Regulation of fission yeast cytokinesis by membrane lipids and septins

La division cellulaire est un processus essentiel requis pour la prolifération des organismes unicellulaires, pour le développement des organismes multicellulaires, ainsi que pour le renouvellement cellulaire dans les tissus et organes. Un contrôle défectueux de la division cellulaire peut conduire à la mort cellulaire ou à une hyper-prolifération cellulaire et contribuer à la progression du cancer. La division cellulaire est donc sous le contrôle de mécanismes de régulation très stricts. Sa dernière étape, la cytocinèse, nécessite un anneau contractile acto-myosique qui se contracte pour permettre le clivage en deux cellules filles. Parallèlement à l'assemblage de l’anneau contractile, des modifications de la composition lipidique de la membrane plasmatique ont lieu avec un impact fonctionnel sur la cytokinèse. S. pombe a été utilisé comme modèle pour disséquer l’impact des lipides dans d`assemblage de l’anneau contractile. Dans la première partie de ma thèse, mon objectif était d’étudier comment les lipides pourraient réguler le positionnement de l’anneau contractile chez la levure S. pombe. J’ai combiné une approche génétique à de l’imagerie sur cellules vivantes de l'assemblage de l’anneau contractile à partir des nœuds précurseurs de l’anneau pour comprendre comment le niveau d'ergostérol affecte le positionnement du plan de division. J'ai constaté que l'augmentation du niveau d'ergostérol empêchait l'assemblage d’actine-F par la formine Cdc12 à partir de nœuds précurseurs, bloquant leur compaction en anneau fin. Comme la stabilité des câbles d’actine n’est pas perturbée globalement et que le phénotype peut être partiellement compensé par inhibition du complexe Arp2/3, en compétition avec les formines, nous proposons que l'augmentation du niveau d'ergostérol dans la membrane plasmique pourrait inhiber l'activité de la formine Cdc12. En plus de l’anneau contractile acto-myosique, la cytocinèse implique un réseau supplémentaire de cytosquelette, les septines, qui forment des filaments au niveau du site de division. Les septines constituent une famille conservée de protéines liant le GTP dont la suppression conduit à des défauts de cytocinèse. Elles servent également d’échafaudages pour les interactions protéine-protéine et/ou de barrières de diffusion pour la compartimentation des protéines pendant la cytocinèse et au-delà. Chez S. pombe, contrairement à S. cerevisiae, les septines arrivent tardivement au site de division et sont seulement impliquées dans les étapes tardives de la cytocinèse, pour promouvoir la séparation des deux cellules filles. Dans la deuxième partie de ma thèse, j’ai décidé d’explorer le comportement dynamique des septines au cours de la progression de la cytocinèse et leurs mécanismes de régulation. En utilisant l’imagerie sur cellules vivantes et des déterminants temporels précis de progression du cycle cellulaire, j’ai identifié une nouvelle étape de recrutement des septines sur le cortex cellulaire à proximité de l’anneau contractile acto-myosique, en un large réseau qui se compacte ensuite en anneau fin. J'ai également trouvé des preuves que Mid2, une protéine de la famille de l'anilline, favorise la compaction des septines et peut agir comme un « bundler » de septines. Cependant, l’analyse de mutants bloqués en mitose indique que cette protéine n’est pas suffisante pour accomplir cette tâche. De plus, j'ai déterminé qu’une activité de Cdk1 élevée permet le recrutement et l’assemblage des septines mais que la voie du SIN est aussi nécessaire pour promouvoir leur recrutement au milieu de la cellule et favoriser leur compaction. Enfin, j’ai trouvé que les niveaux de PIP2 influaient non seulement sur la quantité de septines et de Mid2 associés au cortex médian et leur compaction mais aussi sur le « timing » du recrutement des septines sur le site de division. Ceci démontre que l’organisation des septines dépend d’une série de régulations complexes coordonnées par la machinerie du cycle cellulaire.Cell division is an essential process required for the proliferation of unicellular organisms, for the development of multicellular organisms, as well as for cell renewal within tissues and organs. Defective control of cell division can either lead to cell death, or to cell hyper-proliferation and contribute to cancer progression. Cell division is therefore under the control of very tight regulatory mechanisms. In animals and fungi, its final step, cytokinesis, requires an acto-myosin-based contractile ring that constricts to promote sister cell cleavage. Modifications in the lipid composition of the plasma membrane take place during cell division, with functional impact on cytokinesis.Fission yeast is a simple single cell eukaryotic organism which has been extensively used to study cell division because of its stereotyped rode shape, easy genetics and short generation time. In particular, this model system has proved very powerful for the molecular dissection of contractile ring assembly. However remarkably little is known on how membrane lipids regulate contractile ring assembly. In the first part of my PhD, my objective was to study how lipids could regulate contractile ring positioning in fission yeast. I have combined fission yeast genetics with live-cell imaging of contractile ring assembly from precursor nodes to understand how ergosterol levels affect division plane positioning. I have found that increased ergosterol levels prevent F-actin assembly from cytokinetic precursor nodes by the formin Cdc12, avoiding their compaction into a medially placed contractile ring. Since the stability of F-actin cables was not altered altogether and the phenotype could be partially rescued by inhibition of the Arp2/3 complex which competes with formins, we propose that increasing ergosterol levels in the plasma membrane may inhibit the activity of the formin Cdc12.In addition to the contractile ring, cytokinesis involves an additional component of the cytoskeleton, the septins, which form filaments at the division site. Septins are a family of conserved GTP binding proteins whose deletion leads to cytokinetic defects. They also serve as scaffolds for protein-protein interactions and/or as diffusion barriers for protein compartmentalization in cytokinesis and beyond. In fission yeast, in contrast to budding yeast, septins are late at the division site and are only involved in late stages of cytokinesis, to promote sister cell separation. In the second part of my PhD, I decided to explore the dynamic behavior of septins and decipher how they are regulated. Using live cell imaging and precise cell cycle timers, I have identified a new step in the recruitment of septin to the cell cortex in the proximity of the contractile acto-myosin ring, in a broad meshwork that then compacts into a tight ring. I have also found evidence that the anillin-like protein Mid2 is necessary to promote this compaction and may act as a bundler for septin filaments. However, analysis of mutants blocked in mitosis shows that this protein is not sufficient to accomplish this task. Moreover, I have determined that high Cdk activity allows septins and Mid2 initial recruitment and assembly, but the SIN pathway also plays a role in promoting their recruitment at the cell middle and is then required to drive their compactio. Additionally, I have found that PIP2 levels influence not only the amount of septins and Mid2 filaments associated at the medial cortex together with their compaction but also the timing of septin recruitment to the division site. This demonstrates that septin assembly relies on complex regulations coordinated by the cell cycle machinery

