Functional Comparison Of Cyclin-dependent Kinase Subunits Cks1 And Cks2 In Tetrahymena Thermophila

Cyclin dependent kinase subunits(Cks)are the regulatory subunits of cyclin dependent kinases(Cdk),which are highly conserved in evolution.Cks,together with the corresponding Cdk and Cyclins,can precisely regulate different developmental stages of cells in the form of complexes.Although eukaryotes have numerous cyclins and their cyclin-dependent kinases to form Cyclin-Cdk complexes for precisely regulating the development process of organisms,there is generally only one type of Cks in lower eukaryotes,and there are two kinds of Cks in higher eukaryotes.The details of how the Cks regulating the activity of Cyclin-Cdk in different biological development stages are still unclear.Tetrahymena thermophila is a single cell eukaryote with an intricate developmental processinvolved in a large number of cyclins and cyclin-dependent kinases.We found out two Cks homologous proteins through the alignment of Cks amino acid sequences form Tetrahymena thermophila transcriptome database,named Cksl and Cks2.We analyzed the localization and function of Cks1 and Cks2 during sexual reproduction.The main results are as follows:1.Bioinformatics analysis of Cksl and Cks2 Two CKS genes,CKS1(TTHERM₀0794670)and CKS2(TTHERM₀1248890)in Tetrahymena thermophila were analyzed by bioinformation.The results of qPCR of their expression profiles showed that almost did not express during vegetative growth,express highly during starvation and sexual reproduction,and the highest expression level arose at 2 h.The BLAST results by NCBI website showed that both Cks1 and Cks2 contained a conserved CKS domain.These two proteins are homologous with Cks proteins from Homo,Saccharomyces cerevisiae,Xenopus laevis,Drosophila melanogaster,and Caenorhabditis elegans.The Phylogenetic tree showed that Cks1 from Tetrahymena and Cks from Saccharomyces cerevisiae had similar evolutionary status,and Cks2 protein had similar evolutionary status to that from Hornworm.2.Both Cksl and Cks2 are located in the cytoplasm In order to study the functions of Cks1 and Cks2,their localization signal was analyzed.First,the endogenous expression recombinant plasmids pNeo4-CKS1-3HA and CKS2-3HA mutant strains were constructed.Indirect immunofluorescence assays showed that Cks1 and Cks2 were both localized in the cytoplasm.3.The loss of CKS1 inhibits cellular recognition of different mating cells In order to further study the function of Cks1 and Cks2,CKS1 knockout cell lines were paired with different mating types.The results showed that the loss of CKS1 expression led to the decrease of cell pairing rate of different mating types of Tetrahymena cell.Knockout CKS2 mutant cells were obtained and the sexual development process was analyzed.The results showed that the lack of CKS2 expression did not affect the sexual development of Tetrahymena.4.Overexpression of CKS2 inhibits the mitosis of the gametic and zygotic nucleus of Tetrahymena thermophila In order to further study the localization details and functions of Cks1 and Cks2,an overexpression mutant cell line OE-CKS1 and OE-CKS2 containing a 2HA tag at the N-terminal of the MTT1 promoter was constructed.The results of immunofluorescence localization showed that both overexpression of CKS1 and CKS2 were still localized in the cytoplasm.The analysis of the sexual reproduction progress of OE-CKS1 and OE-CKS2 showed that overexpression of CKS1 did not affect the conjugational stages of Tetrahymena thermophila,but overexpression of CKS2 resulted that the development of the gametic or zygotic nuclei was arrested.While about 20%of the paired cells completed the normal conjugation stages ly,about 80%of the gametic nuclei are delayed in the nuclear exchange stage.Even if 29%of the pairs completed the first mitosis of the zygotic nucleus,the zygotic nucleus failed to undergo the second mitosis,and abnormal swelled new micronuclei occured.Overexpression CKS2 ventually resulted in the abortion of Tetrahymena the sexual reproduction.We supposed that the stable expression of CKS2 in the middle and late conjugational stages of Tetrahymena meight affect the exit of the mitotic S phase of gametic and zygotic nuclei and blocked their entry of mitotic M phase.So that the progeny could not be produced.5.The Analysis of mutual relationship of Cksl and Cks2 In order to answer that whether Cks1 and Cks2 have a functional redundancy,qPCR results showed that the relative expression level of CKS1 was up-regulated when CKS2 was deleted from genome.Similarly,the relative expression level of CKS2 was also up-regulated when CKS2 was destroyed.However,after 2 hours of sexual reproduction,the relative expression of CKS2 was significantly down regulated when CKS1 was deleted from genome.The results showed that the functions of the two CKS in Tetrahymena were partly redundant and had their own unique functions.This study is the first to analyze the function of two cyclin dependent kinase subunits Cks1 and cks2 in Tetrahymena thermophila and the relationship between them.Both CKS1 and CKS2 were highly expressed at 2 h during the initiation of sexual reproduction of Tetrahymena thermophila,and they were both localizated in the cytoplasm.The knockout of CKS1 caused Tetrahymena thermophila to the decrease of cell pairing rate.This study showed that the knockout of CKS2 and Overexpression of CKS1 did not affect the normal sexual reproduction development.Howeve,overexpression of CKS2 caused the failure of the sexual reproduction of Tetrahymena thermophila.Although there is a complementary relationship between CKS1 and CKS2,CKS1 and CKS2 are still necessary for the normal completion of Tetrahymena thermophila sexual reproduction.This study further improved the details of regulation mechanism of Tetrahymena thermophila sexual reproductive development,and laid a scientific foundation for the in-depth understanding of the function of Cks in the sexal development process of eukaryotic organisms.