Controlling Confinement and Topology to Study Collective Cell Behaviors.

Confinement and substrate topology strongly affect the behavior of cell populations and, in particular, their collective migration. In vitro experiments dealing with these aspects require strategies of surface patterning that remain effective over long times (typically several days) and ways to control the surface topology in three dimensions. Here, we describe protocols addressing these two aspects. High-resolution patterning of a robust cell-repellent coating is achieved by etching the coating through a photoresist mask patterned directly on the coated surface. Out-of-plane curvature can be controlled using glass wires or corrugated "wavy" surfaces.

Traveling Pulses for a Two-Species Chemotaxis Model.

Mathematical models have been widely used to describe the collective movement of bacteria by chemotaxis. In particular, bacterial concentration waves traveling in a narrow channel have been experimentally observed and can be precisely described thanks to a mathematical model at the macroscopic scale. Such model was derived in [1] using a kinetic model based on an accurate description of the mesoscopic run-and-tumble process. We extend this approach to study the behavior of the interaction between two populations of E. Coli. Separately, each population travels with its own speed in the channel. When put together, a synchronization of the speed of the traveling pulses can be observed. We show that this synchronization depends on the fraction of the fast population. Our approach is based on mathematical analysis of a macroscopic model of partial differential equations. Numerical simulations in comparison with experimental observations show qualitative agreement.

A multiscale study of bacterial proliferation modes within novel E. coli@Si(HIPE) hybrid macrocellular living foams.

For the first time the study at various length scales of E. coli proliferation modes within Si(HIPE) inorganic macrocellular foams is proposed. Both qualitatively and semi-quantitatively, bacterial proliferation within the foam is not homogeneous and is directly linked to the random distribution of Si(HIPE) macroscopic cells. When inoculated in a nutrient loaded Si(HIPE), the bacterial growth is enhanced within the Si(HIPE) matrices compared to the surrounding LB media. The bacterial growth kinetics tends to be faster and the concentration at saturation is roughly 100% times higher. In the case of a Si(HIPE) host free of nutrients, bacterial motion occurs as an infiltration wave; the peak of this propagation wave moves at a constant speed of 88 µm h-1, while bacterial concentrations within the Si(HIPE) host reach levels far above the ones reached in the presence of nutrients, suggesting a real synergetic relationship between the bacterial colony guests and the Si(HIPE) host. When a nutrient reservoir is present at the opposite position from which bacteria were inoculated, bacterial proliferation is associated with a coalescence process between the growing colonies that were rapidly established within the first hours. When the Si(HIPE) host was fully colonized we found out a specific distance between adjacent colonies of 5 and 15 µm in good correspondence with the repartition of the wall to wall distances of the Si(HIPE) macroscopic cells, meaning that bacterial repartition once colonization occurs is optimum. These results show that Si(HIPE) foams are outstanding candidates for strengthened bacterial proliferation without motion restriction imposed by conventional silica gels.

Biochemical perturbations of the mitotic spindle in Xenopus extracts using a diffusion-based microfluidic assay.

A microfluidic device is a powerful tool to manipulate in a controlled manner at spatiotemporal scales for biological systems. Here, we describe a simple diffusion-based assay to generate and measure the effect of biochemical perturbations within the cytoplasm of cell-free extracts from Xenopus eggs. Our approach comprises a microliter reservoir and a model cytoplasm that are separated by a synthetic membrane containing sub-micrometric pores through which small molecules and recombinant proteins can diffuse. We have used this system to examine the perturbation of elements of the mitotic spindle, which is a microtubule-based bipolar structure involved in the segregation of the replicated genome to daughter cells during cell division. First, we used the small molecule inhibitor monastrol to target kinesin-5, a molecular motor that maintains the microtubule spindle bipolarity. Next, we explored the dynamics of the mitotic spindle by monitoring the exchange between unpolymerized and polymerized tubulin within microtubule fibers. These results show that a simple diffusion-based system can generate biochemical perturbations directly within a cell-free cytoplasm based on Xenopus egg extracts at the time scale of minutes. Our assay is therefore suitable for monitoring the dynamics of supramolecular assemblies within cell-free extracts in response to perturbations. This strategy opens up broad perspectives including phenotype screening or mechanistic studies of biological assembly processes and could be applied to other cell-free extracts such as those derived from mammalian or bacterial cells.

