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Jacek Gaertig - One of the best experts on this subject based on the ideXlab platform.

  • Discovery and Functional Evaluation of Ciliary Proteins in Tetrahymena thermophila
    Methods in Enzymology, 2013
    Co-Authors: Jacek Gaertig, Dorota Wloga, Krishna Kumar Vasudevan, Mayukh Guha, William L. Dentler
    Abstract:

    The ciliate Tetrahymena thermophila is an excellent model system for the discovery and functional studies of ciliary proteins. The power of the model is based on the ease with which cilia can be purified in large quantities for fractionation and proteomic identification, and the ability to knock out any gene by homologous DNA recombination. Here, we include methods used by our laboratories for isolation and fractionation of cilia, in vivo tagging and localization of ciliary proteins, and the evaluation of ciliary mutants.

  • Manipulating Ciliary Protein-Encoding Genes in Tetrahymena thermophila
    Methods in Cell Biology, 2009
    Co-Authors: Drashti Dave, Dorota Wloga, Jacek Gaertig
    Abstract:

    Tetrahymena thermophila has emerged as an excellent protist model for studies on cilia that are based on reverse genetic approaches. In Tetrahymena, genes can be routinely disrupted by the DNA homologous recombination. We present established protocols for the manipulation of genes in either the germline micronucleus or the somatic macronucleus. A detailed protocol is provided for the construction of heterokaryon strains that carry a gene disruption only in the micronucleus. Heterokaryon strain can be propagated like wild-type cells, and ciliary phenotypes can be expressed on demand by mating. We describe methods that can be used for disruption of multiple genes. We include protocols for the generation and maintenance of Tetrahymena cells that either lack cilia or have paralyzed cilia.

  • Septins Stabilize Mitochondria in Tetrahymena thermophila
    Eukaryotic Cell, 2008
    Co-Authors: Dorota Wloga, Jacek Gaertig, I. Strzyżewska-jówko, Maria Jerka-dziadosz
    Abstract:

    We describe phylogenetic and functional studies of three septins in the free-living ciliate Tetrahymena thermophila. Both deletion and overproduction of septins led to vacuolization of mitochondria, destabilization of the nuclear envelope, and increased autophagy. All three green fluorescent protein-tagged septins localized to mitochondria. Specific septins localized to the outer mitochondrial membrane, to septa formed during mitochondrial scission, or to the mitochondrion-associated endoplasmic reticulum. The only other septins known to localize to mitochondria are human ARTS and murine M-septin, both alternatively spliced forms of Sep4 (S. Larisch, Cell Cycle 3:1021-1023, 2004; S. Takahashi, R. Inatome, H. Yamamura, and S. Yanagi, Genes Cells 8:81-93, 2003). It therefore appears that septins have been recruited to mitochondrial functions independently in at least two eukaryotic lineages and in both cases are involved in apoptotic events.

  • metallothionein gene from Tetrahymena thermophila with a copper inducible repressible promoter
    Eukaryotic Cell, 2006
    Co-Authors: Francesco Boldrin, Gianfranco Santovito, Jacek Gaertig, Dorota Wloga, Donna Cassidyhanley, Theodore G Clark, Ester Piccinni
    Abstract:

    We describe a novel metallothionein gene from Tetrahymena thermophila that has a strong copper-inducible promoter. This promoter can be turned on and off rapidly, making it a useful system for induction of ectopic gene expression in Tetrahymena and enhancing its applications in cell and molecular biology, as well as biotechnology.

  • surface display of a parasite antigen in the ciliate Tetrahymena thermophila
    Nature Biotechnology, 1999
    Co-Authors: Jacek Gaertig, Theodore G Clark, Tomer Tishgarten, Harry W Dickerson
    Abstract:

    The ciliated protozoan, Tetrahymena thermophila, offers an attractive medium for the expression of heterologous proteins and could prove particularly useful for the display of foreign proteins on the cell surface. Although progress has been made in transformation of Tetrahymena with heterologous DNA, methods that permit reliable expression of foreign genes have been lacking. Using a mutant strain of T. thermophila carrying a negatively selectable allele of a β-tubulin gene, we have been able to direct foreign genes to this locus by homologous recombination. Transformed cell lines producing foreign proteins were readily identified and, in at least one case, targeting of proteins to the plasma membrane was accomplished.

Martin A. Gorovsky - One of the best experts on this subject based on the ideXlab platform.

