RESUMEN
Thousands of outer-arm dyneins (OADs) are arrayed in the axoneme to drive a rhythmic ciliary beat. Coordination among multiple OADs is essential for generating mechanical forces to bend microtubule doublets (MTDs). Using electron microscopy, we determined high-resolution structures of Tetrahymena thermophila OAD arrays bound to MTDs in two different states. OAD preferentially binds to MTD protofilaments with a pattern resembling the native tracks for its distinct microtubule-binding domains. Upon MTD binding, free OADs are induced to adopt a stable parallel conformation, primed for array formation. Extensive tail-to-head (TTH) interactions between OADs are observed, which need to be broken for ATP turnover by the dynein motor. We propose that OADs in an array sequentially hydrolyze ATP to slide the MTDs. ATP hydrolysis in turn relaxes the TTH interfaces to effect free nucleotide cycles of downstream OADs. These findings lead to a model explaining how conformational changes in the axoneme produce coordinated action of dyneins.
Asunto(s)
Dineínas/química , Dineínas/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Adenosina Trifosfato/metabolismo , Microscopía por Crioelectrón , Modelos Moleculares , Tetrahymena thermophila/citologíaRESUMEN
Non-invasive and label-free calorimetry could become a disruptive technique to study single cell metabolic heat production without altering the cell behavior, but it is currently limited by insufficient sensitivity. Here, we demonstrate microfluidic single-cell calorimetry with 0.2-nW sensitivity, representing more than ten-fold enhancement over previous record, which is enabled by (i) a low-noise thermometry platform with ultralow long-term (10-h) temperature noise (80 µK) and (ii) a microfluidic channel-in-vacuum design allowing cell flow and nutrient delivery while maintaining a low thermal conductance of 2.5 µW K-1. Using Tetrahymena thermophila as an example, we demonstrate on-chip single-cell calorimetry measurement with metabolic heat rates ranging from 1 to 4 nW, which are found to correlate well with the cell size. Finally, we perform real-time monitoring of metabolic rate stimulation by introducing a mitochondrial uncoupling agent to the microchannel, enabling determination of the spare respiratory capacity of the cells.
Asunto(s)
Calorimetría/métodos , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Análisis de la Célula Individual/métodos , Temperatura , Tetrahymena thermophila/metabolismo , Metabolismo Basal , Calorimetría/instrumentación , Microfluídica/instrumentación , Mitocondrias/metabolismo , Consumo de Oxígeno , Análisis de la Célula Individual/instrumentación , Tetrahymena thermophila/citología , Conductividad TérmicaRESUMEN
Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd^(2+) were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.
Asunto(s)
Citocinesis , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Microtúbulos/metabolismo , Mutación , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila , Proteína de Unión al GTP ran/metabolismo , Microtúbulos/patología , Mitosis , Proteínas Protozoarias/genética , Tetrahymena thermophila/citología , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Proteína de Unión al GTP ran/genéticaRESUMEN
During differentiation of the Tetrahymena thermophila somatic nucleus, its germline-derived DNA undergoes extensive reorganization including the removal of â¼50 Mb from thousands of loci called internal eliminated sequences (IESs). IES-associated chromatin is methylated on lysines 9 and 27 of histone H3, marking newly formed heterochromatin for elimination. To ensure that this reorganized genome maintains essential coding and regulatory sequences, the boundaries of IESs must be accurately defined. In this study, we show that the developmentally expressed protein encoded by Lia3-Like 1 (LTL1) (Ttherm_00499370) is necessary to direct the excision boundaries of particular IESs. In ΔLTL1 cells, boundaries of eliminated loci are aberrant and heterogeneous. The IESs regulated by Ltl1 are distinct from those regulated by the guanine-quadruplex binding Lia3 protein. Ltl1 has a general affinity for double stranded DNA (Kd â¼ 350 nM) and binds specifically to a 50 bp A+T rich sequence flanking each side of the D IES (Kd â¼ 43 nM). Together these data reveal that Ltl1 and Lia3 control different subsets of IESs and that their mechanisms for flanking sequence recognition are distinct.