Stimuli-responsive topological change of microstructured surfaces and the resultant variations of wetting properties.

It is now well established that topological microstructures play a key role in the physical properties of surfaces. Stimulus-induced variations of topological microstructure should therefore lead to a change in the physical properties of microstructured responsive surfaces. In this paper, we demonstrate that roughness changes alter the wetting properties of responsive organic surfaces. Oriented nematic liquid crystalline elastomers (LCEs) are used to construct the microstructured surfaces via a replica molding technique. The topological microstructure of the surfaces covered with micropillars changes with temperature, due to the reversible contraction of the LCE pillars along the long axis at the nematic-to-isotropic phase transition. This is directly observed for the first time under environmental scanning electron microscopy (E-SEM). A high boiling point liquid, glycerol, is used to continuously monitor the contact angle change with temperature. The glycerol contact angle of the microstructured surfaces covered with small pillars decreases from 118° at room temperature to 80° at 140 °C, corresponding to a transition from Cassie state to Wenzel state.

Automated velocity mapping of migrating cell populations (AVeMap).

Characterizing the migration of a population of cells remains laborious and somewhat subjective. Advances in genetics and robotics allow researchers to perform many experiments in parallel, but analyzing the large sets of data remains a bottleneck. Here we describe a rapid, fully automated correlation-based method for cell migration analysis, compatible with standard video microscopy. This method allows for the computation of quantitative migration parameters via an extensive dynamic mapping of cell displacements.

Modeling E. coli tumbles by rotational diffusion. Implications for chemotaxis.

The bacterium Escherichia coli in suspension in a liquid medium swims by a succession of runs and tumbles, effectively describing a random walk. The tumbles randomize incompletely, i.e. with a directional persistence, the orientation taken by the bacterium. Here, we model these tumbles by an active rotational diffusion process characterized by a diffusion coefficient and a diffusion time. In homogeneous media, this description accounts well for the experimental reorientations. In shallow gradients of nutrients, tumbles are still described by a unique rotational diffusion coefficient. Together with an increase in the run length, these tumbles significantly contribute to the net chemotactic drift via a modulation of their duration as a function of the direction of the preceding run. Finally, we discuss the limits of this model in propagating concentration waves characterized by steep gradients. In that case, the effective rotational diffusion coefficient itself varies with the direction of the preceding run. We propose that this effect is related to the number of flagella involved in the reorientation process.

Thermal separation: interplay between the Soret effect and entropic force gradient.

Thermophoresis, the Soret effect, depletes a high concentration of a polyethylene glycol polymer solution from the hot region and builds a concentration gradient. In such a solution, solutes of small concentration experience thermophoresis and polyethylene glycol concentration-dependent restoring forces. We report that by using focused laser heating and varying the polyethylene glycol concentration one observes geometrical localizations of solutes like DNA and RNA into patterns such as a ring. For DNA up to 5.6 kbp, the ring size decreases following a behavior analogous to a gel electrophoresis separation. Above 5.6 kbp, the ring diameter increases with the DNA length. Mixtures of DNA and RNA can be separated as well as different RNA lengths. Separation of colloids is also observed. The experiments might be relevant for the separation of small RNA ribozymes in an early stage of life.

Mathematical description of bacterial traveling pulses.

