A novel mitochondrial nuclease-associated protein: a major executor of the programmed nuclear death in Tetrahymena thermophila.

BACKGROUND INFORMATION: Programmed nuclear death (PND) in the ciliate Tetrahymena is an apoptosis-like phenomenon that occurs in a restricted space of cytoplasm during conjugation. In the process, only the parental macronucleus is selectively eliminated from the progeny cytoplasm, in conjunction with differentiation of new macronuclei for the next generation. For the last decade, mitochondria have been elucidated to be a crucial executioner like apoptosis: apoptosis-inducing factor and yet-unidentified nucleases localised in mitochondria are major factors for PND. RESULTS: To identify such nucleases, we performed a DNase assay in a PAGE (SDS-DNA-PAGE) using total mitochondrial proteins. Some proteins showed DNase activity, but particularly a 17 kDa protein exhibited the highest and predominant activity. Mass spectrometric analysis revealed a novel mitochondrial nuclease, named TMN1, whose homologue has been discovered only in the ciliate Paramecium tetraurelia, but not in other eukaryotes. Gene disruption of TMN1 led to a drastic reduction of mitochondrial nuclease activity and blocked nuclear degradation during conjugation, but did not affect accumulation of autophagic and lysosomal machinery around the parental macronucleus. CONCLUSIONS: These observations strongly suggest that the mitochondrial nuclease-associated protein plays a key role in PND as a major executor. Taking the novel protein specific to ciliates in consideration, Tetrahymena would have diverted a different protein from common apoptotic factors shared in eukaryotes to PND in the course of ciliate evolution.

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila.

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible "attack me" signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.

Cytogenetic monitoring of domestic mammals exposed to wastewaters from the localities of Dladla and Boukallou near Settat, Morocco.

The aim of this work was to know the biological impact of wastewaters on livestock in the localities of Dladla and Boukallou (Town of Settat, Morocco), where wastewaters are largely used by the local population for their agricultural activities. We used the micronucleus (MN) test in cytochalasin B blocked binucleated cells (BNC) to assess the possible induced genetic effects. The cellular proliferation index (PI) was also calculated. Blood samples were collected from sheep, bovines and mules and peripheral blood cultures were made according to our laboratories' standard methodology. The results showed a significant increase of micronucleated cells in the lymphocytes of the exposed animals (14+/-8.79 MN/500 BNC) compared to the control (3+/-1.54 MN/500 BNC). The PI showed a nonsignificant decrease in the exposed animals. The highest MN frequency was found in mules which is probably due to their more sedentary life. Direct use of the wastewater by the animals as their source of drinking water significantly increased the frequency of cells with micronuclei. We also established an inverse correlation between the chromosome number and the PI. These results suggest a serious degradation of the ecosystem due to the wastewaters with a direct impact on its inhabitants.