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1. The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis

Author

Quentin Giai Gianetto, Neetu Gupta-Rossi, Mariette Matondo, Stéphane Frémont, Frédérique Cuvelier, Audrey Salles, Julia Chamot-Rooke, Hervé Ménager, Florine Milin, Sandrine Schmutz, Cyril Addi, Murielle Rocancourt, Magalie Duchateau, Arnaud Echard, Thibaut Douché, Adrien Presle, Pascale Zimmermann, Trafic membranaire et Division cellulaire - Membrane Traffic and Cell Division, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège doctoral [Sorbonne universités], Sorbonne Université (SU), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), Institut Pasteur [Paris], Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Plateforme de Protéomique / Proteomics platform, Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Hub Bioinformatique et Biostatistique - Bioinformatics and Biostatistics HUB, BioImagerie Photonique – Photonic BioImaging (UTechS PBI), Centre de Recherche en Cancérologie de Marseille (CRCM), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU), Catholic University of Leuven - Katholieke Universiteit Leuven (KU Leuven), This work has been supported by Institut Pasteur, CNRS, and ANR (AbCyStem, Cytosign) to A.E. C.A. received a fellowship from the Doctoral School Complexité du Vivant ED515, contrat n° 2412/2016 and AMX. A.P. received a fellowship from the Doctoral School Complexité du Vivant ED515, contrat n°2611 bis/2016 and Fondation ARC pour la recherche sur le cancer (DOC20190508876)., We thank R. Basto, G. Hickson, J. Mathieu, J.-R. Huyhn, R. Shaughnessy, M. Serres, and T. Wai for critical reading of the paper, the Echard Lab members for helpful discussions, the Recombinant antibodies platform (TAb-IP, Institut Curie, Paris) and the DSHB (University of Iowa) for antibodies. GFP-MKLP2 cells were from the Hyman Lab MPI-MCBG Dresden45., UTechS PBI is part of the France–BioImaging infrastructure network (FBI) supported by the French National Research Agency (ANR-10-INSB-04, Investments for the Future), and acknowledges support from ANR/FBI and the Région Ile-de-France (program 'Domaine d’Intérêt Majeur-Malinf') for the use of the Zeiss LSM 780 Elyra PS1 microscope. We thank P.H. Commere from the Utechs CB, Institut Pasteur for FACS sorting., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Collège Doctoral, Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Salles, Audrey

Subjects
0301 basic medicine, Syntenins, SYNDECAN, [SDV]Life Sciences [q-bio], General Physics and Astronomy, Cell Cycle Proteins, Plasma protein binding, ABSCISSION, 0302 clinical medicine, ALIX, SYNTENIN, lcsh:Science, Multidisciplinary, Chemistry, MOLECULAR-MECHANISMS, Transmembrane protein, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Multidisciplinary Sciences, Midbody, Science & Technology - Other Topics, MACHINERY, LATE STEPS, Protein Binding, Cell division, Science, BIOGENESIS, SCISSION, HIV Budding, Endosomes, macromolecular substances, General Biochemistry, Genetics and Molecular Biology, ESCRT, Article, 03 medical and health sciences, Abscission, Humans, Cytokinesis, Organelles, MIDBODY, Science & Technology, Endosomal Sorting Complexes Required for Transport, Calcium-Binding Proteins, Cell Membrane, General Chemistry, 030104 developmental biology, Membrane protein, Syndecan-4, lcsh:Q, 030217 neurology & neurosurgery, HeLa Cells
Abstract

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding., ESCRT filaments drive the final abscission between two daughter cells but how they physically interact with the membrane is unclear. Using proteomics, the authors show that syndecan-4/syntenin/ALIX couples the ESCRT machinery to the abscission site and thus promotes efficient abscission.