Régulation de la cytokinèse par les lipides membranaires et les septines chez la levure S. pombe

Cell division is an essential process required for the proliferation of unicellular organisms, for the development of multicellular organisms, as well as for cell renewal within tissues and organs. Defective control of cell division can either lead to cell death, or to cell hyper-proliferation and contribute to cancer progression. Cell division is therefore under the control of very tight regulatory mechanisms. In animals and fungi, its final step, cytokinesis, requires an acto-myosin-based contractile ring that constricts to promote sister cell cleavage. Modifications in the lipid composition of the plasma membrane take place during cell division, with functional impact on cytokinesis.Fission yeast is a simple single cell eukaryotic organism which has been extensively used to study cell division because of its stereotyped rode shape, easy genetics and short generation time. In particular, this model system has proved very powerful for the molecular dissection of contractile ring assembly. However remarkably little is known on how membrane lipids regulate contractile ring assembly. In the first part of my PhD, my objective was to study how lipids could regulate contractile ring positioning in fission yeast. I have combined fission yeast genetics with live-cell imaging of contractile ring assembly from precursor nodes to understand how ergosterol levels affect division plane positioning. I have found that increased ergosterol levels prevent F-actin assembly from cytokinetic precursor nodes by the formin Cdc12, avoiding their compaction into a medially placed contractile ring. Since the stability of F-actin cables was not altered altogether and the phenotype could be partially rescued by inhibition of the Arp2/3 complex which competes with formins, we propose that increasing ergosterol levels in the plasma membrane may inhibit the activity of the formin Cdc12.In addition to the contractile ring, cytokinesis involves an additional component of the cytoskeleton, the septins, which form filaments at the division site. Septins are a family of conserved GTP binding proteins whose deletion leads to cytokinetic defects. They also serve as scaffolds for protein-protein interactions and/or as diffusion barriers for protein compartmentalization in cytokinesis and beyond. In fission yeast, in contrast to budding yeast, septins are late at the division site and are only involved in late stages of cytokinesis, to promote sister cell separation. In the second part of my PhD, I decided to explore the dynamic behavior of septins and decipher how they are regulated. Using live cell imaging and precise cell cycle timers, I have identified a new step in the recruitment of septin to the cell cortex in the proximity of the contractile acto-myosin ring, in a broad meshwork that then compacts into a tight ring. I have also found evidence that the anillin-like protein Mid2 is necessary to promote this compaction and may act as a bundler for septin filaments. However, analysis of mutants blocked in mitosis shows that this protein is not sufficient to accomplish this task. Moreover, I have determined that high Cdk activity allows septins and Mid2 initial recruitment and assembly, but the SIN pathway also plays a role in promoting their recruitment at the cell middle and is then required to drive their compactio. Additionally, I have found that PIP2 levels influence not only the amount of septins and Mid2 filaments associated at the medial cortex together with their compaction but also the timing of septin recruitment to the division site. This demonstrates that septin assembly relies on complex regulations coordinated by the cell cycle machinery.La division cellulaire est un processus essentiel requis pour la prolifération des organismes unicellulaires, pour