Hyperosmotic stress: in situ chromatin phase separation.

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Hyperosmotic stress profoundly impacts cell structure and function.
• Cellular dehydration leads to the collapse of interphase chromatin and mitotic chromosomes, a phenomenon termed 'congelation'.

Purpose of the Study:

• To investigate the structural and functional effects of acute hyperosmotic stress on HL-60/S4 cells.
• To analyze chromosome organization at global, intermediate, and local levels under dehydration conditions.

Main Methods:

• HL-60/S4 cells were subjected to 300mM sucrose hyperosmotic stress.
• Immunostaining confocal and super-resolution (STED) microscopy were employed for imaging.
• In vivo crosslinking was used to assess protein interactions.

Main Results:

• Cells exhibited shrinkage to two-thirds of their original volume while maintaining viability.
• Mitotic chromosomes congealed into a gel-like state with phase separation of proteins like Ki67, CTCF, SMC2, RAD21, H1 histones, and HMG proteins.
• DNA density at the sub-micrometer level and protein crosslinking at the nanometer level remained largely unchanged.

Conclusions:

• Hyperosmotic stress induces significant global structural changes in mitotic chromosomes.
• Despite global reorganization, the local and intermediate-range organization of DNA and protein interactions within chromosomes are surprisingly resilient to dehydration.
• The findings provide insights into chromosome structure and liquid-liquid phase separation under cellular stress.