RESUMO
TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.
Assuntos
Algoritmos , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodos , Processamento de Imagem Assistida por Computador/métodos , Imageamento Tridimensional/métodos , Software , Proteínas Arqueais/química , Proteínas Arqueais/ultraestrutura , Axonema/química , Axonema/ultraestrutura , Endopeptidases/química , Endopeptidases/ultraestrutura , Movimento (Física) , Reprodutibilidade dos Testes , Tetrahymena thermophila/ultraestruturaRESUMO
Telomerase is unique among the reverse transcriptases in containing a noncoding RNA (known as telomerase RNA (TER)) that includes a short template that is used for the processive synthesis of G-rich telomeric DNA repeats at the 3' ends of most eukaryotic chromosomes1. Telomerase maintains genomic integrity, and its activity or dysregulation are critical determinants of human longevity, stem cell renewal and cancer progression2,3. Previous cryo-electron microscopy structures have established the general architecture, protein components and stoichiometries of Tetrahymena and human telomerase, but our understandings of the details of DNA-protein and RNA-protein interactions and of the mechanisms and recruitment involved remain limited4-6. Here we report cryo-electron microscopy structures of active Tetrahymena telomerase with telomeric DNA at different steps of nucleotide addition. Interactions between telomerase reverse transcriptase (TERT), TER and DNA reveal the structural basis of the determination of the 5' and 3' template boundaries, handling of the template-DNA duplex and separation of the product strand during nucleotide addition. The structure and binding interface between TERT and telomerase protein p50 (a homologue of human TPP17,8) define conserved interactions that are required for telomerase activation and recruitment to telomeres. Telomerase La-related protein p65 remodels several regions of TER, bridging the 5' and 3' ends and the conserved pseudoknot to facilitate assembly of the TERT-TER catalytic core.
Assuntos
Microscopia Crioeletrônica , Telomerase/química , Telomerase/metabolismo , Telômero/metabolismo , Tetrahymena thermophila/enzimologia , Motivos de Aminoácidos , Sítios de Ligação , DNA/química , DNA/metabolismo , DNA/ultraestrutura , Humanos , Modelos Moleculares , Nucleotídeos , Ligação Proteica , RNA/química , RNA/metabolismo , RNA/ultraestrutura , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Complexo Shelterina/química , Complexo Shelterina/metabolismo , Telomerase/ultraestrutura , Telômero/genética , Telômero/ultraestrutura , Proteínas de Ligação a Telômeros/química , Proteínas de Ligação a Telômeros/metabolismo , Moldes Genéticos , Tetrahymena thermophila/ultraestruturaRESUMO
Telomerase is a ribonucleoprotein complex that counteracts the shortening of chromosome ends due to incomplete replication. Telomerase contains a catalytic core of telomerase reverse transcriptase (TERT) and telomerase RNA (TER). However, what defines TERT and separates it from other reverse transcriptases remains a subject of debate. A recent cryoelectron microscopy map of Tetrahymena telomerase revealed the structure of a previously uncharacterized TERT domain (TRAP) with unanticipated interactions with the telomerase essential N-terminal (TEN) domain and roles in telomerase activity. Both TEN and TRAP are absent in the putative Tribolium TERT that has been used as a model for telomerase for over a decade. To investigate the conservation of TRAP and TEN across species, we performed multiple sequence alignments and statistical coupling analysis on all identified TERTs and find that TEN and TRAP have coevolved as telomerase-specific domains. Integrating the data from bioinformatic analysis and the structure of Tetrahymena telomerase, we built a pseudoatomic model of human telomerase catalytic core that accounts for almost all of the cryoelectron microscopy density in a published map, including TRAP in previously unassigned density as well as telomerase RNA domains essential for activity. This more complete model of the human telomerase catalytic core illustrates how domains of TER and TERT, including the TEN-TRAP complex, can interact in a conserved manner to regulate telomere synthesis.