Published
2020
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2. Deciphering the transport of elastic filaments by antagonistic motor proteins

Author

J. C. Dallon, Cécile Leduc, Stéphanie Portet, Sandrine Etienne-Manneville, University of Manitoba [Winnipeg], Polarité cellulaire, Migration et Cancer - Cell Polarity, Migration and Cancer, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Brigham Young University (BYU), S.P. was partly supported by an National Sciences and Engineering Research Concil of Canada (NSERC) Discovery Grant. S.E.-M. and C.L. were supported by the Centre National de Recherche Scientifique, La Ligue Contre le Cancer, and the Institut Pasteur., The authors would like to thank the Isaac Newton Institute for Mathematical Sciences for support and hospitality during the program 'Coupling Geometric PDE's with Physics for Cell Morphology, Motility and Pattern Formation' when the work on this paper was undertaken. S.P. would like to thank Institut Pasteur for support of her stay. S.P. would like to thank P. van den Driessche for discussions on similar matrices., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-16-CE13-0019,SiFi2Net,Filaments intermédiaires: du filament unique au réseau(2016)

Subjects
MESH: Biological Transport, Dynein, Intermediate Filaments, MESH: Molecular Motor Proteins, macromolecular substances, Models, Biological, 01 natural sciences, 010305 fluids & plasmas, Slow motion, Protein filament, Motor protein, 0103 physical sciences, Molecular motor, Elasticity (economics), 010306 general physics, Intermediate filament, Physics, MESH: Kinetics, Molecular Motor Proteins, MESH: Models, Biological, Biological Transport, Elasticity, Kinetics, MESH: Intermediate Filaments, Biophysics, MESH: Elasticity, Kinesin, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie
Abstract

International audience; Intermediate filaments are long elastic fibers that are transported by the microtubule-associated motor proteins kinesin and dynein inside the cell. How elastic filaments are efficiently transported by antagonistic motors is not well understood and is difficult to measure with current experimental techniques. Adapting the tug-of-war paradigm for vesiclelike cargos, we develop a mathematical model to describe the motion of an elastic filament punctually bound to antagonistic motors. As observed in cells, up to three modes of transport are obtained; dynein-driven retrograde, kinesin-driven anterograde fast motions, and a slow motion. Motor properties and initial conditions that depend on intracellular context regulate the transport of filaments. Filament elasticity is found to affect both the mode and the efficiency of transport. We further show that the coordination of motors along the filament emerges from the interplay between intracellular context and elastic properties of filaments.

Published
2019
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3. Stochastic modeling reveals how motor protein and filament properties affect intermediate filament transport

Author

Sandrine Etienne-Manneville, Stéphanie Portet, Cécile Leduc, J. C. Dallon, Brigham Young University (BYU), Polarité cellulaire, Migration et Cancer - Cell Polarity, Migration and Cancer, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), University of Manitoba [Winnipeg], This work was supported by: EPSRC grant numbers EP/K032208/1 and EP/R014604/1 and by La Ligue contre le cancer and the Institut Pasteur., The authors would like to thank the Isaac Newton Institute for Mathematical Sciences for support and hospitality during the programme 'Coupling Geometric PDE’s with Physics for Cell Morphology, Motility and Pattern Formation' when the work on this paper was undertaken., Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and ANR-16-CE13-0019,SiFi2Net,Filaments intermédiaires: du filament unique au réseau(2016)

Subjects
0301 basic medicine, Statistics and Probability, Materials science, [SDV]Life Sciences [q-bio], Dynein, Biological Transport, Active, Kinesins, Catch bond, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microtubules, Models, Biological, General Biochemistry, Genetics and Molecular Biology, Quantitative Biology::Cell Behavior, Protein filament, Motor protein, Quantitative Biology::Subcellular Processes, 03 medical and health sciences, 0302 clinical medicine, Molecular motor, Vimentin, Intermediate filaments, Intermediate filament, Cytoskeleton, Stochastic Processes, General Immunology and Microbiology, Applied Mathematics, Dyneins, Molecular motors, General Medicine, Kinesin, Stochastic, 030104 developmental biology, Modeling and Simulation, Biophysics, General Agricultural and Biological Sciences, 030217 neurology & neurosurgery
Abstract

