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International Journal of Molecular... Dec 2023DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in or its eukaryotic homolog, the MutSα (Msh2-Msh6)...
DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6 localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6 only localized in MICs and newly developing MACs. knockout led to aberrant nuclear division during vegetative growth. The mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6 interacted with Msh2 and MMR-independent factors. Downregulation of expression affected the stability of Msh6. In addition, knockout led to the upregulated expression of several homologs at different developmental stages. Msh6 is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in .
Topics: DNA Mismatch Repair; Tetrahymena thermophila; MutS Homolog 2 Protein; Escherichia coli; DNA-Binding Proteins; Meiosis; Gametogenesis
PubMed: 38139447
DOI: 10.3390/ijms242417619 -
Microorganisms Dec 2022Fertilization-initiated development and adult-onset aging are standard features in the life history of eukaryotes. In , the number of cell divisions after the birth of a...
Fertilization-initiated development and adult-onset aging are standard features in the life history of eukaryotes. In , the number of cell divisions after the birth of a new generation is an essential parameter of sexual phase transition and aging. However, the gene driving this process and its evolutionary origin have not yet been elucidated. Here we report several critical outcomes obtained by molecular genetics, immunofluorescence microscopy, transformation by microinjection, and enzymological analysis. The cloned immaturin gene induces sexual rejuvenation in both mature and senescent cells by microinjection. The immaturin gene originated from proteobacteria's glutathione-S-transferase (GST) gene. However, immaturin has been shown to lose GST activity and instead acquire nuclease activity. In vitro substrates for immaturin-nuclease are single- and double-stranded DNA, linear and circular DNA, and single-stranded viral genome RNA such as coronavirus. Anti-immaturin antibodies have shown that the subcellular localizations of immaturin are the macronucleus, cytoplasm, cell surface area, and cilia. The phase transition of sexuality is related to a decrease in the intracellular abundance of immaturin. We propose that sexual maturation and rejuvenation is a process programmed by the immaturin gene, and the sexual function of each age is defined by both the abundance and the intracellular localization mode of the immaturin-nuclease.
PubMed: 36677375
DOI: 10.3390/microorganisms11010082 -
Microorganisms Jan 2020Ciliates form a distinct group of single-celled eukaryotes that host two types of nuclei (micro and macronucleus) in the same cytoplasm and have a special sexual process...
Ciliates form a distinct group of single-celled eukaryotes that host two types of nuclei (micro and macronucleus) in the same cytoplasm and have a special sexual process known as conjugation, which involves mitosis, meiosis, fertilization, nuclear differentiation, and development. Due to their high species diversity, ciliates have evolved different patterns of nuclear events during conjugation. In the present study, we investigate these events in detail in the marine species . Our results indicate that: (i) conjugation lasts for about 50 hours, the longest stage being the development of the new macronucleus (ca. 36 hours); (ii) there are three prezygotic micronuclear divisions (mitosis and meiosis I and II) and two postzygotic synkaryon divisions; and (iii) a fragment of the parental macronucleus fuses with the new developing macronucleus. In addition, we describe for the first time conjugation in amicronucleate cells. When two amicronucleate cells mate, they separate after about 4 hours without evident nuclear changes; when one amicronucleate cell mates with a micronucleate cell, the micronucleus undergoes regular prezygotic divisions to form migratory and stationary pronuclei, but the two pronuclei fuse in the same cell. In the amicronucleate cell, the parental macronucleus breaks into fragments, which are then recovered to form a new functional macronucleus. These results add new information on the process of conjugation in both micronucleate and amicronucleate cells.
PubMed: 31979289
DOI: 10.3390/microorganisms8020162 -
ELife Mar 2021Dynamins are targeted to specific cellular membranes that they remodel via membrane fusion or fission. The molecular basis of conferring specificity to dynamins for...
Dynamins are targeted to specific cellular membranes that they remodel via membrane fusion or fission. The molecular basis of conferring specificity to dynamins for their target membrane selection is not known. Here, we report a mechanism of nuclear membrane recruitment of Drp6, a dynamin member in . Recruitment of Drp6 depends on a domain that binds to cardiolipin (CL)-rich bilayers. Consistent with this, nuclear localization of Drp6 was inhibited either by depleting cellular CL or by substituting a single amino acid residue that abolished Drp6 interactions with CL. Inhibition of CL synthesis, or perturbation in Drp6 recruitment to nuclear membrane, caused defects in the formation of new macronuclei post-conjugation. Taken together, our results elucidate a molecular basis of target membrane selection by a nuclear dynamin and establish the importance of a defined membrane-binding domain and its target lipid in facilitating nuclear expansion.