Assuntos
RNA/ultraestrutura , Telomerase/ultraestrutura , Tetrahymena thermophila/ultraestrutura , Animais , Sítios de Ligação , Domínio Catalítico/genética , Microscopia Crioeletrônica , Humanos , Ligação Proteica , Conformação Proteica , Domínios Proteicos/genética , RNA/genética , Alinhamento de Sequência , Complexo Shelterina , Homologia Estrutural de Proteína , Telomerase/genética , Proteínas de Ligação a Telômeros , Tetrahymena thermophila/enzimologia , Tribolium/enzimologiaRESUMO
Mitochondrial ATP synthases form functional homodimers to induce cristae curvature that is a universal property of mitochondria. To expand on the understanding of this fundamental phenomenon, we characterized the unique type III mitochondrial ATP synthase in its dimeric and tetrameric form. The cryo-EM structure of a ciliate ATP synthase dimer reveals an unusual U-shaped assembly of 81 proteins, including a substoichiometrically bound ATPTT2, 40 lipids, and co-factors NAD and CoQ. A single copy of subunit ATPTT2 functions as a membrane anchor for the dimeric inhibitor IF1. Type III specific linker proteins stably tie the ATP synthase monomers in parallel to each other. The intricate dimer architecture is scaffolded by an extended subunit-a that provides a template for both intra- and inter-dimer interactions. The latter results in the formation of tetramer assemblies, the membrane part of which we determined to 3.1 Å resolution. The structure of the type III ATP synthase tetramer and its associated lipids suggests that it is the intact unit propagating the membrane curvature.
Assuntos
ATPases Mitocondriais Próton-Translocadoras/química , Microscopia Crioeletrônica , Lipídeos de Membrana/química , Membranas Mitocondriais/química , Membranas Mitocondriais/enzimologia , Membranas Mitocondriais/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/classificação , ATPases Mitocondriais Próton-Translocadoras/ultraestrutura , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Proteínas/química , Proteínas/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/ultraestrutura , Tetrahymena thermophila/enzimologia , Tetrahymena thermophila/ultraestrutura , Proteína Inibidora de ATPaseRESUMO
Basal bodies (BBs) are macromolecular complexes required for the formation and cortical positioning of cilia. Both BB assembly and DNA replication are tightly coordinated with the cell cycle to ensure their accurate segregation and propagation to daughter cells, but the mechanisms ensuring coordination are unclear. The Tetrahymena Sas4/CPAP protein is enriched at assembling BBs, localizing to the core BB structure and to the base of BB-appendage microtubules and striated fiber. Sas4 is necessary for BB assembly and cortical microtubule organization, and Sas4 loss disrupts cell division furrow positioning and DNA segregation. The Hippo signaling pathway is known to regulate cell division furrow position, and Hippo molecules localize to BBs and BB-appendages. We find that Sas4 loss disrupts localization of the Hippo activator, Mob1, suggesting that Sas4 mediates Hippo activity by promoting scaffolds for Mob1 localization to the cell cortex. Thus, Sas4 links BBs with an ancient signaling pathway known to promote the accurate and symmetric segregation of the genome.
Assuntos
Corpos Basais/metabolismo , Divisão Celular , Centríolos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Corpos Basais/ultraestrutura , Centríolos/genética , Centríolos/ultraestrutura , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Protozoários/genética , Transdução de Sinais , Tetrahymena thermophila/genética , Tetrahymena thermophila/ultraestrutura , Fatores de TempoRESUMO
Motile cilia generate directed hydrodynamic flow that is important for the motility of cells and extracellular fluids. To optimize directed hydrodynamic flow, motile cilia are organized and oriented into a polarized array. Basal bodies (BBs) nucleate and position motile cilia at the cell cortex. Cytoplasmic BB-associated microtubules are conserved structures that extend from BBs. By using the ciliate, Tetrahymena thermophila, combined with EM-tomography and light microscopy, we show that BB-appendage microtubules assemble coincidently with new BB assembly and that they are attached to the cell cortex. These BB-appendage microtubules are specifically marked by post translational modifications of tubulin, including glycylation. Mutations that prevent glycylation shorten BB-appendage microtubules and disrupt BB positioning and cortical attachment. Consistent with the attachment of BB-appendage microtubules to the cell cortex to position BBs, mutations that disrupt the cellular cortical cytoskeleton disrupt the cortical attachment and positioning of BBs. In summary, BB-appendage microtubules promote the organization of ciliary arrays through attachment to the cell cortex.