International audience; Intermediate filaments are a key component of the cytoskeleton. Their transport along microtubules plays an essential role in the control of the shape and structural organization of cells. To identify the key parameters responsible for the control of intermediate filament transport, we generated a model of elastic filament transport by microtubule-associated dynein and kinesin. The model is also applicable to the transport of any elastically-coupled cargoes. We investigate the effect of filament properties such as number of motor binding sites, length, and elasticity on motion of filaments. Additionally, we consider the effect of motor properties, i.e. off rates, on filament transport. When one motor has a catch bond off rate it dictates the motion, whereas when motors have the same type of off rate filaments can alternate between retrograde and anterograde motions. The elasticity of filaments optimizes the filament transport and the coordination of motors along the length of the filament.

Published
2019
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4. Human candidate gene polymorphisms and risk of severe malaria in children in Kilifi, Kenya: a case-control association study

Author

Ndila, Carolyne M, Uyoga, Sophie, Macharia, Alexander W, Nyutu, Gideon, Peshu, Norbert, Ojal, John, Shebe, Mohammed, Awuondo, Kennedy O, Mturi, Neema, Tsofa, Benjamin, Sepúlveda, Nuno, Clark, Taane G, Band, Gavin, Clarke, Geraldine, Rowlands, Kate, Hubbart, Christina, Jeffreys, Anna, Kariuki, Silvia, Marsh, Kevin, Mackinnon, Margaret, Maitland, Kathryn, Kwiatkowski, Dominic P, Rockett, Kirk A, Williams, Thomas N, Abathina, Amadou, Abubakar, Ismaela, Achidi, Eric, Agbenyega, Tsiri, Aiyegbo, Mohammed, Akoto, Alex, Allen, Angela, Allen, Stephen, Amenga-Etego, Lucas, Amodu, Folakemi, Amodu, Olukemi, Anchang-Kimbi, Judith, Ansah, Nana, Ansah, Patrick, Ansong, Daniel, Antwi, Sampson, Anyorigiya, Thomas, Apinjoh, Tobias, Asafo-Agyei, Emmanuel, Asoala, Victor, Atuguba, Frank, Auburn, Sarah, Bah, Abdou, Bamba, Kariatou, Bancone, Germana, Barnwell, David, Barry, Abdoulaye, Bauni, Evasius, Besingi, Richard, Bojang, Kalifa, Bougouma, Edith, Bull, Susan, Busby, George, Camara, Abdoulie, Camara, Landing, Campino, Susana, Carter, Richard, Carucci, Dan, Casals-Pascual, Climent, Ceesay, Ndey, Ceesay, Pa, Chau, Tran, Chuong, Ly, Clark, Taane, Cole-Ceesay, Ramou, Conway, David, Cook, Katharine, Cook, Olivia, Cornelius, Victoria, Corran, Patrick, Correa, Simon, Cox, Sharon, Craik, Rachel, Danso, Bakary, Davis, Timothy, Day, Nicholas, Deloukas