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1. Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells

Author

Martins, Carla Silva, Taveneau, Cyntia, Castro-Linares, Gerard, Baibakov, Mikhail, Buzhinsky, Nicolas, Eroles, Mar, Milanović, Violeta, Omi, Shizue, Pedelacq, Jean-Denis, Iv, Francois, Bouillard, Léa, Llewellyn, Alexander, Gomes, Maxime, Belhabib, Mayssa, Kuzmić, Mira, Verdier-Pinard, Pascal, Lee, Stacey, Badache, Ali, Kumar, Sanjay, Chandre, Cristel, Brasselet, Sophie, Rico, Felix, Rossier, Olivier, Koenderink, Gijsje H, Wenger, Jerome, Cabantous, Stéphanie, and Mavrakis, Manos

Subjects
Biochemistry and Cell Biology, Biological Sciences, Bioengineering, Nanotechnology, 1.1 Normal biological development and functioning, Underpinning research, Generic health relevance, Humans, Actins, Cell Membrane, Cytoskeleton, Microscopy, Septins, Medical and Health Sciences, Developmental Biology, Biological sciences, Biomedical and clinical sciences
Abstract

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Published
2023

5. Purification of recombinant human and Drosophila septin hexamers for TIRF assays of actin-septin filament assembly

Author

Mavrakis, Manos, Feng-Ching, Tsai, and Koenderink, Gijsje H.

Subjects
Quantitative Biology - Subcellular Processes, Quantitative Biology - Cell Behavior
Abstract

Septins are guanine nucleotide-binding proteins that are conserved from fungi to humans. Septins assemble into heterooligomeric complexes and higher-order structures with key roles in various cellular functions including cell migration and division. The mechanisms by which septins assemble and interact with other cytoskeletal elements like actin remain elusive. A powerful approach to address this question is by cell-free reconstitution of pu- rified cytoskeletal proteins combined with fluorescence microscopy. Here, we describe procedures for the purification of recombinant Drosophila and human septin hexamers from Escherichia coli and reconstitution of actin-septin coassembly. These procedures can be used to compare assembly of Drosophila and human septins and their coassembly with the actin cytoskeleton by total internal reflection fluorescence microscopy.

Published
2016
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6. Visualizing septins in early Drosophila embryos

Author

Mavrakis, Manos

Subjects
Quantitative Biology - Subcellular Processes, Quantitative Biology - Cell Behavior
Abstract

Functional studies in Drosophila have been key for establishing a role for the septin family of proteins in animal cell division and thus extending for the first time observations from the budding yeast to animal cells. Visualizing the distribution of specific septins in different Drosophila tissues and, in particular, in the Drosophila embryo, together with biochemical and mutant phenotype data, has contributed important advances to our understanding of animal septin biology, suggesting roles in processes other than in cytokinesis. Septin localization using immunofluorescence assays has been possible due to the generation of antibodies against different Drosophila septins. The recent availability of lines expressing fluorescent protein fusions of specific septins further promises to facilitate studies on septin dynamics. Here, we provide protocols for preparing early Drosophila embryos to visualize septins using immunofluorescence assays and live fluorescence microscopy. The genetic tractability of the Drosophila embryo together with its amenability to high-resolution fluorescence microscopy promises to provide novel insights into animal septin structure and function.

Published
2016
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7. Genetically encoded reporters of actin filament organization in living cells and tissues

Author

Silva Martins, Carla, primary, Iv, Francois, additional, Kumar Suman, Shashi, additional, Panagiotou, Thomas C., additional, Sidor, Clara, additional, Ruso-Lopez, Maria, additional, Plancke, Camille N., additional, Omi, Shizue, additional, Gomes, Maxime, additional, Llewellyn, Alexander, additional, Bandi, Sourish Reddy, additional, Ramond, Laurie, additional, Arbizzani, Federica, additional, Rimoli, Caio Vaz, additional, Schnorrer, Frank, additional, Robin, Francois, additional, Wilde, Andrew, additional, LeGoff, Loic, additional, Pedelacq, Jean-Denis, additional, Cabantous, Stephanie, additional, Rincon, Sergio A., additional, Chandre, Cristel, additional, Brasselet, Sophie, additional, and Mavrakis, Manos, additional

Published
2024
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8. Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers.