Assuntos
Corpos Basais/metabolismo , Cílios/metabolismo , Microtúbulos/metabolismo , Tetrahymena thermophila/metabolismo , Corpos Basais/ultraestrutura , Cílios/genética , Glicosilação , Microtúbulos/genética , Microtúbulos/ultraestrutura , Mutação , Tetrahymena thermophila/genética , Tetrahymena thermophila/ultraestruturaRESUMO
Meiotic bouquet formation (known as crescent formation in Tetrahymena thermophila) is indispensable for homologous pairing and recombination, but the regulatory mechanism of bouquet formation remains largely unknown. As a conjugation specific cyclin gene, CYC2 knockout mutants failed to form an elongated crescent structure and aborted meiosis progress in T. thermophila. γ-H2A.X staining revealed fewer micronuclear DNA double-strand breaks (DSBs) in cyc2Δ cells than in wild-type cells. Furthermore, cyc2Δ cells still failed to form a crescent structure even though DSBs were induced by exogenous agents, indicating that a lack of DSBs was not completely responsible for failure to enter the crescent stage. Tubulin staining showed that impaired perinuclear microtubule structure may contribute to the blockage in micronuclear elongation. At the same time, expression of microtubule-associated kinesin genes, KIN11 and KIN141, was significantly downregulated in cyc2Δ cells. Moreover, micronuclear specific accumulation of heterochromatin marker trimethylated H3K23 abnormally increased in the cyc2Δ mutants. Together, these results show that cyclin Cyc2p is required for micronuclear bouquet formation via controlling microtubule-directed nuclear elongation in Tetrahymena.
Assuntos
Ciclinas/metabolismo , Tetrahymena thermophila/metabolismo , Segregação de Cromossomos , Quebras de DNA de Cadeia Dupla , Técnicas de Inativação de Genes , Heterocromatina/metabolismo , Meiose/genética , Microtúbulos/metabolismo , Mutação , Tetrahymena thermophila/ultraestruturaRESUMO
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).
Assuntos
Cílios/fisiologia , Flagelos/fisiologia , Organogênese/fisiologia , Tetrahymena thermophila/crescimento & desenvolvimento , Animais , Cílios/ultraestrutura , Flagelos/ultraestrutura , Movimento/fisiologia , Tetrahymena thermophila/citologia , Tetrahymena thermophila/ultraestrutura , Fatores de TempoRESUMO
Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and ß-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule.
Assuntos
Axonema/química , Cílios/química , Microtúbulos/química , Proteínas de Protozoários/química , Tetrahymena thermophila/química , Tubulina (Proteína)/química , Sequência de Aminoácidos , Axonema/ultraestrutura , Sítios de Ligação , Cílios/ultraestrutura , Microscopia Crioeletrônica , Citoesqueleto/química , Citoesqueleto/ultraestrutura , Flagelos/química , Flagelos/ultraestrutura , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tetrahymena thermophila/ultraestrutura , Termodinâmica , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is considered a re-emerging environmental pollutant, and exposure to environmentally relevant concentrations has been shown to cause individual developmental toxicity in zebrafish and the water flea (Daphnia magna). However, multigenerational effects during exposure to TDCIPP and after subsequent recovery were unknown. In the present study, individuals of a model aquatic organism, the ciliated protozoan, T. thermophila were exposed to environmentally-relevant concentrations of TDCIPP (0, 300 or 3000 ng/L) for 60 days (e.g., theoretically 372 generations) followed by a 60-day period of recovery, during which T. thermophila were not exposed to TDCIPP. During exposure and after exposure, effects at the molecular, histological, individual and population levels were examined. Multigenerational exposure to 300 or 3000 ng TDCIPP/L for 60 days significantly decreased numbers of individuals, sizes of individuals, expressed as length and width of bodies, number of cilia, and depth and diameter of basal bodies of cilia, and up-regulated expressions of genes related to assembly and maintenance of cilia. Complete or partial recoveries of theoretical sizes of populations as well as sizes of individuals and expressions of genes were observed during the 60-day recovery period. Effects on number of cilia and depth and diameter of basal body of cilia were not reversible and could still be observed long after cease of TDCIPP exposure. Collectedly, and shown for the first time, multigenerational effects to T. thermophila were caused by exposure to environmentally relevant concentrations of TDCIPP. Also, there were multi-generational effects at the population level that were not caused by carry-over exposure to TDCIPP. The "permanent" alterations and their potential significance are discussed.