le développement des organismes multicellulaires, ainsi que pour le renouvellement cellulaire dans les tissus et organes. Un contrôle défectueux de la division cellulaire peut conduire à la mort cellulaire ou à une hyper-prolifération cellulaire et contribuer à la progression du cancer. La division cellulaire est donc sous le contrôle de mécanismes de régulation très stricts. Sa dernière étape, la cytocinèse, nécessite un anneau contractile acto-myosique qui se contracte pour permettre le clivage en deux cellules filles. Parallèlement à l'assemblage de l’anneau contractile, des modifications de la composition lipidique de la membrane plasmatique ont lieu avec un impact fonctionnel sur la cytokinèse. S. pombe a été utilisé comme modèle pour disséquer l’impact des lipides dans d`assemblage de l’anneau contractile. Dans la première partie de ma thèse, mon objectif était d’étudier comment les lipides pourraient réguler le positionnement de l’anneau contractile chez la levure S. pombe. J’ai combiné une approche génétique à de l’imagerie sur cellules vivantes de l'assemblage de l’anneau contractile à partir des nœuds précurseurs de l’anneau pour comprendre comment le niveau d'ergostérol affecte le positionnement du plan de division. J'ai constaté que l'augmentation du niveau d'ergostérol empêchait l'assemblage d’actine-F par la formine Cdc12 à partir de nœuds précurseurs, bloquant leur compaction en anneau fin. Comme la stabilité des câbles d’actine n’est pas perturbée globalement et que le phénotype peut être partiellement compensé par inhibition du complexe Arp2/3, en compétition avec les formines, nous proposons que l'augmentation du niveau d'ergostérol dans la membrane plasmique pourrait inhiber l'activité de la formine Cdc12. En plus de l’anneau contractile acto-myosique, la cytocinèse implique un réseau supplémentaire de cytosquelette, les septines, qui forment des filaments au niveau du site de division. Les septines constituent une famille conservée de protéines liant le GTP dont la suppression conduit à des défauts de cytocinèse. Elles servent également d’échafaudages pour les interactions protéine-protéine et/ou de barrières de diffusion pour la compartimentation des protéines pendant la cytocinèse et au-delà. Chez S. pombe, contrairement à S. cerevisiae, les septines arrivent tardivement au site de division et sont seulement impliquées dans les étapes tardives de la cytocinèse, pour promouvoir la séparation des deux cellules filles. Dans la deuxième partie de ma thèse, j’ai décidé d’explorer le comportement dynamique des septines au cours de la progression de la cytocinèse et leurs mécanismes de régulation. En utilisant l’imagerie sur cellules vivantes et des déterminants temporels précis de progression du cycle cellulaire, j’ai identifié une nouvelle étape de recrutement des septines sur le cortex cellulaire à proximité de l’anneau contractile acto-myosique, en un large réseau qui se compacte ensuite en anneau fin. J'ai également trouvé des preuves que Mid2, une protéine de la famille de l'anilline, favorise la compaction des septines et peut agir comme un « bundler » de septines. Cependant, l’analyse de mutants bloqués en mitose indique que cette protéine n’est pas suffisante pour accomplir cette tâche. De plus, j'ai déterminé qu’une activité de Cdk1 élevée permet le recrutement et l’assemblage des septines mais que la voie du SIN est aussi nécessaire pour promouvoir leur recrutement au milieu de la cellule et favoriser leur compaction. Enfin, j’ai trouvé que les niveaux de PIP2 influaient non seulement sur la quantité de septines et de Mid2 associés au cortex médian et leur compaction mais aussi sur le « timing » du recrutement des septines sur le site de division. Ceci démontre que l’organisation des septines dépend d’une série de régulations complexes coordonnées par la machinerie du cycle cellulaire

