Characterization of a Biomimetic Mesophase Composed of Nonionic Surfactants and an Aqueous Solvent.

We have investigated the physical and biomimetic properties of a sponge (L3) phase composed of pentaethylene glycol monododecyl ether (C12E5), a nonionic surfactant, an aqueous solvent, and a cosurfactant. The following cosurfactants, commonly used for solubilizing membrane proteins, were incorporated: n-octyl-ß-d-glucopyranoside (ß-OG), n-dodecyl-ß-d-maltopyranoside (DDM), 4-cyclohexyl-1-butyl-ß-d-maltoside (CYMAL-4), and 5-cyclohexyl-1-pentyl-ß-d-maltoside (CYMAL-5). Partial phase diagrams of these systems were created. The L3 phase was characterized using crossed polarizers, diffusion of a fluorescent probe by fluorescence recovery after pattern photobleaching (FRAPP), and freeze fracture electron microscopy (FFEM). By varying the hydration of the phase, we were able to tune the distance between adjacent bilayers. The characteristic distance (db) of the phase was obtained from small angle scattering (SAXS/SANS) as well as from FFEM, which yielded complementary db values. These db values were neither affected by the nature of the cosurfactant nor by the addition of membrane proteins. These findings illustrate that a biomimetic surfactant sponge phase can be created in the presence of several common membrane protein-solubilizing detergents, thus making it a versatile medium for membrane protein studies.

Magnetic engineering of stable rod-shaped stem cell aggregates: circumventing the pitfall of self-bending.

A current challenge for tissue engineering while restoring the function of diseased or damaged tissue is to customize the tissue according to the target area. Scaffold-free approaches usually yield spheroid shapes with the risk of necrosis at the center due to poor nutrient and oxygen diffusion. Here, we used magnetic forces developed at the cellular scale by miniaturized magnets to create rod-shaped aggregates of stem cells that subsequently matured into a tissue-like structure. However, during the maturation process, the tissue-rods spontaneously bent and coiled into sphere-like structures, triggered by the increasing cell-cell adhesion within the initially non-homogeneous tissue. Optimisation of the intra-tissular magnetic forces successfully hindered the transition, in order to produce stable rod-shaped stem cells aggregates.

Interplay of RhoA and mechanical forces in collective cell migration driven by leader cells.

The leading front of a collectively migrating epithelium often destabilizes into multicellular migration fingers where a cell initially similar to the others becomes a leader cell while its neighbours do not alter. The determinants of these leader cells include mechanical and biochemical cues, often under the control of small GTPases. However, an accurate dynamic cartography of both mechanical and biochemical activities remains to be established. Here, by mapping the mechanical traction forces exerted on the surface by MDCK migration fingers, we show that these structures are mechanical global entities with the leader cells exerting a large traction force. Moreover, the spatial distribution of RhoA differential activity at the basal plane strikingly mirrors this force cartography. We propose that RhoA controls the development of these fingers through mechanical cues: the leader cell drags the structure and the peripheral pluricellular acto-myosin cable prevents the initiation of new leader cells.

Orientation and polarity in collectively migrating cell structures: statics and dynamics.

Collective cell migration is often characterized by the spontaneous onset of multicellular protrusions (known as fingers) led by a single leader cell. Working with epithelial Madin-Darby canine kidney monolayers we show that cells within the fingers, as compared with the epithelium, are well oriented and polarized along the main finger direction, which suggests that these cells actively migrate. The cell orientation and polarity decrease continuously from the tip toward the epithelium over a penetration distance of typically two finger lengths. Furthermore, laser photoablation experiments at various locations along these fingers demonstrate that the cells in the fingers are submitted to a tensile stress whose value is larger close to the tip. From a dynamical point of view, cells entering a finger gradually polarize on timescales that depend upon their particular initial position. Selective laser nanosurgery of the leader lamellipodium shows not only that these structures need a leader to progress, but that this leader itself is the consequence of a prior self-organization of the cells forming the finger. These results highlight the complex interplay between the collective orientation within the fingers and the mechanical action of the leader.

Velocity fields in a collectively migrating epithelium.

We report quantitative measurements of the velocity field of collectively migrating cells in a motile epithelium. The migration is triggered by presenting free surface to an initially confluent monolayer by using a microstencil technique that does not damage the cells. To avoid the technical difficulties inherent in the tracking of single cells, the field is mapped using the technique of particle image velocimetry. The main relevant parameters, such as the velocity module, the order parameter, and the velocity correlation function, are then extracted from this cartography. These quantities are dynamically measured on two types of cells (collectively migrating Madin-Darby canine kidney (MDCK) cells and fibroblastlike normal rat kidney (NRK) cells), first as they approach confluence, and then when the geometrical constraints are released. In particular, for MDCK cells filling up the patterns, we observe a sharp decrease in the average velocity after the point of confluence, whereas the densification of the monolayer is much more regular. After the peeling off of the stencil, a velocity correlation length of approximately 200 microm is measured for MDCK cells versus only approximately 40 microm for the more independent NRK cells. Our conclusions are supported by parallel single-cell tracking experiments. By using the biorthogonal decomposition of the velocity field, we conclude that the velocity field of MDCK cells is very coherent in contrast with the NRK cells. The displacements in the fingers arising from the border of MDCK epithelia are very oriented along their main direction. They influence the velocity field in the epithelium over a distance of approximately 200 microm.

Lateral mobility of proteins in liquid membranes revisited.

The biological function of transmembrane proteins is closely related to their insertion, which has most often been studied through their lateral mobility. For >30 years, it has been thought that hardly any information on the size of the diffusing object can be extracted from such experiments. Indeed, the hydrodynamic model developed by Saffman and Delbrück predicts a weak, logarithmic dependence of the diffusion coefficient D with the radius R of the protein. Despite widespread use, its validity has never been thoroughly investigated. To check this model, we measured the diffusion coefficients of various peptides and transmembrane proteins, incorporated into giant unilamellar vesicles of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) or in model bilayers of tunable thickness. We show in this work that, for several integral proteins spanning a large range of sizes, the diffusion coefficient is strongly linked to the protein dimensions. A heuristic model results in a Stokes-like expression for D, (D proportional, variant 1/R), which fits literature data as well as ours. Diffusion measurement is then a fast and fruitful method; it allows determining the oligomerization degree of proteins or studying lipid-protein and protein-protein interactions within bilayers.