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2. Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Author

Faktorová, Drahomíra, Nisbet, R Ellen R, Fernández Robledo, José A, Casacuberta, Elena, Sudek, Lisa, Allen, Andrew E, Ares, Manuel, Aresté, Cristina, Balestreri, Cecilia, Barbrook, Adrian C, Beardslee, Patrick, Bender, Sara, Booth, David S, Bouget, François-Yves, Bowler, Chris, Breglia, Susana A, Brownlee, Colin, Burger, Gertraud, Cerutti, Heriberto, Cesaroni, Rachele, Chiurillo, Miguel A, Clemente, Thomas, Coles, Duncan B, Collier, Jackie L, Cooney, Elizabeth C, Coyne, Kathryn, Docampo, Roberto, Dupont, Christopher L, Edgcomb, Virginia, Einarsson, Elin, Elustondo, Pía A, Federici, Fernan, Freire-Beneitez, Veronica, Freyria, Nastasia J, Fukuda, Kodai, García, Paulo A, Girguis, Peter R, Gomaa, Fatma, Gornik, Sebastian G, Guo, Jian, Hampl, Vladimír, Hanawa, Yutaka, Haro-Contreras, Esteban R, Hehenberger, Elisabeth, Highfield, Andrea, Hirakawa, Yoshihisa, Hopes, Amanda, Howe, Christopher J, Hu, Ian, Ibañez, Jorge, Irwin, Nicholas AT, Ishii, Yuu, Janowicz, Natalia Ewa, Jones, Adam C, Kachale, Ambar, Fujimura-Kamada, Konomi, Kaur, Binnypreet, Kaye, Jonathan Z, Kazana, Eleanna, Keeling, Patrick J, King, Nicole, Klobutcher, Lawrence A, Lander, Noelia, Lassadi, Imen, Li, Zhuhong, Lin, Senjie, Lozano, Jean-Claude, Luan, Fulei, Maruyama, Shinichiro, Matute, Tamara, Miceli, Cristina, Minagawa, Jun, Moosburner, Mark, Najle, Sebastián R, Nanjappa, Deepak, Nimmo, Isabel C, Noble, Luke, Novák Vanclová, Anna MG, Nowacki, Mariusz, Nuñez, Isaac, Pain, Arnab, Piersanti, Angela, Pucciarelli, Sandra, Pyrih, Jan, Rest, Joshua S, Rius, Mariana, Robertson, Deborah, Ruaud, Albane, Ruiz-Trillo, Iñaki, Sigg, Monika A, Silver, Pamela A, Slamovits, Claudio H, Jason Smith, G, Sprecher, Brittany N, Stern, Rowena, Swart, Estienne C, Tsaousis, Anastasios D, Tsypin, Lev, Turkewitz, Aaron, and Turnšek, Jernej

Subjects
Genetics, Biotechnology, Life Below Water, Biodiversity, DNA, Ecosystem, Environment, Eukaryota, Green Fluorescent Proteins, Marine Biology, Models, Biological, Species Specificity, Transformation, Genetic, Biological Sciences, Technology, Medical and Health Sciences, Developmental Biology
Abstract

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Published
2020

3. Publisher Correction: Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Author

Faktorová, Drahomíra, Nisbet, R Ellen R, Fernández Robledo, José A, Casacuberta, Elena, Sudek, Lisa, Allen, Andrew E, Ares, Manuel, Aresté, Cristina, Balestreri, Cecilia, Barbrook, Adrian C, Beardslee, Patrick, Bender, Sara, Booth, David S, Bouget, François-Yves, Bowler, Chris, Breglia, Susana A, Brownlee, Colin, Burger, Gertraud, Cerutti, Heriberto, Cesaroni, Rachele, Chiurillo, Miguel A, Clemente, Thomas, Coles, Duncan B, Collier, Jackie L, Cooney, Elizabeth C, Coyne, Kathryn, Docampo, Roberto, Dupont, Christopher L, Edgcomb, Virginia, Einarsson, Elin, Elustondo, Pía A, Federici, Fernan, Freire-Beneitez, Veronica, Freyria, Nastasia J, Fukuda, Kodai, García, Paulo A, Girguis, Peter R, Gomaa, Fatma, Gornik, Sebastian G, Guo, Jian, Hampl, Vladimír, Hanawa, Yutaka, Haro-Contreras, Esteban R, Hehenberger, Elisabeth, Highfield, Andrea, Hirakawa, Yoshihisa, Hopes, Amanda, Howe, Christopher J, Hu, Ian, Ibañez, Jorge, Irwin, Nicholas AT, Ishii, Yuu, Janowicz, Natalia Ewa, Jones, Adam C, Kachale, Ambar, Fujimura-Kamada, Konomi, Kaur, Binnypreet, Kaye, Jonathan Z, Kazana, Eleanna, Keeling, Patrick J, King, Nicole, Klobutcher, Lawrence A, Lander, Noelia, Lassadi, Imen, Li, Zhuhong, Lin, Senjie, Lozano, Jean-Claude, Luan, Fulei, Maruyama, Shinichiro, Matute, Tamara, Miceli, Cristina, Minagawa, Jun, Moosburner, Mark, Najle, Sebastián R, Nanjappa, Deepak, Nimmo, Isabel C, Noble, Luke, Novák Vanclová, Anna MG, Nowacki, Mariusz, Nuñez, Isaac, Pain, Arnab, Piersanti, Angela, Pucciarelli, Sandra, Pyrih, Jan, Rest, Joshua S, Rius, Mariana, Robertson, Deborah, Ruaud, Albane, Ruiz-Trillo, Iñaki, Sigg, Monika A, Silver, Pamela A, Slamovits, Claudio H, Jason Smith, G, Sprecher, Brittany N, Stern, Rowena, Swart, Estienne C, Tsaousis, Anastasios D, Tsypin, Lev, Turkewitz, Aaron, and Turnšek, Jernej