Increasing ergosterol levels delays formin-dependent assembly of F-actin cables and disrupts division plane positioning in fission yeast

In most eukaryotes, cytokinesis is mediated by the constriction of a contractile acto-myosin ring (CR), which promotes the ingression of the cleavage furrow. Many components of the CR interact with plasma membrane lipids suggesting that lipids may regulate CR assembly and function. Although there is clear evidence that phosphoinositides play an important role in cytokinesis, much less is known about the role of sterols in this process. Here, we studied how sterols influence division plane positioning and CR assembly in fission yeast. We show that increasing ergosterol levels in the plasma membrane blocks the assembly of F-actin cables from cytokinetic precursor nodes, preventing their compaction into a ring. Abnormal F-actin cables form after a delay, leading to randomly placed septa. Since the formin Cdc12 was detected on cytokinetic precursors and the phenotype can be partially rescued by inhibiting the Arp2/3 complex, which competes with formins for F-actin nucleation, we propose that ergosterol may inhibit formin dependent assembly of F-actin cables from cytokinetic precursors.This work was supported by a grant from Fondation ARC pour la Recherche sur le Cancer (PJA 20171206550) to A.P., which also provided a. received a fourth year PhD fellowship to F.A. A.P. is a member of the LabEx CelTisPhyBi

Crosslinked Chitosan Binder for Sustainable Aqueous Batteries

none5noThe increased percentage of renewable power sources involved in energy production highlights the importance of developing systems for stationary energy storage that satisfy the requirements of safety and low costs. Na ion batteries can be suitable candidates, specifically if their components are economic and safe. This study focuses on the development of aqueous processes and binders to prepare electrodes for sodium ion cells operating in aqueous solutions. We demonstrated the feasibility of a chitosan-based binder to produce freestanding electrodes for Na ion cells, without the use of organic solvents and current collectors in electrode processing. To our knowledge, it is the first time that water-processed, freestanding electrodes are used in aqueous Na ion cells, which could also be extended to other types of aqueous batteries. This is a real breakthrough in terms of sustainability, taking into account low risks for health and environment and low costs.openLuca Bargnesi; Federica Gigli; Nicolò Albanelli; Christina Toigo; Catia ArbizzaniLuca Bargnesi; Federica Gigli; Nicolò Albanelli; Christina Toigo; Catia Arbizzan

Progettare in vivo la rigenerazione urbana

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Septin Filament Compaction Into Rings Requires the Anillin Mid2 and Contractile Ring Constriction

International audienceSeptin filaments assemble into high-order molecular structures that associate with membranes, acting as diffusion barriers and scaffold proteins crucial for many cellular processes. However, how septin filaments organize in such structures is still not well understood. In this study, we used fission yeast to explore septin filament organization during cell division and decipher key factors responsible for their regulation. Live-imaging and polarization microscopy analysis uncovered that septin filaments are initially recruited as a diffuse meshwork surrounding the acto-myosin contractile ring (CR) in anaphase, which undergoes compaction into two rings when CR constriction is initiated. We found evidence that the anillin-like protein Mid2 is necessary to promote this novel compaction step, possibly acting as a bundler for septin filaments. We also found that Mid2-driven septin compaction requires inputs from the Septation Initiation Network (SIN) as well as CR constriction or the β-1-3 glucan synthase Bgs1. This work highlights the complex regulations that allow the coordination between septin ring assembly and cell cycle progression

Architettura e Tecnologie per l’abitare. Upcycling degli edifici ERP di Tor Bella Monaca

La pubblicazione raccoglie gli esiti di RE-LIVE2020.Architettura e Tecnologia per l’abitare - Valle Giulia _ Facoltà di Architettura_ Sapienza Università di Roma 19-21 febbraio 2020, Workshop Progettuale under 40_ Upcycling degli edifici ERP di Tor Bella Monaca a Roma Responsabili scientifici: Prof.ssa Maria Teresa Lucarelli, Prof.ssa Elena Mussinelli Coordinatori: Eugenio Arbizzani, Adolfo Baratta, Eliana Cangelli, Laura Daglio, Federica Ottone, Donatella Radogn