  • Tetrahymena thermophila contains a conventional γ tubulin that is differentially required for the maintenance of different microtubule organizing centers
    Journal of Cell Biology, 2002
    Co-Authors: Yuhua Shang, Bing Li, Martin A. Gorovsky
    Abstract:

    The gene ( GTU1 ) encoding Tetrahymena thermophila γ-tubulin was cloned and analyzed. GTU1 is a single-copy, essential gene encoding a conventional γ-tubulin. HA-tagged GTU1p localizes to four microtubule-organizing centers (MTOCs) in vegetative cells: basal bodies (BBs), macronuclear envelopes,

  • Proposed Genetic Nomenclature Rules for Tetrahymena thermophila, Paramecium primaurelia and Paramecium tetraurelia
    Genetics, 1998
    Co-Authors: Sally Lyman Allen, F. Paul Doerder, Eduardo Orias, Jacek Gaertig, Peter J. Bruns, Marsha I. Altschuler, Jean Cohen, Martin A. Gorovsky, Aaron P. Turkewitz
    Abstract:

    The genetics of the ciliated protozoa Tetrahymena thermophila and certain species of Paramecium ( P. primaurelia and P. tetraurelia ) have reached a level of maturity such that rules for genetic nomenclature for micronuclear and macronuclear genetics need to be clarified for workers in the field as well as for other geneticists. After a short introduction, the rules follow.

  • germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment
    Genetics, 1997
    Co-Authors: Donna Cassidyhanley, Jacek Gaertig, Martin A. Gorovsky, Josephine Bowen, Eric S Cole, Lynn A Verplank, Peter J. Bruns
    Abstract:

    Mating Tetrahymena thermophila were bombarded with ribosomal DNA-coated particles at various times in development. Both macronuclear and micronuclear transformants were recovered. Optimal developmental stages for transformation occurred during meiosis for the micronucleus and during anlagen formation for the macronucleus. Evidence is given for transient retention of the introduced plasmid. Genetic and molecular tests confirmed that sexually heritable transformation was associated with integration at the homologous site in the recipient micronuclear chromosome.

  • efficient mass transformation of Tetrahymena thermophila by electroporation of conjugants
    Proceedings of the National Academy of Sciences of the United States of America, 1992
    Co-Authors: Jacek Gaertig, Martin A. Gorovsky
    Abstract:

    Abstract Conjugating cells of the ciliate Tetrahymena thermophila were electroporated in the presence of plasmid DNA containing a paromomycin-resistant ribosomal RNA gene (rDNA). Cells were selected with paromomycin following 12-24 hr of growth on nonselective medium. Resistant cells appeared after 2-3 days. Processing vectors containing the micronuclear rDNA and somatic vectors containing the macronuclear gene transformed the cells, with the former yielding frequencies up to 900 transformants per microgram of plasmid DNA. A ribosomal protein gene (rpL29) conferring cycloheximide resistance also transformed conjugating cells. The transformation efficiency of the plasmid containing only the rpL29 gene was increased by insertion of an rDNA replication origin and by cotransformation and preselection with an rDNA vector. These results indicate that electroporation can be used for the production of large numbers of transformed Tetrahymena.

  • Transcriptional regulation of gene expression in Tetrahymena thermophila
    Nucleic Acids Research, 1990
    Co-Authors: Laurie A. Stargell, Kathleen M. Karrer, Martin A. Gorovsky
    Abstract:

    Abstract The only well-characterized study of gene expression in Tetrahymena thermophila (1) demonstrates that the temperature dependent expression of the Ser H3 gene is regulated at the level of mRNA stability. A run-on transcription assay was developed to determine if regulation of RNA stability was a major mechanism regulating gene expression in Tetrahymena or if transcriptional regulation dominates. The relative transcriptional activities of 14 Tetrahymena genes were determined in different physiological/developmental states (growing, starved and conjugating) in which many of the genes showed striking differences in RNA abundance. In every case except Ser H3, changes in transcription accompanied changes in RNA abundance. Thus differential transcription, not differential RNA degradation, is the major mechanism regulating RNA abundance in Tetrahymena.

Heather G Kuruvilla - One of the best experts on this subject based on the ideXlab platform.