Assuntos
Monitoramento Ambiental/métodos , Compostos Organofosforados/toxicidade , Tetrahymena thermophila/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Animais , Humanos , Modelos Teóricos , Tetrahymena thermophila/crescimento & desenvolvimento , Tetrahymena thermophila/ultraestruturaRESUMO
Fluorescence microscopy (FM) is a powerful tool for observing specific molecular components in living cells, but its spatial resolution is relatively low. In contrast, electron microscopy (EM) provides high-resolution information about cellular structures, but it cannot provide temporal information in living cells. To achieve molecular selectivity in imaging at high resolution, a method combining EM imaging with live-cell fluorescence imaging, known as live correlative light-EM (CLEM), has been developed. In this method, living cells are first observed by FM, fixed in situ during the live observation and then subjected to EM observation. Various fluorescence techniques and tools can be applied for FM, resulting in the generation of various modified methods that are useful for understanding cellular structure in high resolution. Here, we review the methods of CLEM and live-cell imaging associated with CLEM (live CLEM). Such methods can greatly advance the understanding of the function of cellular structures on a molecular level, and thus are useful for medical fields as well as for basic biology.
Assuntos
Microscopia Eletrônica/métodos , Microscopia de Fluorescência/métodos , Saccharomyces cerevisiae/ultraestrutura , Schizosaccharomyces/ultraestrutura , Tetrahymena thermophila/ultraestrutura , Linhagem Celular Tumoral , Corantes Fluorescentes , Células HeLa , Humanos , Nanopartículas Metálicas , Simulação de Dinâmica Molecular , Pontos QuânticosRESUMO
The activities of Tetrahymena corlissi, Tetrahymena thermophila, and Tetrahymena canadensis were studied in coculture with cell lines of insects, fish, amphibians, and mammals. These ciliates remained viable regardless of the animal cell line partner. All three species could engulf animal cells in suspension. However, if the animal cells were monolayer cultures, the monolayers were obliterated by T. corlissi and T. thermophila. Both fibroblast and epithelial monolayers were destroyed but the destruction of human cell monolayers was done more effectively by T. thermophila. By contrast, T. canadensis was unable to destroy any monolayer. At 4 °C T. thermophila and T. corlissi did not carryout phagocytosis and did not destroy monolayers, whereas T. canadensis was able to carryout phagocytosis but still could not destroy monolayers. Therefore, monolayer destruction appeared to require phagocytosis, but by itself this was insufficient. In addition, the ciliates expressed a unique swimming behavior. Tetrahymena corlissi and T. thermophila swam vigorously and repeatedly into the monolayer, which seemed to loosen or dislodge cells, whereas T. canadensis swam above the monolayer. Therefore, differences in swimming behavior might explain why T. corlissi has been reported to be a pathogen but T. canadensis has not.