Subjects
Biological Sciences, Technology, Medical and Health Sciences, Developmental Biology
Abstract

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Published
2020

5. An Alveolata secretory machinery adapted to parasite host cell invasion

Author

Aquilini, Eleonora, Cova, Marta Mendonça, Mageswaran, Shrawan Kumar, Dos Santos Pacheco, Nicolas, Sparvoli, Daniela, Penarete-Vargas, Diana Marcela, Najm, Rania, Graindorge, Arnault, Suarez, Catherine, Maynadier, Marjorie, Berry-Sterkers, Laurence, Urbach, Serge, Fahy, Pilar Ruga, Guérin, Amandine N., Striepen, Boris, Dubremetz, Jean-François, Chang, Yi-Wei, Turkewitz, Aaron P., and Lebrun, Maryse

Published
2021
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8. Macronuclear genome sequence of the ciliate Tetrahymena thermophila, a model eukaryote.

Author

Eisen, Jonathan A, Coyne, Robert S, Wu, Martin, Wu, Dongying, Thiagarajan, Mathangi, Wortman, Jennifer R, Badger, Jonathan H, Ren, Qinghu, Amedeo, Paolo, Jones, Kristie M, Tallon, Luke J, Delcher, Arthur L, Salzberg, Steven L, Silva, Joana C, Haas, Brian J, Majoros, William H, Farzad, Maryam, Carlton, Jane M, Smith, Roger K, Garg, Jyoti, Pearlman, Ronald E, Karrer, Kathleen M, Sun, Lei, Manning, Gerard, Elde, Nels C, Turkewitz, Aaron P, Asai, David J, Wilkes, David E, Wang, Yufeng, Cai, Hong, Collins, Kathleen, Stewart, B Andrew, Lee, Suzanne R, Wilamowska, Katarzyna, Weinberg, Zasha, Ruzzo, Walter L, Wloga, Dorota, Gaertig, Jacek, Frankel, Joseph, Tsao, Che-Chia, Gorovsky, Martin A, Keeling, Patrick J, Waller, Ross F, Patron, Nicola J, Cherry, J Michael, Stover, Nicholas A, Krieger, Cynthia J, del Toro, Christina, Ryder, Hilary F, Williamson, Sondra C, Barbeau, Rebecca A, Hamilton, Eileen P, and Orias, Eduardo

Abstract

The ciliate Tetrahymena thermophila is a model organism for molecular and cellular biology. Like other ciliates, this species has separate germline and soma functions that are embodied by distinct nuclei within a single cell. The germline-like micronucleus (MIC) has its genome held in reserve for sexual reproduction. The soma-like macronucleus (MAC), which possesses a genome processed from that of the MIC, is the center of gene expression and does not directly contribute DNA to sexual progeny. We report here the shotgun sequencing, assembly, and analysis of the MAC genome of T. thermophila, which is approximately 104 Mb in length and composed of approximately 225 chromosomes. Overall, the gene set is robust, with more than 27,000 predicted protein-coding genes, 15,000 of which have strong matches to genes in other organisms. The functional diversity encoded by these genes is substantial and reflects the complexity of processes required for a free-living, predatory, single-celled organism. This is highlighted by the abundance of lineage-specific duplications of genes with predicted roles in sensing and responding to environmental conditions (e.g., kinases), using diverse resources (e.g., proteases and transporters), and generating structural complexity (e.g., kinesins and dyneins). In contrast to the other lineages of alveolates (apicomplexans and dinoflagellates), no compelling evidence could be found for plastid-derived genes in the genome. UGA, the only T. thermophila stop codon, is used in some genes to encode selenocysteine, thus making this organism the first known with the potential to translate all 64 codons in nuclear genes into amino acids. We present genomic evidence supporting the hypothesis that the excision of DNA from the MIC to generate the MAC specifically targets foreign DNA as a form of genome self-defense. The combination of the genome sequence, the functional diversity encoded therein, and the presence of some pathways missing from other model organisms makes T. thermophila an ideal model for functional genomic studies to address biological, biomedical, and biotechnological questions of fundamental importance.

