Structure of the mitotic apparatus and chromosomes after hypotonic treatment of mammalian cells in vitro

Abstract

Cultured mammalian cells were exposed briefly to culture media made hypotonic with various dilutions of distilled water. Electron microscopic observations and tubulin immunofluorescence were carried out on mitotic cells that had been briefly exposed to hypotonic conditions. Similar observations were made on cells that had been exposed to hypotonic medium and then returned to isotonic conditions. These experiments provided new insight into the structure and physical properties of the mitotic apparatus and chromosomes. Spindle micro tubules were reversibly depolymerized by hypotonic treatments. Spindle fibers disappeared after a 15-min exposure to hypotonic medium, leaving only two brightly fluorescent spindle poles. When hypotonically swollen cells were returned to isotonic medium for 15 min prior to fixation, the spindle was reassembled. Several types of spindle aberrations were noted in cells recovering from hypotonic treatment. Chromosomes were greatly expanded by hypotonic treatment, and the kinetochores were disrupted. When treated cells were returned to isotonic culture medium, the chromosomes recondensed and the kinetochores reappeared. Brief hypotonic treatments, described herein, had no adverse effect on cell viability and may prove useful in investigations of the structure and physical properties of the mitotic apparatus.