Assuntos
Tetrahymena/fisiologia , Tetrahymena/patogenicidade , Anfíbios/parasitologia , Animais , Cultura Axênica , Técnicas de Cultura de Células , Linhagem Celular , Peixes/parasitologia , Células HeLa , Humanos , Insetos/citologia , Insetos/parasitologia , Mamíferos/parasitologia , Fagocitose , Natação , Temperatura , Tetrahymena/classificação , Tetrahymena thermophila/patogenicidade , Tetrahymena thermophila/fisiologia , Tetrahymena thermophila/ultraestruturaRESUMO
Using serial-section transmission electron microscopy and three-dimensional (3D) electron tomography, we characterized membrane dynamics that accompany the construction of a nuclear exchange junction between mating cells in the ciliate Tetrahymena thermophila. Our methods revealed a number of previously unknown features. (i) Membrane fusion is initiated by the extension of hundreds of 50-nm-diameter protrusions from the plasma membrane. These protrusions extend from both mating cells across the intercellular space to fuse with membrane of the mating partner. (ii) During this process, small membrane-bound vesicles or tubules are shed from the plasma membrane and into the extracellular space within the junction. The resultant vesicle-filled pockets within the extracellular space are referred to as junction lumens. (iii) As junction lumens fill with extracellular microvesicles and swell, the plasma membrane limiting these swellings undergoes another deformation, pinching off vesicle-filled vacuoles into the cytoplasm (reclamation). (iv) These structures (resembling multivesicular bodies) seem to associate with autophagosomes abundant near the exchange junction. We propose a model characterizing the membrane-remodeling events that establish cytoplasmic continuity between mating Tetrahymena cells. We also discuss the possible role of nonvesicular lipid transport in conditioning the exchange junction lipid environment. Finally, we raise the possibility of an intercellular signaling mechanism involving microvesicle shedding and uptake.
Assuntos
Membrana Celular/metabolismo , Extensões da Superfície Celular/metabolismo , Junções Intercelulares/metabolismo , Tetrahymena thermophila/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Espaço Extracelular/metabolismo , Junções Intercelulares/ultraestrutura , Metabolismo dos Lipídeos , Vesículas Secretórias/metabolismo , Tetrahymena thermophila/fisiologia , Tetrahymena thermophila/ultraestruturaRESUMO
Cilia-organizing basal bodies (BBs) are microtubule scaffolds that are visibly asymmetrical because they have attached auxiliary structures, such as striated fibers. In multiciliated cells, BB orientation aligns to ensure coherent ciliary beating, but the mechanisms that maintain BB orientation are unclear. For the first time in Tetrahymena thermophila, we use comparative whole-genome sequencing to identify the mutation in the BB disorientation mutant disA-1. disA-1 abolishes the localization of the novel protein DisAp to T. thermophila striated fibers (kinetodesmal fibers; KFs), which is consistent with DisAp's similarity to the striated fiber protein SF-assemblin. We demonstrate that DisAp is required for KFs to elongate and to resist BB disorientation in response to ciliary forces. Newly formed BBs move along KFs as they approach their cortical attachment sites. However, because they contain short KFs that are rotated, BBs in disA-1 cells display aberrant spacing and disorientation. Therefore, DisAp is a novel KF component that is essential for force-dependent KF elongation and BB orientation in multiciliary arrays.
Assuntos
Cílios/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/ultraestrutura , Fenômenos Biomecânicos , Cílios/ultraestrutura , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Filogenia , Proteínas de Protozoários/genética , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismoRESUMO
Telomerase adds telomeric repeats to chromosome ends using an internal RNA template and a specialized telomerase reverse transcriptase (TERT), thereby maintaining genome integrity. Little is known about the physical relationships among protein and RNA subunits within a biologically functional holoenzyme. Here we describe the architecture of Tetrahymena thermophila telomerase holoenzyme determined by electron microscopy. Six of the seven proteins and the TERT-binding regions of telomerase RNA (TER) have been localized by affinity labelling. Fitting with high-resolution structures reveals the organization of TERT, TER and p65 in the ribonucleoprotein (RNP) catalytic core. p50 has an unanticipated role as a hub between the RNP catalytic core, p75-p19-p45 subcomplex, and the DNA-binding Teb1. A complete in vitro holoenzyme reconstitution assigns function to these interactions in processive telomeric repeat synthesis. These studies provide the first view of the extensive network of subunit associations necessary for telomerase holoenzyme assembly and physiological function.
