AN ULTRAVIOLET MICROBEAM STUDY OF ROLES OF NUCLEUS AND CYTOPLASM IN DIVISION DELAY, KILLING, AND PHOTOREACTIVATION OF AMOEBA PROTEUS

These experiments were directed primarily to the question of the relative roles of nucleus and cytoplasm in whole-cell ultraviolet irradiation effects. Starved G2 cells of Amoeba proteus were flattened to a thickness of 20 μ and irradiated with microbeams of polychromatic ultraviolet radiation (UV). The technique permits irradiation either of all the nucleus or of all the cytoplasm with little overlap. Whole-cell irradiations were also performed, both with the microbeam and with the germicidal lamp, and photoreactivation was investigated. We find that interactions between the effects of UV damage on nucleus and cytoplasm are small and possibly insignificant. This does not mean that one organelle does not possess recovery potential for the other, but merely that such factors, if they exist, are not UV-sensitive. In flattened cells, for a standard division delay (doubling of time to complete the second division after irradiation), nuclear irradiation requires 6 times as large a dose (ergs/mm2) as cytoplasmic irradiation; for killing (lysis within 3 weeks), nuclear irradiation requires about the same dose as cytoplasmic irradiation. From this behavior and the lack of interactions noted above, we conclude that killing of a flattened A. proteus with UV results about equally from damage to the nucleus and to the cytoplasm, whereas division delay results almost entirely from cytoplasmic damage. When one irradiates a free (unflattened) ameba with UV below 300 mμ, the effects appear to be due almost solely to cytoplasmic damage, owing to shielding of the nucleus by overlying cytoplasm. A. proteus shows a small but significant photoreactivation of both division delay and killing after nuclear irradiation, but a much larger photoreactivation of both effects after cytoplasmic irradiation (photoreactivable sectors of 0.45 for killing and 0.6 for division delay). This appears to be the first clear-cut demonstration of cytoplasmic photoreactivation in a whole cell. In general, these experiments confirm the suggestion by others (see introduction) that the ameba cytoplasm plays an important role in radiation response. The present experiments have eliminated the possibility that this unusual response is due only to shielding of the nucleus by the cytoplasm. They have also shown that the principal site of damage in the cytoplasm is apparently nucleic acid, since this damage is highly photoreactivable. © 1969.