, Panos, Dembele, Awa, Devries, Jantina, Dewasurendra, Rajika, Diakite, Mahamadou, Diarra, Elizabeth, Dibba, Yaya, Diss, Andrea, Djimdé, Abdoulaye, Dolo, Amagana, Doumbo, Ogobara, Doyle, Alan, Drakeley, Chris, Drury, Eleanor, Duffy, Patrick, Dunstan, Sarah, Ebonyi, Augustine, Elhassan, Ahmed, Elhassan, Ibrahim, Elzein, Abier, Enimil, Anthony, Esangbedo, Pamela, Evans, Jennifer, Evans, Julie, Farrar, Jeremy, Fernando, Deepika, Fitzpatrick, Kathryn, Fullah, Janet, Garcia, Jacob, Ghansah, Anita, Gottleib, Michael, Green, Angie, Hart, Lee, Hennsman, Meike, Hien, Tran, Hieu, Nguyen, Hilton, Eliza, Hodgson, Abraham, Horstmann, Rolf, Hughes, Catherine, Hussein, Ayman, Hutton, Robert, Ibrahim, Muntaser, Ishengoma, Deus, Jaiteh, Jula, Jallow, Mariatou, Jallow, Muminatou, Jammeh, Kebba, Jasseh, Momodou, Jobarteh, Amie, Johnson, Kimberly, Joseph, Sarah, Jyothi, Dushyanth, Kachala, David, Kamuya, Dorcas, Kanyi, Haddy, Karunajeewa, Harin, Karunaweera, Nadira, Keita, Momodou, Kerasidou, Angeliki, Khan, Aja, Kivinen, Katja, Kokwaro, Gilbert, Konate, Amadou, Konate, Salimata, Koram, Kwadwo, Kwiatkowski, Dominic, Laman, Moses, Si, Le, Leffler, Ellen, Lemnge, Martha, Lin, Enmoore, Alioune, Ly, Macharia, Alexander, Macinnis, Bronwyn, Mai, Nguyen, Makani, Julie, Malangone, Cinzia, Mangano, Valentina, Manjurano, Alphaxard, Manneh, Lamin, Manning, Laurens, Manske, Magnus, Marsh, Vicki, Maslen, Gareth, Maxwell, Caroline, Mbunwe, Eric, Mccreight, Marilyn, Mead, Daniel, Mendy, Alieu, Mendy, Anthony, Mensah, Nathan, Michon, Pascal, Miles, Alistair, Miotto, Olivo, Modiano, David, Mohamed, Hiba, Molloy, Sile, Molyneux, Malcolm, Molyneux, Sassy, Moore, Mike, Moyes, Catherine, Mtei, Frank, Mtove, George, Mueller, Ivo, Mugri, Regina, Munthali, Annie, Mutabingwa, Theonest, Nadjm, Behzad, Ndi, Andre, Ndila, Carolyne, Newton, Charles, Niangaly, Amadou, Njie, Haddy, Njie, Jalimory, Njie, Madi, Njie, Malick, Njie, Sophie, Njiragoma, Labes, Nkrumah, Francis, Ntunthama, Neema, Nyika, Aceme, Nyirongo, Vysaul, O'Brien, John, Obu, Herbert, Oduro, Abraham, Ofori, Alex, Olaniyan, Subulade, Olaosebikan, Rasaq, Oluoch, Tom, Omotade, Olayemi, Oni, Olajumoke, Onykwelu, Emmanuel, Opi, Daniel, Orimadegun, Adebola, O'Riordan, Sean, Ouedraogo, Issa, Oyola, Samuel, Parker, Michael, Pearson, Richard, Pensulo, Paul, Phiri, Ajib, Phu, Nguyen, Pinder, Margaret, Pirinen, Matti, Plowe, Chris, Potter, Claire, Poudiougou, Belco, Puijalon, Odile, Quyen, Nguyen, Ragoussis, Ioannis, Ragoussis, Jiannis, Rasheed, Oba, Reeder, John, Reyburn, Hugh, Riley, Eleanor, Risley, Paul, Rockett, Kirk, Rodford