“…Why and how the different condensins are enriched at these chromosomal regions is unknown. Although the interior of mitotic chromosomes is highly crowded (Hancock, 2012;Wachsmuth et al, 2008), at the same time, it is also a network of well-solvated chromatin that is held together by noncovalent crosslinking proteins (Poirier and Marko, 2002) and is readily accessible by macromolecules (Hihara et al, 2012). Thus, the interior of chromosomes could share some of the physico-chemical properties of a hydrogel.…”

Mitotic Chromosome Assembly In Vitro: Functional Cross Talk between Nucleosomes and Condensins

“…It will be possible to further advance the reaction by supplementing it with additional factors such as linker histones, chromatin remodeling factors, and condensin II. Equally important, some agents that mimic macromolecular crowding in crude extracts could further improve the reconstitution reaction (Hancock 2012). Moreover, we must admit that we are still largely ignorant of ion atmospheres (Mg 2+ , Ca 2+ , Na + , and K + ) that contribute to large-scale chromosome assembly in vivo (Mathieson and Olayemi 1975;Strick et al 2001;Hudson et al 2003;Phengchat et al 2016;Ono et al 2017).…”