Author

Kuzmić, Mira, Castro Linares, Gerard, Leischner Fialová, Jindřiška, Iv, François, Salaün, Daniele, Llewellyn, Alex, Gomes, Maxime, Belhabib, Mayssa, Yuxiang Liu, Keisuke Asano, Rodrigues, Magda, Isnardon, Daniel, Tachibana, Taro, Koenderink, Gijsje H., Badache, Ali, Mavrakis, Manos, and Verdier-Pinard, Pascal

Subjects
MICROTUBULES, BRACHIAL plexus neuropathies, MOLECULAR pathology, G proteins, SEPTINS, MICROTUBULE-associated proteins, FIBERS
Abstract

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering themechanisms underlying septin-associated pathologies. [ABSTRACT FROM AUTHOR]

Published
2024
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10. Supplementary Video 2 from Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

Author

Meoli, Elise, primary, Bossis, Ioannis, primary, Cazabat, Laure, primary, Mavrakis, Manos, primary, Horvath, Anelia, primary, Stergiopoulos, Sotiris, primary, Shiferaw, Miriam L., primary, Fumey, Glawdys, primary, Perlemoine, Karine, primary, Muchow, Michael, primary, Robinson-White, Audrey, primary, Weinberg, Frank, primary, Nesterova, Maria, primary, Patronas, Yianna, primary, Groussin, Lionel, primary, Bertherat, Jérôme, primary, and Stratakis, Constantine A., primary

Published
2023
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11. Data from Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

Author

Meoli, Elise, primary, Bossis, Ioannis, primary, Cazabat, Laure, primary, Mavrakis, Manos, primary, Horvath, Anelia, primary, Stergiopoulos, Sotiris, primary, Shiferaw, Miriam L., primary, Fumey, Glawdys, primary, Perlemoine, Karine, primary, Muchow, Michael, primary, Robinson-White, Audrey, primary, Weinberg, Frank, primary, Nesterova, Maria, primary, Patronas, Yianna, primary, Groussin, Lionel, primary, Bertherat, Jérôme, primary, and Stratakis, Constantine A., primary

Published
2023
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12. sSupplementary Video 3 from Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

Author

Meoli, Elise, primary, Bossis, Ioannis, primary, Cazabat, Laure, primary, Mavrakis, Manos, primary, Horvath, Anelia, primary, Stergiopoulos, Sotiris, primary, Shiferaw, Miriam L., primary, Fumey, Glawdys, primary, Perlemoine, Karine, primary, Muchow, Michael, primary, Robinson-White, Audrey, primary, Weinberg, Frank, primary, Nesterova, Maria, primary, Patronas, Yianna, primary, Groussin, Lionel, primary, Bertherat, Jérôme, primary, and Stratakis, Constantine A., primary

Published
2023
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13. Supplementary Video 1 from Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

Author

Meoli, Elise, primary, Bossis, Ioannis, primary, Cazabat, Laure, primary, Mavrakis, Manos, primary, Horvath, Anelia, primary, Stergiopoulos, Sotiris, primary, Shiferaw, Miriam L., primary, Fumey, Glawdys, primary, Perlemoine, Karine, primary, Muchow, Michael, primary, Robinson-White, Audrey, primary, Weinberg, Frank, primary, Nesterova, Maria, primary, Patronas, Yianna, primary, Groussin, Lionel, primary, Bertherat, Jérôme, primary, and Stratakis, Constantine A., primary

Published
2023
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14. Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells

