Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation.

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.

Forcing cells into shape: the mechanics of actomyosin contractility.

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.

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. :

Passive coupling of membrane tension and cell volume during active response of cells to osmosis.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Transverse Fluctuations Control the Assembly of Semiflexible Filaments.

The kinetics of the assembly of semiflexible filaments through end-to-end annealing is key to the structure of the cytoskeleton, but is not understood. We analyze this problem through scaling theory and simulations, and uncover a regime where filaments' ends find each other through bending fluctuations without the need for the whole filament to diffuse. This results in a very substantial speedup of assembly in physiological regimes, and could help with understanding the dynamics of actin and intermediate filaments in biological processes such as wound healing and cell division.

Generic stress rectification in nonlinear elastic media.

Stress propagation in nonlinear media is crucial in cell biology, where molecular motors exert anisotropic force dipoles on the fibrous cytoskeleton. While the force dipoles can be either contractile or expansile, a medium made of fibers which buckle under compression rectifies these stresses towards a biologically crucial contraction. A general understanding of this rectification phenomenon as a function of the medium's elasticity is however lacking. Here we use theoretical continuum elasticity to show that rectification is actually a very general effect in nonlinear materials subjected to anisotropic internal stresses. We analytically show that both bucklable and constitutively linear materials subjected to geometrical nonlinearities rectify small forces towards contraction, while granular-like materials rectify towards expansion. Using simulations, we moreover show that these results extend to larger forces. Beyond fiber networks, these results could shed light on the propagation of stresses in brittle or granular materials following a local plastic rearrangement.

Local structure of DNA toroids reveals curvature-dependent intermolecular forces.

In viruses and cells, DNA is closely packed and tightly curved thanks to polyvalent cations inducing an effective attraction between its negatively charged filaments. Our understanding of this effective attraction remains very incomplete, partly because experimental data is limited to bulk measurements on large samples of mostly uncurved DNA helices. Here we use cryo electron microscopy to shed light on the interaction between highly curved helices. We find that the spacing between DNA helices in spermine-induced DNA toroidal condensates depends on their location within the torus, consistent with a mathematical model based on the competition between electrostatic interactions and the bending rigidity of DNA. We use our model to infer the characteristics of the interaction potential, and find that its equilibrium spacing strongly depends on the curvature of the filaments. In addition, the interaction is much softer than previously reported in bulk samples using different salt conditions. Beyond viruses and cells, our characterization of the interactions governing DNA-based dense structures could help develop robust designs in DNA nanotechnologies.

A super-resolution platform for correlative live single-molecule imaging and STED microscopy.

Super-resolution microscopy offers tremendous opportunities to unravel the complex and dynamic architecture of living cells. However, current super-resolution microscopes are well suited for revealing protein distributions or cell morphology, but not both. We present a super-resolution platform that permits correlative single-molecule imaging and stimulated emission depletion microscopy in live cells. It gives nanoscale access to the positions and movements of synaptic proteins within the morphological context of growth cones and dendritic spines.

Neuromesodermal progenitors are a conserved source of spinal cord with divergent growth dynamics.

During gastrulation, embryonic cells become specified into distinct germ layers. In mouse, this continues throughout somitogenesis from a population of bipotent stem cells called neuromesodermal progenitors (NMps). However, the degree of self-renewal associated with NMps in the fast-developing zebrafish embryo is unclear. Using a genetic clone-tracing method, we labelled early embryonic progenitors and found a strong clonal similarity between spinal cord and mesoderm tissues. We followed individual cell lineages using light-sheet imaging, revealing a common neuromesodermal lineage contribution to a subset of spinal cord tissue across the anterior-posterior body axis. An initial population subdivides at mid-gastrula stages and is directly allocated to neural and mesodermal compartments during gastrulation. A second population in the tailbud undergoes delayed allocation to contribute to the neural and mesodermal compartment only at late somitogenesis. Cell tracking and retrospective cell fate assignment at late somitogenesis stages reveal these cells to be a collection of mono-fated progenitors. Our results suggest that NMps are a conserved population of bipotential progenitors, the lineage of which varies in a species-specific manner due to vastly different rates of differentiation and growth.

A nonequilibrium force can stabilize 2D active nematics.

Suspensions of actively driven anisotropic objects exhibit distinctively nonequilibrium behaviors, and current theories predict that they are incapable of sustaining orientational order at high activity. By contrast, here we show that nematic suspensions on a substrate can display order at arbitrarily high activity due to a previously unreported, potentially stabilizing active force. This force moreover emerges inevitably in theories of active orientable fluids under geometric confinement. The resulting nonequilibrium ordered phase displays robust giant number fluctuations that cannot be suppressed even by an incompressible solvent. Our results apply to virtually all experimental assays used to investigate the active nematic ordering of self-propelled colloids, bacterial suspensions, and the cytoskeleton and have testable implications in interpreting their nonequilibrium behaviors.