Topics: Amino Acid Sequence; Cardiolipins; Dynamins; Nuclear Envelope; Protein Binding; Protozoan Proteins; Sequence Alignment; Tetrahymena thermophila
PubMed: 33661098
DOI: 10.7554/eLife.64416 -
Protist Apr 2021Spirostomum is a widely distributed heterotrichean genus composed of well-known species with described ecology and phylogenetic affinities. The morphological...
Macronuclear Plasticity in Two South American Populations of Spirostomum (Ciliophora, Heterotrichea) Warns About its Use for Species Classification: Revision and New Insights.
Spirostomum is a widely distributed heterotrichean genus composed of well-known species with described ecology and phylogenetic affinities. The morphological classification of Spirostomum species is mostly based on the body size/shape, number of cortical granule rows and macronuclear characteristics. These features along with molecular phylogenies based on ribosomal genes divide the genus into two phylogroups, one including species with a compact macronucleus, and another including species with a moniliform macronucleus. Here, we present our observations on atypical Spirostomum specimens with unusually two distinct macronuclei and shortened adoral zone of membranelles. These atypical forms appeared in the cultures of S. minus and S. yagiui, sampled at different sites in South America (Chile and Brazil) and associated with unrelated substrate types. Morphological observations of living and stained cells, 18S rRNA gene analyses, and a thorough investigation of the literature suggest that the atypical phenotype may be a result of uncommon pathways during the conjugative process. Thus, we demonstrate that studies of ciliate natural populations and their morphological variations, especially from undersampled biogeographical regions, can reveal the boundaries of widely used morphological characters for Spirostomum taxonomy and species identification.
Topics: Ciliophora; Classification; Genetic Variation; Macronucleus; South America; Species Specificity
PubMed: 33940500
DOI: 10.1016/j.protis.2021.125803 -
European Journal of Protistology Apr 2023Although the river nerite Theodoxus fluviatilis (Gastropoda: Neritimorpha) has an exceptionally broad geographical and ecological distribution, it carries only four...
Re-discovery and novel contributions to morphology and multigene phylogeny of Protospirella mazurica (Raabe, 1968) Aescht, 2001 (Ciliophora: Pleuronematida), an obligate symbiont of the river nerite Theodoxus fluviatilis Linnaeus, 1758 (Mollusca: Gastropoda).
Although the river nerite Theodoxus fluviatilis (Gastropoda: Neritimorpha) has an exceptionally broad geographical and ecological distribution, it carries only four ciliate species: Hypocomella quatuor, Protospirella mazurica, Scyphidia sp., and Trichodina baltica. After more than a half-century gap, we re-discovered P. mazurica in a Danubian population of T. fluviatilis (haplotypes F31 and F34) and characterized it using an integrative morpho-molecular approach. Protospirella mazurica is distinguished by (i) a small, elongate-ellipsoidal to ovoidal body, (ii) a broadly ellipsoidal macronucleus accompanied by a single globular micronucleus, (iii) a subterminal contractile vacuole, (iv) about 24 somatic kineties, (v) thigmotactic ciliature composed of about 10 kineties shortened posteriorly to form a parenthetical system, and (vi) a long inverted J-shaped paroral membrane associated with three unequally long membranelles. According to the present phylogenetic analyses of two mitochondrial and three nuclear markers, P. mazurica robustly clusters within the order Pleuronematida (Oligohymenophorea: Scuticociliatia) along with other symbiotic members of the families Hemispeiridae and Thigmophryidae as well as free-living representatives of the paraphyletic family Cyclidiidae. In light of the present phylogenetic analyses, we consider the family Ancistridae to be a junior synonym of the family Hemispeiridae, which collates 14 genera in our classification framework.
Topics: Humans; Animals; Phylogeny; Gastropoda; Rivers; Oligohymenophorea; Ciliophora; Mollusca; China
PubMed: 36805973
DOI: 10.1016/j.ejop.2023.125956 -
Marine Life Science & Technology Aug 2023During a study on the diversity of ciliated protists in Lake Weishan Wetland, the largest wetland in northern China, four epibiotic sessilid peritrichs were isolated...
On four epibiotic peritrichous ciliates (Protozoa, Ciliophora) found in Lake Weishan Wetland: morphological and molecular data support the establishment of a new genus, gen. nov., and two new species.