  • netrin 1 peptide is a chemorepellent in Tetrahymena thermophila
    International Journal of Peptides, 2016
    Co-Authors: Heather G Kuruvilla, Bradley Schmidt, Stephanie E Song, Marian A Bhajjan, Matthew S Merical, Caleb Alley, Christopher T Griffin, David Yoder, Josephine Hein, Daniel Kohl
    Abstract:

    Netrin-1 is a highly conserved, pleiotropic signaling molecule that can serve as a neuronal chemorepellent during vertebrate development. In vertebrates, chemorepellent signaling is mediated through the tyrosine kinase, src-1, and the tyrosine phosphatase, shp-2. Tetrahymena thermophila has been used as a model system for chemorepellent signaling because its avoidance response is easily characterized under a light microscope. Our experiments showed that netrin-1 peptide is a chemorepellent in T. thermophila at micromolar concentrations. T. thermophila adapts to netrin-1 over a time course of about 10 minutes. Netrin-adapted cells still avoid GTP, PACAP-38, and nociceptin, suggesting that netrin does not use the same signaling machinery as any of these other repellents. Avoidance of netrin-1 peptide was effectively eliminated by the addition of the tyrosine kinase inhibitor, genistein, to the assay buffer; however, immunostaining using an anti-phosphotyrosine antibody showed similar fluorescence levels in control and netrin-1 exposed cells, suggesting that tyrosine phosphorylation is not required for signaling to occur. In addition, ELISA indicates that a netrin-like peptide is present in both whole cell extract and secreted protein obtained from Tetrahymena thermophila. Further study will be required in order to fully elucidate the signaling mechanism of netrin-1 peptide in this organism.

  • nociceptin signaling involves a calcium based depolarization in Tetrahymena thermophila
    International Journal of Peptides, 2013
    Co-Authors: Thomas J Lampert, Cheryl Nugent, John Weston, Nathanael Braun, Heather G Kuruvilla
    Abstract:

    Tetrahymena thermophila are free-living, ciliated eukaryotes. Their behavioral response to stimuli is well characterized and easily observable, since cells swim toward chemoattractants and avoid chemorepellents. Chemoattractant responses involve increased swim speed or a decreased change in swim direction, while chemorepellent signaling involves ciliary reversal, which causes the organism to jerk back and forth, swim in small circles, or spin in an attempt to get away from the repellent. Many food sources, such as proteins, are chemoattractants for these organisms, while a variety of compounds are repellents. Repellents in nature are thought to come from the secretions of predators or from ruptured organisms, which may serve as “danger” signals. Interestingly, several peptides involved in vertebrate pain signaling are chemorepellents in Tetrahymena, including substances P, ACTH, PACAP, VIP, and nociceptin. Here, we characterize the response of Tetrahymena thermophila to three different isoforms of nociceptin. We find that G-protein inhibitors and tyrosine kinase inhibitors do not affect nociceptin avoidance. However, the calcium chelator, EGTA, and the SERCA calcium ATPase inhibitor, thapsigargin, both inhibit nociceptin avoidance, implicating calcium in avoidance. This result is confirmed by electrophysiology studies which show that 50 M nociceptin-NH2 causes a sustained depolarization of approximately 40 mV, which is eliminated by the addition of extracellular EGTA.

  • Vertebrate Neuronal Chemorepellents, Semaphorin 3C and Netrin-1, Are Chemorepellents in Tetrahymena thermophila
    2013
    Co-Authors: Caleb Alley, Christopher T Griffin, David Yoder, Josie Hein, Daniel B. Kohl, Brad Schmidt, Heather G Kuruvilla
    Abstract:

    During vertebrate development, neuronal growth is guided by chemical signals. Chemoattractants encourage neuronal growth cones to form, while chemorepellents cause growth cone collapse. Integration of these signals allows for proper neural positioning in the developing organism. Not all signaling pathways are clear-cut, however. Netrin-1, for example, can function as either a chemoattractant or a chemorepellent, depending upon the cell type involved, the signals the cell has previously received, and the concentration of the signal. Netrin-1 primarily signals through a G-protein mediated receptor via the adenylyl cyclase pathway. The semaphorins mainly serve as chemorepellents and as immune signals in vertebrates. However, signaling through the semaphorins is not well understood. Tetrahymena thermophila are free-living, ciliated eukaryotic organisms that are often used as a model for showing behavioral responses to both chemoattractants and chemorepellents. A number of polycationic peptides function as chemorepellents in this organism, including lysozyme, VIP, PACAP, nociceptin, substance P, and ACTH derivatives. In the current study, we used two polycationic peptides derived from the vertebrate neuronal chemorepellents, semaphorin 3C and netrin-1, in order to determine whether they were chemorepellents in Tetrahymena thermophila. Both peptides were chemorepellents in Tetrahymena thermophila. Semaphorin 3C peptide showed chemorepellent activity with an EC100 of approximately 10 μM. Netrin-1 peptide showed chemorepellent activity over a range of concentrations from micromolar to nanomolar. However, there was a great deal of variability in the response to netrin-1. Further characterization of the pathways involved in the signaling of these repellents will allow comparison between ciliate and vertebrate signaling, and may help us better understand vertebrate development. Introduction Materials and Methods Behavioral assays were conducted using a dissection microscope, a 3-well microtiter plate, and a modified Pasteur pipette as described in Mace et al., 2000, and as pictured below.