The Keller-Segel system has been widely proposed as a model for bacterial waves driven by chemotactic processes. Current experiments on Escherichia coli have shown the precise structure of traveling pulses. We present here an alternative mathematical description of traveling pulses at the macroscopic scale. This modeling task is complemented with numerical simulations in accordance with the experimental observations. Our model is derived from an accurate kinetic description of the mesoscopic run-and-tumble process performed by bacteria. This can account for recent experimental observations with E. coli. Qualitative agreements include the asymmetry of the pulse and transition in the collective behaviour (clustered motion versus dispersion). In addition, we can capture quantitatively the traveling speed of the pulse as well as its characteristic length. This work opens several experimental and theoretical perspectives since coefficients at the macroscopic level are derived from considerations at the cellular scale. For instance, the particular response of a single cell to chemical cues turns out to have a strong effect on collective motion. Furthermore, the bottom-up scaling allows us to perform preliminary mathematical analysis and write efficient numerical schemes. This model is intended as a predictive tool for the investigation of bacterial collective motion.

Strength dependence of cadherin-mediated adhesions.

Traction forces between adhesive cells play an important role in a number of collective cell processes. Intercellular contacts, in particular cadherin-based intercellular junctions, are the major means of transmitting force within tissues. We investigated the effect of cellular tension on the formation of cadherin-cadherin contacts by spreading cells on substrates with tunable stiffness coated with N-cadherin homophilic ligands. On the most rigid substrates, cells appear well-spread and present cadherin adhesions and cytoskeletal organization similar to those classically observed on cadherin-coated glass substrates. However, when cells are cultured on softer substrates, a change in morphology is observed: the cells are less spread, with a more disorganized actin network. A quantitative analysis of the cells adhering on the cadherin-coated surfaces shows that forces are correlated with the formation of cadherin adhesions. The stiffer the substrates, the larger are the average traction forces and the more developed are the cadherin adhesions. When cells are treated with blebbistatin to inhibit myosin II, the forces decrease and the cadherin adhesions disappear. Together, these findings are consistent with a mechanosensitive regulation of cadherin-mediated intercellular junctions through the cellular contractile machinery.

Effect of surface pattern on the adhesive friction of elastomers.

We present experimental results for the friction of a flat surface against a hexagonally patterned surface, both being made of PolyDiMethylSiloxane. We simultaneously measure forces of range 10 mN and observe the contact under sliding velocities of about 100 µm/s. We observe adhesive friction on three different pattern heights (80, 310, and 2100 nm). Two kinds of contacts have been observed: the flat surface is in close contact with the patterned one (called intimate contact, observed for 80 nm) or only suspended on the tops on the asperities (called laid contact, observed for 2100 nm). In the range of velocities used, the contact during friction is similar to the static one. Furthermore, our experimental system presents a contact transition during friction for h=310 nm.

Micron-sized main-chain liquid crystalline elastomer actuators with ultralarge amplitude contractions.

Responsive surfaces composed of cylindrical or square micrometer-sized thermoresponsive pillars made of thiol-ene nematic main-chain liquid crystalline elastomers (LCEs) are produced by replica molding. The individual pillars behave as microactuators, showing ultralarge and reversible contractions of around 300-400% at the nematic to isotropic phase transition. The nematic main-chain LCE microactuators described here present contractions as large as the best macroscopic systems reported in the literature. Moreover, the contraction observed for this new system outperforms the best values already reported for other LCE microsystems.

Efficiency of a self-aminoacylating ribozyme: effect of the length and base-composition of its 3' extension.

Variants of a previously described small self-aminoacylating ribozyme are tested in order to uncover the potentialities of a 3' extension responsible for the esterification. The base-composition and the length of this specific part of the ribozyme are investigated. Very short extensions can still reach the active site, reflecting the small persistence length of RNA. The yield of aminoacylation is particularly high for ribozymes with extensions made up of a poly-U, for which a maximum of efficiency is observed for a total length of about 10 nucleotides. A simple model describing the behavior of this region of the ribozyme can account for the data.

Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates.