Asunto(s)
ADN Protozoario/genética , Proteínas de Unión al ADN/genética , Heterocromatina/genética , Proteínas Protozoarias/genética , Tetrahymena thermophila/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Línea Celular , ADN Protozoario/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Heterocromatina/metabolismo , Unión Proteica , Proteínas Protozoarias/metabolismo , Homología de Secuencia de Aminoácido , Tetrahymena thermophila/citología , Tetrahymena thermophila/metabolismoRESUMEN
Eukaryotic motile cilia/flagella play vital roles in various physiological processes in mammals and some protists. Defects in cilia formation underlie multiple human disorders, known as ciliopathies. The detailed processes of cilia growth and development are still far from clear despite extensive studies. In this study, we characterized the process of cilium formation (ciliogenesis) by investigating the newly developed motile cilia of deciliated protists using complementary techniques in electron microscopy and image analysis. Our results demonstrated that the distal tip region of motile cilia exhibit progressive morphological changes as cilia develop. This developmental process is time-dependent and continues after growing cilia reach their full lengths. The structural analysis of growing ciliary tips revealed that B-tubules of axonemal microtubule doublets terminate far away from the tip end, which is led by the flagellar tip complex (FTC), demonstrating that the FTC might not directly mediate the fast turnover of intraflagellar transport (IFT).
Asunto(s)
Cilios/fisiología , Flagelos/fisiología , Organogénesis/fisiología , Tetrahymena thermophila/crecimiento & desarrollo , Animales , Cilios/ultraestructura , Flagelos/ultraestructura , Movimiento/fisiología , Tetrahymena thermophila/citología , Tetrahymena thermophila/ultraestructura , Factores de TiempoRESUMEN
Condensin is a protein complex with diverse functions in chromatin packaging and chromosome condensation and segregation. We studied condensin in the evolutionarily distant protist model Tetrahymena, which features noncanonical nuclear organization and divisions. In Tetrahymena, the germline and soma are partitioned into two different nuclei within a single cell. Consistent with their functional specializations in sexual reproduction and gene expression, condensins of the germline nucleus and the polyploid somatic nucleus are composed of different subunits. Mitosis and meiosis of the germline nucleus and amitotic division of the somatic nucleus are all dependent on condensins. In condensin-depleted cells, a chromosome condensation defect was most striking at meiotic metaphase, when Tetrahymena chromosomes are normally most densely packaged. Live imaging of meiotic divisions in condensin-depleted cells showed repeated nuclear stretching and contraction as the chromosomes failed to separate. Condensin depletion also fundamentally altered chromosome arrangement in the polyploid somatic nucleus: multiple copies of homologous chromosomes tended to cluster, consistent with a previous model of condensin suppressing default somatic pairing. We propose that failure to form discrete chromosome territories is the common cause of the defects observed in the absence of condensins.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Segregación Cromosómica , Proteínas de Unión al ADN/fisiología , Complejos Multiproteicos/fisiología , Tetrahymena thermophila/fisiología , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Cromatina/metabolismo , Hibridación Fluorescente in Situ , Meiosis/fisiología , Microscopía Fluorescente , Mitosis/fisiología , Poliploidía , Tetrahymena thermophila/citologíaRESUMEN
Ciliates such as Tetrahymena thermophila have two distinct nuclei within one cell: the micronucleus that undergoes mitosis and meiosis and the macronucleus that undergoes amitosis, a type of nuclear division that does not involve a bipolar spindle, but still relies on intranuclear microtubules. Ciliates provide an opportunity for the discovery of factors that specifically contribute to chromosome segregation based on a bipolar spindle, by identification of factors that affect the micronuclear but not the macronuclear division. Kinesin-14 is a conserved minus-end directed microtubule motor that cross-links microtubules and contributes to the bipolar spindle sizing and organization. Here, we use homologous DNA recombination to knock out genes that encode kinesin-14 orthologues (KIN141, KIN142) in Tetrahymena. A loss of KIN141 led to severe defects in the chromosome segregation during both mitosis and meiosis but did not affect amitosis. A loss of KIN141 altered the shape of the meiotic spindle in a way consistent with the KIN141's contribution to the organization of the spindle poles. EGFP-tagged KIN141 preferentially accumulated at the spindle poles during the meiotic prophase and metaphase I. Thus, in ciliates, kinesin-14 is important for nuclear divisions that involve a bipolar spindle.
