The Reissner fiber under tension in vivo shows dynamic interaction with ciliated cells contacting the cerebrospinal fluid.

The Reissner fiber (RF) is an acellular thread positioned in the midline of the central canal that aggregates thanks to the beating of numerous cilia from ependymal radial glial cells (ERGs) generating flow in the central canal of the spinal cord. RF together with cerebrospinal fluid (CSF)-contacting neurons (CSF-cNs) form an axial sensory system detecting curvature. How RF, CSF-cNs and the multitude of motile cilia from ERGs interact in vivo appears critical for maintenance of RF and sensory functions of CSF-cNs to keep a straight body axis, but is not well-understood. Using in vivo imaging in larval zebrafish, we show that RF is under tension and resonates dorsoventrally. Focal RF ablations trigger retraction and relaxation of the fiber's cut ends, with larger retraction speeds for rostral ablations. We built a mechanical model that estimates RF stress diffusion coefficient D at 5 mm2/s and reveals that tension builds up rostrally along the fiber. After RF ablation, spontaneous CSF-cN activity decreased and ciliary motility changed, suggesting physical interactions between RF and cilia projecting into the central canal. We observed that motile cilia were caudally-tilted and frequently interacted with RF. We propose that the numerous ependymal motile monocilia contribute to RF's heterogenous tension via weak interactions. Our work demonstrates that under tension, the Reissner fiber dynamically interacts with motile cilia generating CSF flow and spinal sensory neurons.

Compressibility and porosity modulate the mechanical properties of giant gas vesicles.

Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here the formation of perfluoro-n-butane microbubbles coated with surface-active proteins that are produced by filamentous fungi (hydrophobin HFBI from Trichoderma reesei). Using pendant drop and pipette aspiration techniques, we show that these giant gas vesicles behave like glassy polymersomes, and we discover novel gas extraction regimes. We develop a model to analyze the micropipette aspiration of these compressible gas vesicles and compare them to incompressible liquid-filled vesicles. We introduce a sealing parameter to characterize the leakage of gas under aspiration through the pores of the protein coating. Utilizing this model, we can determine the elastic dilatation modulus, surface viscosity, and porosity of the membrane. These results demonstrate the engineering potential of protein-coated bubbles for echogenic and therapeutic applications and extend the use of the pipette aspiration technique to compressible and porous systems.

Fusion Dynamics of Hybrid Cell-Microparticle Aggregates: A Jelly Pearl Model.

We study the fusion of homogeneous cell aggregates and of hybrid aggregates combining cells and microparticles. In all cases, we find that the contact area does not vary linearly over time, as observed for liquid drops, but rather it follows a power law in t2/3. This result is interpreted by generalizing the fusion model of soft viscoelastic solid balls to viscoelastic liquid balls, akin to jelly pearls. We also explore the asymmetric fusion between a homogeneous aggregate and a hybrid aggregate. This latter experiment allows the determination of the self-diffusion coefficient of the cells in a tissue by following the spatial distribution of internalized particles in the cells.

Spreading of Cell Aggregates on Zwitterion-Modified Chitosan Films.

The sulfobetaine (SB) moiety, which comprises a quaternary ammonium group linked to a negatively charged sulfonate ester, is known to impart nonfouling properties to interfaces coated with polysulfobetaines or grafted with SB-polymeric brushes. Increasingly, evidence emerges that the SB group is, overall, a better antifouling group than the phosphorylcholine (PC) moiety extensively used in the past. We report here the synthesis of a series of SB-modified chitosans (CH-SB) carrying between 20 and 40 mol % SB per monosaccharide unit. Chitosan (CH) itself is a naturally derived copolymer of glucosamine and N-acetyl-glucosamine linked with a ß-1,4 bond. Analysis by quartz crystal microbalance with dissipation (QCM-D) indicates that CH-SB films (thickness ∼ 20 nm) resist adsorption of bovine serum albumin (BSA) with increasing efficiency as the SB content of the polymer augments (surface coverage ∼ 15 µg cm-2 for films of CH with 40 mol % SB). The cell adhesivity of CH-SB films coated on glass was assessed by determining the spreading dynamics of CT26 cell aggregates. When placed on chitosan films, known to be cell-adhesive, the CT26 cell aggregates spread by forming a cell monolayer around them. The spreading of CT26 cell aggregates on zwitterion-modified chitosans films is thwarted remarkably. In the cases of CH-SB30 and CH-SB40 films, only a few isolated cells escape from the aggregates. The extent of aggregate spreading, quantified based on the theory of liquid wetting, provides a simple in vitro assay of the nonfouling properties of substrates toward specific cell lines. This assay can be adopted to test and compare the fouling characteristics of substrates very different from the chemical viewpoint.

