Dynamin-mediated membrane fission

Membrane fission is required for vesicular traffic between intracellular compartments. Dynamin is a GTPase implicated in vesicle scission during Clathrin-mediated endocytosis. It polymerizes into a helix at the neck of endocytic buds. Upon GTP hydrolysis, conformational changes reduce the helical radius and pitch showing that fission proceeds through a constriction mechanism. We show that the deformation of Dynamin helices is highly concerted and damped by the friction between membrane and Dynamin. To further understand fission, Dynamin polymerization and fission are studied on lipid tubes extruded from Giant Unilamellar Vesicles. Fission occurs at the edge of the helix, where the membrane is strongly curved. A statistical analysis of fission time reveals that the fission reaction can be modeled by a single step energy barrier. The fission time dependence on membrane tension, membrane rigidity and torque is established theoretically and validated experimentally. This work gives a quantitative picture of the energy landscape of Dynamin-mediated fission: the height of the energy barrier of fission is estimated around 70 kBT

MECANISME DE FISSION MEMBRANAIRE : APPROCHES MECANIQUE ET ENERGETIQUE DU CAS DE LA DYNAMINE.

The eukaryotic cell is organized in several compartments, named organelles, delimited by lipid membranes. The fission of these membranes is required for vesicular traffic between organelles. Endocytosis is the mechanism of vesicular traffic from the plasma membrane towards other organelles inside the cell. Dynamin is a guanosine triphosphatase (GTPase) implicated in vesicle scission during Clathrin-mediated endocytosis. It polymerizes into a helix at the neck of endocytic buds. Upon GTP hydrolysis, conformational changes modify the helical structure: the inner radius decreases from 10 to 5 nm and the helical pitch reduces from 13 to 9 nm. These modifications show that fission proceeds through a constriction mechanism. The dynamics of constriction is investigated by monitoring the rotations of microbeads attached along Dynamin-coated tubes after GTP addition. The deformation of Dynamin helices is highly concerted and damped by the friction between membrane and Dynamin. However constriction is not enough to trigger fission. To further understand fission, Dynamin polymerization and fission are studied on lipid tubes extruded from Giant Unilamellar Vesicles. It is shown that fission occurs at the edge of the helix, where the membrane is strongly curved. A statistical analysis of fission time reveals that the fission reaction can be modeled by a single step energy barrier. The fission time dependence on membrane tension, membrane rigidity and torque is established theoretically and validated experimentally. This work gives a quantitative picture of the energy landscape of Dynamin-mediated fission: the height of the energy barrier of fission is estimated around 70 kT.La cellule eukaryote est organisée en plusieurs compartiments, appelés organelles, délimités par des membranes. La fission des membranes est nécessaire pour le transport intracellulaire entre organelles. L'endocytose est un mécanisme de transport depuis la membrane plasmique vers les autres organelles. La Dynamine est une guanosine triphosphatase (GTPase) impliquée dans la fission des vésicules pendant l'endocytose médiée par la Clathrine. Elle polymérise en hélice au coup des bourgeons endocytiques. Après hydrolyse du GTP, la structure de l'hélice est modifiée : le rayon interne diminue de 10 à 5 nm et le pas hélical de 13 à 9 nm. Ces modifications indiquent un mécanisme de constriction. La dynamique de constriction est étudiée en suivant la rotation de microbilles attachées à des tubes lipidiques recouverts de Dynamine. La déformation des hélices de Dynamine est concertée et amortie par la friction entre membrane et Dynamine. Cependant la constriction ne suffit pas pour la fission. Pour comprendre davantage son mécanisme, la fission par la Dynamine est étudiée à l'aide de tubes lipidiques extraits de vésicules unilamellaires géantes. La fission se produit au bord de l'hélice, où la membrane est fortement courbée. D'après l'analyse statistique des temps de fission, la réaction de fission peut être modélisée par une unique barrière énergétique. La dépendance du temps de fission en rigidité de membrane, en tension de membrane et en couple est établie théoriquement et validée expérimentalement. Ce travail établit le profil énergétique de la réaction de fission membranaire et évalue à 70 kT la barrière énergétique

Mechanics of dynamin-mediated membrane fission

In eukaryotic cells, membrane compartments are split into two by membrane fission. This ensures discontinuity of membrane containers and thus proper compartmentalization. The first proteic machinery implicated in catalyzing membrane fission was dynamin. Dynamin forms helical collars at the neck of endocytic buds. This structural feature suggested that the helix of dynamin could constrict in order to promote fission of the enclosed membrane. However, verifying this hypothesis revealed itself to be a challenge, which inspired many in vitro and in vivo studies. The primary goal of this review is to discuss recent structural and physical data from biophysical studies that have refined our understanding of the dynamin mechanism. In addition to the constriction hypothesis, other models have been proposed to explain how dynamin induces membrane fission. We present experimental data supporting these various models and assess which model is the most probable

Mécanisme de fission membranaire (approches mécanique et énergétique du cas de la dynamine)