Assuntos
Telomerase/química , Telomerase/ultraestrutura , Tetrahymena thermophila/enzimologia , Domínio Catalítico , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/ultraestrutura , Microscopia Eletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Maleabilidade , Estrutura Terciária de Proteína , Subunidades Proteicas/análise , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/ultraestrutura , RNA/química , RNA/metabolismo , RNA/ultraestrutura , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/ultraestrutura , Telomerase/genética , Telomerase/metabolismo , Tetrahymena thermophila/química , Tetrahymena thermophila/genética , Tetrahymena thermophila/ultraestruturaRESUMO
Alveolins are a recently described class of proteins common to all members of the superphylum Alveolata that are characterized by conserved charged repeat motifs (CRMs) but whose exact function remains unknown. We have analyzed the smaller of the two alveolins of Tetrahymena thermophila, TtALV2. The protein localizes to dispersed, broken patches arranged between the rows of the longitudinal microtubules. Macronuclear knockdown of Ttalv2 leads to multinuclear cells with no apparent cell polarity and randomly occurring cell protrusions, either by interrupting pellicle integrity or by disturbing cytokinesis. Correct association of TtALV2 with the alveoli or the pellicle is complex and depends on both the termini as well as the charged repeat motifs of the protein. Proteins containing similar CRMs are a dominant part of the ciliate membrane cytoskeleton, suggesting that these motifs may play a more general role in mediating membrane attachment and/or cytoskeletal association. To better understand their integration into the cytoskeleton, we localized a range of CRM-based fusion proteins, which suggested there is an inherent tendency for proteins with CRMs to be located in the peripheral cytoskeleton, some nucleating as filaments at the basal bodies. Even a synthetic protein, mimicking the charge and repeat pattern of these proteins, directed a reporter protein to a variety of peripheral cytoskeletal structures in Tetrahymena. These motifs might provide a blueprint for membrane and cytoskeleton affiliation in the complex pellicles of Alveolata.
Assuntos
Membrana Celular/genética , Citoesqueleto/genética , Metaloendopeptidases/genética , Proteínas de Protozoários/genética , Tetrahymena thermophila/genética , Motivos de Aminoácidos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Polaridade Celular , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Expressão Gênica , Metaloendopeptidases/metabolismo , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Tetrahymena thermophila/metabolismo , Tetrahymena thermophila/ultraestruturaRESUMO
Recent studies have implicated the phosducin-like protein-2 (PHLP2) in regulation of CCT, a chaperonin whose activity is essential for folding of tubulin and actin. However, the exact molecular function of PHLP2 is unclear. Here we investigate the significance of PHLP2 in a ciliated unicellular model, Tetrahymena thermophila, by deleting its single homolog, Phlp2p. Cells lacking Phlp2p became larger and died within 96 h. Overexpressed Phlp2p-HA localized to cilia, basal bodies, and cytosol without an obvious change in the phenotype. Despite similar localization, overexpressed GFP-Phlp2p caused a dominant-negative effect. Cells overproducing GFP-Phlp2p had decreased rates of proliferation, motility and phagocytosis, as compared to wild type cells or cells overproducing a non-tagged Phlp2p. Growing GFP-Phlp2p-overexpressing cells had fewer cilia and, when deciliated, failed to regenerate cilia, indicating defects in cilia assembly. Paclitaxel-treated GFP-Phlp2p cells failed to elongate cilia, indicating a change in the microtubules dynamics. The pattern of ciliary and cytosolic tubulin isoforms on 2D gels differed between wild type and GFP-Phlp2p-overexpressing cells. Thus, in Tetrahymena, PhLP2 is essential and under specific experimental conditions its activity affects tubulin and microtubule-dependent functions including cilia assembly.
