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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. 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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11. 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

14. 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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16. 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

17. 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

18. 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

22. 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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24. 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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30. 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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32. 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

33. 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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41. Genome plasticity in response to stress in Tetrahymena thermophila: selective and reversible chromosome amplification and paralogous expansion of metallothionein genes.

Author

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

Subjects
TETRAHYMENA thermophila, METALLOTHIONEIN genetics, GENE amplification, POLYPLOIDY, METAL toxicology
Abstract

Summary: Extreme stress situations can induce genetic variations including genome reorganization. In ciliates like Tetrahymena thermophila, the approximately 45‐fold ploidy of the somatic macronucleus may enable adaptive responses that depend on genome plasticity. To identify potential genome‐level adaptations related to metal toxicity, we isolated three Tetrahymena thermophila strains after an extended adaptation period to extreme metal concentrations (Cd2+, Cu2+ or Pb2+). In the Cd‐adapted strain, we found a approximately five‐fold copy number increase of three genes located in the same macronuclear chromosome, including two metallothionein genes, MTT1 and MTT3. The apparent amplification of this macronuclear chromosome was reversible and reproducible, depending on the presence of environmental metal. We also analysed three knockout (KO) and/or knockdown (KD) strains for MTT1 and/or MTT5. In the MTT5KD strain, we found at least two new genes arising from paralogous expansion of MTT1, which encode truncated variants of MTT1. The expansion can be explained by a model based on somatic recombination between MTT1 genes on pairs of macronuclear chromosomes. At least two of the new paralogs are transcribed and upregulated in response to Cd2+. Altogether, we have thus identified two distinct mechanisms, both involving genomic plasticity in the polyploid macronucleus that may represent adaptive responses to metal‐related stress. [ABSTRACT FROM AUTHOR]

Published
2018
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43. Secretion of Polypeptide Crystals from Tetrahymena thermophilaSecretory Organelles (Mucocysts) Depends on Processing by a Cysteine Cathepsin, Cth4p

Author

Kumar, Santosh, Briguglio, Joseph S., and Turkewitz, Aaron P.

Abstract

ABSTRACTIn many organisms, sophisticated mechanisms facilitate release of peptides in response to extracellular stimuli. In the ciliate Tetrahymena thermophila, efficient peptide secretion depends on specialized vesicles called mucocysts that contain dense crystalline cores that expand rapidly during exocytosis. Core assembly depends of endoproteolytic cleavage of mucocyst proproteins by an aspartyl protease, cathepsin 3 (CTH3). Here, we show that a second enzyme identified by expression profiling, Cth4p, is also required for processing of proGrl proteins and for assembly of functional mucocysts. Cth4p is a cysteine cathepsin that localizes partially to endolysosomal structures and appears to act downstream of, and may be activated by, Cth3p. Disruption of CTH4results in cells (?cth4) that show aberrant trimming of Grl proproteins, as well as grossly aberrant mucocyst exocytosis. Surprisingly, ?cth4cells succeed in assembling crystalline mucocyst cores. However, those cores do not undergo normal directional expansion during exocytosis, and they thus fail to efficiently extrude from the cells. We could phenocopy the ?cth4defects by mutating conserved catalytic residues, indicating that the in vivofunction of Cth4p is enzymatic. Our results indicate that as for canonical proteins packaged in animal secretory granules, the maturation of mucocyst proproteins involves sequential processing steps. The ?cth4defects uncouple, in an unanticipated way, the assembly of mucocyst cores and their subsequent expansion and thereby reveal a previously unsuspected aspect of polypeptide secretion in ciliates.

