The observation of high hypotonicity manipulating cell division

Abstract

We report a morphological manipulation of cell division which was achieved by changing the environment from isotonic to highly hypotonic. Cells at telophase were observed to undergo a morphological reversal to anaphase, with the contractile ring being reopened and the cell shape reversing from dumb-bell back to spherical. Once restored to isosmotic environment, the reversed cells would either continue to divide or instead to form binuclear cells that further proliferated in runaway fashions. The immunofluorescent staining of tubulins and myosin II indicated that the hypotonic stress affected the accumulation of tubulins and myosin II at the contractile ring. Distinct from previous studies using specific chemical reagents, the present study provides a simple method to manipulate cell division. The morphological reversal is the adaption of dividing cells to the environmental change. The observation opens a new window to understand cell division mechanisms and runaways.

Keywords: Binuclear; Bioengineering; Cytokinesis; Division; Hypotonicity; Osmotic.

Fig. 1

The morphological change of HeLa cells undergoing division in highly hypotonic environment. The red arrows indicate the retracting of the cytokinetic furrow: (a) The HeLa cell was cultured in isotonic DMEM till its telophase and then the medium was replaced by 5% PBS at time 0 min. Morphological reversal occurred. At 62 min the cell was cultured again in isotonic DMEM and the cell finally became binuclear. (b) The HeLa cell was cultured in isotonic DMEM till its telophase and then the medium was replaced by 5% PBS at time 0 min. Morphological reversal occurred. At 46 min the cell was cultured again in isotonic DMEM and the cell started a secondary division and finally finished the division.

Fig. 2

A culture process in isotonic DMEM of a binuclear cell after the morphological reversal in 5% PBS. The culture was started at 0 h. The binuclear cell was growing and proliferating in a disordered fashion. Multinucleate cells were produced. Scale bar = 100 μm.

Fig. 3

HeLa cells treated with 5% PBS with 5 uM flavopiridol being added. The cytokinetic furrow got wider at the beginning and then stopped and contracted later, indicating that low concentration flavopiridol might have a competition with the hypotonic stress on the cell cycle. Scale bar = 20 um.

Fig. 4

Immunofluorescence results of tubulins (a-f, red) and myosin II (g-l, red) and DNA (blue) of HeLa cells. Normal HeLa cells were taken as the control (a, b, g, h). The timing when the reversed cells were just back to isotonic DMEM culture was taken as time point 1, then 40 min after was taken as time point 2. The symbol “*” represents results of binuclear cells and “**” represents results of resuming-to-divide cells. Scale bar = 20 μm.

Fig. 5

Typical AFM result of cell stiffness change during hypotonic treatment. The error bar stands for the standard deviation of the data points.