Aurélien Roux

GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission

Dynamin, a crucial factor in endocytosis, is a member of a family of GTPases that participates in membrane fission. It was initially proposed to act as a machine that constricts and cuts the neck of nascent vesicles in a GTP-hydrolysis-dependent reaction, but subsequent studies suggested alternative models. Here we monitored the effect of nucleotides on dynamin-coated lipid tubules in real time. Addition of GTP, but not of GDP or GTP-γS, resulted in twisting of the tubules and supercoiling, suggesting a rotatory movement of the helix turns relative to each other during GTP hydrolysis. Rotation was confirmed by the movement of beads attached to the tubules. Twisting activity produced a longitudinal tension that was released by tubule breakage when both ends of the tubule were anchored. Fission also occurred when dynamin and GTP were added to lipid tubules that had been generated from liposomes by the motor activity of kinesin on microtubules. No fission events were observed in the absence of longitudinal tension. These findings demonstrate a mechanoenzyme activity of dynamin in endocytosis, but also imply that constriction is not sufficient for fission. At the short necks of endocytic vesicles, other factors leading to tension may cooperate with the constricting activity of dynamin to induce fission.

Structural Basis of Membrane Invagination by F-BAR Domains

BAR superfamily domains shape membranes through poorly understood mechanisms. We solved structures of F-BAR modules bound to flat and curved bilayers using electron (cryo)microscopy. We show that membrane tubules form when F-BARs polymerize into helical coats that are held together by lateral and tip-to-tip interactions. On gel-state membranes or after mutation of residues along the lateral interaction surface, F-BARs adsorb onto bilayers via surfaces other than their concave face. We conclude that membrane binding is separable from membrane bending, and that imposition of the modules concave surface forces fluid-phase bilayers to bend locally. Furthermore, exposure of the domains lateral interaction surface through a change in orientation serves as the crucial trigger for assembly of the helical coat and propagation of bilayer bending. The geometric constraints and sequential assembly of the helical lattice explain how F-BAR and classical BAR domains segregate into distinct microdomains, and provide insight into the spatial regulation of membrane invagination.

A minimal system allowing tubulation with molecular motors pulling on giant liposomes

The elucidation of physical and molecular mechanisms by which a membrane tube is generated from a membrane reservoir is central to the understanding of the structure and dynamics of intracellular organelles and of transport intermediates in eukaryotic cells. Compelling evidence exists that molecular motors of the dynein and kinesin families are involved in the tubulation of organelles. Here, we show that lipid giant unilamellar vesicles (GUVs), to which kinesin molecules have been attached by means of small polystyrene beads, give rise to membrane tubes and to complex tubular networks when incubated in vitro with microtubules and ATP. Similar tubes and networks are obtained with GUVs made of purified Golgi lipids, as well as with Golgi membranes. No tube formation was observed when kinesins were directly bound to the GUV membrane, suggesting that it is critical to distribute the load on both lipids and motors by means of beads. A kinetic analysis shows that network growth occurs in two phases: a phase in which membrane-bound beads move at the same velocity than free beads, followed by a phase in which the tube growth rate decreases and strongly fluctuates. Our work demonstrates that the action of motors bound to a lipid bilayer is sufficient to generate membrane tubes and opens the way to well controlled experiments aimed at the understanding of basic mechanisms in intracellular transport.

Membrane curvature controls dynamin polymerization

The generation of membrane curvature in intracellular traffic involves many proteins that can curve lipid bilayers. Among these, dynamin-like proteins were shown to deform membranes into tubules, and thus far are the only proteins known to mechanically drive membrane fission. Because dynamin forms a helical coat circling a membrane tubule, its polymerization is thought to be responsible for this membrane deformation. Here we show that the force generated by dynamin polymerization, 18 pN, is sufficient to deform membranes yet can still be counteracted by high membrane tension. Importantly, we observe that at low dynamin concentration, polymer nucleation strongly depends on membrane curvature. This suggests that dynamin may be precisely recruited to membrane buds’ necks because of their high curvature. To understand this curvature dependence, we developed a theory based on the competition between dynamin polymerization and membrane mechanical deformation. This curvature control of dynamin polymerization is predicted for a specific range of concentrations (∼0.1–10 μM), which corresponds to our measurements. More generally, we expect that any protein that binds or self-assembles onto membranes in a curvature-coupled way should behave in a qualitatively similar manner, but with its own specific range of concentration.

Nature of curvature coupling of amphiphysin with membranes depends on its bound density

Cells are populated by a vast array of membrane-binding proteins that execute critical functions. Functions, like signaling and intracellular transport, require the abilities to bind to highly curved membranes and to trigger membrane deformation. Among these proteins is amphiphysin 1, implicated in clathrin-mediated endocytosis. It contains a Bin-Amphiphysin-Rvs membrane-binding domain with an N-terminal amphipathic helix that senses and generates membrane curvature. However, an understanding of the parameters distinguishing these two functions is missing. By pulling a highly curved nanotube of controlled radius from a giant vesicle in a solution containing amphiphysin, we observed that the action of the protein depends directly on its density on the membrane. At low densities of protein on the nearly flat vesicle, the distribution of proteins and the mechanical effects induced are described by a model based on spontaneous curvature induction. The tube radius and force are modified by protein binding but still depend on membrane tension. In the dilute limit, when practically no proteins were present on the vesicle, no mechanical effects were detected, but strong protein enrichment proportional to curvature was seen on the tube. At high densities, the radius is independent of tension and vesicle protein density, resulting from the formation of a scaffold around the tube. As a consequence, the scaling of the force with tension is modified. For the entire density range, protein was enriched on the tube as compared to the vesicle. Our approach shows that the strength of curvature sensing and mechanical effects on the tube depends on the protein density.

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. :

Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation

Summary ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.

Membrane fission by dynamin: what we know and what we need to know.

The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.

BAR Domain Scaffolds in Dynamin-Mediated Membrane Fission

Biological membranes undergo constant remodeling by membrane fission and fusion to change their shape and to exchange material between subcellular compartments. During clathrin-mediated endocytosis, the dynamic assembly and disassembly of protein scaffolds comprising members of the bin-amphiphysin-rvs (BAR) domain protein superfamily constrain the membrane into distinct shapes as the pathway progresses toward fission by the GTPase dynamin. In this Review, we discuss how BAR domain protein assembly and disassembly are controlled in space and time and which structural and biochemical features allow the tight regulation of their shape and function to enable dynamin-mediated membrane fission.

Arf1-GTP-induced tubule formation suggests a function of Arf family proteins in curvature acquisition at sites of vesicle budding.

ADP-ribosylation factor (Arf) and related small GTPases play crucial roles in membrane traffic within the exo- and endocytic pathways. Arf proteins in their GTP-bound state are associated with curved membrane buds and tubules, frequently together with effector coat proteins to which they bind. Here we report that Arf1 is found on membrane tubules originating from the Golgi complex where it colocalizes with COPI and GGA1 vesicle coat proteins. Arf1 also induces tubulation of liposomes in vitro. Mutations within the amino-terminal amphipathic helix (NTH) of Arf1 affect the number of Arf1-positive tubules in vivo and its property to tubulate liposomes. Moreover, hydrophilic substitutions within the hydrophobic part of its NTH impair Arf1-catalyzed budding of COPI vesicles in vitro. Our data indicate that GTP-controlled local induction of high curvature membranes is an important property of Arf1 that might be shared by a subgroup of Arf/Arl family GTPases.