Published
2006

12. Structure and dynamics of the contractile vacuole complex in Tetrahymena thermophila.

Author

Chao-Yin Cheng, Romero, Daniel P., Zoltner, Martin, Meng-Chao Yao, and Turkewitz, Aaron P.

Subjects
TETRAHYMENA, OSMOREGULATION, ORGANELLE formation, BLADDER, MITOSIS
Abstract

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprising a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying their structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, whereas the lipid scramblase Scr7p colocalizes with Vps8Dp. The H+-ATPase subunit Vma4 is spongiome specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools versus lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. Although the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome might be isolated from bulk endocytic input. [ABSTRACT FROM AUTHOR]

Published
2023
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16. An apical membrane complex controls rhoptry exocytosis and invasion in Toxoplasma

Author

Sparvoli, Daniela, Delabre, Jason, Penarete-Vargas, Diana Marcela, Mageswaran, Shrawan Kumar, Tsypin, Lev M., Heckendorn, Justine, Theveny, Liam, Maynadier, Marjorie, Cova, Marta Mendonça, Berry-Sterkers, Laurence, Guérin, Amandine, Dubremetz, Jean-François, Urbach, Serge, Striepen, Boris, Turkewitz, Aaron P., Chang, Yi-Wei, Lebrun, Maryse, Sparvoli, Daniela, Delabre, Jason, Penarete-Vargas, Diana Marcela, Mageswaran, Shrawan Kumar, Tsypin, Lev M., Heckendorn, Justine, Theveny, Liam, Maynadier, Marjorie, Cova, Marta Mendonça, Berry-Sterkers, Laurence, Guérin, Amandine, Dubremetz, Jean-François, Urbach, Serge, Striepen, Boris, Turkewitz, Aaron P., Chang, Yi-Wei, and Lebrun, Maryse

Abstract

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. In contrast to previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associated with the exocytic site - prior to invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarity to GPCR proteins. We propose that the CRMP complex acts as host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Published
2022

19. An apical membrane complex controls rhoptry exocytosis and invasion in Toxoplasma

Author

Sparvoli, Daniela, primary, Delabre, Jason, additional, Penarete-Vargas, Diana Marcela, additional, Mageswaran, Shrawan Kumar, additional, Tsypin, Lev M., additional, Heckendorn, Justine, additional, Theveny, Liam, additional, Maynadier, Marjorie, additional, Cova, Marta Mendonça, additional, Berry-Sterkers, Laurence, additional, Guérin, Amandine, additional, Dubremetz, Jean-François, additional, Urbach, Serge, additional, Striepen, Boris, additional, Turkewitz, Aaron P., additional, Chang, Yi-Wei, additional, and Lebrun, Maryse, additional

Published
2022
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23. Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Author

Nisbet, R. Ellen R., Casacuberta, Elena, Sudek, Lisa, Allen, Andrew E., Ares, Manuel, Balestreri, Cecilia, Barbrook, Adrian C., Beardslee, Patrick, Bender, Sara, Booth, David S., Bowler, Chris, Breglia, Susana A., Brownlee, Colin, Burger, Gertraud, Cerutti, Heriberto, Cesaroni, Rachele, Chiurillo, Miguel A., Clemente, Thomas, Coles, Duncan B., Collier, Jackie L., Cooney, Elizabeth C., Coyne, Kathryn, Docampo, Roberto, Dupont, Christopher L., Edgcomb, Virginia, Einarsson, Elin, Federici, Fernan, Freire-Beneitez, Veronica, Freyria, Nastasia J., Fukuda, Kodai, Girguis, Peter R., Gomaa, Fatma, Gornik, Sebastian G., Guo, Jian, Hanawa, Yutaka, Haro-Contreras, Esteban R., Hehenberger, Elisabeth, Highfield, Andrea, Hirakawa, Yoshihisa, Hopes, Amanda, Howe, Christopher J., Hu, Ian, Irwin, Nicholas A. T., Ishii, Yuu, Janowicz, Natalia Ewa, Jones, Adam C., Kachale, Ambar, Fujimura-Kamada, Konomi, Kaur, Binnypreet, Kaye, Jonathan Z., Kazana, Eleanna, Keeling, Patrick J., King, Nicole, Klobutcher, Lawrence A., Lander, Noelia, Lassadi, Imen, Li, Zhuhong, Lin, Senjie, Lozano, Jean-Claude, Luan, Fulei, Maruyama, Shinichiro, Matute, Tamara, Miceli, Cristina, Minagawa, Jun, Moosburner, Mark, Nanjappa, Deepak, Nimmo, Isabel C., Noble, Luke, Nowacki, Mariusz, Pain, Arnab, Piersanti, Angela, Pucciarelli, Sandra, Pyrih, Jan, Rest, Joshua S., Rius, Mariana, Robertson, Deborah, Ruaud, Albane, Sigg, Monika A., Silver, Pamela A., Slamovits, Claudio H., Jason Smith, G., Sprecher, Brittany N., Stern, Rowena, Swart, Estienne C., Tsaousis, Anastasios D., Tsypin, Lev, Turkewitz, Aaron, Valach, Matus, von Dassow, Peter, von der Haar, Tobias, Waller, Ross F., Wang, Lu, Wen, Xiaoxue, Wheeler, Glen, Woods, April, Zhang, Huan, Mock, Thomas, and Worden, Alexandra Z.