Author

Martins, Carla Silva, primary, Taveneau, Cyntia, additional, Castro-Linares, Gerard, additional, Baibakov, Mikhail, additional, Buzhinsky, Nicolas, additional, Eroles, Mar, additional, Milanović, Violeta, additional, Omi, Shizue, additional, Pedelacq, Jean-Denis, additional, Iv, Francois, additional, Bouillard, Léa, additional, Llewellyn, Alexander, additional, Gomes, Maxime, additional, Belhabib, Mayssa, additional, Kuzmić, Mira, additional, Verdier-Pinard, Pascal, additional, Lee, Stacey, additional, Badache, Ali, additional, Kumar, Sanjay, additional, Chandre, Cristel, additional, Brasselet, Sophie, additional, Rico, Felix, additional, Rossier, Olivier, additional, Koenderink, Gijsje H., additional, Wenger, Jerome, additional, Cabantous, Stéphanie, additional, and Mavrakis, Manos, additional

Published
2022
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15. Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers

Author

Kuzmić, Mira, Castro Linares, Gerard, Leischner Fialová, Jindřiška, Iv, Francois, Salaün, Danièle, Llewellyn, Alex, Gomes, Maxime, Belhabib, Mayssa, Liu, Yuxiang, Asano, Keisuke, Rodrigues, Magda, Isnardon, Daniel, Tachibana, Taro, Koenderink, Gijsje, Badache, Ali, Mavrakis, Manos, Verdier-Pinard, Pascal, Centre de Recherche en Cancérologie de Marseille (CRCM), 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), Delft University of Technology (TU Delft), Kavli Institute of Nanosciences [Delft] (KI-NANO), Institut FRESNEL (FRESNEL), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), MOSAIC (MOSAIC), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), University of Shanghai [Shanghai], Osaka City University (OCU), ANR-17-CE13-0014,SEPTIMORF,Rôle des septines dans la morphogenèse des cellules animales(2017), 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), Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU), Mavrakis, Manos, and Rôle des septines dans la morphogenèse des cellules animales - - SEPTIMORF2017 - ANR-17-CE13-0014 - AAPG2017 - VALID

Subjects
[SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, fungi, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], [SDV.BBM.BP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BDD.MOR]Life Sciences [q-bio]/Development Biology/Morphogenesis, macromolecular substances, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], [SDV.BC.IC] Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], biological phenomena, cell phenomena, and immunity, [SDV.BDD.MOR] Life Sciences [q-bio]/Development Biology/Morphogenesis
Abstract

International audience; Septins, a family of GTP-binding proteins assembling into higher order structures, interface with the membrane, actin filaments and microtubules, which positions them as important regulators of cytoarchitecture. Septin 9 (Sept9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short MAP-like motif unique to Sept9 isoform 1 (Sept9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring Sept9_i1. Although outside of the MAP-like motif, HNA mutations abrogates this association, identifying a putative regulatory domain. Removal of this domain from Sept9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin associated pathologies.

Published
2022
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16. Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Author

Chauvin, Brieuc, primary, Nakazawa, Koyomi, primary, Beber, Alexandre, primary, Di Cicco, Aurélie, primary, Hajj, Bassam, primary, Iv, François, primary, Mavrakis, Manos, primary, Koenderink, Gijsje H., primary, Cabral, João T., primary, Trichet, Michaël, primary, Mangenot, Stéphanie, primary, and Bertin, Aurélie, primary

Published
2022
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18. Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Author

Castro Linares, G. (author), den Haan, J.C.A. (author), Iv, Francois (author), Martins, Carla Silva (author), Bertin, Aurélie (author), Mavrakis, Manos (author), Koenderink, G.H. (author), Castro Linares, G. (author), den Haan, J.C.A. (author), Iv, Francois (author), Martins, Carla Silva (author), Bertin, Aurélie (author), Mavrakis, Manos (author), and Koenderink, G.H. (author)

Abstract

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers., uilding a Synthetic Cell' Gravitation grant (024.003.019) and from the Agence Nationale pour la Recherche (ANR grants ANR-17-CE13-0014: “SEPTIMORF”; ANR-13-JSV8-0002-01: “SEPTIME”; and ANR-20-CE11-0014-01: “SEPTSCORT”). Publisher Copyright: © 2022 JoVE., BN/Gijsje Koenderink Lab

