Hyperosmotic stress: in situ chromatin phase separation.

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 (mu m), 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-mu m), radial distribution analysis of STED images revealed a most probable peak DNA density separation of 0.16 mu m, 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.