La cellule eukaryote est organisée en plusieurs compartiments, appelés organelles, délimités par des membranes; La fission des membranes est nécessaire pour le transport intracellulaire entre organelles. L'endocytose est un mécanisme de transport depuis la membrane plasmique vers les autres organelles. La Dynamine est une guanosine triphosphatase (GTPase) impliquée dans la fission des vésicules pendant l'endocytose médiée par la Clathrine. Elle polymerise en hélice au coup des bourgeons endocytiques. Après hydrolyse du GTP, la structure de l'hélice est modifiée : le rayon interne diminue de 10 à 5 nm et le pas hélical de 13 à 9 nm. Ces modifications indiquent un mécanisme de constriction. La dynamique de constriction est étudiée en suivant la rotation de microbilles attachées à des tubes lipidiques recouverts de Dynamine. La déformation des hélices de Dynamine est concertée et amortie par la friction entre membrane et Dynamine. Cependant la constriction ne suffit pas pour la fission. Pour comprendre davantage son mécanisme, la fission par la Dynamine est étudiée à l'aide de tubes lipidiques extraits de vésicules unilamellaires géantes. La fission se produit au bord de l'hélice, où la membrane est fortement courbée. D'après l'analyse statistique des temps de fission, la réaction de fission peut être modélisée par une unique barrière énergétique. La dépendance du temps de fission en rigidité de membrane, en tension de membrane et en couple est établie théoriquement et validée expérimentalement. Ce travail établit le profil énergétique de la réaction de fission membranaire et évalue à 70 KBT la barrière énergétique.The eukaryotic cell is organized in several compartments, named organelles, delimited by lipid membranes. The fission of these membranes is required for vesicular traffic between organelles. Endocytosis is the mechanism of vesicular traffic from the plasma membrane towards other organelles inside the cell. Dynamin is a guanosise triphosphatase (GTPase) implicated in vesicle scission during Clathrin-mediated endocytosis. It polymerizes into a helix at the neck of endocytic buds. Upon GTP hydrolysis, conformational changes modify the helical structure : the inner radius decreases from 10 to 5 nm and the helical pitch reduces from 13 to 9 nm. These modifications show that fission proceeds through a constriction mechanism. The dynamics of constriction is investigated by monitoring the rotations of microbeads attached along Dynamin-coated tubes after GTP addition. The deformation of Dynamic helices is highly concerted and damped by the friction between membrane and Dynamin. However constriction is not enough to trigger fission. To further understand fission, Dynamin polymerization and fission are studied on lipid tubes extruded from Giant Unilamellar Vesicles. It is shown that fission occurs at the edge of the helix, where the membrane is strongly curved. A statistical analysis of fission time reveals that the fission reaction can be modeledby a single step energy barrier. The fission time dependence on membrane tension, membrane rigidity and torque is established theoretically and validated experimentally. This work gives a quantitative picture of the energy landscape of Dynamin-mediated fission : the height of the energy barrier of fission is estimated around 70 KBT.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Proteostasis collapse, a hallmark of aging, hinders the chaperone-Start network and arrests cells in G1

Loss of proteostasis and cellular senescence are key hallmarks of aging, but direct cause-effect relationships are not well understood. We show that most yeast cells arrest in G1 before death with low nuclear levels of Cln3, a key G1 cyclin extremely sensitive to chaperone status. Chaperone availability is seriously compromised in aged cells, and the G1 arrest coincides with massive aggregation of a metastable chaperone-activity reporter. Moreover, G1-cyclin overexpression increases lifespan in a chaperone-dependent manner. As a key prediction of a model integrating autocatalytic protein aggregation and a minimal Start network, enforced protein aggregation causes a severe reduction in lifespan, an effect that is greatly alleviated by increased expression of specific chaperones or cyclin Cln3. Overall, our data show that proteostasis breakdown, by compromising chaperone activity and G1-cyclin function, causes an irreversible arrest in G1, configuring a molecular pathway postulating proteostasis decay as a key contributing effector of cell senescence.This work was funded by the Ministry of Economy and Competitiveness of Spain, Consolider-Ingenio 2010, and the European Union (FEDER) to MA. KJ was supported by the EPSRC Centre for Doctoral Training in Cross-Disciplinary Approaches to Non-Equilibrium Systems (CANES, EP/L015854/1). DFM received an FI fellow of Generalitat de Catalunya

A balance between membrane elasticity and polymerization energy sets the shape of spherical clathrin coats

In endocytosis, scaffolding is one of the mechanisms to create membrane curvature by moulding the membrane into the spherical shape of the clathrin cage. However, the impact of membrane elastic parameters on the assembly and shape of clathrin lattices has never been experimentally evaluated. Here, we show that membrane tension opposes clathrin polymerization. We reconstitute clathrin budding in vitro with giant unilamellar vesicles (GUVs), purified adaptors and clathrin. By changing the osmotic conditions, we find that clathrin coats cause extensive budding of GUVs under low membrane tension while polymerizing into shallow pits under moderate tension. High tension fully inhibits polymerization. Theoretically, we predict the tension values for which transitions between different clathrin coat shapes occur. We measure the changes in membrane tension during clathrin polymerization, and use our theoretical framework to estimate the polymerization energy from these data. Our results show that membrane tension controls clathrin-mediated budding by varying the membrane budding energy

Excessive rDNA Transcription Drives the Disruption in Nuclear Homeostasis during Entry into Senescence in Budding Yeast

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