, Joanne, Rogers, Jane, Rogers, William, Ruano-Rubio, Valentín, Sabally-Ceesay, Kumba, Sadiq, Abubacar, Saidy-Khan, Momodou, Saine, Horeja, Sakuntabhai, Anavaj, Sall, Abdourahmane, Sambian, David, Sambou, Idrissa, Sanjoaquin, Miguel, Shah, Shivang, Shelton, Jennifer, Siba, Peter, Silva, Nilupa, Simmons, Cameron, Simpore, Jaques, Singhasivanon, Pratap, Sinh, Dinh, Sirima, Sodiomon, Sirugo, Giorgio, Sisay-Joof, Fatoumatta, Sissoko, Sibiry, Small, Kerrin, Somaskantharajah, Elilan, Spencer, Chris, Stalker, Jim, Stevens, Marryat, Suriyaphol, Prapat, Sylverken, Justice, Taal, Bintou, Tall, Adama, Taylor, Terrie, Teo, Yik, Thai, Cao, Thera, Mahamadou, Titanji, Vincent, Toure, Ousmane, Troye-Blomberg, Marita, Usen, Stanley, Vanderwal, Aaron, Wangai, Hannah, Watson, Renee, Williams, Thomas, Wilson, Michael, Wrigley, Rebecca, Yafi, Clarisse, Yamoah, Lawrence, The Wellcome Trust Centre for Human Genetics [Oxford], University of Oxford, London School of Hygiene and Tropical Medicine (LSHTM), The Wellcome Trust Sanger Institute [Cambridge], St Mary's Hospital, Imperial College, TNW and MM are funded through awards from the Wellcome Trust (grants 091758 and 202800 [to TNW] and grant 088634 [to MM]) and DPK and TGC receive support from the Medical Research Council (grant G19/9 [to DPK] and grants MR/K000551/1, MR/M01360X/1, MR/N010469/1, and MC_PC_15103 [to TGC]). The research leading to these results received funding from the European Community's Seventh Framework Programme (FP7/2007-2013, under grant agreement 242095) and from the Medical Research Council (grant G0600718). MalariaGEN is supported by the Wellcome Trust (WT077383/Z/05/Z) and by the Foundation for the National Institutes of Health (grant 566) as part of the Bill & Melinda Gates' Grand Challenges in Global Health Initiative. The Resource Centre for Genomic Epidemiology of Malaria is supported by the Wellcome Trust (grant 090770/Z/09/Z). Support was also provided by the Medical Research Council (grant G0600718). The Wellcome Trust also provides core awards to the Wellcome Trust Centre for Human Genetics (grant 090532/Z/09/Z) and to the Wellcome Trust Sanger Institute (grant 098051). This work forms part of a larger collaboration with the MalariaGEN Consortium, whose members are listed at http://www.malariagen.net/projects/host/consortium-members. This paper is published with permission from the Director of the Kenya Medical Research Institute (KEMRI)., MalariaGEN Consortium (Anavaj Sakuntabhai), and European Project: 242095,EC:FP7:HEALTH,FP7-HEALTH-2009-single-stage,EVIMALAR(2009)