The physical properties of the cellular environment are involved in regulating the formation and maintenance of tissues. In particular, substrate rigidity appears to be a key factor dictating cell response on culture surfaces. Here we study the behavior of epithelial cells cultured on microfabricated substrates engineered to exhibit an anisotropic stiffness. The substrate consists of a dense array of micropillars of oval cross-section, so that one direction is made stiffer than the other. We demonstrate how such an anisotropic rigidity can induce directional epithelial growth and guide cell migration along the direction of greatest rigidity. Regions of high tractional stress and large cellular deformations within the sheets of cells are concentrated at the edges, in particular at the two poles of the islands along their long axis, in correlation with the orientation of actin stress fibers and focal adhesions. By inducing scattering activity of epithelial cells, we show that isolated cells also migrate along the direction of greatest stiffness. Taken together, these findings show that the mechanical interactions of cells with their microenvironment can be tuned to engineer particular tissue properties.

Traction forces exerted through N-cadherin contacts.

BACKGROUND INFORMATION: Mechanical forces play an important role in the organization, growth and function of living tissues. The ability of cells to transduce mechanical signals is governed by two types of microscale structures: focal adhesions, which link cells to the extracellular matrix, and adherens junctions, which link adjacent cells through cadherins. Although many studies have examined forces induced by focal adhesions, there is little known about the role of adherens junctions in force-regulation processes. The present study focuses on the determination of force transduction through cadherins at a single cell level. RESULTS: We characterized for the first time the distribution of forces developed by the cell through cadherin contacts. A N-cadherin (neural cadherin)-Fc chimaera, which mimicks the cell adhesion molecule N-cadherin, was immobilized on a muFSA (micro-force sensor array), comprising a dense array of vertical elastomer pillars, which were used both as a cell culture support for N-cadherin-expressing C2 myogenic cells and as detectors for force mapping. We coated the top of the pillars on which cells adhere and recruit adhesion complexes and F-actin. Individual pillar bending allowed the measurement of forces that mainly developed at the cell edge and directed toward their centre. Similar force distribution and amplitude were detected with an unrelated cell line of neuronal origin. Further comparison with forces applied by cells on pillars coated with fibronectin indicates that mechanical stresses transduced through both types of adhesions were comparable in distribution, orientation and amplitude. CONCLUSIONS: These results present a versatile method to measure and map forces exerted by cell-cell adhesion complexes. They show that cells transduce mechanical stress through cadherin contacts which are of the same order as magnitude of those previously characterized for focal adhesions. Altogether, they emphasize the mechanotransduction role of cytoskeleton-linked adhesion receptors of the cadherin family in tissue cohesion and reshaping.

Nonmuscle myosin IIA-dependent force inhibits cell spreading and drives F-actin flow.

Nonmuscle myosin IIA (NMM-IIA) is involved in the formation of focal adhesions and neurite retraction. However, the role of NMM-IIA in these functions remains largely unknown. Using RNA interference as a tool to decrease NMM-IIA expression, we have found that NMM-IIA is the major myosin involved in traction force generation and retrograde F-actin flow in mouse embryonic fibroblast cells. Quantitative analyses revealed that approximately 60% of traction force on fibronectin-coated surfaces is contributed by NMM-IIA and approximately 30% by NMM-IIB. The retrograde F-actin flow decreased dramatically in NMM-IIA-depleted cells, but seemed unaffected by NMM-IIB deletion. In addition, we found that depletion of NMM-IIA caused cells to spread at a higher rate and to a greater area on fibronectin substrates during the early spreading period, whereas deletion of NMM-IIB appeared to have no effect on spreading. The distribution of NMM-IIA was concentrated on the dorsal surface and approached the ventral surface in the periphery, whereas NMM-IIB was primarily concentrated around the nucleus and to a lesser extent at the ventral surface in cell periphery. Our results suggest that NMM-IIA is involved in generating a coherent cytoplasmic contractile force from one side of the cell to the other through the cross-linking and the contraction of dorsal actin filaments.

Micro-actuators: when artificial muscles made of nematic liquid crystal elastomers meet soft lithography.

The production, via soft lithography, of micron-sized thermoresponsive pillars made of side-on nematic LCE leads to the formation of surface-responsive structures. The individual, monodisperse pillars, when cut out from the surface, behave as micro-actuators, showing contraction of around 35% at the nematic to isotropic phase transition.