Subjects
Cell Biology, Biochemistry, Molecular Biology, Biotechnology
Abstract

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Published
2020

24. ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms

Author

Casler, Jason C., Zajac, Allison L., Valbuena, Fernando M., Sparvoli, Daniela, Jeyifous, Okunola, Turkewitz, Aaron, Horne-Badovinac, Sally, Green, William N., Glick, Benjamin S., Casler, Jason C., Zajac, Allison L., Valbuena, Fernando M., Sparvoli, Daniela, Jeyifous, Okunola, Turkewitz, Aaron, Horne-Badovinac, Sally, Green, William N., and Glick, Benjamin S.

Abstract

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Casler, J. C., Zajac, A. L., Valbuena, F. M., Sparvoli, D., Jeyifous, O., Turkewitz, A. P., Horne-Badovinac, S., Green, W. N., & Glick, B. S. ESCargo: a regulatable fluorescent secretory cargo for diverse model organisms. Molecular Biology of the Cell, (2020): mbcE20090591, doi:10.1091/mbc.E20-09-0591., Membrane traffic can be studied by imaging a cargo protein as it transits the secretory pathway. The best tools for this purpose initially block export of the secretory cargo from the endoplasmic reticulum (ER), and then release the block to generate a cargo wave. However, previously developed regulatable secretory cargoes are often tricky to use or specific for a single model organism. To overcome these hurdles for budding yeast, we recently optimized an artificial fluorescent secretory protein that exits the ER with the aid of the Erv29 cargo receptor, which is homologous to mammalian Surf4. The fluorescentsecretory protein forms aggregates in the ER lumen and can be rapidly disaggregated by addition of a ligand to generate a nearly synchronized cargo wave. Here we term this regulatable secretory proteinESCargo (Erv29/Surf4-dependent Secretory Cargo) and demonstrate its utility not only in yeast cells, but also in cultured mammalian cells, Drosophila cells, and the ciliate Tetrahymena thermophila. Kinetic studies indicate that rapid export from the ER requires recognition by Erv29/Surf4. By choosing an appropriate ER signal sequence and expression vector, this simple technology can likely be used withmany model organisms., This work was supported by NIH grant R01 GM104010 to BSG, by NIH grant R01 GM105783 to APT, by NIH grant R01 GM136961 and American Cancer Society grant RSG-14-176 to SHB, and by NIH grant R01 DA044760 to WNG. JCC was supported by NIH training grant T32 GM007183. AZ was supported by American Heart Association fellowship 16POST2726018 and American Cancer Society fellowship 132123-PF-18-025-01-CSM. Thanks for assistance with fluorescence microscopy to Vytas Bindokas and Christine Labno at the Integrated Microscopy Core Facility, which is supported by the NIH-funded Cancer Center Support Grant P30 CA014599. The pUASt-ManII-eGFP plasmid was a gift from Bing Ye, and the Ubi-Gal4 plasmid was a gift from Rick Fehon., 2020-12-28