Assuntos
Cílios/metabolismo , Microtúbulos/metabolismo , Organogênese , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Cílios/ultraestrutura , Técnicas de Inativação de Genes , Genes Dominantes , Proteínas de Fluorescência Verde/metabolismo , Filogenia , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tetrahymena thermophila/citologia , Tetrahymena thermophila/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
Centrins are a ubiquitous family of small Ca(2+)-binding proteins found at basal bodies that are placed into two groups based on sequence similarity to the human centrins 2 and 3. Analyses of basal body composition in different species suggest that they contain a centrin isoform from each group. We used the ciliate protist Tetrahymena thermophila to gain a better understanding of the functions of the two centrin groups and to determine their potential redundancy. We have previously shown that the Tetrahymena centrin 1 (Cen1), a human centrin 2 homologue, is required for proper basal body function. In this paper, we show that the Tetrahymena centrin 2 (Cen2), a human centrin 3 homologue, has functions similar to Cen1 in basal body orientation, maintenance, and separation. The two are, however, not redundant. A further examination of human centrin 3 homologues shows that they function in a manner distinct from human centrin 2 homologues. Our data suggest that basal bodies require a centrin from both groups in order to function correctly.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Centríolos/metabolismo , Proteínas de Protozoários/metabolismo , Tetrahymena thermophila/metabolismo , Sequência de Aminoácidos , Western Blotting , Proteínas de Ligação ao Cálcio/classificação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centríolos/ultraestrutura , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Eletrônica , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Filogenia , Isoformas de Proteínas/classificação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Tetrahymena thermophila/genética , Tetrahymena thermophila/ultraestruturaRESUMO
Tetrahymena thermophila is both a cell and an organism, which combines great intracellular complexity with a remarkable accessibility to investigation using many different approaches. In this review, we start with a description of the elaborate cortical organization of the Tetrahymena cell, and then proceed inward to consider the mitochondria and then the nuclei. For each of these cellular organelles and organelle-systems, first we familiarize the reader with its location in the cell and its structure and ultrastructure, and then we analyze the molecular mechanisms associated with organelle assembly, function, and subdivision. This analysis includes a molecular inventory of the organelle or organelle system, as well as a review of the consequences of modification, disruption or overexpression of important molecular components of each structure or system. Relevant comparisons to results obtained with other well-studied organisms, from Paramecium to Homo sapiens, are also included. Our goal is to provide investigators, in particular those who are new to this organism, both the background and the motivation to work with this model system and achieve further insight into its organization and dynamics.
Assuntos
Genes de Protozoários , Genoma Mitocondrial , Proteínas de Protozoários/química , Tetrahymena thermophila/ultraestrutura , Fator de Indução de Apoptose/química , Fator de Indução de Apoptose/genética , Membrana Celular/química , Forma do Núcleo Celular , Cílios/química , Fragmentação do DNA , Células Eucarióticas/química , Células Eucarióticas/ultraestrutura , Macronúcleo/química , Macronúcleo/genética , Micronúcleo Germinativo/química , Micronúcleo Germinativo/genética , Microscopia Eletrônica de Transmissão , Mitocôndrias/química , Mitocôndrias/genética , Transporte Proteico , Proteínas de Protozoários/genética , Tetrahymena thermophila/química , Tetrahymena thermophila/genéticaRESUMO
Mob1 is a component of both the mitotic exit network and Hippo pathway, being required for cytokinesis, control of cell proliferation and apoptosis. Cell division accuracy is crucial in maintaining cell ploidy and genomic stability and relies on the correct establishment of the cell division axis, which is under the control of the cell's environment and its intrinsic polarity. The ciliate Tetrahymena thermophila possesses a permanent anterior-posterior axis, left-right asymmetry and divides symmetrically. These unique features of Tetrahymena prompted us to investigate the role of Tetrahymena Mob1. Unexpectedly, we found that Mob1 accumulated in basal bodies at the posterior pole of the cell, and is the first molecular polarity marker so far described in Tetrahymena. In addition, Mob1 depletion caused the abnormal establishment of the cell division plane, providing clear evidence that Mob1 is important for its definition. Furthermore, cytokinesis was arrested and ciliogenesis delayed in Tetrahymena cells depleted of Mob1. This is the first evidence for an involvement of Mob1 in cilia biology. In conclusion, we show that Mob1 is an important cell polarity marker that is crucial for correct division plane placement, for cytokinesis completion and for normal cilia growth rates.