Dynamin-mediated Membrane Fission Research Articles

Abstract 1311 Snapshots of dynamin-mediated membrane fission by Cryo-EM

Abstract 1311 Snapshots of dynamin-mediated membrane fission by Cryo-EM

Snapshots of dynamin-mediated membrane fission by cryoEM

Snapshots of dynamin-mediated membrane fission by cryoEM

Cryo-EM Studies of Dynamin Assembled on Membranes In Vitro and within Cells

Dynamin GTPases enable endocytosis by wrapping around the neck of budding vesicles to catalyze membrane fission. While structural and biophysical studies have contributed to elucidating the mechanism of dynamin-mediated membrane fission, questions remain about how dynamin forms the final pre-fission super-constricted state and how dynamin assembles in cells. To address these questions, we have applied cryo-electron microscopy (cryo-EM) helical reconstruction methods, time-resolved cryo-EM, and cryo-electron tomography (cryo-ET) to elucidate the assembly of dynamin in vitro on lipid tubules and in vivo on membranes within HeLa cells.

GTP Concentration Bursts Locally at Endocytic Sites for Dynamin-Dependent Membrane Fisson

Endocytosis is a basic property of cells and is required for a variety of cellular processes including migration, hormone signaling, and neurotransmission. During endocytosis, a part of plasma membrane is buckled inwards to form a vesicle. These vesicles are severed from the plasma membrane by dynamin GTPases. However, this reaction requires a large amount of GTP. The global elevation in the GTP level within cytoplasm would be toxic to the cells, and thus, how its level is regulated for dynamin activity during endocytosis remains enigmatic. Here, we measure the GTP concentrations around endocytic sites using GTP sensors and show that the GTP concentration increases acutely around endocytic pits to provide a sufficient amount of GTP for the dynamin activity. This local enrichment of GTP precedes dynamin recruitment by ∼500 ms, suggesting that GTP bursting is prerequisite for dynamin assembly. The generated GTP does not diffuse out from endocytic sites, indicating that GTP is constantly consumed by dynamin. GTP bursting is most likely regulated by nucleoside-diphosphate kinases, which convert ATP to GTP. Our results demonstrate that GTP concentration is spatiotemporally regulated to provide sufficient energy for dynamin-mediated membrane fission.

Structural inhibition of dynamin-mediated membrane fission by endophilin.

Dynamin, which mediates membrane fission during endocytosis, binds endophilin and other members of the Bin-Amphiphysin-Rvs (BAR) protein family. How endophilin influences endocytic membrane fission is still unclear. Here, we show that dynamin-mediated membrane fission is potently inhibited in vitro when an excess of endophilin co-assembles with dynamin around membrane tubules. We further show by electron microscopy that endophilin intercalates between turns of the dynamin helix and impairs fission by preventing trans interactions between dynamin rungs that are thought to play critical roles in membrane constriction. In living cells, overexpression of endophilin delayed both fission and transferrin uptake. Together, our observations suggest that while endophilin helps shape endocytic tubules and recruit dynamin to endocytic sites, it can also block membrane fission when present in excess by inhibiting inter-dynamin interactions. The sequence of recruitment and the relative stoichiometry of the two proteins may be critical to regulated endocytic fission.

Rupturing the hemi-fission intermediate in membrane fission under tension: Reaction coordinates, kinetic pathways, and free-energy barriers.

Membrane fission is a fundamental process in cells, involved inter alia in endocytosis, intracellular trafficking, and virus infection. Its underlying molecular mechanism, however, is only incompletely understood. Recently, experiments and computer simulation studies have revealed that dynamin-mediated membrane fission is a two-step process that proceeds via a metastable hemi-fission intermediate (or wormlike micelle) formed by dynamin's constriction. Importantly, this hemi-fission intermediate is remarkably metastable, i.e., its subsequent rupture that completes the fission process does not occur spontaneously but requires additional, external effects, e.g., dynamin's (unknown) conformational changes or membrane tension. Using simulations of a coarse-grained, implicit-solvent model of lipid membranes, we investigate the molecular mechanism of rupturing the hemi-fission intermediate, such as its pathway, the concomitant transition states, and barriers, as well as the role of membrane tension. The membrane tension is controlled by the chemical potential of the lipids, and the free-energy landscape as a function of two reaction coordinates is obtained by grand canonical Wang-Landau sampling. Our results show that, in the course of rupturing, the hemi-fission intermediate undergoes a "thinning → local pinching → rupture/fission" pathway, with a bottle-neck-shaped cylindrical micelle as a transition state. Although an increase of membrane tension facilitates the fission process by reducing the corresponding free-energy barrier, for biologically relevant tensions, the free-energy barriers still significantly exceed the thermal energy scale kBT.

