Ezrin, radixin, and moesin are dispensable for macrophage migration and cellular cortex mechanics.

The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity.

Mechanical stiffness of reconstituted actin patches correlates tightly with endocytosis efficiency.

Clathrin-mediated endocytosis involves the sequential assembly of more than 60 proteins at the plasma membrane. An important fraction of these proteins regulates the assembly of an actin-related protein 2/3 (Arp2/3)-branched actin network, which is essential to generate the force during membrane invagination. We performed, on wild-type (WT) yeast and mutant strains lacking putative actin crosslinkers, a side-by-side comparison of in vivo endocytic phenotypes and in vitro rigidity measurements of reconstituted actin patches. We found a clear correlation between softer actin networks and a decreased efficiency of endocytosis. Our observations support a chain-of-consequences model in which loss of actin crosslinking softens Arp2/3-branched actin networks, directly limiting the transmission of the force. Additionally, the lifetime of failed endocytic patches increases, leading to a larger number of patches and a reduced pool of polymerizable actin, which slows down actin assembly and further impairs endocytosis.

Correction: Non-linear elastic properties of actin patches to partially rescue yeast endocytosis efficiency in the absence of the cross-linker Sac6.

Correction for 'Non-linear elastic properties of actin patches to partially rescue yeast endocytosis efficiency in the absence of the cross-linker Sac6' by Belbahri Reda et al., Soft Matter, 2022, 18, 1479-1488, https://doi.org/10.1039/D1SM01437D.

Impact of branching on the elasticity of actin networks.

Actin filaments play a fundamental role in cell mechanics: assembled into networks by a large number of partners, they ensure cell integrity, deformability, and migration. Here we focus on the mechanics of the dense branched network found at the leading edge of a crawling cell. We develop a new technique based on the dipolar attraction between magnetic colloids to measure mechanical properties of branched actin gels assembled around the colloids. This technique allows us to probe a large number of gels and, through the study of different networks, to access fundamental relationships between their microscopic structure and their mechanical properties. We show that the architecture does regulate the elasticity of the network: increasing both capping and branching concentrations strongly stiffens the networks. These effects occur at protein concentrations that can be regulated by the cell. In addition, the dependence of the elastic modulus on the filaments' flexibility and on increasing internal stress has been studied. Our overall results point toward an elastic regime dominated by enthalpic rather than entropic deformations. This result strongly differs from the elasticity of diluted cross-linked actin networks and can be explained by the dense dendritic structure of lamellipodium-like networks.

The third dimension of the actin cortex.

The actin cortex, commonly described as a thin 2-dimensional layer of actin filaments beneath the plasma membrane, is beginning to be recognized as part of a more dynamic and three-dimensional composite material. In this review, we focus on the elements that contribute to the three-dimensional architecture of the actin cortex. We also argue that actin-rich structures such as filopodia and stress fibers can be viewed as specialized integral parts of the 3D actin cortex. This broadens our definition of the cortex, shifting from its simplified characterization as a thin, two-dimensional layer of actin filaments.

A Magnetic Pincher for the Dynamic Measurement of the Actin Cortex Thickness in Live Cells.

The actin cortex is an essential element of the cytoskeleton allowing cells to control and modify their shape. It is involved in cell division and migration. However, probing precisely the physical properties of the actin cortex has proved to be challenging: it is a thin and dynamic material, and its location in the cell-directly under the plasma membrane-makes it difficult to study with standard light microscopy and cell mechanics techniques. In this chapter, we present a novel protocol to probe dynamically the thickness of the cortex and its fluctuations using superparamagnetic microbeads in a uniform magnetic field. A bead ingested by the cell and another outside the cell attract each other due to dipolar forces. By tracking their position with nanometer precision, one can measure the thickness of the cortex pinched between two beads and monitor its evolution in time. We first present the set of elements necessary to realize this protocol: a magnetic field generator adapted to a specific imaging setup and the aforementioned superparamagnetic microbeads. Then we detail the different steps of a protocol that can be used on diverse cell types, adherent or not.

Studying membrane fusion using supported lipid bilayers on superparamagnetic beads.