Published
2015
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45. Genetic tool development in marine protists: Emerging model organisms for experimental cell biology

Author

Deepak Nanjappa, Iñaki Ruiz-Trillo, Christopher J. Howe, Chris Bowler, Mariusz Nowacki, Anastasios D. Tsaousis, Noelia Lander, Christopher L. Dupont, Shinichiro Maruyama, Fulei Luan, Andrew E. Allen, Anna M. G. Novák Vanclová, Pamela A. Silver, Nastasia J. Freyria, Peter von Dassow, Elisabeth Hehenberger, Konomi Fujimura-Kamada, Julius Lukeš, Roberto Docampo, Deborah L. Robertson, Thomas E. Clemente, Sebastián R. Najle, Kathryn J. Coyne, Cristina Aresté, Brittany N. Sprecher, Lu Wang, Eleanna Kazana, Verónica Freire-Benéitez, Vladimír Hampl, Cecilia Balestreri, Isabel C. Nimmo, Mariana Rius, Yoshihisa Hirakawa, Arnab Pain, Jorge Ibañez, Elin Einarsson, Lev Tsypin, Heriberto Cerutti, Adrian C. Barbrook, G. Jason Smith, Alexandra Z. Worden, Jian Guo, Jean Claude Lozano, Virginia P. Edgcomb, Huan Zhang, Andrea Highfield, Isaac Núñez, Fatma Gomaa, Jan Pyrih, Natalia Ewa Janowicz, Sara J. Bender, Ross F. Waller, Tobias von der Haar, François-Yves Bouget, Matus Valach, Amanda Hopes, Luke M. Noble, Paulo A. Garcia, Nicholas A.T. Irwin, Tamara Matute, Jernej Turnšek, Ian Hu, Sandra Pucciarelli, Fernán Federici, Valérie Vergé, R. Ellen R. Nisbet, Miguel Angel Chiurillo, Mark Moosburner, Monika Abedin Sigg, Aaron P. Turkewitz, Albane Ruaud, Angela Piersanti, Sebastian G. Gornik, Peter R. Girguis, Adam C. Jones, Lawrence A. Klobutcher, Claudio H. Slamovits, Elena Casacuberta, Imen Lassadi, Elizabeth C. Cooney, Kodai Fukuda, Nicole King, Manuel Ares, Jonathan Z. Kaye, Lisa Sudek, Patrick Beardslee, Cristina Miceli, Xiaoxue Wen, Estienne C. Swart, Yuu Ishii, Ambar Kachale, Pia A. Elustondo, Esteban R. Haro-Contreras, Patrick J. Keeling, José A. Fernández Robledo, Glen L. Wheeler, Colin Brownlee, Rowena Stern, Joshua S. Rest, Susana A. Breglia, Senjie Lin, Duncan B. Coles, Jackie L. Collier, Drahomíra Faktorová, David S. Booth, April Woods, Binnypreet Kaur, Thomas Mock, Gertraud Burger, Jun Minagawa, Yutaka Hanawa, Zhu-Hong Li, Rachele Cesaroni, Laboratoire d'Océanographie Microbienne (LOMIC), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire océanologique de Banyuls (OOB), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Faktorová, Drahomíra [0000-0001-9623-2233], Nisbet, R. Ellen R. [0000-0003-4487-196X], Allen, Andrew E. [0000-0001-5911-6081], Ares, Manuel, Jr [0000-0002-2552-9168], Bowler, Chris [0000-0003-3835-6187], Burger, Gertraud [0000-0002-8679-8812], Collier, Jackie L. [0000-0001-8774-5715], Cooney, Elizabeth C. [0000-0001-7435-7609], Docampo, Roberto [0000-0003-4229-8784], Dupont, Christopher L. [0000-0002-0896-6542], Edgcomb, Virginia [0000-0001-6805-381X], Freyria, Nastasia J. [0000-0002-4972-9383], Gornik, Sebastian G. [0000-0002-8026-1336], Howe, Christopher J. [0000-0002-6975-8640], Hu, Ian [0000-0002-2287-031X], Ishii, Yuu [0000-0003-1735-9557], Jones, Adam C. [0000-0001-7521-1863], Lin, Senjie [0000-0001-8831-6111], Maruyama, Shinichiro [0000-0002-1128-5916], Minagawa, Jun [0000-0002-3028-3203], Najle, Sebastián R. [0000-0002-0654-9147], Nowacki, Mariusz [0000-0003-4894-2905], Pain, Arnab [0000-0002-1755-2819], Silver, Pamela A. [0000-0002-7856-4071], Jason Smith, G. [0000-0003-1258-4800], Sprecher, Brittany N. [0000-0001-5032-3834], Tsaousis, Anastasios D. [0000-0002-5424-1905], Tsypin, Lev [0000-0002-0642-8468], Turkewitz, Aaron [0000-0003-3531-5806], Turnšek, Jernej [0000-0002-9056-3565], Valach, Matus [0000-0001-8689-0080], von der Haar, Tobias [0000-0002-6031-9254], Mock, Thomas [0000-0001-9604-0362], Worden, Alexandra Z. [0000-0002-9888-9324], Lukeš, Julius [0000-0002-0578-6618], Apollo - University of Cambridge Repository, Nisbet, R Ellen R [0000-0003-4487-196X], Allen, Andrew E [0000-0001-5911-6081], Ares, Manuel [0000-0002-2552-9168], Collier, Jackie L [0000-0001-8774-5715], Cooney, Elizabeth C [0000-0001-7435-7609], Dupont, Christopher L [0000-0002-0896-6542], Freyria, Nastasia J [0000-0002-4972-9383], Gornik, Sebastian G [0000-0002-8026-1336], Howe, Christopher J [0000-0002-6975-8640], Jones, Adam C [0000-0001-7521-1863], Najle, Sebastián R [0000-0002-0654-9147], Silver, Pamela A [0000-0002-7856-4071], Jason Smith, G [0000-0003-1258-4800], Sprecher, Brittany N [0000-0001-5032-3834], Tsaousis, Anastasios D [0000-0002-5424-1905], Worden, Alexandra Z [0000-0002-9888-9324], Gordon and Betty Moore Foundation, Leverhulme Trust, Ministry of Education, Youth and Sports (Czech Republic), and European Commission