Asunto(s)
Segregación Cromosómica , Cilióforos/genética , Cinesinas/genética , Cinesinas/fisiología , Meiosis , Mitosis , Tetrahymena thermophila/genética , Animales , Núcleo Celular , Cilióforos/citología , Técnicas de Inactivación de Genes , Cinesinas/clasificación , Cinesinas/ultraestructura , Macronúcleo , Profase Meiótica I , Metafase , Microtúbulos , Mutación , Filogenia , Proteínas Recombinantes , Huso Acromático , Polos del Huso , Tetrahymena/genética , Tetrahymena thermophila/citología , Tetrahymena thermophila/metabolismoRESUMEN
Members of the E2F family of transcription factors have been reported to regulate the expression of genes involved in cell cycle control, DNA replication, and DNA repair in multicellular eukaryotes. Here, E2FL1, a meiosis-specific E2F transcription factor gene, was identified in the model ciliate Tetrahymena thermophila. Loss of this gene resulted in meiotic arrest prior to anaphase I. The cytological experiments revealed that the meiotic homologous pairing was not affected in the absence of E2FL1, but the paired homologous chromosomes did not separate and assumed a peculiar tandem arrangement. This is the first time that an E2F family member has been shown to regulate meiotic events. Moreover, BrdU incorporation showed that DSB processing during meiosis was abnormal upon the deletion of E2FL1. Transcriptome sequencing analysis revealed that E2FL1 knockout decreased the expression of genes involved in DNA replication and DNA repair in T. thermophila, suggesting that the function of E2F is highly conserved in eukaryotes. In addition, E2FL1 deletion inhibited the expression of related homologous chromosome segregation genes in T. thermophila. The result may explain the meiotic arrest phenotype at anaphase I. Finally, by searching for E2F DNA-binding motifs in the entire T. thermophila genome, we identified 714 genes containing at least one E2F DNA-binding motif; of these, 235 downregulated represent putative E2FL1 target genes.
Asunto(s)
Meiosis/genética , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/citología , Tetrahymena thermophila/metabolismo , Factores de Transcripción/metabolismo , Anafase , Segregación Cromosómica/genética , ADN/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Recombinación Homóloga/genética , Mutación/genética , Motivos de Nucleótidos/genética , Fenotipo , Reproducción/genética , Tetrahymena thermophila/genéticaRESUMEN
Meiosis is an important process in sexual reproduction. Meiosis initiation has been found to be highly diverse among species. In yeast, it has been established that cyclin-dependent kinases (Cdks) and cyclins are essential components in the meiosis initiation pathway. In this study, we identified 4 Cdks in the model ciliate, Tetrahymena thermophila, and we found one of them, Cdk3, which is specifically expressed during early conjugation, to be essential for meiosis initiation. Cdk3 deletion led to arrest at the pair formation stage of conjugation. We then confirmed that Cdk3 acts upstream of double-strand break (DSB) formation. Moreover, we detected that Cdk3 is necessary for the expression of many genes involved in early meiotic events. Through proteomic quantification of phosphorylation, co-expression analysis and RNA-Seq analyses, we identified a conjugation-specific cyclin, Cyc2, which most likely partners with Cdk3 to initiate meiosis.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Meiosis , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/citología , Tetrahymena thermophila/enzimología , Secuencia de Aminoácidos , Puntos de Control del Ciclo Celular , Quinasas Ciclina-Dependientes/química , Roturas del ADN de Doble Cadena , Reparación del ADN , Mutación/genética , Proteínas Protozoarias/químicaRESUMEN
Although the role of cyclins in controlling nuclear division is well established, their function in ciliate meiosis remains unknown. In ciliates, the cyclin family has undergone massive expansion which suggests that diverse cell cycle systems exist, and this warrants further investigation. A screen for cyclins in the model ciliate Tetrahymena thermophila showed that there are 34 cyclins in this organism. Only 1 cyclin, Cyc17, contains the complete cyclin core and is specifically expressed during meiosis. Deletion of CYC17 led to meiotic arrest at the diakinesis-like metaphase I stage. Expression of genes involved in DNA metabolism and chromosome organization (chromatin remodeling and basic chromosomal structure) was repressed in cyc17 knockout matings. Further investigation suggested that Cyc17 is involved in regulating spindle pole attachment, and is thus essential for chromosome segregation at meiosis. These findings suggest a simple model in which chromosome segregation is influenced by Cyc17.
