Hyperosmotic stress:in situchromatin phase separation

Abstract

Dehydration of cells by acute hyperosmotic stress has profound effects upon cell structure and function. Interphase chromatin and mitotic chromosomes collapse ("congelation"). HL-60/S4 cells remain~100% viable for, at least, 1 hour, exhibiting shrinkage to~2/3 their original volume, when placed in 300mM sucrose in tissue culture medium. Fixed cells were imaged by immunostaining confocal and STED microscopy. At a "global" structural level (μm), mitotic chromosomes congeal into a residual gel with apparent (phase) separations of Ki67, CTCF, SMC2, RAD21, H1 histones and HMG proteins. At an "intermediate" level (sub-μm), radial distribution analysis of STED images revealed a most probable peak DNA density separation of~0.16 μm, essentially unchanged by hyperosmotic stress. At a "local" structural level (~1-2 nm), in vivo crosslinking revealed essentially unchanged crosslinked products between H1, HMG and inner histones. Hyperosmotic cellular stress is discussed in terms of concepts of mitotic chromosome structure and liquid-liquid phase separation.

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles

“…While a majority of previously reported condensation responses occur over a timespan of minutes to hours, it is notable that our observation of HOPS, along with evidence from Cai et al, suggests that cellular response by condensation may also occur much more rapidly, at the timescale of seconds (49,91). In addition to being ubiquitous and extremely rapid, sustained HOPS influences translation of mRNA targets of microRNAs (69) and impacts cleavage and polyadenylation of nascent transcripts (49), among other gene regulatory processes (92).…”

Condensed chromatin behaves like a solid on the mesoscale in vitro and in living cells

“…This reveals that, while the volume is significantly altered, the relative geometric orientation of the replication-labeled chromatin remained unchanged within individual chromocenters across these three conditions ( Figure 5B). Although condensates that form by liquid-liquid phase separation usually show a sensitivity towards osmotic changes of the surrounding environment (Olins et al, 2020), the isometric contraction and expansion-behavior of the chromatin domains in this experiment resembled that of a hydrogel -not that of a liquid.…”

Optical quantification of intracellular mass density and cell mechanics in 3D mechanical confinement

“…Indeed, it was recently reported that inhibition of chromatin condensation in HeLa cells on 2D substrates results in a reduction of RI and r, while inducing chromatin condensation results in the opposite effect. 28 It has been widely reported that external osmotic pressure modulates chromatin structure and aberrations [67][68][69] and more recently intracellular mass density. 15 In addition, it was shown that cancer cells under mechanical compression, actively efflux ions to decrease their intracellular tonicity in order to improve their chances of survival.…”