Subjects
Case-Control Studies, Child, Child, Preschool, Female, Gene Frequency, Genetic Predisposition to Disease, Humans, Kenya, Malaria, Male, Polymorphism, Genetic, Hematology, macromolecular substances, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Article, Genetic, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, parasitic diseases, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.SPEE]Life Sciences [q-bio]/Santé publique et épidémiologie, Polymorphism, Preschool
Abstract

Summary Background Human genetic factors are important determinants of malaria risk. We investigated associations between multiple candidate polymorphisms—many related to the structure or function of red blood cells—and risk for severe Plasmodium falciparum malaria and its specific phenotypes, including cerebral malaria, severe malaria anaemia, and respiratory distress. Methods We did a case-control study in Kilifi County, Kenya. We recruited as cases children presenting with severe malaria to the high-dependency ward of Kilifi County Hospital. We included as controls infants born in the local community between Aug 1, 2006, and Sept 30, 2010, who were part of a genetics study. We tested for associations between a range of candidate malaria-protective genes and risk for severe malaria and its specific phenotypes. We used a permutation approach to account for multiple comparisons between polymorphisms and severe malaria. We judged p values less than 0·005 significant for the primary analysis of the association between candidate genes and severe malaria. Findings Between June 11, 1995, and June 12, 2008, 2244 children with severe malaria were recruited to the study, and 3949 infants were included as controls. Overall, 263 (12%) of 2244 children with severe malaria died in hospital, including 196 (16%) of 1233 with cerebral malaria. We investigated 121 polymorphisms in 70 candidate severe malaria-associated genes. We found significant associations between risk for severe malaria overall and polymorphisms in 15 genes or locations, of which most were related to red blood cells: ABO, ATP2B4, ARL14, CD40LG, FREM3, INPP4B, G6PD, HBA (both HBA1 and HBA2), HBB, IL10, LPHN2 (also known as ADGRL2), LOC727982, RPS6KL1, CAND1, and GNAS. Combined, these genetic associations accounted for 5·2% of the variance in risk for developing severe malaria among individuals in the general population. We confirmed established associations between severe malaria and sickle-cell trait (odds ratio [OR] 0·15, 95% CI 0·11–0·20; p=2·61 × 10−58), blood group O (0·74, 0·66–0·82; p=6·26 × 10−8), and –α3·7-thalassaemia (0·83, 0·76–0·90; p=2·06 × 10−6). We also found strong associations between overall risk of severe malaria and polymorphisms in both ATP2B4 (OR 0·76, 95% CI 0·63–0·92; p=0·001) and FREM3 (0·64, 0·53–0·79; p=3·18 × 10−14). The association with FREM3 could be accounted for by linkage disequilibrium with a complex structural mutation within the glycophorin gene region (comprising GYPA, GYPB, and GYPE) that encodes for the rare Dantu blood group antigen. Heterozygosity for Dantu was associated with risk for severe malaria (OR 0·57, 95% CI 0·49–0·68; p=3·22 × 10−11), as was homozygosity (0·26, 0·11–0·62; p=0·002). Interpretation Both ATP2B4 and the Dantu blood group antigen are associated with the structure and function of red blood cells. ATP2B4 codes for plasma membrane calcium-transporting ATPase 4 (the major calcium pump on red blood cells) and the glycophorins are ligands for parasites to invade red blood cells. Future work should aim at uncovering the mechanisms by which these polymorphisms can result in severe malaria protection and investigate the implications of these associations for wider health. Funding Wellcome Trust, UK Medical Research Council, European Union, and Foundation for the National Institutes of Health as part of the Bill & Melinda Gates Grand Challenges in Global Health Initiative.

Published
2018
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5. Actin activates Pseudomonas aeruginosa ExoY nucleotidyl cyclase toxin and ExoY-like effector domains from MARTX toxins

Author

Undine Mechold, Abdelkader Namane, Cosmin Saveanu, Vasily Ogryzko, Alexander Belyy, Daniel Ladant, Lina Worpenberg, Souad Fellous, Dorothée Raoux-Barbot, Christien Merrifield, Elodie Assayag, Violaine David, Louis Renault, Véronique Henriot, Biochimie des Interactions Macromoléculaires / Biochemistry of Macromolecular Interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Gamaleya Research Center, Génétique des Interactions Macromoléculaires, Signalisation, noyaux et innovations en cancérologie (UMR8126), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), This project was funded by the Institut Pasteur under #PTR425 to U.M. and C.S. and by CNRS. A.B. was supported by a stipend from the Pasteur—Paris University (PPU) International PhD Program. We acknowledge the core facilities of Imagerie-Gif,(http://www.i2bc.paris-saclay.fr), supported by ‘France-BioImaging’ (ANR-10-INBS-04-01). This paper is dedicated to the memory of Vasily Ogryzko (1960–2016)., ANR-10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée(2010), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Biochimie des Interactions Macromoléculaires, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Signalisation, noyaux et innovations en cancérologie ( UMR8126 ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut Gustave Roussy ( IGR ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), and ANR-10-INBS-04-01/10-INBS-0004,France-BioImaging,Développment d'une infrastructure française distribuée coordonnée ( 2010 )