Published
2022
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19. Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins

Author

Chauvin, Brieuc (author), Nakazawa, Koyomi (author), Beber, Alexandre (author), Di Cicco, Aurélie (author), Hajj, Bassam (author), Iv, François (author), Mavrakis, Manos (author), Koenderink, G.H. (author), Cabral, João T. (author), Chauvin, Brieuc (author), Nakazawa, Koyomi (author), Beber, Alexandre (author), Di Cicco, Aurélie (author), Hajj, Bassam (author), Iv, François (author), Mavrakis, Manos (author), Koenderink, G.H. (author), and Cabral, João T. (author)

Abstract

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature., Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public., BN/Gijsje Koenderink Lab

Published
2022
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20. Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring

Author

Martins, Carla Silva, primary, Taveneau, Cyntia, additional, Castro-Linares, Gerard, additional, Baibakov, Mikhail, additional, Buzhinsky, Nicolas, additional, Eroles, Mar, additional, Milanović, Violeta, additional, Iv, Francois, additional, Bouillard, Léa, additional, Llewellyn, Alex, additional, Gomes, Maxime, additional, Belhabib, Mayssa, additional, Kuzmić, Mira, additional, Verdier-Pinard, Pascal, additional, Lee, Stacey, additional, Badache, Ali, additional, Kumar, Sanjay, additional, Chandre, Cristel, additional, Brasselet, Sophie, additional, Rico, Felix, additional, Rossier, Olivier, additional, Koenderink, Gijsje H., additional, Wenger, Jerome, additional, Cabantous, Stéphanie, additional, and Mavrakis, Manos, additional

Published
2022
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21. Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers

Author

Kuzmić, Mira, primary, Castro Linares, Gerard, additional, Leischner Fialová, Jindřiška, additional, Iv, François, additional, Salaün, Danièle, additional, Llewellyn, Alex, additional, Gomes, Maxime, additional, Belhabib, Mayssa, additional, Liu, Yuxiang, additional, Asano, Keisuke, additional, Rodrigues, Magda, additional, Isnardon, Daniel, additional, Tachibana, Taro, additional, Koenderink, Gijsje H., additional, Badache, Ali, additional, Mavrakis, Manos, additional, and Verdier-Pinard, Pascal, additional

Published
2022
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23. Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers.

Author

Kuzmić, Mira, Linares, Gerard Castro, Fialovÿ, Jindřiška Leischner, Iv, François, Salaün, Danièle, Llewellyn, Alex, Gomes, Maxime, Belhabib, Mayssa, Yuxiang Liu, Asano, Keisuke, Rodrigues, Magda, Isnardon, Daniel, Tachibana, Taro, Koenderink, Gijsje H., Badache, Ali, Mavrakis, Manos, and Verdier-Pinard, Pascal

Subjects
MICROTUBULES, G proteins, MOLECULAR interactions, SEPTINS, BRACHIAL plexus neuropathies, MICROTUBULE-associated proteins, CYTOSKELETON
Abstract

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering themechanisms underlying septin-associated pathologies. [ABSTRACT FROM AUTHOR]

Published
2023
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24. Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms

Author

Iv, Francois, primary, Martins, Carla Silva, additional, Castro-Linares, Gerard, additional, Taveneau, Cyntia, additional, Barbier, Pascale, additional, Verdier-Pinard, Pascal, additional, Camoin, Luc, additional, Audebert, Stéphane, additional, Tsai, Feng-Ching, additional, Ramond, Laurie, additional, Llewellyn, Alex, additional, Belhabib, Mayssa, additional, Nakazawa, Koyomi, additional, Di Cicco, Aurélie, additional, Vincentelli, Renaud, additional, Wenger, Jerome, additional, Cabantous, Stéphanie, additional, Koenderink, Gijsje H., additional, Bertin, Aurélie, additional, and Mavrakis, Manos, additional

Published
2021
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25. Membrane binding controls ordered self-assembly of animal septins

Author

Szuba, Agata, Bano, Fouzia, Linares, Gerard Castro, Iv, Francois, Mavrakis, Manos, Richter, Ralf P., Bertin, Aurélie, Koenderink, Gijsje H., Szuba, Agata, Bano, Fouzia, Linares, Gerard Castro, Iv, Francois, Mavrakis, Manos, Richter, Ralf P., Bertin, Aurélie, and Koenderink, Gijsje H.