Polymeric Nanoparticles Limit the Collective Migration of Cellular Aggregates.

Controlling the propagation of primary tumors is fundamental to avoiding the epithelial to mesenchymal transition process leading to the dissemination and seeding of tumor cells throughout the body. Here we demonstrate that nanoparticles (NPs) limit the propagation of cell aggregates of CT26 murine carcinoma cells used as tumor models. The spreading behavior of these aggregates incubated with NPs is studied on fibronectin-coated substrates. The cells spread with the formation of a cell monolayer, the precursor film, around the aggregate. We study the effect of NPs added either during or after the formation of aggregates. We demonstrate that, in both cases, the spreading of the cell monolayer is slowed down in the presence of NPs and occurs only above a threshold concentration that depends on the size and surface chemistry of the NPs. The density of cells in the precursor films, measured by confocal microscopy, shows that the NPs stick cells together. The mechanism of slowdown is explained by the increase in cell-cell interactions due to the NPs adsorbed on the membrane of the cells. The present results demonstrate that NPs can modulate the collective migration of cells; therefore, they may have important implications for cancer treatment.

Spontaneous migration of cellular aggregates from giant keratocytes to running spheroids.

Despite extensive knowledge on the mechanisms that drive single-cell migration, those governing the migration of cell clusters, as occurring during embryonic development and cancer metastasis, remain poorly understood. Here, we investigate the collective migration of cell on adhesive gels with variable rigidity, using 3D cellular aggregates as a model system. After initial adhesion to the substrate, aggregates spread by expanding outward a cell monolayer, whose dynamics is optimal in a narrow range of rigidities. Fast expansion gives rise to the accumulation of mechanical tension that leads to the rupture of cell-cell contacts and the nucleation of holes within the monolayer, which becomes unstable and undergoes dewetting like a liquid film. This leads to a symmetry breaking and causes the entire aggregate to move as a single entity. Varying the substrate rigidity modulates the extent of dewetting and induces different modes of aggregate motion: "giant keratocytes," where the lamellipodium is a cell monolayer that expands at the front and retracts at the back; "penguins," characterized by bipedal locomotion; and "running spheroids," for nonspreading aggregates. We characterize these diverse modes of collective migration by quantifying the flows and forces that drive them, and we unveil the fundamental physical principles that govern these behaviors, which underscore the biological predisposition of living material to migrate, independent of length scale.

Adhesion to nanofibers drives cell membrane remodeling through one-dimensional wetting.

The shape of cellular membranes is highly regulated by a set of conserved mechanisms that can be manipulated by bacterial pathogens to infect cells. Remodeling of the plasma membrane of endothelial cells by the bacterium Neisseria meningitidis is thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unclear. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a physical mechanism that we term 'one-dimensional' membrane wetting. We provide a theoretical model that describes the physical basis of one-dimensional wetting and show that this mechanism occurs in model membranes interacting with nanofibers, and in human cells interacting with extracellular matrix meshworks. We propose one-dimensional wetting as a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococci during infection.

ALCAM shedding at the invasive front of the tumor is a marker of myometrial infiltration and promotes invasion in endometrioid endometrial cancer.

Endometrial cancer (EC) is the sixth deadliest cancer in women. The depth of myometrial invasion is one of the most important prognostic factors, being directly associated with tumor recurrence and mortality. In this study, ALCAM, a previously described marker of EC recurrence, was studied by immunohistochemistry at the superficial and the invasive tumor areas from 116 EC patients with different degree of myometrial invasion and related to a set of relevant epithelial and mesenchymal markers. ALCAM expression presented a heterogeneous functionality depending on its localization, it correlated with epithelial markers (E-cadherin/ß-catenin) at the superficial area, and with mesenchymal markers at the invasive front (COX-2, SNAIL, ETV5, and MMP-9). At the invasive front, ALCAM-negativity was an independent marker of myometrial invasion. This negativity, together with an increase of soluble ALCAM in uterine aspirates from patients with an invasive EC, and its positive correlation with MMP-9 levels, suggested that ALCAM shedding by MMP-9 occurs at the invasive front. In vivo and in vitro models of invasive EC were generated by ETV5-overexpression. In those, we demonstrated that ALCAM shedding was related to a more invasive pattern and that full-ALCAM recovery reverted most of the ETV5-cells mesenchymal abilities, partially through a p-ERK dependent-manner.

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.

How gluttonous cell aggregates clear substrates coated with microparticles.

