Marc-Antoine Fardin

Temporary increase in plasma membrane tension coordinates the activation of exocytosis and contraction during cell spreading

Cell migration and spreading involve the coordination of membrane trafficking, actomyosin contraction, and modifications to plasma membrane tension and area. The biochemical or biophysical basis for this coordination is however unknown. In this study, we show that during cell spreading, lamellipodia protrusion flattens plasma membrane folds and blebs and, once the plasma membrane area is depleted, there is a temporary increase in membrane tension by over twofold that is followed by activation of exocytosis and myosin contraction. Further, an artificial increase in plasma membrane tension stopped lamellipodia protrusion and activated an exocytotic burst. Subsequent decrease in tension restored spreading with activation of contraction. Conversely, blebbistatin inhibition of actomyosin contraction resulted in an even greater increase in plasma membrane tension and exocytosis activation. This spatiotemporal synchronization indicates that membrane tension is the signal that coordinates membrane trafficking, actomyosin contraction, and plasma membrane area change. We suggest that cells use plasma membrane tension as a global physical parameter to control cell motility.

Cytoskeletal coherence requires myosin-IIA contractility

Maintaining a physical connection across cytoplasm is crucial for many biological processes such as matrix force generation, cell motility, cell shape and tissue development. However, in the absence of stress fibers, the coherent structure that transmits force across the cytoplasm is not understood. We find that nonmuscle myosin-II (NMII) contraction of cytoplasmic actin filaments establishes a coherent cytoskeletal network irrespective of the nature of adhesive contacts. When NMII activity is inhibited during cell spreading by Rho kinase inhibition, blebbistatin, caldesmon overexpression or NMIIA RNAi, the symmetric traction forces are lost and cell spreading persists, causing cytoplasm fragmentation by membrane tension that results in ‘C’ or dendritic shapes. Moreover, local inactivation of NMII by chromophore-assisted laser inactivation causes local loss of coherence. Actin filament polymerization is also required for cytoplasmic coherence, but microtubules and intermediate filaments are dispensable. Loss of cytoplasmic coherence is accompanied by loss of circumferential actin bundles. We suggest that NMIIA creates a coherent actin network through the formation of circumferential actin bundles that mechanically link elements of the peripheral actin cytoskeleton where much of the force is generated during spreading.

Force generated by actomyosin contraction builds bridges between adhesive contacts

Extracellular matrices in vivo are heterogeneous structures containing gaps that cells bridge with an actomyosin network. To understand the basis of bridging, we plated cells on surfaces patterned with fibronectin (FN)‐coated stripes separated by non‐adhesive regions. Bridges developed large tensions where concave cell edges were anchored to FN by adhesion sites. Actomyosin complexes assembled near those sites (both actin and myosin filaments) and moved towards the centre of the non‐adhesive regions in a treadmilling network. Inhibition of myosin‐II (MII) or Rho‐kinase collapsed bridges, whereas extension continued over adhesive areas. Inhibition of actin polymerization (latrunculin‐A, jasplakinolide) also collapsed the actomyosin network. We suggest that MII has distinct functions at different bridge regions: (1) at the concave edges of bridges, MIIA force stimulates actin filament assembly at adhesions and (2) in the body of bridges, myosin cross‐links actin filaments and stimulates actomyosin network healing when breaks occur. Both activities ensure turnover of actin networks needed to maintain stable bridges from one adhesive region to another.

Shear Banding of Complex Fluids

Even in simple geometries, many complex fluids display nontrivial flow fields, with regions where shear is concentrated. The possibility for such shear banding has been known for several decades, but in recent years, we have seen an upsurge in studies offering an ever-more precise understanding of the phenomenon. The development of new techniques to probe the flow on multiple scales with increasing spatial and temporal resolution has opened the possibility for a synthesis of the many phenomena that could only have been thought of separately before. In this review, we bring together recent research on shear banding in polymeric and soft glassy materials and highlight their similarities and disparities.

Elastic Turbulence in Shear Banding Wormlike Micelles

We study the dynamics of the Taylor-Couette flow of shear banding wormlike micelles. We focus on the high shear rate branch of the flow curve and show that for sufficiently high Weissenberg numbers, this branch becomes unstable. This instability is strongly subcritical and is associated with a shear stress jump. We find that this increase of the flow resistance is related to the nucleation of turbulence. The flow pattern shows similarities with the elastic turbulence, so far only observed for polymer solutions. The unstable character of this branch led us to propose a scenario that could account for the recent observations of Taylor-like vortices during the shear banding flow of wormlike micelles.

Taylor-like Vortices in Shear-Banding Flow of Giant Micelles

Using flow visualizations in Couette geometry, we demonstrate the existence of Taylor-like vortices in the shear-banding flow of a giant micelles system. We show that vortices stacked along the vorticity direction develop concomitantly with interfacial undulations. These cellular structures are mainly localized in the induced band and their dynamics is fully correlated with that of the interface. As the control parameter increases, we observe a transition from a steady vortex flow to a state where pairs of vortices are continuously created and destroyed. Normal stress effects are discussed as potential mechanisms driving the three-dimensional flow.