The fusion between two lipid membranes is a ubiquitous mechanism in cell traffic and pathogens invasion. Yet it is not well understood how two distinct bilayers overcome the energy barriers towards fusion and reorganize themselves to form a unique continuous bilayer. The magnitudes and numbers of these energy barriers are themselves an open question. To tackle these issues, we developed a new tool that allows to control the forces applied between two supported lipid bilayers (SLBs) deposited on superparamagnetic beads. By applying a magnetic field, the beads self-organize along field lines in chains of beads and compress the two membranes on the contact zone. Using the diffusion of fluorescently labelled lipids from one bilayer to the other allows us to identify fusion of the bilayers in contact. We applied increasing forces on SLBs and increased the occurrence of fusion. This experimental system allows the simultaneous study of tens of facing bilayers in a single experiment and mitigates the stochasticity of the fusion process. It is thus a powerful tool to test the various parameters involved in the membrane fusion process.

Photo-dynamic induction of oxidative stress within cholesterol-containing membranes: shape transitions and permeabilization.

Photochemical internalization is a drug delivery technology employing a photo-destabilization of the endosomes and the photo-controlled release of endocyted macromolecules into the cytosol. This effect is based on the ability of some photosensitizers to interact with endosomal membranes and to photo-induce damages leading to its breakdown. The permeabilization efficiency is not quantitatively related to the importance of the damages, but to their asymmetric repartition within the leaflets. Using unilamellar vesicles and a chlorin, we studied the effect of the membrane's cholesterol content on its photo-permeabilization. First, the affinity of the chlorin for membranes was studied. Then, we asymmetrically oxidized the membranes. For DOPC/CHOL GUVs, we observed different shape transitions, in accordance with an increase followed by a decrease of the membrane effective curvature. These modifications are delayed by the cholesterol. Finally, the photo-permeabilization of GUVs occurs, corresponding to a pore formation due to the membrane tension, resulting from vesicles buddings. Cholesterol-rich GUVs permeabilization occurs after a lag, and is less important. These results are interpreted regarding both (i) the cholesterol-induced tightening of the lipids, its consequences on physical parameters of the membrane and on oxidation rate and (ii) the suggested ability of cholesterol to flip rapidly and then to relax the differential density-based stress accumulated during membrane bending.

Programmable Design and Performance of Modular Magnetic Microswimmers.

Synthetic biomimetic microswimmers are promising agents for in vivo healthcare and important frameworks to advance the understanding of locomotion strategies and collective motion at the microscopic scale. Nevertheless, constructing these devices with design flexibility and in large numbers remains a challenge. Here, a step toward meeting this challenge is taken by assembling such swimmers via the programmed shape and arrangement of superparamagnetic micromodules. The method's capacity for design flexibility is demonstrated through the assembly of a variety of swimmer architectures. On their actuation, strokes characterized by a balance of viscous and magnetic forces are found in all cases, but swimmers formed from a series of size-graded triangular modules swim quicker than more traditional designs comprising a circular "head" and a slender tail. Linking performance to design, rules are extracted informing the construction of a second-generation swimmer with a short tail and an elongated head optimized for speed. Its fast locomotion is attributed to a stroke that better breaks beating symmetry and an ability to beat fully with flex at high frequencies. Finally, production at scale is demonstrated through the assembly and swimming of a flock of the triangle-based architectures to reveal four types of swimmer couplings.

Pinching the cortex of live cells reveals thickness instabilities caused by myosin II motors.

The cell cortex is a contractile actin meshwork, which determines cell shape and is essential for cell mechanics, migration, and division. Because its thickness is below optical resolution, there is a tendency to consider the cortex as a thin uniform two-dimensional layer. Using two mutually attracted magnetic beads, one inside the cell and the other in the extracellular medium, we pinch the cortex of dendritic cells and provide an accurate and time-resolved measure of its thickness. Our observations draw a new picture of the cell cortex as a highly dynamic layer, harboring large fluctuations in its third dimension because of actomyosin contractility. We propose that the cortex dynamics might be responsible for the fast shape-changing capacity of highly contractile cells that use amoeboid-like migration.

Asymmetric oxidation of giant vesicles triggers curvature-associated shape transition and permeabilization.

Oxidation of unsaturated lipids is a fundamental process involved in cell bioenergetics as well as in cell death. Using giant unilamellar vesicles and a chlorin photosensitizer, we asymmetrically oxidized the outer or inner monolayers of lipid membranes. We observed different shape transitions such as oblate to prolate and budding, which are typical of membrane curvature modifications. The asymmetry of the shape transitions is in accordance with a lowered effective spontaneous curvature of the leaflet being targeted. We interpret this effect as a decrease in the preferred area of the targeted leaflet compared to the other, due to the secondary products of oxidation (cleaved-lipids). Permeabilization of giant vesicles by light-induced oxidation is observed after a lag and is characterized in relation with the photosensitizer concentration. We interpret permeabilization as the opening of a pore above a critical membrane tension, resulting from the budding of vesicles. The evolution of photosensitized giant vesicle lysis tension was measured and yields an estimation of the effective spontaneous curvature at lysis. Additionally photo-oxidation was shown to be fusogenic.

