Reentrant wetting transition in the spreading of cellular aggregates.

We study spreading on soft substrates of cellular aggregates using CT26 cells that produce an extracellular matrix (ECM). Compared to our previous work on the spreading of S180 cellular aggregates, which did not secrete ECMs, we found that the spreading velocity of the precursor film is also maximal for intermediate rigidities, but new striking features show up. First, we observed a cascade of liquid-gas-liquid (L/G/L) transitions of the precursor film as the substrate rigidity is decreased. We attribute the L/G transition to a decrease of cell/cell adhesion resulting from the weakening of the cell/substrate adhesion. We attribute the reentrant liquid phase (G/L) observed on soft substrates to the slow spreading of the aggregates on ultra-soft substrates, which gives time to the cells to secrete more ECM proteins and stick together. Second, a nematic order appears in the cohesive (liquid) states of the precursor film, attributed to the gradient of cell's velocities.

Interplay of packing and flip-flop in local bilayer deformation. How phosphatidylglycerol could rescue mitochondrial function in a cardiolipin-deficient yeast mutant.

In a previous work, we have shown that a spatially localized transmembrane pH gradient, produced by acid micro-injection near the external side of cardiolipin-containing giant unilamellar vesicles, leads to the formation of tubules that retract after the dissipation of this gradient. These tubules have morphologies similar to mitochondrial cristae. The tubulation effect is attributable to direct phospholipid packing modification in the outer leaflet, that is promoted by protonation of cardiolipin headgroups. In this study, we compare the case of cardiolipin-containing giant unilamellar vesicles with that of giant unilamellar vesicles that contain phosphatidylglycerol (PG). Local acidification also promotes formation of tubules in the latter. However, compared with cardiolipin-containing giant unilamellar vesicles the tubules are longer, exhibit a visible pearling, and have a much longer lifetime after acid micro-injection is stopped. We attribute these differences to an additional mechanism that increases monolayer surface imbalance, namely inward PG flip-flop promoted by the local transmembrane pH gradient. Simulations using a fully nonlinear membrane model as well as geometrical calculations are in agreement with this hypothesis. Interestingly, among yeast mutants deficient in cardiolipin biosynthesis, only the crd1-null mutant, which accumulates phosphatidylglycerol, displays significant mitochondrial activity. Our work provides a possible explanation of such a property and further emphasizes the salient role of specific lipids in mitochondrial function.

How cells flow in the spreading of cellular aggregates.

Like liquid droplets, cellular aggregates, also called "living droplets," spread onto adhesive surfaces. When deposited onto fibronectin-coated glass or polyacrylamide gels, they adhere and spread by protruding a cellular monolayer (precursor film) that expands around the droplet. The dynamics of spreading results from a balance between the pulling forces exerted by the highly motile cells at the periphery of the film, and friction forces associated with two types of cellular flows: (i) permeation, corresponding to the entry of the cells from the aggregates into the film; and (ii) slippage as the film expands. We characterize these flow fields within a spreading aggregate by using fluorescent tracking of individual cells and particle imaging velocimetry of cell populations. We find that permeation is limited to a narrow ring of width ξ (approximately a few cells) at the edge of the aggregate and regulates the dynamics of spreading. Furthermore, we find that the subsequent spreading of the monolayer depends heavily on the substrate rigidity. On rigid substrates, the migration of the cells in the monolayer is similar to the flow of a viscous liquid. By contrast, as the substrate gets softer, the film under tension becomes unstable with nucleation and growth of holes, flows are irregular, and cohesion decreases. Our results demonstrate that the mechanical properties of the environment influence the balance of forces that modulate collective cell migration, and therefore have important implications for the spreading behavior of tissues in both early development and cancer.

Binding of moesin and ezrin to membranes containing phosphatidylinositol (4,5) bisphosphate: a comparative study of the affinity constants and conformational changes.

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Moesin and ezrin, proteins from the ezrin/radixin/moesin (ERM) family, provide a direct linkage between the cytoskeleton and the membrane via their interaction with phosphatidylinositol 4,5-bisphosphate (PIP(2)). PIP(2) binding is considered as a prerequisite step in ERM activation. The main objective of this work was to compare moesin and ezrin interaction with PIP(2)-containing membranes in terms of affinity and to analyze secondary structure modifications leading eventually to ERM activation. For this purpose, we used two types of biomimetic model membranes, large and giant unilamellar vesicles. The dissociation constant between moesin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles was found to be very similar to that between ezrin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles. In addition, both proteins were found to undergo conformational changes after binding to PIP(2)-containing large unilamellar vesicles. Changes were evidenced by an increased sensitivity to proteolysis, modifications in the fluorescence intensity of the probe attached to the C-terminus and in the proportion of secondary structure elements.

Amyloid-ß and the failure to form mitochondrial cristae: a biomimetic study involving artificial membranes.