Mechanisms controlling cell size and shape during isotropic cell spreading.

Cell motility is important for many developmental and physiological processes. Motility arises from interactions between physical forces at the cell surface membrane and the biochemical reactions that control the actin cytoskeleton. To computationally analyze how these factors interact, we built a three-dimensional stochastic model of the experimentally observed isotropic spreading phase of mammalian fibroblasts. The multiscale model is composed at the microscopic levels of three actin filament remodeling reactions that occur stochastically in space and time, and these reactions are regulated by the membrane forces due to membrane surface resistance (load) and bending energy. The macroscopic output of the model (isotropic spreading of the whole cell) occurs due to the movement of the leading edge, resulting solely from membrane force-constrained biochemical reactions. Numerical simulations indicate that our model qualitatively captures the experimentally observed isotropic cell-spreading behavior. The model predicts that increasing the capping protein concentration will lead to a proportional decrease in the spread radius of the cell. This prediction was experimentally confirmed with the use of Cytochalasin D, which caps growing actin filaments. Similarly, the predicted effect of actin monomer concentration was experimentally verified by using Latrunculin A. Parameter variation analyses indicate that membrane physical forces control cell shape during spreading, whereas the biochemical reactions underlying actin cytoskeleton dynamics control cell size (i.e., the rate of spreading). Thus, during cell spreading, a balance between the biochemical and biophysical properties determines the cell size and shape. These mechanistic insights can provide a format for understanding how force and chemical signals together modulate cellular regulatory networks to control cell motility.

Cell spreading as a hydrodynamic process

Many cell types have the ability to move themselves by crawling on extra-cellular matrices. Although cell motility is governed by actin and myosin filament assembly, the pattern of the movement follows the physical properties of the network ensemble average. The first step of motility, cell spreading on matrix substrates, involves a transition from round cells in suspension to polarized cells on substrates. Here we show that the spreading dynamics on 2D surfaces can be described as a hydrodynamic process. In particular, we show that the transition from isotropic spreading at early time to anisotropic spreading is reminiscent of the fingering instability observed in many spreading fluids. During cell spreading, the main driving force is the polymerization of actin filaments that push the membrane forward. From the equilibrium between the membrane force and the cytoskeleton, we derive a first order expression of the polymerization stress that reproduces the observed behavior. Our model also allows an interpretation of the effects of pharmacological agents altering the polymerization of actin. In particular we describe the influence of Cytochalasin D on the nucleation of the fingering instability.

Shear-banding in surfactant wormlike micelles: elastic instabilities and wall slip

We report on the flow dynamics of a wormlike micellar system (CPCl/NaSal/brine) undergoing a shear-banding transition using a combination of global rheology, 1D ultrasonic velocimetry and 2D optical visualisation. The different measurements being performed in a single Taylor–Couette geometry, we find a strong correlation between the induced turbid band observed optically and the high shear rate band. This correspondence reveals that fluctuations observed in the 1D velocity profiles are related to elastic instabilities triggered in the high shear rate band: 3D coherent (laminar) flow and 3D turbulent flow successively develop as the applied shear rate is increased. The specific characteristics of the resulting complex dynamics are found to depend on subtle changes in the sample, due to temporary light exposure. The CPCl molecules exhibit a photochemistry mainly influenced by the photo-induced cleavage of the pyridine ring that yields an unstable aldehyde enamine, which further decays by thermally activated processes. The products of the reaction possibly build up a lubrication layer responsible for pathological flow dynamics. Overall, our results bridge the gap between previous independent optical and local velocity measurements and explain most of the observed fluctuations in terms of a sequence of elastic instabilities which turns out to be widespread among semidilute wormlike micellar systems.

Interface dynamics in shear-banding flow of giant micelles

We report on the non trivial dynamics of the interface between shear-bands following the initiation of flow in a semi-dilute wormlike micellar system investigated using a combination of mechanical and optical measurements under strain-controlled conditions. During build up of the banding structure, we observed the stages of formation and migration of the interface between bands and finally the destabilization of this interface along the vorticity axis. The mechanical signature of these processes has been identified in the time series of the shear stress. Interface instability occurs all along the stress plateau, the asymptotic wavelength of the patterns increasing with the control parameter, typically from a fraction of the gap width to about four times the gap width. Three main dynamics regimes are highlighted: a spatially stable oscillating mode approximately in the middle of the coexistence zone flanked by two regions where the dynamics appears more exotic with propagative and chaotic events respectively at low and high shear rates. The distribution of small particles seeded in the solution strongly suggests that the flow is three-dimensional. Finally, we demonstrate that the shear-banding scenario described in this paper is not specific to our system.