Published
2020

25. Genetic tool development in marine protists: emerging model organisms for experimental cell biology

Author

Gordon and Betty Moore Foundation, Leverhulme Trust, Ministry of Education, Youth and Sports (Czech Republic), European Commission, Faktorová, Drahomíra, Nisbet, R. Ellen R, Robledo, José A. F., Casacuberta, Elena, Sudek, Lisa, Allen, Andrew E., Ares, Manuel, Aresté, Cristina, Balestreri, Cecilia, Barbrook, Adrian C., Beardslee, Patrick, Ruaud, Albane, Ruiz-Trillo, Iñaki, Sigg, Monika A., Silver, Pamela A., Slamovits, Claudio H., Smith, G. Jason, Sprecher, Brittany N., Stern, Rowena, Swart, Estienne C., Tsaousis, Anastasios D., Valach, Matus, Tsypin, Lev, Turkewitz, Aaron, Turnšek, Jernej, Vergé, Valérie, von Dassow, Peter, von der Haar, Tobias, Waller, Ross F., Wang, Lu, Wen, Xiaoxue, Wheeler, Glen, Woods, April, Zhang, Huan, Bender, Sara, Mock, Thomas, Worden, Alexandra Z., Lukeš, Julius, Booth, David S., Bouget, François-Yves, Bowler, Chris, Breglia, Susana A., Brownlee, Colin, Burger, Gertraud, Cerutti, Heriberto, Cesaroni, Rachele, Chiurillo, Miguel A., Clemente, Thomas, Coles, Duncan B., Collier, Jackie L., Cooney, Elizabeth C., Coyne, Kathryn, Docampo, Roberto, Dupont, Christopher L., Edgcomb, Virginia, Einarsson, Elin, Elustondo, Pía A., Federici, Fernan, Freire-Beneitez, Verónica, Freyria, Nastasia J., Fukuda, Kodai, García, Paulo A., Girguis, Peter R., Gomaa, Fatma, Gornik, Sebastian G., Guo, Jian, Hampl, Vladimir, Hanawa, Yutaka, Haro-Contreras, Esteban R., Hehenberger, Elisabeth, Highfield, Andrea, Hirakawa, Yoshihisa, Hopes, Amanda, Howe, Christopher J., Hu, Ian, Ibañez, Jorge, Irwin, Nicholas A. T., Ishii, Yuu, Janowicz, Natalia Ewa, Jones, Adam C., Kachale, Ambar, Fujimura-Kamada, Konomi, Kaur, Binnypreet, Kaye, Jonathan Z., Kazana, Eleanna, Keeling, Patrick J., King, Nicole, Klobutcher, Lawrence A., Lander, Noelia, Lassadi, Imen, Li, Zhuhong, Lin, Senjie, Lozano, Jean-Claude, Luan, Fulei, Maruyama, Shinichiro, Matute, Tamara, Miceli, Cristina, Minagawa, Jun, Moosburner, Mark, Najle, Sebastián R., Nanjappa, Deepak, Nimmo, Isabel C., Noble, Luke, Novák Vanclová, Anna M. G., Nowacki, Mariusz, Núñez, Isaac, Pain, Arnab, Piersanti, Angela, Pucciarelli, Sandra, Pyrih, Jan, Rest, Joshua S., Rius, Mariana, Robertson, Deborah, Gordon and Betty Moore Foundation, Leverhulme Trust, Ministry of Education, Youth and Sports (Czech Republic), European Commission, Faktorová, Drahomíra, Nisbet, R. Ellen R, Robledo, José A. F., Casacuberta, Elena, Sudek, Lisa, Allen, Andrew E., Ares, Manuel, Aresté, Cristina, Balestreri, Cecilia, Barbrook, Adrian C., Beardslee, Patrick, Ruaud, Albane, Ruiz-Trillo, Iñaki, Sigg, Monika A., Silver, Pamela A., Slamovits, Claudio H., Smith, G. Jason, Sprecher, Brittany N., Stern, Rowena, Swart, Estienne C., Tsaousis, Anastasios D., Valach, Matus, Tsypin, Lev, Turkewitz, Aaron, Turnšek, Jernej, Vergé, Valérie, von Dassow, Peter, von der Haar, Tobias, Waller, Ross F., Wang, Lu, Wen, Xiaoxue, Wheeler, Glen, Woods, April, Zhang, Huan, Bender, Sara, Mock, Thomas, Worden, Alexandra Z., Lukeš, Julius, Booth, David S., Bouget, François-Yves, Bowler, Chris, Breglia, Susana A., Brownlee, Colin, Burger, Gertraud, Cerutti, Heriberto, Cesaroni, Rachele, Chiurillo, Miguel A., Clemente, Thomas, Coles, Duncan B., Collier, Jackie L., Cooney, Elizabeth C., Coyne, Kathryn, Docampo, Roberto, Dupont, Christopher L., Edgcomb, Virginia, Einarsson, Elin, Elustondo, Pía A., Federici, Fernan, Freire-Beneitez, Verónica, Freyria, Nastasia J., Fukuda, Kodai, García, Paulo A., Girguis, Peter R., Gomaa, Fatma, Gornik, Sebastian G., Guo, Jian, Hampl, Vladimir, Hanawa, Yutaka, Haro-Contreras, Esteban R., Hehenberger, Elisabeth, Highfield, Andrea, Hirakawa, Yoshihisa, Hopes, Amanda, Howe, Christopher J., Hu, Ian, Ibañez, Jorge, Irwin, Nicholas A. T., Ishii, Yuu, Janowicz, Natalia Ewa, Jones, Adam C., Kachale, Ambar, Fujimura-Kamada, Konomi, Kaur, Binnypreet, Kaye, Jonathan Z., Kazana, Eleanna, Keeling, Patrick J., King, Nicole, Klobutcher, Lawrence A., Lander, Noelia, Lassadi, Imen, Li, Zhuhong, Lin, Senjie, Lozano, Jean-Claude, Luan, Fulei, Maruyama, Shinichiro, Matute, Tamara, Miceli, Cristina, Minagawa, Jun, Moosburner, Mark, Najle, Sebastián R., Nanjappa, Deepak, Nimmo, Isabel C., Noble, Luke, Novák Vanclová, Anna M. G., Nowacki, Mariusz, Núñez, Isaac, Pain, Arnab, Piersanti, Angela, Pucciarelli, Sandra, Pyrih, Jan, Rest, Joshua S., Rius, Mariana, and Robertson, Deborah

Abstract

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Published
2020

29. Analysis of a mutant exhibiting conditional sorting to dense core secretory granules in tetrahymena thermophila

Author

Bowman, Grant R. and Turkewitz, Aaron P.

