Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines

Abstract

We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies (Dounce AL, Chanda SK, Townes PL (1973) J Theor Biol 42:275-285, Sumner AT (1984) J Cell Sci 70:177-188). Human chromosomes isolated into buffer unfold when exposed to DTT or TCEP. In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.

DNA Cross-Bridging Shapes a Single Nucleus from a Set of Mitotic Chromosomes

“…The initial support for reforming nuclear membranes is hence provided directly by mitotic chromosomes (Anderson et al, 2009; Lu et al, 2011). Mitotic chromosomes consist of a mechanically loose chromatin gel with a Young’s modulus of only 0.5 kPa (Eastland et al, 2016). Moreover, as a set of separate bodies (Cuylen et al, 2016), they do not intrinsically provide a coherent nuclear surface.…”

The mechanics of mitotic chromosomes

“…This is considerably smaller than the loop size of 70–80 kb estimated from structural data (Gibcus et al ., 2018; Walther et al ., 2018). Treatment with reducing agents was found to dramatically soften the chromosome (Eastland et al ., 2016). However, this non-specific approach did not allow deciphering of the contribution of different proteins since numerous proteins were equally affected.…”

Genome organization during the cell cycle: unity in division

“…Chromosome compaction provides the mechanical rigidity needed to organize discrete chromosomes at the metaphase plate and to maintain chromosomal integrity during anaphase. When key protein crosslinks in the compact mitotic chromosome were removed by reducing agents, force application led to dramatic and irreversible elongations in chromosomes . If only the physical shape and stiffness of the chromosome mattered, any mechanism that created this level of compaction would be sufficient, but this is not the case.…”