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

Asymmetric Oxidation of Giant Vesicles triggers Curvature-associated Shape Transition and Permeabilization. Running title: Oxidation of Vesicles produces Curvature

International audience(200 words) 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

Hémifusion de vésicules géantes (caractérisation d'un état intermédiaire vers la fusion, en présence de forces spécifiques)

Nous avons étudié l'interaction entre deux vésicules géantes pour modéliser la fusion membranaire, un processus biologique impliqué entre autres dans l'infection virale et la communication intra- et extracellulaire. La fusion dans la cellule est déclenchée par l'action de protéines fusogènes qui, dans une des étapes, rapprochent deux membranes opposées jusqu'à une faible distance intermembranaire. Dans notre système, le rapprochement des membranes est induit par l'attraction spécifique entre lipides portant des nucléotides adénine et thymine sur leur tête. Nous avons estimé numériquement la distance d'équilibre entre deux bicouches, elle décroît de 2,6 nm sans forces spécifiques à 1,4 nm en présence de forces spécifiques. Deux vésicules sont micromanipulées afin d'être placées en contact. Un mélange partiel des lipides et une indépendance des milieux internes sont observés en microscopie de fluorescence. Le mélange partiel des lipides est quantifié; il correspond à un mélange complet des monocouches externes avec une indépendance des monocouches internes. Ces observations caractérisent un état intermédiaire vers la fusion: l'hémifusion. La cinétique de redistribution des lipides entre les deux vésicules est mesurée; elle est indépendante de la proportion de lipides fonctionnalisés, mais s'accorde bien dans la plupart des cas à un modèle de diffusion sur une "cacahouète ". Cet accord est compatible avec la présence d'au moins quelques pédoncules à l'interface entre les vésicules. Des événements de fusion complète sont parfois observés, ce qui nous ouvre des perspectives pour obtenir une fusion complète de manière reproductible.We studied the interaction between two giant vesicles to model membrane fusion, a biological process involved in viral infection and in intra- and extra-cellular communication. Fusion in the cell is induced by the action of fusogenic proteins which bring closer two opposing membranes to a short intermembrane distance. In our system, the bringing together of the membranes is induced by a specific attraction between functionalized lipids carrying adenine or thymine nucleotides on their head. We numerically estimated the equilibrium distance between two membranes, which decreases from 2.6 nm without specific forces to 1.4 nm when specific forces are present. Two vesicles are micromanipulated in order to place them into contact. A partial mixing of the lipids and an independence of the inner mediums are observed by fluorescence microscopy. The partial mixing of the lipids is quantified; it corresponds to a complete mixing of the external monolayers with an independence of the internal monolayers. These observations are characteristic of an intermediate state towards fusion: hemifusion. The kinetics of redistribution of the lipids between the two vesicles is measured; it is independent of the proportion of functionalized lipids, but agrees well in most cases with a model of diffusion on a "peanut". This agreement is consistent with the presence of at least a few stalks at the interface between the vesicles. Some full-fusion events are sometimes observed, which open up new prospects for obtaining reproducible full-fusion in our systemORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

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

International audienceClathrin mediated endocytosis is an essential and complex cellular process involving more than 60 proteins. In yeast, successful endocytosis requires counteracting a large turgor pressure. To this end, yeasts assemble actin patches, which accumulate elastic energy during their assembly. We investigated the material properties of reconstituted actin patches from a wild-type (WT) strain and a mutant strain lacking the cross-linker Sac6 (sac6), which has reduced endocytosis efficiency in live cells. We hypothesized that a change in the viscous properties of the actin patches, which would dissipate more mechanical energy, could explain this reduced efficiency. There was however no significant difference in the viscosity of both types of patches. However, we discovered a significantly different non-linear elastic response. While WT patches had a constant elastic modulus at different stresses, sac6 patches had a lower elastic modulus at low stresses, before stiffening at higher ones, up to values similar to WT patches. To understand the consequences of this discovery, we performed, in-vivo, a precise analysis of actin patch dynamics. Our analysis reveals that a small fraction of actin patches successfully complete endocytosis in sac6 cells, provided that those assemble an excess of actin at the membrane compared to WT. This observation indicates that non-linear elastic properties of actin networks in sac6 cells contribute to rescue endocytosis, requiring nevertheless more actin material to build-up the necessary stored elastic energy

Role of cross-linkers in yeast branched actin networks: Linking biochemistry and mechanics

Role of cross-linkers in yeast branched actin networks: Linking biochemistry and mechanics. 60. Annual Meeting of the Biophysical-Societ

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

La rigidité de patchs reconstituées de filaments d'actine est fortement corrélée à l'efficacité de l'endocytose

International audienceClathrin-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

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

International audienceThe 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 timeresolved 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