Subjects
Genetic research -- Analysis, Biological sciences
Abstract

The formation of dense core granules (DCGs) requires both the sorting of granule contents from other secretory proteins and a postsorting maturation process. The Tetrahymena thermophila strain SB281 fails to synthesize DCGs, and previous analysis suggested that the defect lay at or near the sorting step. Because this strain represents one of the very few mutants in this pathway, we have undertaken a more complete study of the phenotype. Genetic epistasis analysis places the defect upstream of those in two other characterized Tetrahymena mutants. Using immunofluorescent detection of granule content proteins, as well as GFP tagging, we describe a novel cytoplasmic compartment to which granule contents can be sorted in growing SB281 cells. Cell fusion experiments indicate that this compartment is not a biosynthetic intermediate in DCG synthesis. Sorting in SB281 is strongly conditional with respect to growth. When cells are starved, the storage compartment is degraded and de novo synthesized granule proteins are rapidly secreted. The mutation in SB281 therefore appears to affect DCG synthesis at the level of both sorting and maturation.

Published
2001

32. Proposed genetic nomenclature rules for Tetrahymena thermophila, Paramecium primaurelia and Paramecium tetraurelia

Author

Allen, Sally Lyman, Altschuler, Marsha I., Bruns, Peter J., Cohen, Jean, Doerder, F. Paul, Gaertig, Jacek, Gorovsky, Martin, Orias, Eduardo, and Turkewitz, Aaron

Subjects
Ciliata -- Genetic aspects, Protozoa -- Genetic aspects, Biological sciences
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.

Published
1998

36. Additional file 1: of N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA

Author

Guan-Zheng Luo, Ziyang Hao, Liangzhi Luo, Mingren Shen, Sparvoli, Daniela, Yuqing Zheng, Zijie Zhang, Xiaocheng Weng, Chen, Kai, Cui, Qiang, Turkewitz, Aaron, and He, Chuan

Abstract

Figure S1. Preferred sequence motifs for Tetrahymena 6mA sites. Figure S2. Agarose gel of MNase digested nuclei. Figure S3. Nucleosome assembly favors 6mA-free regions in vitro. Figure S4. Changes of mean value after 6mA modification in the intra-base pair (A-F) and inter-base pair (G-L) parameters as a function of distance from the center of the modification site. Figure S5. Changes of standard deviation (s.d.) after 6mA modification in the intra-base pair (A-F) and inter-base pair (G-L) parameters as a function of distance from the center of AT. Figure S6. Multiple sequence alignment and phylogenetic distribution analysis of MTA70 family. Figure S7. Knockout of two methyltransferases in Tarahymena and in vitro methylation activity characterization of methytransferaseTAMT-1. Figure S8. Effects of tamt-1 knockout. (DOC 11096 kb)

Published
2018
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38. Genetic tool development in marine protists: Emerging model organisms for experimental cell biology

