Functional Analysis Of Ran GTPase During Macronuclear Amitosis And Programmed Nuclear Death In Tetrahymena Thermophila

Ran (Ras-like nuclear protein), a member of the small GTPase superfamily, is abundant in the nucleus of eukaryotic cells. Ran is an important cell proliferation regulatory factor that coordinates multiple cellular activities, including nucleocytoplasmic transport, nuclear envelope assembly, spindle assembly, DNA replication, RNA transcription, mRNA processing and transport and cell cycle regulation. Although very diverse, all these processes depend on the GDP-and GTP-bound forms of Ran interacting differentially with downstream targets. Since homologous proteins of Ran play multiple roles in mitosis, meiosis and apoptosis, the study of Ran in evolutionarily different species helps further clarify the biological roles of the protein.The ciliated protozoan Tetrahymena thermophila has all the necessary functions for life sustaining and continuation of offspring in the single cell, and undergoes complicated cell cycle progression and nuclear development process throughout the life cycle. As a unicellular eukaryote, Tetrahymena is an ideal model organism for the research on the roles of proteins. The life cycle of Tetrahymena includes both asexual and sexual reproductive stages. During amitosis of asexual reproduction, intra-macronuclear microtubules are reorganized into specified arrays which assist in separation of macronucleus, despite lack of a bipolar spindle. During the sexual reproduction, when the new micro-and macronuclei differentiate, parental macronucleus degenerates through programmed nuclear death (PND). The molecular mechanisms underlying macronuclear amitosis and parental macronuclear PND are not clear. To study the roles of Ran in macronuclear amitosis and parental macronuclear PND of Tetrahymena, the following results are obtained:1. Bioinformatics analysis of Tetrahymena RAN1gene RAN1, the Tetrahymena Ran protein-coding gene, is1443bp in full-length, which encodes for225amino acids with the predicted molecular weight of25.6kD. Tetrahymena Ran1has high sequence identity to Ran proteins of animal and plant cells. Phylogenetic analysis shows that the evolutionary relationship between Tetrahymena Ran1and Paramecium Ran is the most recent. Tetrahymena Ran1contains the GDP/GTP binding domain and switch regions which both are structurally the same for all members of the small GTPase superfamily. Ran1also contais the accessory factors-binding domains and acidic C-terminal tail which both are specific for the members of Ran subfamily. Ran1is sustained high expression throughout all stages of Tetrahymena life cycle. The functional domains of Tetrahymena Ran1accessory proteins are divergent to other Ran sequences indicating that the functions of Tetrahymena Ran1and other homologous proteins may be similar, but there are differences.2. Ran1coordinates amitotic progression during asexual reproduction of Tetrahym ena The localizations of HA-tagged Ran1and its mutants that mimic the GDP-and GTP bound states, Ran1T25N and Ran1Q70L throughout the cell cycle of asexual reproduction were different. And the accessory factors-binding domains of Ran1contributed to the macronuclear localizations of distinct nucleotide bound states of Ran1. RAN1incomplete knockout strain, iARAN1was obtained through homologous recombination. Incomplete knockout of RAN1resulted in the decrease of cell proliferation rate and abnormal macronuclear amitosis. Immunofluorescence analysis showed that incomplete knockout of RAN1resulted in aberrant intramacronuclear microtubule array formation, missegregation of macronuclear chromosomes and ultimately blocked macronuclei proliferation. Overexpression of Ran1WT/T25N/Q70L disrupted the Ran1cycle and affected the macronuclear amitotic progress leading to a decline in cell proliferation rate. Ran1and its GTP-and GDP-bound mimetic mutants overexpression levels were positively correlated with abnormal amitotic cells ratio. Overexpression of Ran1T25N elicited more pronounced inhibition of macronuclear amitosis and intra-macronuclear microtubule assembly than Ran1WT and Ran1Q70L. Moreover, overexpression of Ran1and its mutants destructed the distribution patterns of endogenous Ran1GDP and Ran1GTP. These results suggest that Ran GTPase pathway is involved in assembly of a specialized intramacronuclear microtubule network and coordinates amitotic progression in Tetrahymena.3. Ran1regulates parental macronuclear PND during sexual reproduction of Tetrahym ena We first observed the nuclear developmental stages during conjugation especially the stages of nuclear PND in conjugating iARANl cells, and found that conjugating iARAN1cells initiated normal nuclear events during early stages of conjugation, and the inhibition of three haploid meiotic micronuclear and parental macronuclear PND was occurred at late conjugation stages. Whereas, the suppression of PND progression recovered to some degree in mating cells between iARANl and OERan1cells. Immunofluorescence analysis of the dynamic localizations of HA-Ranl WT/T25N/Q70L during conjugation showed that Ran1GDP/GTP cycle may occur across the degenerating parental nucleoplasm barrier. We also determined if Ran1was involved in translocation of apoptosis-inducing factor (AIF) from mitochondria to parental macronucleus. The iARANl mutant failed to show parental macronuclear accumulation of AIF. Moreover, the Ran1GDP mimetic prompted AIF macronuclear import, while Ran1GTP mimetic inhibited AIF import, suggesting that Ran1GDP/GTP cycle may regulate import of AIF to the degenerating parental macronucleus. Artificial accumulation AIF in the parental macronucleus by the Ran1-independent and improtin a-mediated nuclear transport system, restored nuclear development and process of parental macronuclear PND to a certain extent in the iARAN1mutant. These results indicate a novel role of Ran1in regulation of programmed nuclear death through controlling nuclear import of AIF.This is the first study designed to assess the localization and role of Ran1in the protozoan cell. And we found that Ran1regulates macronuclear amitosis during asexual reproduction of Tetrahymena thermophila, and plays an important role in parental macronuclear programmed nuclear death during sexual reproduction through regulation of AIF signal. These results suggest that Ran GDP/GTP cycle is a conservative regulatory mechanism in eukaryotic cells.