Enucleation of CHO cells by means of cytochalasin B and centrifugation: the topography of enucleation

Scanning electron microscopy has been used to observe the changes in morphology associated with the enucleation of Chinese hamster ovary cells using cytochalasin B and centrifugal force. Cells grown in monolayer culture on glass coverslips were inserted into centrifuge tubes with growth medium or growth medium containing cytochalasin B and subsequently centrifuged for various periods up to 60 min at 3 000 g in a prewarmed RC-2B centrifuge containing an SS-34 rotor. The cells spun without cytochalasin B were not enucleated and, when fixed and examined, were found not to differ in their morphology from cells of control cultures not subjected to centrifugal force. In contrast, cells centrifuged in growth medium containing cytochalasin B immediately show gross morphological changes. Initially the nucleus of each cell protrudes as an outpacketing on the cell's free surface. As the outpacketing extends from the cell body, a stalk of cytoplasm is formed, which gradually decreases in diameter. Finally, the stalk breaks and the nucleus and small amounts of associated cytoplasm (karyoplast) sediments to the bottom of the centrifuge tube. When placed in fresh growth medium without cytochalasin B the greatly distorted enucleated cells (cytoplasts) which remain attached to the glass coverslip, rapidly recover a morphology similar to that of the normal nucleated cells.