Asunto(s)
Anafase , Segregación Cromosómica , Ciclinas/metabolismo , Meiosis , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/citología , Tetrahymena thermophila/metabolismo , Secuencia de Aminoácidos , Ciclinas/química , Metafase , Mutación/genética , Proteínas Protozoarias/químicaRESUMEN
Tcb2 is a putative calcium-binding protein from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila. It has been hypothesized to participate in several calcium-mediated processes in Tetrahymena, including ciliary movement, cell cortex signaling, and pronuclear exchange. Sequence analysis suggests that the protein belongs to the calmodulin family, with N- and C-terminal domains connected by a central linker, and two helix-loop-helix motifs in each domain. However, its calcium-binding properties, structure and precise biological function remain unknown. Interestingly, Tcb2 is a major component of unique contractile fibers isolated from the Tetrahymena cytoskeleton; in these fibers, addition of calcium triggers an ATP-independent type of contraction. Here we report the (1)H, (13)C and (15)N backbone and side-chain chemical shift assignments of the C-terminal domain of the protein (Tcb2-C) in the absence and presence of calcium ions. (1)H-(15)N HSQC spectra show that the domain is well folded both in the absence and presence of calcium, and undergoes a dramatic conformational change upon calcium addition. Secondary structure prediction from chemical shifts reveals an architecture encountered in other calcium-binding proteins, with paired EF-hand motifs connected by a flexible linker. These studies represent a starting point for the determination of the high-resolution solution structure of Tcb2-C at both low and high calcium levels, and, together with additional structural studies on the full-length protein, will help establish the molecular basis of Tcb2 function and unique contractile properties.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Citoesqueleto/metabolismo , Resonancia Magnética Nuclear Biomolecular , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Tetrahymena thermophila/citología , Secuencia de Aminoácidos , Dominios ProteicosRESUMEN
Spermiogenesis is the differentiation of spermatids into motile sperm consisting of a head and a tail. The head harbors a condensed elongated nucleus partially covered by the acrosome-acroplaxome complex. Defects in the acrosome-acroplaxome complex are associated with abnormalities in sperm head shaping. The head-tail coupling apparatus (HTCA), a complex structure consisting of two cylindrical microtubule-based centrioles and associated components, connects the tail or flagellum to the sperm head. Defects in the development of the HTCA cause sperm decapitation and disrupt sperm motility, two major contributors to male infertility. Here, we provide data indicating that mutations in the gene Coiled-coil domain containing 42 (Ccdc42) is associated with malformation of the mouse sperm flagella. In contrast to many other flagella and motile cilia genes, Ccdc42 expression is only observed in the brain and developing sperm. Male mice homozygous for a loss-of-function Ccdc42 allele (Ccdc42(KO)) display defects in the number and location of the HTCA, lack flagellated sperm, and are sterile. The testes enriched expression of Ccdc42 and lack of other phenotypes in mutant mice make it an ideal candidate for screening cases of azoospermia in humans.
Asunto(s)
Fertilidad/genética , Proteínas/genética , Cabeza del Espermatozoide/metabolismo , Cola del Espermatozoide/metabolismo , Espermatozoides/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Cabeza del Espermatozoide/ultraestructura , Motilidad Espermática/genética , Cola del Espermatozoide/ultraestructura , Espermátides/crecimiento & desarrollo , Espermátides/metabolismo , Espermátides/ultraestructura , Espermatogénesis/genética , Espermatozoides/crecimiento & desarrollo , Espermatozoides/ultraestructura , Testículo/citología , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Tetrahymena thermophila/citología , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismoRESUMEN
The information about adverse effects of emerging contaminants on aquatic protozoa is very scarce. The growth inhibition effect, cell viability, genotoxicity and multixenobiotic resistance (MXR) responses of two commonly used antimicrobial agents, triclosan (TCS) and triclocarban (TCC) to protozoan Tetrahymena thermophila were investigated in this study. The results revealed that TCS and TCC can inhibit the growth of T. thermophila with 24h EC50 values of 1063 and 295µgL(-1), respectively. The impairment of plasma membrane was observed after 2h exposure of TCS or TCC at the level of mg/L. Furthermore, it is noticeable that at environmentally relevant concentration (1.0µgL(-1)), both TCS and TCC can lead to statistically significant DNA damage in T. thermophila, while the inhibition of growth and change of cell viability cannot be observed. Our results firstly provide the evidence for genotoxic effects of TCS and TCC on the freshwater protozoan. Additionally, both TCS and TCC were found to inhibit the efflux transporter activities, with the inhibitory potencies of 39% and 40% (using verapamil as a model inhibitor), respectively. Particularly, TCC could significantly down-regulate the expression of MXR related gene Abcb15, which encodes the membrane efflux protein that acting as P-gp in T. thermophila. The results raise the awareness of potential aquatic ecological and human health risks from the exposure of TCS and TCC, as they might potentiate the toxic effects by chemosensitizing with co-existing toxicants.