Subjects
0301 basic medicine, Science, Bacterial Toxins, General Physics and Astronomy, macromolecular substances, Saccharomyces cerevisiae, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, [ SDV.MP.BAC ] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Filamentous actin, Cyclase, Article, General Biochemistry, Genetics and Molecular Biology, Type three secretion system, Adenylyl cyclase, 03 medical and health sciences, Cyclic nucleotide, chemistry.chemical_compound, Bacterial Proteins, ExoY, Type III Secretion Systems, Adenylate cyclase, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Actin, Multidisciplinary, Virulence, Mass spectrometry, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, Chemistry, Effector, General Chemistry, Transfection, Guanylate cyclase, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Actins, 3. Good health, Cell biology, 030104 developmental biology, Glucosyltransferases, Mutation, Pseudomonas aeruginosa, Protein Binding
Abstract

The nucleotidyl cyclase toxin ExoY is one of the virulence factors injected by the Pseudomonas aeruginosa type III secretion system into host cells. Inside cells, it is activated by an unknown eukaryotic cofactor to synthesize various cyclic nucleotide monophosphates. ExoY-like adenylate cyclases are also found in Multifunctional-Autoprocessing Repeats-in-ToXin (MARTX) toxins produced by various Gram-negative pathogens. Here we demonstrate that filamentous actin (F-actin) is the hitherto unknown cofactor of ExoY. Association with F-actin stimulates ExoY activity more than 10,000 fold in vitro and results in stabilization of actin filaments. ExoY is recruited to actin filaments in transfected cells and alters F-actin turnover. Actin also activates an ExoY-like adenylate cyclase MARTX effector domain from Vibrio nigripulchritudo. Finally, using a yeast genetic screen, we identify actin mutants that no longer activate ExoY. Our results thus reveal a new sub-group within the class II adenylyl cyclase family, namely actin-activated nucleotidyl cyclase (AA-NC) toxins., The ExoY toxin is injected by Pseudomonas aeruginosa into host cells, where it is activated by an unknown host factor. Here the authors identify such factor as filamentous actin.

Published
2016
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6. Coordinated recruitment of Spir actin nucleators and myosin V motors to Rab11 vesicle membranes

Author

Andreas T. Grasskamp, Martin Kollmar, Janine Tittel, Margaret A. Titus, Petra Schwille, Eugen Kerkhoff, Florian Chardon, Bruno Goud, Thomas Weidemann, Gilles Malherbe, Olena Pylypenko, Annette Samol-Wolf, Bruno Baron, Patrick England, Carina Ida Luise Michel, Sabine Weiss, Alistair N. Hume, Anne Houdusse, Tobias Welz, Biologie Cellulaire et Cancer, Institut Curie [Paris]-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), University Hospital Regensburg, Max-Planck-Institut für Biochemie = Max Planck Institute of Biochemistry (MPIB), Max-Planck-Gesellschaft, Max-Planck-Institut für Biophysikalische Chemie - Max Planck Institute for Biophysical Chemistry [Göttingen], Motilité structurale, Compartimentation et dynamique cellulaires (CDC), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), University of Nottingham, UK (UON), Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), University of Minnesota [Twin Cities] (UMN), University of Minnesota System, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), This paper was supported by the following grants:Centre National de la Recherche Scientifique to Anne Houdusse, Olena Pylypenko., Deutsche Forschungsgemeinschaft SPP 1464: KO 2251/13-1 to Martin Kollmar., Bayerische Forschungsstiftung WFKMS Nr. F2 - 5121.4.3.1 ‐10c (BayGene) to Eugen Kerkhoff., Fondation de la Recherche Medicale ING20140129255 to Anne Houdusse., Agence Nationale de la Recherche ANR-13-BSV8-0019-01 to Anne Houdusse., National Science Foundation MCB-1244235 to Margaret A Titus., University of Minnesota Medical School Award, E-0915-04 to Margaret A Titus., Deutsche Forschungsgemeinschaft SPP 1464: SCHW 716/11-1, -2 to Petra Schwille., Ligue Contre le Cancer RS16/75-67 to Anne Houdusse., Fondation ARC pour la Recherche sur le Cancer SFI20121205398 to Anne Houdusse., Deutsche Forschungsgemeinschaft SPP 1464: KE 447/10-1, -2 to Eugen Kerkhoff., We acknowledge SOLEIL for provision of synchrotron radiation facilities (proposal 20141015) and we would like to thank Pierre Legrand and Beatriz Guimares for assistance in using beamline PX1. We thank Sandra Lemke, Carsten Grashoff, Yilmaz Niyaz (Zeiss) and Volker Buschmann (Picoquant) for their generous support in FLIM. We thank Mitsuo Ikebe for providing a MyoVb cDNA. We acknowledge Wikayatou Attanda and Charles Gauquelin for support in recombinant protein production. We also acknowledge Damarys Loew and Vanessa Masson (Curie Institute Protein Mass Spectrometry Platform) for the peptide fingerprint analysis of Spir-2 constructs. BG, AHo, OP, GM, FC are part of the Labex CelTisPhyBio 11-LBX-0038, which is part of the IDEX PSL (ANR-10-IDEX-0001-02 PSL)., Max Planck Institute of Biochemistry (MPIB), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)