Subjects
Resource, Molecular biology, Range (biology), Tree of life (biology), [SDV]Life Sciences [q-bio], Green Fluorescent Proteins, ved/biology.organism_classification_rank.species, Marine Biology, Environment, Diversification (marketing strategy), Biology, Models, Biological, Biochemistry, Genome, 03 medical and health sciences, Transformation, Genetic, 0302 clinical medicine, Species Specificity, Genome editing, 631/1647/767/722, Ecosystem, 14. Life underwater, Model organism, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Dna delivery, 030306 microbiology, ved/biology, Cellular pathways, Eukaryota, Genetic systems, Biodiversity, DNA, Cell Biology, Genetic models, biology.organism_classification, Cell biology, QR, Transformation (genetics), Eukaryote, 030217 neurology & neurosurgery, 631/337, 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., This collaborative effort was supported by the Gordon and Betty Moore Foundation EMS Program of the Marine Microbiology Initiative (grant nos. GBMF4972 and 4972.01 to F.-Y.B.; GBMF4970 and 4970.01 to M.A. and A.Z.W.; GBMF3788 to A.Z.W.; GBMF 4968 and 4968.01 to H.C.; GBMF4984 to V.H.; GBMF4974 and 4974.01 to C. Brownlee; GBMF4964 to Y. Hirakawa; GBMF4961 to T. Mock; GBMF4958 to P.S.; GBMF4957 to A.T.; GBMF4960 to G.J.S.; GBMF4979 to K.C.; GBMF4982 and 4982.01 to J.L.C.; GBMF4964 to P.J.K.; GBMF4981 to P.v.D.; GBMF5006 to A.E.A.; GBMF4986 to C.M.; GBMF4962 to J.A.F.R.; GBMF4980 and 4980.01 to S.L.; GBMF 4977 and 4977.01 to R.F.W.; GBMF4962.01 to C.H.S.; GBMF4985 to J.M.; GBMF4976 and 4976.01 to C.H.; GBMF4963 and 4963.01 to V.E.; GBMF5007 to C.L.D.; GBMF4983 and 4983.01 to J.L.; GBMF4975 and 4975.01 to A.D.T.; GBMF4973 and 4973.01 to I.R.-T. and GBMF4965 to N.K.), by The Leverhulme Trust (RPG-2017-364) to T. Mock and A. Hopes, and by ERD funds (16_019/0000759) from the Czech Ministry of Education to J.L.