Alzheimer's disease (AD) is a degenerative disease of the central nervous system which causes irreversible damage to neuron structure and function. The main hypothesis concerning the cause of AD is excessive accumulation of amyloid-ß peptides (Aß). There has recently been a surge in studies on neuronal morphological and functional pathologies related to Aß-induced mitochondrial dysfunctions and morphological alternations. What is the relation between the accumulation of Aß in mitochondria, decreased production of ATP, and the large number of mitochondria with broken or scarce cristae observed in AD patients' neurons? The problem is complex, as it is now widely recognized that mitochondria function determines mitochondrial inner membrane (IM) morphology and, conversely, that IM morphology can influence mitochondrial functions. In our previous work, we designed an artificial mitochondrial IM, a minimal model system (giant unilamellar vesicle) mimicking the IM. We showed experimentally that modulation of the local pH gradient at the membrane level of cardiolipin-containing vesicles induces dynamic membrane invaginations similar to the mitochondrial cristae. In the present work we show, using our artificial IM, that Aß renders the membrane unable to support the formation of cristae-like structures when local pH gradient occurs, leading to the failure of this cristae-like morphology. Fluorescent probe studies suggest that the dramatic change of membrane mechanical properties is due to Aß-induced lipid bilayer dehydration, increased ordering of lipids, loss of membrane fluidity, and possibly to Aß-induced changes in dynamic friction between the two leaflets of the lipid membrane.

Lipid packing variations induced by pH in cardiolipin-containing bilayers: the driving force for the cristae-like shape instability.

Cardiolipin is a four-tailed acidic lipid found predominantly within the inner membrane of mitochondria, and is thought to be a key component in determining inner membrane properties and potential. Thus, cardiolipin may be involved in the dynamics of the inner membrane characteristic invaginations (named cristae) that protrude into the matrix space. In previous studies, we showed the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid model membrane mimicking the inner mitochondrial membrane. In addition, we reported a theoretical model describing the dynamics of a chemically driven membrane shape instability caused by a modification of the plane-shape equilibrium density of the lipids in the membrane. In the present work, we focus on the lipid-packing modifications observed in a model cardiolipin-containing lipid membrane submitted to pH decrease because this is the driving force of the instability. Laurdan fluorescence and ζ-potential measurements show that under pH decrease, membrane surface charge decreases, but that significant modification of the lipid packing is observed only for CL-containing membranes. Our giant unilamellar vesicle experiments also indicate that cristae-like morphologies are only observed for CL-containing lipid membranes. Taken together, these results highlight the fact that only a strong modulation of the lipid packing of the exposed monolayer leads to membrane shape instability and suggest that mitochondrial lipids, in particular the cardiolipin, play a specific role under pH modulation in inner mitochondrial membrane morphology and dynamics.

Phosphatidylinositol 4,5-bisphosphate-induced conformational change of ezrin and formation of ezrin oligomers.

The plasma membrane-cytoskeleton interface is a dynamic structure involved in a variety of cellular events. Ezrin, a protein from the ERM family, provides a direct linkage between the cytoskeleton and the membrane via its interaction with phosphatidylinositol 4,5-bisphosphate (PIP2). In this paper, we investigate the interaction between PIP2 and ezrin in vitro using PIP2 dispersed in a unimolecular way in buffer. We compared the results obtained with full-length ezrin to those obtained with an ezrin mutant, which was previously found not to be localized at the cell membrane, and with the N-terminal membrane binding domain (FERM domain) of ezrin. We show that PIP2 induced a conformational change in full-length ezrin. PIP2 was also found to induce, in vitro, the formation of oligomers of wild-type ezrin, but not of mutant ezrin. These oligomers had previously been observed in vivo, but their role is yet to be clarified. Our finding hints at a possible role for PIP2 in the formation of ezrin oligomers.

Membrane deformation under local pH gradient: mimicking mitochondrial cristae dynamics.

Mitochondria are cell substructures (organelles) critical for cell life, because biological fuel production, the ATP synthesis by oxidative phosphorylation, occurs in them driven by acidity (pH) gradients. Mitochondria play a key role as well in the cell death and in various fatigue and exercise intolerance syndromes. It is clear now that mitochondria present an astonishing variety of inner membrane morphologies, dynamically correlated with their functional state, coupled with the rate of the ATP synthesis, and characteristic for normal as well as for pathological cases. Our work offers some original insights into the factors that determine the dynamical tubular structures of the inner membrane cristae. We show the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid membrane. We designed a minimal membrane system (GUV) and experimentally showed that the directional modulation of local pH gradient at membrane level of cardiolipin-containing vesicles induces dynamic cristae-like membrane invaginations. We propose a mechanism and theoretical model to explain the observed tubular membrane morphology and suggest the underlying role of cardiolipin. Our results support the hypothesis of localized bioenergetic transduction and contribute to showing the inherent capacity of cristae morphology to become self-maintaining and to optimize the ATP synthesis.