Subjects
0301 basic medicine, Models, Molecular, Protein Conformation, none, [SDV]Life Sciences [q-bio], Arp2/3 complex, Microfilament, Crystallography, X-Ray, vesicle transport, Myosin head, Mice, 0302 clinical medicine, MESH: Protein Conformation, biophysics, Myosin, cell biology, structural biology, MESH: Animals, Biology (General), Actin nucleation, MESH: Protein Multimerization, General Neuroscience, Microfilament Proteins, General Medicine, Biophysics and Structural Biology, MESH: rab GTP-Binding Proteins/chemistry, Cell biology, MESH: rab GTP-Binding Proteins/metabolism, Medicine, MESH: Cytoplasmic Vesicles/metabolism, MESH: Membranes/metabolism, MESH: Models, Molecular, Research Article, Protein Binding, Myosin light-chain kinase, QH301-705.5, Science, Myosin Type V, MESH: Microfilament Proteins/chemistry, MESH: Microfilament Proteins/metabolism, macromolecular substances, Biology, General Biochemistry, Genetics and Molecular Biology, actin motor proteins, 03 medical and health sciences, MESH: Myosin Type V/metabolism, Animals, MESH: Protein Binding, Actin-binding protein, MESH: Mice, MESH: Myosin Type V/chemistry, Membranes, Spire, General Immunology and Microbiology, Cytoplasmic Vesicles, myosin V, Actin remodeling, MESH: Crystallography, X-Ray, 030104 developmental biology, rab GTP-Binding Proteins, Rab11, biology.protein, Protein Multimerization, actin nucleation, 030217 neurology & neurosurgery
Abstract

There is growing evidence for a coupling of actin assembly and myosin motor activity in cells. However, mechanisms for recruitment of actin nucleators and motors on specific membrane compartments remain unclear. Here we report how Spir actin nucleators and myosin V motors coordinate their specific membrane recruitment. The myosin V globular tail domain (MyoV-GTD) interacts directly with an evolutionarily conserved Spir sequence motif. We determined crystal structures of MyoVa-GTD bound either to the Spir-2 motif or to Rab11 and show that a Spir-2:MyoVa:Rab11 complex can form. The ternary complex architecture explains how Rab11 vesicles support coordinated F-actin nucleation and myosin force generation for vesicle transport and tethering. New insights are also provided into how myosin activation can be coupled with the generation of actin tracks. Since MyoV binds several Rab GTPases, synchronized nucleator and motor targeting could provide a common mechanism to control force generation and motility in different cellular processes. DOI: http://dx.doi.org/10.7554/eLife.17523.001

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