Abstract

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

Published
2021
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26. Homeostatic mechanisms for iron storage revealed by genetic manipulations and live imaging of Drosophila ferritin

Author

Missirlis, Fanis, Kosmidis, Stylianos, Brody, Tom, Mavrakis, Manos, Holmberg, Sara, Odenwald, Ward F., Skoulakis, Efthimios M.C., and Rouault, Tracey A.

Subjects
Ferritin -- Properties, Drosophila -- Genetic aspects, Homeostasis -- Genetic aspects, Genetic research, Biological sciences
Abstract

Ferritin is a symmetric, 24-subunit iron-storage complex assembled of H and L chains. It is found in bacteria, plants, and animals and in two classes of mutations in the human L-chain gene, resulting in hereditary hyperferritinemia cataract syndrome or in neuroferritinopathy. Here, we examined systemic and cellular ferritin regulation and trafficking in the model organism Drosophila melanogaster. We showed that ferritin H and L transcripts are coexpressed during embryogenesis and that both subunits are essential for embryonic development. Ferritin overexpression impaired the survival of iron-deprived flies. In vivo expression of GFP-tagged holoferritin confirmed that iron-loaded ferritin molecules traffic through the Golgi organelle and are secreted into hemolymph. A constant ratio of ferritin H and L subunits, secured via tight post-transcriptional regulation, is characteristic of the secreted ferritin in flies. Differential cellular expression, conserved post-transcriptional regulation via the iron regulatory element, and distinct subcellular localization of the ferritin subunits prior to the assembly of holoferritin are all important steps mediating iron homeostasis. Our study revealed both conserved features and insect-specific adaptations of ferritin nanocages and provides novel imaging possibilities for their in vivo characterization.

Published
2007

27. The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei

Author

Frescas, David, Mavrakis, Manos, Lorenz, Holger, DeLotto, Robert, and Lippincott-Schwartz, Jennifer

Subjects
Drosophila -- Genetic aspects, Drosophila -- Research, Embryology, Experimental, Biological sciences
Abstract

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.

Published
2006

29. Septin-microtubule association requires a MAP-like motif unique to Sept9 isoform 1 embedded into septin octamers

Author

Kuzmić, Mira, primary, Linares, Gerard Castro, additional, Fialová, Jindřiška Leischner, additional, Iv, François, additional, Salaün, Danièle, additional, Llewellyn, Alex, additional, Gomes, Maxime, additional, Belhabib, Mayssa, additional, Liu, Yuxiang, additional, Asano, Keisuke, additional, Tachibana, Taro, additional, Koenderink, Gijsje, additional, Badache, Ali, additional, Mavrakis, Manos, additional, and Verdier-Pinard, Pascal, additional

Published
2021
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38. Super resolution polarized imaging of the organization of protein assemblies in cells

Author

Brasselet, Sophie, Mavrakis, Manos, vaz Rimoli, caio, Institut FRESNEL (FRESNEL), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), MOSAIC (MOSAIC), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), Brasselet, Sophie, Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU), and Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU)

Subjects
[SPI.OPTI] Engineering Sciences [physics]/Optics / Photonic, [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019

39. Super resolution polarized imaging of the Structural Organization of Actin Filaments in cells

Author

Brasselet, Sophie, Mavrakis, Manos, MOSAIC (MOSAIC), Institut FRESNEL (FRESNEL), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-École Centrale de Marseille (ECM)-Aix Marseille Université (AMU), and Brasselet, Sophie

Subjects
[SPI.OPTI] Engineering Sciences [physics]/Optics / Photonic, [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic, ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2019

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