  • Pacap-38 signaling in Tetrahymena thermophila involves NO and cGMP
    Acta Protozoologica, 2004
    Co-Authors: John Lucas, John Riddle, Janine N. Bartholomew, Brendan Thomas, Jason Forni, L. Emery Nickerson, Bradley Van Heukelum, Joshua Paulick, Heather G Kuruvilla
    Abstract:

    Summary. Chemorepellents are signaling molecules, which have been shown to be important for mammalian neuronal development, and are presumed to have a role in protozoan defense. Tetrahymena thermophila represent a good model system in which to study repellents because of their ease of use in biochemical, behavioral, electrophysiological, and genetic analyses. In this study, we have used Tetrahymena as a model in which to study the chemorepellent, PACAP. Using behavioral and biochemical (EIA) assays, we have found that the NO/cGMP pathway plays an important role in PACAP signaling. An increase in intracellular calcium is also critical for PACAP avoidance, which appears to be mediated through a pertussis toxin-sensitive G-protein.

Marcus Hartmann - One of the best experts on this subject based on the ideXlab platform.

  • a recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila
    BMC Microbiology, 2007
    Co-Authors: Thomas Weide, Ulrike Bockau, Lutz Herrmann, Angelika Rave, Marcus Hartmann
    Abstract:

    Background Tetrahymena thermophila is one of the best characterized unicellular eukaryotes and its genome is sequenced in its entirety. However, the AT-richness of the genome and an unusual codon usage cause problems in cloning and expression of the ciliate DNA. To overcome these technical hiatuses we developed a Cre-dependent recombinase system.

  • secretion of functional human enzymes by Tetrahymena thermophila
    BMC Biotechnology, 2006
    Co-Authors: Thomas Weide, Ulrike Bockau, Ingo Aldag, Lutz Herrmann, Nadine Niebur, Wouter Laroy, Roland Contreras, Arno Tiedtke, Marcus Hartmann
    Abstract:

    Background The non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen.

Ulrike Bockau - One of the best experts on this subject based on the ideXlab platform.

  • A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila
    PLoS ONE, 2014
    Co-Authors: Graeme J.m. Cowan, Ulrike Bockau, Janna Eleni-muus, Ingo Aldag, Kay Samuel, Marcus W.w. Hartmann, Alison M Creasey, David R Cavanagh
    Abstract:

    Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.

  • a recombinase system facilitates cloning of expression cassettes in the ciliate Tetrahymena thermophila
    BMC Microbiology, 2007
    Co-Authors: Thomas Weide, Ulrike Bockau, Lutz Herrmann, Angelika Rave, Marcus Hartmann
    Abstract:

    Background Tetrahymena thermophila is one of the best characterized unicellular eukaryotes and its genome is sequenced in its entirety. However, the AT-richness of the genome and an unusual codon usage cause problems in cloning and expression of the ciliate DNA. To overcome these technical hiatuses we developed a Cre-dependent recombinase system.

  • secretion of functional human enzymes by Tetrahymena thermophila
    BMC Biotechnology, 2006
    Co-Authors: Thomas Weide, Ulrike Bockau, Ingo Aldag, Lutz Herrmann, Nadine Niebur, Wouter Laroy, Roland Contreras, Arno Tiedtke, Marcus Hartmann
    Abstract:

    Background The non-pathogenic ciliate Tetrahymena thermophila is one of the best-characterized unicellular eucaryotes used in various research fields. Previous work has shown that this unicellular organism provides many biological features to become a high-quality expression system, like multiplying to high cell densities with short generation times in bioreactors. In addition, the expression of surface antigens from the malaria parasite Plasmodium falciparum and the ciliate Ichthyophthirius multifiliis suggests that T. thermophila might play an important role in vaccine development. However, the expression of functional mammalian or human enzymes remains so far to be seen.