During a study on the diversity of ciliated protists in Lake Weishan Wetland, the largest wetland in northern China, four epibiotic sessilid peritrichs were isolated from aquatic host animals. Two of them, i.e., Kellicott, 1885 and Fauré-Fremiet, 1943, were known species whereas the other two, i.e., gen. nov., sp. nov. and sp. nov., are new to science. The new genus gen. nov. is characterized by its branched non-contractile stalk, everted peristomial lip, obconical macronucleus and transverse silverlines. Two species are assigned to the new genus, namely sp. nov. and (Fauré-Fremiet, 1905) comb. nov. Morphologically, sp. nov. is recognized by its goblet-shaped zooids, single-layered peristomial lip, dichotomously branched stalk, and infundibular polykinety 3 (P3) containing three equal-length rows. sp. nov. is characterized by its slender zooid, curved macronucleus, and three equal-length rows in infundibular P3. Improved diagnoses and redescriptions of and are provided including, for the first time, data on the ciliature of . Phylogenetic analyses based on SSU rDNA, ITS1-5.8S rDNA -ITS2, and LSU rDNA sequence data strongly support the assertion that the family Epistylididae comprises morphospecies with different evolutionary lineages and indicate that gen. nov. may represent a new taxon at family level.
PubMed: 37637257
DOI: 10.1007/s42995-023-00184-z -
Microorganisms Feb 2023The conjusome plays an important role in the conjugation events that occur in . The conjusome appears in the anterior of conjugant pairs during the early stages of new...
The conjusome plays an important role in the conjugation events that occur in . The conjusome appears in the anterior of conjugant pairs during the early stages of new macronuclei (anlagen) development. It lacks a membrane, and is composed of a network of fibrous, electron dense material, containing background cytoplasm and ribosomes. Several proteins localize to this organelle, including Pdd1p, a chromodomain protein that participates in the formation of chromatin-containing structures in developing macronuclear anlagen, and is associated with the elimination of specific germ-line sequences from developing macronuclei. Conjugants lacking the allele in the parental macronucleus do not show Pdd1p antibody staining in conjusomes. Investigations were performed using mutant cell lines, uniparental cytogamy and drug treatment, and show that the conjusome appears to be dependent on parental macronuclei condensation, and is a transitory organelle that traffics nuclear determinants from the parental macronucleus to the developing anlagen. These data, taken together with Pdd1p knockout experiments, suggest the conjusome is involved in the epigenetic phenomena that occur during conjugation and sexual reorganization. This is likely a conserved organelle. Conjusome-like structures were also observed in another Ciliate, . In general, conjusomes have features that resemble germ line P-granules.
PubMed: 36838383
DOI: 10.3390/microorganisms11020418 -
Frontiers in Cell and Developmental... 2019In the ciliate , a 631-amino acid -MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key...
In the ciliate , a 631-amino acid -MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key component of an autocrine, cell-growth promoting self-signaling mechanism. While its 283-amino acid N-terminal domain includes all the structural specificities of the mitogen-activated protein kinases required for a catalytic function, the 348-amino acid C-terminal domain is structurally unique with undetermined functions. By expressing the two -MAPK1 domains tagged with the green fluorescent protein in mammalian fibroblasts, the yeast and the ciliate , evidence was obtained that the C-terminal domain contains all the sequence information responsible for the -MAPK1 subcellular localization. However, in fibroblasts and this information determined a nucleolar localization of the GFP-tagged C-terminal domain, and a ciliary localization in . In the light of these findings, the -MAPK1 localization in was re-examined via immunoreactions and shown to be ciliary besides that nuclear, as is the case for the mammalian intestinal cell kinase with which the -MAPK1 N-terminal domain shares a strong sequence identity and a catalytic function.
PubMed: 31681773
DOI: 10.3389/fcell.2019.00244 -
Zootaxa Feb 2020The morphologies of the three freshwater stentorid ciliates in Korea, Stentor coeruleus (Pallas, 1766); Stentor muelleri Ehrenberg, 1831, and Stentor tartari Murthy Bai,...
The morphologies of the three freshwater stentorid ciliates in Korea, Stentor coeruleus (Pallas, 1766); Stentor muelleri Ehrenberg, 1831, and Stentor tartari Murthy Bai, 1974, were investigated based on live observations and protargol impregnation. The Korean population of S. tartari exhibits the following characteristics: body size 200-355 × 85-135 µm in vivo, 62-106 somatic kineties, 8-13 peristomial kineties, 110-180 adoral membranelles, mostly two macronuclear nodules and 5-18 micronuclei, reddish and colorless cortical granules and the presence of symbiotic algae. We identified S. tartari based on unique characteristics compared to close congeners. Korean populations of S. coeruleus and S. muelleri are congruent with previously described populations in most aspects of their morphologies. Here, for the first time, we report molecular gene sequence information for S. tartari. Small subunit (SSU) rRNA gene sequence-based phylogeny indicates that S. tartari, which has multiple macronuclei, forms a monophyletic group with other Stentor species having a single macronucleus. Our findings based on morphology and SSU rRNA gene sequence information corroborate the hypothesis that the elongated macronucleus evolved from the compact single or multi macronucleus state.
Topics: Animals; China; Ciliophora; DNA, Protozoan; Phylogeny; Republic of Korea
PubMed: 32230251
DOI: 10.11646/zootaxa.4732.3.6