1. Hyperosmotic stress: in situ chromatin phase separation.
- Author
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Olins AL, Gould TJ, Boyd L, Sarg B, and Olins DE
- Subjects
- Chromatin chemistry, Chromatin genetics, Chromosomes chemistry, Chromosomes genetics, HL-60 Cells, Humans, Microscopy, Confocal, Microscopy, Fluorescence, Mitosis, Optical Imaging, Tumor Cells, Cultured, Chromatin metabolism, Chromosomes metabolism, Osmotic Pressure
- 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.
- Published
- 2020
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