Published
2020

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

Author

Cheng CY, Romero DP, Zoltner M, Yao MC, and Turkewitz AP

Subjects
Endosomes, Proteins metabolism, Mitosis, Vacuoles metabolism, Tetrahymena thermophila
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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Published
2023
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47. VPS8D, a CORVET subunit, is required to maintain the contractile vacuole complex in Tetrahymena thermophila .

Author

Cheng CY, Hernández J, and Turkewitz AP

Abstract

Contractile vacuole complexes (CVCs) are complex osmoregulatory organelles, with vesicular (bladder) and tubular (spongiome) subcompartments. The mechanisms that underlie their formation and maintenance within the eukaryotic endomembrane network are poorly understood. In the Ciliate Tetrahymena thermophila , six differentiated CORVETs (class C core vacuole/endosome tethering complexes), with Vps8 subunits designated A-F, are likely to direct endosomal trafficking. Vps8Dp localizes to both bladder and spongiome. We show by inducible knockdown that VPS8D is essential to CVC organization and function. VPS8D knockdown increased susceptibility to osmotic shock, tolerated in the wildtype but triggering irreversible lethal swelling in the mutant. The knockdown rapidly triggered contraction of the spongiome and lengthened the period of the bladder contractile cycle. More prolonged knockdown resulted in disassembly of both the spongiome and bladder, and dispersal of proteins associated with those compartments. In stressed cells where the normally singular bladder is replaced by numerous vesicles bearing bladder markers, Vps8Dp concentrated conspicuously at long-lived inter-vesicle contact sites, consistent with tethering activity. Similarly, Vps8Dp in cell-free preparations accumulated at junctions formed after vacuoles came into close contact. Also consistent with roles for Vps8Dp in tethering and/or fusion were the emergence in knockdown cells of multiple vacuole-related structures, replacing the single bladder., Competing Interests: Competing interests The authors declare no competing or financial interests.

Published
2023
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48. Whole Genome Sequencing Identifies a Novel Factor Required for Secretory Granule Maturation in Tetrahymena thermophila.

Author

Kontur C, Kumar S, Lan X, Pritchard JK, and Turkewitz AP

Subjects
Cytosol metabolism, Exocytosis genetics, Gene Expression Regulation, High-Throughput Nucleotide Sequencing, Mutagenesis, Proteins genetics, Secretory Vesicles metabolism, Tetrahymena thermophila physiology, Genome, Proteins metabolism, Secretory Vesicles genetics, Tetrahymena thermophila genetics
Abstract

Unbiased genetic approaches have a unique ability to identify novel genes associated with specific biological pathways. Thanks to next generation sequencing, forward genetic strategies can be expanded to a wider range of model organisms. The formation of secretory granules, called mucocysts, in the ciliate Tetrahymena thermophila relies, in part, on ancestral lysosomal sorting machinery, but is also likely to involve novel factors. In prior work, multiple strains with defects in mucocyst biogenesis were generated by nitrosoguanidine mutagenesis, and characterized using genetic and cell biological approaches, but the genetic lesions themselves were unknown. Here, we show that analyzing one such mutant by whole genome sequencing reveals a novel factor in mucocyst formation. Strain UC620 has both morphological and biochemical defects in mucocyst maturation-a process analogous to dense core granule maturation in animals. Illumina sequencing of a pool of UC620 F2 clones identified a missense mutation in a novel gene called MMA1 (Mucocyst maturation). The defects in UC620 were rescued by expression of a wild-type copy of MMA1, and disrupting MMA1 in an otherwise wild-type strain phenocopies UC620. The product of MMA1, characterized as a CFP-tagged copy, encodes a large soluble cytosolic protein. A small fraction of Mma1p-CFP is pelletable, which may reflect association with endosomes. The gene has no identifiable homologs except in other Tetrahymena species, and therefore represents an evolutionarily recent innovation that is required for granule maturation., (Copyright © 2016 Kontur et al.)

Published
2016
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