Reconstitution of an actin cortex inside a liposome.

The composite and versatile structure of the cytoskeleton confers complex mechanical properties on cells. Actin filaments sustain the cell membrane and their dynamics insure cell shape changes. For example, the lamellipodium moves by actin polymerization, a mechanism that has been studied using simplified experimental systems. Much less is known about the actin cortex, a shell-like structure underneath the membrane that contracts for cell movement. We have designed an experimental system that mimicks the cell cortex by allowing actin polymerization to nucleate and assemble at the inner membrane of a liposome. Actin shell growth can be triggered inside the liposome, which offers a useful system for a controlled study. The observed actin shell thickness and estimated mesh size of the actin structure are in good agreement with cellular data. Such a system paves the way for a thorough characterization of cortical dynamics and mechanics.

Assembly Modulated by Particle Position and Shape: A New Concept in Self-Assembly.

In this communication we outline how the bespoke arrangements and design of micron-sized superparamagnetic shapes provide levers to modulate their assembly under homogeneous magnetic fields. We label this new approach, 'assembly modulated by particle position and shape' (APPS). Specifically, using rectangular lattices of superparamagnetic micron-sized cuboids, we construct distinct microstructures by adjusting lattice pitch and angle of array with respect to a magnetic field. Broadly, we find two modes of assembly: (1) immediate 2D jamming of the cuboids as they rotate to align with the applied field (rotation-induced jamming) and (2) aggregation via translation after their full alignment (dipole-dipole assembly). The boundary between these two assembly pathways is independent on field strength being solely a function of the cuboid's dimensions, lattice pitch, and array angle with respect to field-a relationship which we capture, along with other features of the assembly process, in a 'phase diagram'. In doing so, we set out initial design rules to build custom made assemblies. Moreover, these assemblies can be made flexible thanks to the hinged contacts of their particle building blocks. This flexibility, combined with the superparamagnetic nature of the architectures, renders our assembly method particularly appropriate for the construction of complex actuators at a scale hitherto not possible.

A new method to measure mechanics and dynamic assembly of branched actin networks.

We measured mechanical properties and dynamic assembly of actin networks with a new method based on magnetic microscopic cylinders. Dense actin networks are grown from the cylinders' surfaces using the biochemical Arp2/3-machinery at play in the lamellipodium extension and other force-generating processes in the cell. Under a homogenous magnetic field the magnetic cylinders self-assemble into chains in which forces are attractive and depend on the intensity of the magnetic field. We show that these forces, from piconewtons to nanonewtons, are large enough to slow down the assembly of dense actin networks and controlled enough to access to their non linear mechanical responses. Deformations are measured with nanometer-resolution, well below the optical resolution. Self-assembly of the magnetic particles into chains simplifies experiments and allows for parallel measurements. The combination of accuracy and good throughput of measurements results in a method with high potential for cell and cytoskeleton mechanics. Using this method, we observed in particular a strong non linear mechanical behavior of dense branched actin networks at low forces that has not been reported previously.

ARF1-mediated actin polymerization produces movement of artificial vesicles.

Vesicular trafficking and actin dynamics on Golgi membranes are both regulated by ADP-ribosylation factor 1 (ARF1) through the recruitment of various effectors, including vesicular coats. Actin assembly on Golgi membranes contributes to the architecture of the Golgi complex, vesicle formation, and trafficking and is mediated by ARF1 through a cascade that leads to Arp2/3 complex activation. Here we addressed the role of Golgi actin downstream of ARF1 by using a biomimetic assay consisting of liposomes of defined lipid composition, carrying an activated form of ARF1 incubated in cytosolic cell extracts. We observed actin polymerization around the liposomes resulting in thick actin shells and actin comet tails that pushed the ARF1 liposomes forward. The assay was used to characterize the ARF1-dependent pathway, leading to actin polymerization, and confirmed a dependency on CDC42 and its downstream effector N-WASP. Overall, this study demonstrates that actin polymerization driven by the complex multicomponent signaling cascade of the Golgi apparatus can be reproduced with a biomimetic system. Moreover, our results are consistent with the view that actin-based force generation at the site of vesicle formation contributes to the mechanism of fission. In addition to its well established function in coat recruitment, the ARF1 machinery also might produce movement- and fission-promoting forces through actin polymerization.