Structural insights into the centronuclear myopathy-associated functions of BIN1 and dynamin 2.

Centronuclear myopathies (CNMs) are genetic diseases whose symptoms are muscle weakness and atrophy (wasting) and centralised nuclei. Recent human genetic studies have isolated several groups of mutations. Among them, many are found in two interacting proteins essential to clathrin-mediated endocytosis, dynamin and the BIN-Amphiphysin-Rvs (BAR) protein BIN1/amphiphysin 2. In this review, by using structural and functional data from the study of endocytosis mainly, we discuss how the CNM mutations could affect the structure and the function of these ubiquitous proteins and cause the muscle-specific phenotype. The literature shows that both proteins are involved in the plasma membrane tubulation required for T-tubule biogenesis. However, this system also requires the regulation of the dynamin-mediated membrane fission, and the formation of a stable protein-scaffold to maintain the T-tubule structure. We discuss how the specific functions, isoforms and partners (myotubularin in particular) of these two proteins can lead to the establishment of muscle-specific features.

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.

Coordination of Bending and Wedging in Membrane Fission

Protein complexes controlling intracellular membrane scission generally utilize multiple membrane insertions to enforce structural rearrangements of lipid monolayers. In general, the deformation fields created by individual insertions might interact destructively or constructively so that the resulting stress distribution depends strongly on the spatial organization and orientation of the individual insertions. We show here that in cylindrical topology the interaction between two ring-like membrane insertions can be both attractive and repulsive, dependently on the ratio between the curvatures of the membrane and the ring as well as on the insertion parameters. The attractive interaction drives clusterization of the rings leading to substantial stored elastic stress. This stored energy can be further used for localized membrane remodeling if the rings can interact changing the system geometry and boundary constraints. The model was applied to calculate energy barriers for dynamin-mediated membrane fission, substantial reduction of the barrier was found for a particular parameters range, indicating that the molecular design of the fission machinery involves optimization of the membrane insertion geometry.

Essential Elastic and Shape Parameters Govern the Dynamics and Energetics of Dynamin-Mediated Membrane Fission

Membrane fission is the final step enabling the release of vesicles during endocytosis. Dynamin is a protein required for this crucial process in clathrin-mediated endocytosis. It polymerizes into a helix at the neck between the plasma membrane and the endocytic bud. A constriction of the helix occurs upon guanosine triphosphate (GTP) hydrolysis leading to fission. Here we show how the fission mechanism is regulated temporally, spatially and energetically. We perform in vitro experiments where all mechanical and chemical parameters are controlled. Membrane nanotubes are pulled from giant unilamellar vesicles (GUV) using optical tweezers. 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. We show that the probability of fission depends on GTP concentration. Membrane tension and bending rigidity are key parameters controlling fission times in a non trivial way. Saddle-like shape favors membrane fission supporting the idea that negative Gaussian curvature plays an important role in the process.

Membrane Insertion of the Pleckstrin Homology Domain Variable Loop 1 Is Critical for Dynamin-catalyzed Vesicle Scission

The GTPase dynamin catalyzes the scission of deeply invaginated clathrin-coated pits at the plasma membrane, but the mechanisms governing dynamin-mediated membrane fission remain poorly understood. Through mutagenesis, we have altered the hydrophobic nature of the membrane-inserting variable loop 1 (VL1) of the pleckstrin homology (PH) domain of dynamin-1 and demonstrate that its stable insertion into the lipid bilayer is critical for high membrane curvature generation and subsequent membrane fission. Dynamin PH domain mutants defective in curvature generation regain function when assayed on precurved membrane templates in vitro, but they remain defective in the scission of clathrin-coated pits in vivo. These results demonstrate that, in concert with dynamin self-assembly, PH domain membrane insertion is essential for fission and vesicle release in vitro and for clathrin-mediated endocytosis in vivo.