Cell contraction induces long-ranged stress stiffening in the extracellular matrix.

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.

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.

Chiral Active Hexatics: Giant Number Fluctuations, Waves, and Destruction of Order.

Active materials, composed of internally driven particles, have properties that are qualitatively distinct from matter at thermal equilibrium. However, the most spectacular departures from equilibrium phase behavior are thought to be confined to systems with polar or nematic asymmetry. In this Letter, we show that such departures are also displayed by more symmetric phases such as hexatics if, in addition, the constituent particles have chiral asymmetry. We show that chiral active hexatics whose rotation rate does not depend on density have giant number fluctuations. If the rotation rate depends on density, the giant number fluctuations are suppressed due to a novel orientation-density sound mode with a linear dispersion which propagates even in the overdamped limit. However, we demonstrate that beyond a finite but large length scale, a chirality and activity-induced relevant nonlinearity invalidates the predictions of the linear theory and destroys the hexatic order. In addition, we show that activity modifies the interactions between defects in the active chiral hexatic phase, making them nonmutual. Finally, to demonstrate the generality of a chiral active hexatic phase we show that it results from the melting of chiral active crystals in finite systems.

Swimmer Suspensions on Substrates: Anomalous Stability and Long-Range Order.

We present a comprehensive theory of the dynamics and fluctuations of a two-dimensional suspension of polar active particles in an incompressible fluid confined to a substrate. We show that, depending on the sign of a single parameter, a state with polar orientational order is anomalously stable (or anomalously unstable), with a nonzero relaxation (or growth) rate for angular fluctuations, not parallel to the ordering direction, at zero wave number. This screening of the broken-symmetry mode in the stable state does lead to conventional rather than giant number fluctuations as argued by Bricard et al., Nature 503, 95 (2013), but their bend instability in a splay-stable flock does not exist and the polar phase has long-range order in two dimensions. Our theory also describes confined three-dimensional thin-film suspensions of active polar particles as well as dense compressible active polar rods, and predicts a flocking transition without a banding instability.

Fiber plucking by molecular motors yields large emergent contractility in stiff biopolymer networks.

The mechanical properties of the cell depend crucially on the tension of its cytoskeleton, a biopolymer network that is put under stress by active motor proteins. While the fibrous nature of the network is known to strongly affect the transmission of these forces to the cellular scale, our understanding of this process remains incomplete. Here we investigate the transmission of forces through the network at the individual filament level, and show that active forces can be geometrically amplified as a transverse motor-generated force "plucks" the fiber and induces a nonlinear tension. In stiff and densely connected networks, this tension results in large network-wide tensile stresses that far exceed the expectation drawn from a linear elastic theory. This amplification mechanism competes with a recently characterized network-level amplification due to fiber buckling, suggesting that that fiber networks provide several distinct pathways for living systems to amplify their molecular forces.

Stress-dependent amplification of active forces in nonlinear elastic media.

The production of mechanical stresses in living organisms largely relies on localized, force-generating active units embedded in filamentous matrices. Numerical simulations of discrete fiber networks with fixed boundaries have shown that buckling in the matrix dramatically amplifies the resulting active stresses. Here we extend this result to a continuum elastic medium prone to buckling subjected to an arbitrary external stress, and derive analytical expressions for the active, nonlinear constitutive relations characterizing the full active medium. Inserting these relations into popular "active gel" descriptions of living tissues and the cytoskeleton will enable investigations into nonlinear regimes previously inaccessible due to the phenomenological nature of these theories.

Fiber networks amplify active stress.

Large-scale force generation is essential for biological functions such as cell motility, embryonic development, and muscle contraction. In these processes, forces generated at the molecular level by motor proteins are transmitted by disordered fiber networks, resulting in large-scale active stresses. Although these fiber networks are well characterized macroscopically, this stress generation by microscopic active units is not well understood. Here we theoretically study force transmission in these networks. We find that collective fiber buckling in the vicinity of a local active unit results in a rectification of stress towards strongly amplified isotropic contraction. This stress amplification is reinforced by the networks' disordered nature, but saturates for high densities of active units. Our predictions are quantitatively consistent with experiments on reconstituted tissues and actomyosin networks and shed light on the role of the network microstructure in shaping active stresses in cells and tissue.

Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy.

Super-resolution techniques that localize single molecules in three dimensions through point spread function (PSF) engineering are very sensitive to aberrations and optical alignment. Here we show how double-helix point spread function is affected by such mis-alignment and aberration. Specifically, we demonstrate through simulation and experiment how misplacement of phase masks in infinity corrected systems is a common source of significant loss of accuracy. We also describe an optimal alignment and calibration procedure to correct for these errors. In combination, these optimizations allow for a maximal field of view with high accuracy and precision. Though discussed with reference to double-helix point spread function (DHPSF), the optimization techniques are equally applicable to other engineered PSFs.