Asunto(s)
Carbanilidas/toxicidad , Resistencia a Medicamentos/efectos de los fármacos , Tetrahymena thermophila/citología , Tetrahymena thermophila/genética , Triclosán/toxicidad , Xenobióticos/toxicidad , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Supervivencia Celular/efectos de los fármacos , Interacciones Farmacológicas , Monitoreo del Ambiente/métodos , Agua Dulce/química , Mutágenos/toxicidad , Tetrahymena thermophila/efectos de los fármacos , Tetrahymena thermophila/crecimiento & desarrollo , Contaminantes Químicos del Agua/toxicidad , Xenobióticos/metabolismoRESUMEN
Deoxyribonucleases (DNases) play a major role in apoptotic DNA fragmentation/degradation, and apoptotic-like DNA degradation is also observed during conjugation of the ciliate Tetrahymena thermophila; however, the characteristics of neutral and acidic DNases are still undefined in its life stages. Here, we report the biochemical characterization of DNase activities displayed in three different Tetrahymena life stages in a comparative manner. Maximum DNase activity of Tetrahymena was observed under acidic conditions, indicating that Tetrahymena has strong DNase II-like activities. Zymography revealed that Tetrahymena has at least five distinct DNase activity bands at 28, 32, 33.8, 35.5, and 69-kDa, and that the activities at 32 and 33.8-kDa were also secreted into starvation buffer. Cofactor analysis demonstrated that Mg(2+) exerted inhibitory effects on neutral DNase activities. Unexpectedly, Mg(2+) and Ca(2+) had favorable effects on acidic DNase activities. The DNase activity profile of conjugating Tetrahymena cells revealed that the 32 and 33.8-kDa activities at pH 5.0 increased from 14 to 18 h of conjugation, corresponding to the final resorption of the old macronucleus by lysosomal enzymes during programmed nuclear death (PND). Overall, we found that Tetrahymena DNases exhibit different biochemical properties and a possible involvement of DNase II-like activities in PND.
Asunto(s)
Desoxirribonucleasas/metabolismo , Tetrahymena thermophila/enzimología , Apoptosis , Núcleo Celular/enzimología , Endodesoxirribonucleasas/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Macronúcleo/metabolismo , Tetrahymena thermophila/citología , Tetrahymena thermophila/efectos de los fármacosRESUMEN
Over time and repeated asexual divisions, many ciliate species display the characteristics of senescence, reduced fecundity and increased mortality. Their only path to recovery is sexual conjugation or autogamy. While more traditional models of cellular aging have been proposed, one of the most accepted explanations relies on the faulty mechanism by which ciliates duplicate their somatic nucleus, a process referred to as amitosis. Amitosis involves the random segregation of chromosomes with no consideration for homology. Over subsequent divisions, chromosome copy numbers will fluctuate until an entire chromosome is lost, resulting in death. Via simulations of this process, we find that senescence and death via chromosome loss is not the only possible result of amitosis. Random chromosome loss is less damaging to populations than previously thought, and strict adherence to the model predicts that Paramecium tetraurelia would not senesce. A combination of the reciprocal nature of amitosis and lethal selection against low-copy number chromosomes is responsible for this startling prediction. Additionally, our results provide an alternate explanation to recent evidence for selection on chromosome copy number in Tetrahymena thermophila and peculiar patterns of senescence in Tetrahymena pyriformis.