Author

Faktorová, Drahomíra, primary, Nisbet, R. Ellen R., additional, Robledo, José A. Fernández, additional, Casacuberta, Elena, additional, Sudek, Lisa, additional, Allen, Andrew E., additional, Ares, Manuel, additional, Aresté, Cristina, additional, Balestreri, Cecilia, additional, Barbrook, Adrian C., additional, Beardslee, Patrick, additional, Bender, Sara, additional, Booth, David S., additional, Bouget, François-Yves, additional, Bowler, Chris, additional, Breglia, Susana A., additional, Brownlee, Colin, additional, Burger, Gertraud, additional, Cerutti, Heriberto, additional, Cesaroni, Rachele, additional, Chiurillo, Miguel A., additional, Clemente, Thomas, additional, Coles, Duncan B., additional, Collier, Jackie L., additional, Cooney, Elizabeth C., additional, Coyne, Kathryn, additional, Docampo, Roberto, additional, Dupont, Christopher L., additional, Edgcomb, Virginia, additional, Einarsson, Elin, additional, Elustondo, Pía A., additional, Federici, Fernan, additional, Freire-Beneitez, Veronica, additional, Freyria, Nastasia J., additional, Fukuda, Kodai, additional, García, Paulo A., additional, Girguis, Peter R., additional, Gomaa, Fatma, additional, Gornik, Sebastian G., additional, Guo, Jian, additional, Hampl, Vladimír, additional, Hanawa, Yutaka, additional, Haro-Contreras, Esteban R., additional, Hehenberger, Elisabeth, additional, Highfield, Andrea, additional, Hirakawa, Yoshihisa, additional, Hopes, Amanda, additional, Howe, Christopher J., additional, Hu, Ian, additional, Ibañez, Jorge, additional, Irwin, Nicholas A.T., additional, Ishii, Yuu, additional, Janowicz, Natalia Ewa, additional, Jones, Adam C., additional, Kachale, Ambar, additional, Fujimura-Kamada, Konomi, additional, Kaur, Binnypreet, additional, Kaye, Jonathan Z., additional, Kazana, Eleanna, additional, Keeling, Patrick J., additional, King, Nicole, additional, Klobutcher, Lawrence A., additional, Lander, Noelia, additional, Lassadi, Imen, additional, Li, Zhuhong, additional, Lin, Senjie, additional, Lozano, Jean-Claude, additional, Luan, Fulei, additional, Maruyama, Shinichiro, additional, Matute, Tamara, additional, Miceli, Cristina, additional, Minagawa, Jun, additional, Moosburner, Mark, additional, Najle, Sebastián R., additional, Nanjappa, Deepak, additional, Nimmo, Isabel C., additional, Noble, Luke, additional, Vanclová, Anna M.G. Novák, additional, Nowacki, Mariusz, additional, Nuñez, Isaac, additional, Pain, Arnab, additional, Piersanti, Angela, additional, Pucciarelli, Sandra, additional, Pyrih, Jan, additional, Rest, Joshua S., additional, Rius, Mariana, additional, Robertson, Deborah, additional, Ruaud, Albane, additional, Ruiz-Trillo, Iñaki, additional, Sigg, Monika A., additional, Silver, Pamela A., additional, Slamovits, Claudio H., additional, Smith, G. Jason, additional, Sprecher, Brittany N., additional, Stern, Rowena, additional, Swart, Estienne C., additional, Tsaousis, Anastasios D., additional, Tsypin, Lev, additional, Turkewitz, Aaron, additional, Turnšek, Jernej, additional, Valach, Matus, additional, Vergé, Valérie, additional, von Dassow, Peter, additional, von der Haar, Tobias, additional, Waller, Ross F., additional, Wang, Lu, additional, Wen, Xiaoxue, additional, Wheeler, Glen, additional, Woods, April, additional, Zhang, Huan, additional, Mock, Thomas, additional, Worden, Alexandra Z., additional, and Lukeš, Julius, additional

Published
2019
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44. Remodeling the Specificity of an Endosomal CORVET Tether Underlies Formation of Regulated Secretory Vesicles in the Ciliate Tetrahymena thermophila

Author

Sparvoli, Daniela, primary, Richardson, Elisabeth, additional, Osakada, Hiroko, additional, Lan, Xun, additional, Iwamoto, Masaaki, additional, Bowman, Grant R., additional, Kontur, Cassandra, additional, Bourland, William A., additional, Lynn, Denis H., additional, Pritchard, Jonathan K., additional, Haraguchi, Tokuko, additional, Dacks, Joel B., additional, and Turkewitz, Aaron P., additional

Published
2018
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46. Extreme metal adapted, knockout and knockdown strains reveal a coordinated gene expression among different Tetrahymena thermophila metallothionein isoforms

Author

Francisco Martínez, Patricia de, Martín González, Ana María, Turkewitz, Aaron P., Gutiérrez Fernández, Juan Carlos, Francisco Martínez, Patricia de, Martín González, Ana María, Turkewitz, Aaron P., and Gutiérrez Fernández, Juan Carlos

Abstract

Metallothioneins (MT) constitute a superfamily of small cytosolic proteins that are able to bind metal cations through numerous cysteine (Cys) residues. Like other organisms the ciliate Tetrahymena thermophila presents several MT isoforms, which have been classified into two subfamilies (Cd- and Cu-metallothioneins). The main aim of this study was to examine the specific functions and transcriptional regulation of the five MT isoforms present in T. thermophila, by using several strains of this ciliate. After a laboratory evolution experiment over more than two years, three different T. thermophila strains adapted to extreme metal stress (Cd2+, Cu2+ or Pb2+) were obtained. In addition, three knockout and/or knockdown strains for different metallothionein (MT) genes were generated. These strains were then analyzed for expression of the individual MT isoforms. Our results provide a strong basis for assigning differential roles to the set of MT isoforms. MTT1 appears to have a key role in adaptation to Cd. In contrast, MTT2/4 are crucial for Cu-adaptation and MTT5 appears to be important for Pb-adaptation and might be considered as an “alarm” MT gene for responding to metal stress. Moreover, results indicate that likely a coordinated transcriptional regulation exists between the MT genes, particularly among MTT1, MTT5 and MTT2/4. MTT5 appears to be an essential gene, a first such report in any organism of an essential MT gene., Ministerio de Economía y Competitividad (MINECO), NIH, Ministerio de Educación, Cultura y Deporte (MECD), Depto. de Genética, Fisiología y Microbiología, Fac. de Ciencias Biológicas, TRUE, pub