We study the spreading of cell aggregates deposited on adhesive substrates decorated with microparticles (MPs). A cell monolayer expands around the aggregate. The cells on the periphery of the monolayer take up the MPs, clearing the substrate as they progress and forming an aureole of cells filled with MPs. We study the dynamics of spreading and determine the width of the aureole and the level of MP internalization in cells as a function of MP size, composition, and density. From the radius and width of the aureole, we quantify the volume fraction of MPs within the cell, which leads to an easy, fast, and inexpensive measurement of the cell - particle internalization.

Activated leukocyte cell adhesion molecule (ALCAM) is a marker of recurrence and promotes cell migration, invasion, and metastasis in early-stage endometrioid endometrial cancer.

Endometrial cancer is the most common gynaecological cancer in western countries, being the most common subtype of endometrioid tumours. Most patients are diagnosed at an early stage and present an excellent prognosis. However, a number of those continue to suffer recurrence, without means of identification by risk classification systems. Thus, finding a reliable marker to predict recurrence becomes an important unmet clinical issue. ALCAM is a cell-cell adhesion molecule and member of the immunoglobulin superfamily that has been associated with the genesis of many cancers. Here, we first determined the value of ALCAM as a marker of recurrence in endometrioid endometrial cancer by conducting a retrospective multicentre study of 174 primary tumours. In early-stage patients (N = 134), recurrence-free survival was poorer in patients with ALCAM-positive compared to ALCAM-negative tumours (HR 4.237; 95% CI 1.01-17.76). This difference was more significant in patients with early-stage moderately-poorly differentiated tumours (HR 9.259; 95% CI 2.12-53.47). In multivariate analysis, ALCAM positivity was an independent prognostic factor in early-stage disease (HR 6.027; 95% CI 1.41-25.74). Then we demonstrated in vitro a role for ALCAM in cell migration and invasion by using a loss-of-function model in two endometrial cancer cell lines. ALCAM depletion resulted in a reduced primary tumour size and reduced metastatic local spread in an orthotopic murine model. Gene expression analysis of ALCAM-depleted cell lines pointed to motility, invasiveness, cellular assembly, and organization as the most deregulated functions. Finally, we assessed some of the downstream effector genes that are involved in ALCAM-mediated cell migration; specifically FLNB, TXNRD1, and LAMC2 were validated at the mRNA and protein level. In conclusion, our results highlight the potential of ALCAM as a recurrent biomarker in early-stage endometrioid endometrial cancer and point to ALCAM as an important molecule in endometrial cancer dissemination by regulating cell migration, invasion, and metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Nanostickers for cells: a model study using cell-nanoparticle hybrid aggregates.

We present direct evidence that nanoparticles (NPs) can stick together cells that are inherently non-adhesive. Using cadherin-depleted S180 murine cells lines, which exhibit very low cell-cell adhesion, we show that NPs can assemble dispersed single cells into large cohesive aggregates. The dynamics of aggregation, which is controlled by diffusion and collision, can be described as a second-order kinetic law characterized by a rate of collision that depends on the size, concentration, and surface chemistry of the NPs. We model the cell-cell adhesion induced by the "nanostickers" using a three-state dynamical model, where the NPs are free, adsorbed on the cell membrane or internalized by the cells. We define a "sticking efficiency parameter" to compare NPs and look for the most efficient type of NP. We find that 20 nm carboxylated polystyrene NPs are more efficient nanostickers than 20 nm silica NPs which were reported to induce fast wound healing and to glue soft tissues. Nanostickers, by increasing the cohesion of tissues and tumors, may have important applications for tissue engineering and cancer treatment.

Spreading of porous vesicles subjected to osmotic shocks: the role of aquaporins.

Aquaporin 0 (AQP0) is a transmembrane protein specific to the eye lens, involved as a water carrier across the lipid membranes. During eye lens maturation, AQP0s are truncated by proteolytic cleavage. We investigate in this work the capability of truncated AQP0 to conduct water across membranes. We developed a method to accurately determine water permeability across lipid membranes and across proteins from the deflation under osmotic pressure of giant unilamellar vesicles (GUVs) deposited on an adhesive substrate. Using reflection interference contrast microscopy (RICM), we measure the spreading area of GUVs during deswelling. We interpret these results using a model based on hydrodynamic, binder diffusion towards the contact zone, and Helfrich's law for the membrane tension, which allows us to relate the spread area to the vesicle internal volume. We first study the specific adhesion of vesicles coated with biotin spreading on a streptavidin substrate. We then determine the permeability of a single functional AQP0 and demonstrate that truncated AQP0 is no more a water channel.

Mechanics of Biomimetic Liposomes Encapsulating an Actin Shell.