Asunto(s)
Cromosomas/genética , Cilióforos/genética , Núcleo Celular/metabolismo , Cilióforos/citología , Simulación por Computador , Ploidias , Reproducción Asexuada , Tetrahymena pyriformis/citología , Tetrahymena pyriformis/genética , Tetrahymena thermophila/citología , Tetrahymena thermophila/genéticaRESUMEN
Ciliate nuclear architecture, in particular the sequestration of a transcriptionally silent germline genome, allows for the accumulation of mutations that are "hidden" from selection during many rounds of asexual reproduction. After sexual conjugation, these mutations are expressed, potentially resulting in highly variable phenotypes. Morphological traits are widely used in ciliate taxonomy, however, the extent to which the values of these traits are robust to change in the face of mutation remains largely unknown. In this study, we examine the effects of mutations accumulated in the germline genome to test the mutational robustness of four traits commonly used in ciliate morphological taxonomy (number of somatic kineties, number of postoral kineties, macronuclear size, and cell size). We found that the number of postoral kineties is robust to mutation, confirming that it should be preferentially used in taxonomy. By contrast, we found that, as in other unicellular and multicellular species, cell and macronucleus sizes change in response to mutation. Thus, we argue that cell and macronucleus sizes, which are widely used in taxonomy, should be treated cautiously for species identification. Finally, we found evidence of correlations between cell and macronucleus sizes and fitness, suggesting possible mutational pleiotropy. This study demonstrates the importance of, and methods for, determining mutational robustness to guide morphological taxonomy in ciliates.
Asunto(s)
Mutación , Tetrahymena thermophila/citología , Tetrahymena thermophila/genética , Pleiotropía Genética , Genoma de ProtozoosRESUMEN
The eukaryotic reverse transcriptase telomerase copies its internal RNA template to synthesize telomeric DNA repeats at chromosome ends in balance with sequence loss during cell proliferation. Previous work has established several factors involved in telomerase recruitment to telomeres in yeast and mammalian cells; however, it remains unclear what determines the association of telomerase with telomeres in other organisms. Here we investigate the cell cycle dependence of telomere binding by each of the seven Tetrahymena thermophila telomerase holoenzyme proteins TERT, p65, Teb1, p50, p75, p45, and p19. We observed coordinate cell cycle-regulated recruitment and release of all of the subunits, including the telomeric-repeat DNA-binding subunit Teb1. Using domain truncation and mutagenesis approaches, we investigated which subunits govern the interaction of telomerase holoenzyme with telomeres. Our results show that Teb1 is critical for telomere interaction of other holoenzyme subunits and demonstrate that high-affinity Teb1 DNA-binding activity is necessary and sufficient for cell cycle-regulated telomere association. Overall, these and additional findings indicate that in the ciliate Tetrahymena, telomerase recruitment to telomeres requires direct binding to single-stranded DNA, unlike the indirect DNA recognition through telomere-bound proteins essential in yeast and mammalian cells.
Asunto(s)
ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Protozoarias/metabolismo , Telomerasa/metabolismo , Telómero/metabolismo , Tetrahymena thermophila/metabolismo , Animales , Ciclo Celular , Proteínas de Unión al ADN/análisis , Modelos Moleculares , Unión Proteica , Proteínas Protozoarias/análisis , Telomerasa/análisis , Tetrahymena thermophila/citologíaRESUMEN
BACKGROUND: By segregating somatic and germinal functions into large, compound macronuclei and small diploid micronuclei, respectively, ciliates can explore sexuality in ways other eukaryotes cannot. Sex, for instance, is not for reproduction but for nuclear replacement in the two cells temporarily joined in conjugation. With equal contributions from both conjugants, there is no cost of sex which theory predicts should favor asexuality. Yet ciliate asexuality is rare. The exceptional Tetrahymena has abandoned sex through loss of the micronucleus; its amicronucleates are abundant in nature where they reproduce by binary fission but never form conjugating pairs. A possible reason for their abundance is that the Tetrahymena macronucleus does not accumulate mutations as proposed by Muller's ratchet. As such, Tetrahymena amicronucleates have the potential to be very old. This study used cytochrome oxidase-1 barcodes to determine the phylogenetic origin and relative age of amicronucleates isolated from nature. RESULTS: Amicronucleates constituted 25% of Tetrahymena-like wild isolates. Of the 244 amicronucleates examined for cox1 barcodes, 237 belonged to Tetrahymena, seven to other genera. Sixty percent originated from 12 named species or barcoded strains, including the model Tetrahymena thermophila, while the remaining 40% represent 19 putative new species, eight of which have micronucleate counterparts and 11 of which are known only as amicronucleates. In some instances, cox1 haplotypes were shared among micronucleate and amicronucleates collected from the same source. Phylogenetic analysis showed that most amicronucleates belong to the "borealis" clade in which mating type is determined by gene rearrangement. Some amicronucleate species were clustered on the SSU phylogenetic tree and had longer branch lengths, indicating more ancient origin. CONCLUSIONS: Naturally occurring Tetrahymena amicronucleates have multiple origins, arising from numerous species. Likely many more new species remain to be discovered. Shared haplotypes indicate that some are of contemporary origin, while phylogeny indicates that others may be millions of years old. The apparent success of amicronucleate Tetrahymena may be because macronuclear assortment and recombination allow them to avoid Muller's ratchet, incorporate beneficial mutations, and evolve independently of sex. The inability of amicronucleates to mate may be the result of error(s) in mating type gene rearrangement.
