Valérie M. Laurent

Measurement of the elasticity of the actin tail of Listeria monocytogenes

Abstract We report biophysical experiments performed on the bacterium Listeria monocytogenes, a model system to study actin-based motility. Using optical tweezers and electrophoresis experiments, we find that the bacterium is firmly attached to its tail, and we demonstrate that the tail responds as an elastic gel when deformed. We have measured its elastic modulus at a value of 103–104u2009Pa, which is 10 times higher than the rigidity of the eukaryotic cytoplasm. These results demonstrate that the bacterium and its tail form a very robust system, consistent with the steadyness of the motion observed in vivo. We propose an elastic model for the propulsion mechanism which takes into account the connection and thus the interaction between the actin filaments. It provides a generic description of the various aspects of actin-tail based movements.

Time-dependent traction force microscopy for cancer cells as a measure of invasiveness

The migration of tumor cells of different degrees of invasivity is studied, on the basis of the traction forces exerted in time on soft substrates (Young modulus ∼ 10 kPa). It is found that the outliers of the traction stresses can be an effective indicator to distinguish cancer cell lines of different invasiveness. Here, we test two different epithelial bladder cancer cell lines, one invasive (T24), and a less invasive one (RT112). Invasive cancer cells move in a nearly periodic motion, with peaks in velocity corresponding to higher traction forces exerted on the substrate, whereas less invasive cells develop traction stresses almost constant in time. The dynamics of focal adhesions (FAs) as well as cytoskeleton features reveals that different mechanisms are activated to migrate: T24 cells show an interconnected cytoskeleton linked to mature adhesion sites, leading to small traction stresses, whereas less invasive cells (RT112) show a less‐structured cytoskeleton and unmature adhesions corresponding to higher traction stresses. Migration velocities are smaller in the case of less invasive cells. The mean squared displacement shows super‐diffusive motion in both cases with higher exponent for the more invasive cancer cells. Further correlations between traction forces and the actin cytoskeleton reveal an unexpected pattern of a large actin rim at the RT112 cell edge where higher forces are colocalized, whereas a more usual cytoskeleton structure with stress fibers and FAs are found for T24 cancer cells. We conjecture that this kind of analysis can be useful to classify cancer cell invasiveness.

Atomic Force microscopy reveals a role for endothelial cell ICAM-1 expression in bladder cancer cell adherence

Cancer metastasis is a complex process involving cell-cell interactions mediated by cell adhesive molecules. In this study we determine the adhesion strength between an endothelial cell monolayer and tumor cells of different metastatic potentials using Atomic Force Microscopy. We show that the rupture forces of receptor-ligand bonds increase with retraction speed and range between 20 and 70 pN. It is shown that the most invasive cell lines (T24, J82) form the strongest bonds with endothelial cells. Using ICAM-1 coated substrates and a monoclonal antibody specific for ICAM-1, we demonstrate that ICAM-1 serves as a key receptor on endothelial cells and that its interactions with ligands expressed by tumor cells are correlated with the rupture forces obtained with the most invasive cancer cells (T24, J82). For the less invasive cancer cells (RT112), endothelial ICAM-1 does not seem to play any role in the adhesion process. Moreover, a detailed analysis of the distribution of rupture forces suggests that ICAM-1 interacts preferentially with one ligand on T24 cancer cells and with two ligands on J82 cancer cells. Possible counter receptors for these interactions are CD43 and MUC1, two known ligands for ICAM-1 which are expressed by these cancer cells.

Physical properties of polyacrylamide gels probed by AFM and rheology

Polymer gels have been shown to behave as viscoelastic materials but only a small amount of data is usually provided in the glass transition. In this paper, the dynamic moduli and of polyacrylamide hydrogels are investigated using both an AFM in contact force modulation mode and a classical rheometer. The validity is shown by the matching of the two techniques. Measurements are carried out on gels of increasing polymer concentration in a wide frequency range. A model based on fractional derivatives is successfully used, covering the whole frequency range. , the plateau modulus, as well as several other parameters are obtained at low frequencies. The model also predicts the slope a of both moduli in the glass transition, and a transition frequency is introduced to separate the gel-like behavior with the glassy state. Its variation with polymer content c gives a dependence , in good agreement with previous theories. Therefore, the AFM data provides new information on the physics of polymer gels.

Tumor response assessment by MRI following stereotactic body radiation therapy for hepatocellular carcinoma

Background To evaluate the MRI features of a tumor response, local control, and predictive factors of local control after stereotactic body radiation therapy (SBRT) for hepatocellular carcinoma (HCC). Methods Thirty-five consecutive patients with 48 HCCs who were treated by SBRT were included in this retrospective study. All patients provided written informed consent to be treated by SBRT, and prior to inclusion they authorized use of the treatment data for further studies. The assessment was made using MRI, with determination of local and hepatic responses according to Response Evaluation Criteria in Solid Tumors (RECIST) and modified RECIST (mRECIST) criteria during a two-year follow-up. Results The local response rate according to mRECIST was higher than with RECIST. A tumor diameter less than 20 mm at baseline was an independent predictive factor for RECIST and mRECIST responses, as was diffusion-weighted signal for RECIST. During follow-up, a tumor diameter of <20 mm (p = 0.034) and absence of a high intensity on T2-weighted (p = 0.006) and diffusion-weighted images (p = 0.039) were associated with a better response according to RECIST. Post-treatment changes include peritumoral ring-like enhanced changes with high intensity on T2-weighted images. Conclusions SBRT is a promising technique for the treatment of inoperable HCC. Post-treatment changes on MRI images can resemble tumor progression and as such must be adequately distinguished. The regression of tumorous enhancement is variable over time, although diffusion-weighted and T2-weighted intensities are predictive factors for tumor RECIST responses on subsequent MRIs. They hence provide a way to reliably predict treatment responses.