Published
2017

47. Whole‐cell biosensors for detection of heavy metal ions in environmental samples based on metallothionein promoters from Tetrahymena thermophila

Author

Amaro, Francisco, Turkewitz, Aaron P., Martín‐González, Ana, and Gutiérrez, Juan‐Carlos

Subjects
Ions, Time Factors, technology, industry, and agriculture, macromolecular substances, Biosensing Techniques, Sensitivity and Specificity, Artificial Gene Fusion, Tetrahymena thermophila, Metals, Heavy, Environmental Pollutants, Metallothionein, Luciferases, Promoter Regions, Genetic, Research Articles
Abstract

Summary Heavy metals are among the most serious pollutants, and thus there is a need to develop sensitive and rapid biomonitoring methods for heavy metals in the environment. Critical parameters such as bioavailability, toxicity and genotoxicity cannot be tested using chemical analysis, but only can be assayed using living cells. A whole‐cell biosensor uses the whole cell as a single reporter incorporating both bioreceptor and transducer elements. In the present paper, we report results with two gene constructs using the Tetrahymena thermophila MTT1 and MTT5 metallothionein promoters linked with the eukaryotic luciferase gene as a reporter. This is the first report of a ciliated protozoan used as a heavy metal whole‐cell biosensor. T. thermophila transformed strains were created as heavy metal whole‐cell biosensors, and turn on bioassays were designed to detect, in about 2 h, the bioavailable heavy metals in polluted soil or aquatic samples. Validation of these whole‐cell biosensors was carried out using both artificial and natural samples, including methods for detecting false positives and negatives. Comparison with other published cell biosensors indicates that the Tetrahymena metallothionein promoter‐based biosensors appear to be the most sensitive eukaryotic metal biosensors and compare favourably with some prokaryotic biosensors as well.

Published
2011

48. Biogenesis of Dense-Core Secretory Granules

Author

Bowman, Grant R., Cowan, Andrew T., and Turkewitz, Aaron P.

Subjects
Secretory Pathway, Secretory Vesicle, Secretory Granule, AtT20 Cell, health care economics and organizations, Article, PC12 Cell
Abstract

Dense core granules (DCGs) are vesicular organelles derived from outbound traffic through the eukaryotic secretory pathway. As DCGs are formed, the secretory pathway can also give rise to other types of vesicles, such as those bound for endosomes, lysosomes, and the cell surface. DCGs differ from these other vesicular carriers in both content and function, storing highly concentrated cores’ of condensed cargo in vesicles that are stably maintained within the cell until a specific extracellular stimulus causes their fusion with the plasma membrane. These unique features are imparted by the activities of membrane and lumenal proteins that are specifically delivered to the vesicles during synthesis. This chapter will describe the DCG biogenesis pathway, beginning with the sorting of DCG proteins from proteins that are destined for other types of vesicle carriers. In the trans-Golgi network (TGN), sorting occurs as DCG proteins aggregate, causing physical separation from non-DCG proteins. Recent work addresses the nature of interactions that produce these aggregates, as well as potentially important interactions with membranes and membrane proteins. DCG proteins are released from the TGN in vesicles called immature secretory granules (ISGs). The mechanism of ISG formation is largely unclear but is not believed to rely on the assembly of vesicle coats like those observed in other secretory pathways. The required cytosolic factors are now beginning to be identified using in vitro systems with purified cellular components. ISG transformation into a mature fusion-competent, stimulus-dependent DCG occurs as endoproteolytic processing of many DCG proteins causes continued condensation of the lumenal contents. At the same time, proteins that fail to be incorporated into the condensing core are removed by a coat-mediated budding mechanism, which also serves to remove excess membrane and membrane proteins from the maturing vesicle. This chapter will summarize the work leading to our current view of granule synthesis, and will discuss questions that need to be addressed in order to gain a more complete understanding of the pathway.

Published
2010

49. An evolutionary switch in the specificity of an endosomal CORVET tether underlies formation of regulated secretory vesicles in the ciliateTetrahymena thermophila

Author

Sparvoli, Daniela, primary, Richardson, Elisabeth, additional, Osakada, Hiroko, additional, Lan, Xun, additional, Iwamoto, Masaaki, additional, Bowman, Grant R., additional, Kontur, Cassandra, additional, Bourland, William A., additional, Lynn, Denis H., additional, Pritchard, Jonathan K., additional, Haraguchi, Tokuko, additional, Dacks, Joel B., additional, and Turkewitz, Aaron P., additional

Published
2017
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