Sandrine Morlot

Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction.

The GTPase dynamin polymerizes into a helical coat that constricts membrane necks of endocytic pits to promote their fission. However, the dynamin mechanism is still debated because constriction is necessary but not sufficient for fission. Here, we show that fission occurs at the interface between the dynamin coat and the uncoated membrane. At this location, the considerable change in membrane curvature increases the local membrane elastic energy, reducing the energy barrier for fission. Fission kinetics depends on tension, bending rigidity, and the dynamin constriction torque. Indeed, we experimentally find that the fission rate depends on membrane tension in vitro and during endocytosis in vivo. By estimating the energy barrier from the increased elastic energy at the edge of dynamin and measuring the dynamin torque, we show that the mechanical energy spent on dynamin constriction can reduce the energy barrier for fission sufficiently to promote spontaneous fission. :

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.

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.

Mechanical requirements for membrane fission: Common facts from various examples

Membrane fission is the last step of membrane carrier formation. As fusion, it is a very common process in eukaryotic cells, and participates in the integrity and specificity of organelles. Although many proteins have been isolated to participate in the various membrane fission reactions, we are far from understanding how membrane fission is mechanically triggered. Here we aim at reviewing the well‐described examples of dynamin and lipid phase separation, and try to extract the essential requirements for fission. Then, we survey the recent knowledge obtained on other fission reactions, analyzing the similarities and differences with previous examples.

Deformation of dynamin helices damped by membrane friction.

Dynamin and other proteins of the dynamin superfamily are widely used by cells to sever lipid bilayers. During this process, a short helical dynamin polymer (one to three helical turns) assembles around a membrane tubule and reduces its radius and pitch upon guanosine triphosphate hydrolysis. This deformation is thought to be crucial for dynamins severing action and results in an observable twisting of the helix. Here, we quantitatively characterize the dynamics of this deformation by studying long dynamin helices (many helical turns). We perform in vitro experiments where we attach small beads to the dynamin helix and track their rotation in real time, thus collecting information about the space and time dependence of the deformation. We develop a theoretical formalism to predict the dynamics of a mechanically continuous helix deforming on long timescales. Longer helices deform more slowly, as predicted by theory. This could account for the previously reported observation that they are less fission-competent. Comparison between experiments and our model indicates that the deformation dynamics is dominated by the draining of the membrane out of the helix, allowing quantification of helix-membrane interactions.

Nucleolar stress triggers the irreversible cell cycle slow down leading to cell death during replicative aging in Saccharomyces cerevisiae

Asymmetric division in Saccharomyces cerevisiae generates an aging mother cell and a rejuvenated daughter cell. The accumulation of Extrachromosomal rDNA Circles (ERCs) and their specific retention in mothers have been hypothesized to be responsible for replicative aging. However, it remains unclear by which molecular mechanisms ERCs would trigger the cell cycle slow-down occurring during replicative aging and leading to cell death. In this study, we show that ERCs accumulation is initiated within the 5 divisions preceding the onset of cell cycle decline. The generation of ERCs is also concomitant with a nucleolar stress characterized by an up-regulation of RNA polymerase I activity and an accumulation of pre-rRNAs in the nucleolus which do not lead neither to a higher production of ribosomes, nor to an increased growth rate. We further demonstrate that this nucleolar stress observed in old mothers is not inherited by daughters, which recover basal RNA polymerase I activity and normal cell cycle durations following an asymmetrical nuclear division. In the long-lived mutant fob1Δ, we identified a sub-population, with a further extended longevity, which does not present neither a nucleolar stress nor a cell cycle slow down prior to cell death. Altogether, these findings support a causal role for the nucleolar stress in entering cellular senescence.The accumulation of Extrachromosomal rDNA Circles (ERCs) and their asymmetric segregation upon division have been hypothesized to be responsible for replicative senescence in mother yeasts and rejuvenation in daughter cells. However, it remains unclear by which molecular mechanisms ERCs would trigger the irreversible cell cycle slow-down leading to cell death. We show that ERCs accumulation is concomitant with a nucleolar stress, characterized by a massive accumulation of pre-rRNAs in the nucleolus, leading to a loss of nucleus-to-cytoplasm ratio, decreased growth rate and cell-cycle slow-down. This nucleolar stress, observed in old mothers, is not inherited by rejuvenated daughters. Unlike WT, in the long-lived mutant fob1∆, a majority of cells is devoid of nucleolar stress and does not experience replicative senescence before death. Our study provides a unique framework to order the successive steps that govern the transition to replicative senescence and highlights the causal role of nucleolar stress in cellular aging.

Quantitative Analysis of Membrane Deformation and Fission Induced by Dynamin GTPase Activity

Dynamin is widely used by cells to sever lipid bilayers. During this process a short helical dynamin polymer (1 to 3 helical turns) assembles around a membrane tubule and reduces its radius and pitch upon GTP hydrolysis. This deformation is thought to be crucial for dynamins severing action and results in an observable twisting of the helix [1]. Here we quantitatively study the factors determining the dynamics of this deformation by studying long dynamin. We perform in vitro experiments where we attach small beads to the dynamin helix and track their rotation in real time, thus collecting information about the space and time dependence of the deformation. Longer helices deform more slowly as predicted by a generalized hydrodynamics theoretical model [2]. Further agreement between experiments and theory indicates that the concerted deformation dynamics is dominated by the draining of the membrane out of the helix, allowing us to quantitatively characterize helix-membrane interactions [3]. We also study the dynamics of tube fission induced by dynamin GTPase activity. Membrane nanotubes are pulled from Giant Unilamellar Vesicles (GUV) using optical tweezers and membrane tension is set by aspirating the GUVs within a micropipette. Dynamin and GTP are injected near the tube. Tubes always break few seconds after dynamin starts polymerizing around the tube. We show that probability of fission depends on GTP concentration, no global depolymerization occurs during GTP hydrolysis and membrane geometry affects fission.[1] Aurelien Roux, Katherine Uyhazi, Adam Frost, and Pietro De Camilli. Nature 2006.[2] Martin Lenz, Jacques Prost, and Jean-Francois Joanny. Phys. Rev. E 2008.[3] Sandrine Morlot, Martin Lenz, Jacques Prost, Jean-Francois Joanny, Aurelien Roux. submitted