Actomyosin-mediated contractility is a highly conserved mechanism for generating mechanical stress in animal cells and underlies muscle contraction, cell migration, cell division and tissue morphogenesis. Whereas actomyosin-mediated contractility in striated muscle is well understood, the regulation of such contractility in non-muscle and smooth muscle cells is less certain. Our increased understanding of the mechanics of actomyosin arrays that lack sarcomeric organization has revealed novel modes of regulation and force transmission. This work also provides an example of how diverse mechanical behaviours at cellular scales can arise from common molecular components, underscoring the need for experiments and theories to bridge the molecular to cellular length scales.

SummaryESCRT-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.

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.

Red blood cells are amazingly deformable structures able to recover their initial shape even after large deformations as when passing through tight blood capillaries. The reason for this exceptional property is found in the composition of the membrane and the membrane-cytoskeleton interaction. We investigate the mechanics and the dynamics of RBCs by a unique noninvasive technique, using weak optical tweezers to measure membrane fluctuation amplitudes with s temporal and sub nm spatial resolution. This enhanced edge detection method allows to span over >4 orders of magnitude in frequency. Hence, we can simultaneously measure red blood cell membrane mechanical properties such as bending modulus ‫؍‬ 2.8 ؎ 0.3 ؋ 10 ؊19 J ‫؍‬ 67.6 ؎ 7.2 kBT, tension ‫؍‬ 6.5 ؎ 2.1 ؋ 10 ؊7 N/m, and an effective viscosity eff ‫؍‬ 81 ؎ 3.7 ؋ 10 ؊3 Pa s that suggests unknown dissipative processes. We furthermore show that cell mechanics highly depends on the membrane-spectrin interaction mediated by the phosphorylation of the interconnection protein 4.1R. Inhibition and activation of this phosphorylation significantly affects tension and effective viscosity. Our results show that on short time scales (slower than 100 ms) the membrane fluctuates as in thermodynamic equilibrium. At time scales longer than 100 ms, the equilibrium description breaks down and fluctuation amplitudes are higher by 40% than predicted by the membrane equilibrium theory. Possible explanations for this discrepancy are influences of the spectrin that is not included in the membrane theory or nonequilibrium fluctuations that can be accounted for by defining a nonthermal effective energy of up to Eeff ‫؍‬ 1.4 ؎ 0.1 kBT, that corresponds to an actively increased effective temperature.erythrocyte ͉ membrane fluctuations ͉ nonequilibrium ͉ optical tweezer ͉ spectrin T he extraordinary deformability of RBCs is vital for their proper function, as it enables the cells to be elongated by more than twice their size when passing through m-sized capillaries. The elastic properties of RBCs are dominated by the interaction between the cell membrane and the underlying cytoskeleton, mainly consisting of spectrin, a long heterodimer that aligns tail to tail forming a 200-nm-long tetramer. Each spectrin filament interconnects to up to 5 other spectrins and is bound to the cell membrane via a protein complex consisting of protein 4.1R, actin, and glycophorin C (1). The plasma membrane fluctuations are related to RBCs' mechanical properties that have been extensively studied over the past three decades (2-6). It is well known that fluctuations of the membrane depend on its bending rigidity and its membrane tension (7). Furthermore, RBCs gain their elasticity from the elastic shear modulus of the cytoskeletal spectrin network (8, 9). Recent theoretical analysis (10) suggest that the spectrin cytoskeleton acts as a steric barrier restricting membrane undulations toward the network.Experiments by Tuvia and coworkers showed an ATP dependent effect by monitoring the static fluctuation amplit...

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.force | nucleation | fission | endocytosis | dynamin-like proteins M embrane remodeling is an essential task of proteins involved in membrane traffic (1, 2). Dynamin is a large GTPase that has been shown to polymerize into a helical collar at the neck of endocytic buds (3), where it subsequently plays a key role in the formation of endocytic vesicles through fission (4-8). This function is fundamental, as the knockout of the dynamin neuronal isoform leads to striking defects in synapse organization and results in a strong dysfunction of neuronal activity (9). The recruitment of dynamin to endocytic buds is thought to depend on the local synthesis of phosphatidylinositol(4,5)bisphosphate (PIP 2 ), as dynamin has a PIP 2 binding pleckstrin homology (PH) domain (10). Dynamin is recruited late in clathrin-coated vesicle formation, as seen by total internal reflection fluorescence (TIRF) microscopy (11-13). Because PIP 2 is also responsible for the binding of clathrin coats, it is expected to be present at the clathrin bud from the beginning of its formation. Thus, another explanation was suggested: Proteins that interact with dynamin and possess a curvature-sensing Bin-Amphiphysin-Rvs (BAR) domain (such as endophilin and amphiphysin) were proposed to sense the high curvature of the neck and recruit dynamin (14). Because the curvature of the neck is increasing during clathrin-coated vesicle formation to finally reach the range needed for BAR recruitment, this could explain the arrival of dynamin at the very late stage of clathrin-coated vesicle formation. However, in solution, dynamin spontaneously associates into helices and rings (15) with an internal radius of ≈10 nm. Dynamin can also polymerize around pre...

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. :

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.

Animal cells in tissues are supported by biopolymer matrices, which typically exhibit highly nonlinear mechanical properties. While the linear elasticity of the matrix can significantly impact cell mechanics and functionality, it remains largely unknown how cells, in turn, affect the nonlinear mechanics of their surrounding matrix. Here, we show that living contractile cells are able to generate a massive stiffness gradient in three distinct 3D extracellular matrix model systems: collagen, fibrin, and Matrigel. We decipher this remarkable behavior by introducing nonlinear stress inference microscopy (NSIM), a technique to infer stress fields in a 3D matrix from nonlinear microrheology measurements with optical tweezers. Using NSIM and simulations, we reveal large long-ranged cell-generated stresses capable of buckling filaments in the matrix. These stresses give rise to the large spatial extent of the observed cell-induced matrix stiffness gradient, which can provide a mechanism for mechanical communication between cells.