Asunto(s)
Tetrahymena/clasificación , Tetrahymena/fisiología , Núcleo Celular/genética , Conjugación Genética , Ciclooxigenasa 1/genética , ADN Protozoario/genética , Filogenia , Reproducción , Reproducción Asexuada , Tetrahymena/citología , Tetrahymena/genética , Tetrahymena thermophila/citología , Tetrahymena thermophila/genéticaRESUMEN
In Tetrahymena thermophila, peptides secreted via dense-core granules, called mucocysts, are generated by proprotein processing. We used expression profiling to identify candidate processing enzymes, which localized as cyan fluorescent protein fusions to mucocysts. Of note, the aspartyl cathepsin Cth3p plays a key role in mucocyst-based secretion, since knockdown of this gene blocked proteolytic maturation of the entire set of mucocyst proproteins and dramatically reduced mucocyst accumulation. The activity of Cth3p was eliminated by mutation of two predicted active-site mutations, and overexpression of the wild-type gene, but not the catalytic-site mutant, partially rescued a Mendelian mutant defective in mucocyst proprotein processing. Our results provide the first direct evidence for the role of proprotein processing in this system. Of interest, both localization and the CTH3 disruption phenotype suggest that the enzyme provides non-mucocyst-related functions. Phylogenetic analysis of the T. thermophila cathepsins, combined with prior work on the role of sortilin receptors in mucocyst biogenesis, suggests that repurposing of lysosomal enzymes was an important step in the evolution of secretory granules in ciliates.
Asunto(s)
Carboxipeptidasas/metabolismo , Catepsinas/metabolismo , Exocitosis , Vesículas Secretoras/metabolismo , Tetrahymena thermophila/citología , Catepsinas/genética , Mutación , Filogenia , Tetrahymena thermophila/genéticaRESUMEN
When nanoparticles can enter a unicellular organism directly, how may they affect the bioaccumulation and toxicity of other pollutants already present in the environment? To answer this question, we conducted experiments with a protozoan Tetrahymena thermophila. The well-dispersed polyacrylate-coated TiO2 nanoparticles (PAA-TiO2-NPs) were used as a representative nanomaterial, and Cd as a conventional pollutant. We found that PAA-TiO2-NPs could get into Tetrahymena cells directly. Such internalization was first induced by low concentrations of Cd, but later suppressed when Cd concentrations were higher than 1 µg/L. Considering its significant adsorption on PAA-TiO2-NPs, Cd could be taken up by T. thermophila in the form of free ion or metal-nanoparticle complexes. The latter route accounted for 46.3% of Cd internalization. During the 5 h depuration period, 4.34-22.1% of Cd was excreted out, which was independent of the concentrations of intracellular Cd and PAA-TiO2-NPs. On the other hand, both free and intracellular Cd concentrations only partly predicted its toxicity at different levels of PAA-TiO2-NPs. This may have resulted from PAA-TiO2-NPs' synergistic effects and the distinct subcellular distribution of Cd taken up via the two routes above. Overall, we should pay attention to the carrier effects of nanoparticles when assessing their environmental risks.