Cell-shape changes are insured by a thin, dynamic, cortical layer of cytoskeleton underneath the plasma membrane. How this thin cortical structure impacts the mechanical properties of the whole cell is not fully understood. Here, we study the mechanics of liposomes or giant unilamellar vesicles, when a biomimetic actin cortex is grown at the inner layer of the lipid membrane via actin-nucleation-promoting factors. Using a hydrodynamic tube-pulling technique, we show that tube dynamics is clearly affected by the presence of an actin shell anchored to the lipid bilayer. The same force pulls much shorter tubes in the presence of the actin shell compared to bare membranes. However, in both cases, we observe that the dynamics of tube extrusion has two distinct features characteristic of viscoelastic materials: rapid elastic elongation, followed by a slower elongation phase at a constant rate. We interpret the initial elastic regime by an increase of membrane tension due to the loss of lipids into the tube. Tube length is considerably shorter for cortex liposomes at comparable pulling forces, resulting in a higher spring constant. The presence of the actin shell seems to restrict lipid mobility, as is observed in the corral effect in cells. The viscous regime for bare liposomes corresponds to a leakout of the internal liquid at constant membrane tension. The presence of the actin shell leads to a larger friction coefficient. As the tube is pulled from a patchy surface, membrane tension increases locally, leading to a Marangoni flow of lipids. As a conclusion, the presence of an actin shell is revealed by its action that alters membrane mechanics.

Formation of Tethers from Spreading Cellular Aggregates.

Membrane tubes are commonly extruded from cells and vesicles when a point-like force is applied on the membrane. We report here the unexpected formation of membrane tubes from lymph node cancer prostate (LNCaP) cell aggregates in the absence of external applied forces. The spreading of LNCaP aggregates deposited on adhesive glass substrates coated with fibronectin is very limited because cell-cell adhesion is stronger than cell-substrate adhesion. Some cells on the aggregate periphery are very motile and try to escape from the aggregate, leading to the formation of membrane tubes. Tethered networks and exchange of cargos between cells were observed as well. Growth of the tubes is followed by either tube retraction or tube rupture. Hence, even very cohesive cells are successful in escaping aggregates, which may lead to epithelial mesenchymal transition and tumor metastasis. We interpret the dynamics of formation and retraction of tubes in the framework of membrane mechanics.

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.

Nanopore-Based Characterization of Branched Polymers.

We propose a novel characterization method of randomly branched polymers based on the geometrical property of such objects in confined spaces. The central idea is that randomly branched polymers exhibit a passing/clogging transition across the nanochannel as a function of the channel size. This critical channel size depends on the degree of the branching, whereby allowing the extraction of the branching information of the molecule.

Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins.

Dewetting is the spontaneous withdrawal of a liquid film from a non-wettable surface by nucleation and growth of dry patches. Two recent reports now propose that the principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, referred to as cellular dewetting. This was discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For both liquid and cellular dewetting, the growth of holes is governed by a competition between surface forces and line tension. We also discuss how the dynamics of TEM opening and closure represent remarkable systems to investigate actin cytoskeleton regulation by sensors of plasma membrane curvature and investigate the impact on membrane tension and the role of TEM in vascular dysfunctions.

Soft matter models of developing tissues and tumors.

Analogies with inert soft condensed matter--such as viscoelastic liquids, pastes, foams, emulsions, colloids, and polymers--can be used to investigate the mechanical response of soft biological tissues to forces. A variety of experimental techniques and biophysical models have exploited these analogies allowing the quantitative characterization of the mechanical properties of model tissues, such as surface tension, elasticity, and viscosity. The framework of soft matter has been successful in explaining a number of dynamical tissue behaviors observed in physiology and development, such as cell sorting, tissue spreading, or the escape of individual cells from a tumor. However, living tissues also exhibit active responses, such as rigidity sensing or cell pulsation, that are absent in inert soft materials. The soft matter models reviewed here have provided valuable insight in understanding morphogenesis and cancer invasion and have set bases for using tissue engineering within medicine.

Spreading dynamics of cellular aggregates confined to adhesive bands.

We examine the spreading of cellular aggregates deposited on adhesive striated glass surfaces consisting of 100 µm large bands alternatively coated with fibronectin and with PolyEthyleneGlycol-Poly-L-lysine (PEG-PLL). The aggregates spread confined to the adhesive fibronectin bands. A front of cells expands from the aggregate at constant velocity. In comparison, the radial spreading of an aggregate on the uniform fibronectin coated glass surface obeys a diffusive law. We develop a common theoretical model in agreement with our experimental observations to describe the apparently disparate spreading kinetics of cellular aggregates. These results demonstrate the dominant role of the permeation in the expansion of the precursor film of cells around the aggregate.