Traction forces of cancer cells

Cell motility of cancer cells is a fundamental problem, and requires precise correlation between cell adhesive and rheological properties. The migration of cancer cells is studied in two dimensions, as cells are seeded onto functionnalized polyacrylamide gels. They migrate by developping focal adhesion sites and modulate their cytoskeleton dynamics by regulating actin and myosin. This research aims at understanding the forces developped by different cancer cells of moderate to high invasiveness in order to investigate their contractility as well as the adhesion molecules necessary for cell migration.

Unraveling the Receptor-Ligand Interactions between Bladder Cancer Cells and the Endothelium Using AFM

Adhesion of cancer cells to endothelial cells is a key step in cancer metastasis; therefore, identifying the key molecules involved during this process promises to aid in efforts to block the metastatic cascade. We have previously shown that intercellular adhesion molecule-1 (ICAM-1) expressed by endothelial cells is involved in the interactions of bladder cancer cells (BCs) with the endothelium. However, the ICAM-1 ligands have never been investigated. In this study, we combined adhesion assays and atomic force microscopy (AFM) to identify the ligands involved and to quantify the forces relevant in such interactions. We report the expression of MUC1 and CD43 on BCs, and demonstrate that these ligands interact with ICAM-1 to mediate cancer cell-endothelial cell adhesion in the case of the more invasive BCs. This was achieved with the use of adhesion assays, which showed a strong decrease in the attachment of BCs to endothelial cells when MUC1 and CD43 were blocked by antibodies. In addition, AFM measurements showed a similar decrease, by up to 70%, in the number of rupture events that occurred when MUC1 and CD43 were blocked. When we applied a Gaussian mixture model to the AFM data, we observed a distinct force range for receptor-ligand bonds, which allowed us to precisely identify the interactions of ICAM-1 with MUC1 or CD43. Furthermore, a detailed analysis of the rupture events suggested that CD43 is strongly connected to the cytoskeleton and that its interaction with ICAM-1 mainly corresponds to force ramps followed by sudden jumps. In contrast, MUC1 seems to be weakly connected to the cytoskeleton, as its interactions with ICAM-1 are mainly associated with the formation of tethers. This analysis is quite promising and may also be applied to other types of cancer cells.

Microrheology of complex systems and living cells using AFM

Biological cells display viscoelastic mechanical properties that are a key factor in the regulation of cell processes, such as migration, adhesion and deformability. One interesting tool to probe the mechanical properties of living cells and other complex systems is the Atomic Force Microscopy (AFM). Rheological properties of the sample are obtained usually from the force-indentation (F-δ) measurements and by fitting with the Sneddons modification of the Hertz model. This model describes the relationship between the force applied by a stiff cone, a purely elastic indentation in a flat and soft sample and the Youngs modulus E. AFM uses a flexible cantilever with a pyramidal tip (or sphere) at the end that will locally indent the viscoelastic material. Another approach is to superpose low-amplitude sinusoidal oscillations to an initial identation δ0 (polyacrylamide gel or cells). Then one can determine the complex shear modulus G*...

Prediction of traction forces of motile cells

When crawling on a flat substrate, living cells exert forces on it via adhesive contacts, enabling them to build up tension within their cytoskeleton and to change shape. The measurement of these forces has been made possible by traction force microscopy (TFM), a technique which has allowed us to obtain time-resolved traction force maps during cell migration. This cell ‘footprint’ is, however, not sufficient to understand the details of the mechanics of migration, that is how cytoskeletal elements (respectively, adhesion complexes) are put under tension and reinforce or deform (respectively, mature and/or unbind) as a result. In a recent paper, we have validated a rheological model of actomyosin linking tension, deformation and myosin activity. Here, we complement this model with tentative models of the mechanics of adhesion and explore how closely these models can predict the traction forces that we recover from experimental measurements during cell migration. The resulting mathematical problem is a PDE set on the experimentally observed domain, which we solve using a finite-element approach. The four parameters of the model can then be adjusted by comparison with experimental results on a single frame of an experiment, and then used to test the predictive power of the model for following frames and other experiments. It is found that the basic pattern of traction forces is robustly predicted by the model and fixed parameters as a function of current geometry only.

Mechanosensitivity of Cancer Cells in Contact with Soft Substrates Using AFM

Cancer cells are usually found to be softer than normal cells, but their stiffness changes when they are in contact with different environments because of mechanosensitivity. For example, they adhere to a given substrate by tuning their cytoskeleton, thus affecting their rheological properties. This mechanism could become efficient when cancer cells invade the surrounding tissues, and they have to remodel their cytoskeleton in order to achieve particular deformations. Here we use an atomic force microscope in force modulation mode to study how local rheological properties of cancer cells are affected by a change of the environment. Cancer cells were plated on functionalized polyacrylamide substrates of different stiffnesses as well as on an endothelium substrate. A new correction of the Hertz model was developed because measurements require one to account for the precise properties of the thin, layered viscoelastic substrates. The main results show the influence of local cell rheology (the nucleus, perinuclear region, and edge locations) and the role of invasiveness. A general mechanosensitive trend is found by which the cell elastic modulus and transition frequency increase with substrate elasticity, but this tendency breaks downxa0with a real endothelium substrate. These effects are investigated further during cell transmigration, when the actin cytoskeleton undergoes a rapid reorganization process necessary to push through the endothelial gap, in agreement with the local viscoelastic changes measured by atomic force microscopy. Taken together, these results introduce a paradigm for a new-to our knowledge-possible extravasation mechanism.