Live 3D imaging and mapping of shear stresses within tissues using incompressible elastic beads.

To investigate the role of mechanical constraints in morphogenesis and development, we have developed a pipeline of techniques based on incompressible elastic sensors. These techniques combine the advantages of incompressible liquid droplets, which have been used as precise in situ shear stress sensors, and of elastic compressible beads, which are easier to tune and to use. Droplets of a polydimethylsiloxane mix, made fluorescent through specific covalent binding to a rhodamin dye, are produced by a microfluidics device. The elastomer rigidity after polymerization is adjusted to the tissue rigidity. Its mechanical properties are carefully calibrated in situ, for a sensor embedded in a cell aggregate submitted to uniaxial compression. The local shear stress tensor is retrieved from the sensor shape, accurately reconstructed through an active contour method. In vitro, within cell aggregates, and in vivo, in the prechordal plate of the zebrafish embryo during gastrulation, our pipeline of techniques demonstrates its efficiency to directly measure the three dimensional shear stress repartition within a tissue.

The desmin network is a determinant of the cytoplasmic stiffness of myoblasts.

BACKGROUND INFORMATION: The mechanical properties of cells are essential to maintain their proper functions, and mainly rely on their cytoskeleton. A lot of attention has been paid to actin filaments, demonstrating their central role in the cells mechanical properties, but much less is known about the participation of intermediate filament (IF) networks. Indeed the contribution of IFs, such as vimentin, keratins and lamins, to cell mechanics has only been assessed recently. We study here the involvement of desmin, an IF specifically expressed in muscle cells, in the rheology of immature muscle cells. Desmin can carry mutations responsible for a class of muscle pathologies named desminopathies. RESULTS: In this study, using three types of cell rheometers, we assess the consequences of expressing wild-type (WT) or mutated desmin on the rheological properties of single myoblasts. We find that the mechanical properties of the cell cortex are not correlated to the quantity, nor the quality of desmin expressed. On the contrary, the overall cell stiffness increases when the amount of WT or mutated desmin polymerised in cytoplasmic networks increases. However, myoblasts become softer when the desmin network is partially depleted by the formation of aggregates induced by the expression of a desmin mutant. CONCLUSIONS: We demonstrate that desmin plays a negligible role in the mechanical properties of the cell cortex but is a determinant of the overall cell stiffness. More particularly, desmin participates to the cytoplasm viscoelasticity. SIGNIFICANCE: Desminopathies are associated with muscular weaknesses attributed to a disorganisation of the structure of striated muscle that impairs the active force generation. The present study evidences for the first time the key role of desmin in the rheological properties of myoblasts, raising the hypothesis that desmin mutations could also alter the passive mechanical properties of muscles, thus participating to the lack of force build up in muscle tissue.

Impact of a mechanical shear stress on intracellular trafficking.

Intracellular trafficking mainly takes place along the microtubules, and its efficiency depends on the local architecture and organization of the cytoskeletal network. In this work, the cytoplasm of stem cells is subjected to mechanical vortexing at a frequency of up to 1 Hz, by using magnetic chains of endosomes embedded in the cell body, in order to locally perturb the network structure. The consequences are evaluated on the directionality and processivity of the spontaneous motion of endosomes. When the same chains are used both to shear the cell medium and to probe the intracellular traffic, a substantial decrease in transport efficiency is detected after applying the mechanical shear. Interestingly, when using different objects to apply the shear and to probe the spontaneous motion, no alteration of the transport efficiency can be detected. We conclude that shaking the vesicles mainly causes their unbinding from the cytoskeletal tracks, but has little influence on the integrity of the network itself. This is corroborated by active microrheology measurements, performed with chains actuated by a magnetic field, and showing that the mechanical compliance of the cytoplasm is similar before and after slow vortexing.

Negative feedback from integrins to cadherins: a micromechanical study.

The coupling between cell-cell and cell-matrix adhesion systems is known to affect the stability of the adhesive status of cells, as well as tissue cohesion. In this work, we perform quantitative assays of integrin-cadherin cross talk in controlled and reproducible conditions. This is achieved by plating cells on microprinted fibronectin patterns of different sizes, and simulating the formation of an intercellular contact with a microbead coated with E-cadherin extracellular domains and brought to the cell membrane. Using an optical trap, we measure the average rigidity modulus of the E-cadherin bead-cell contact as a function of the contact incubation time and of the cell spreading area. For a given incubation time, this rigidity modulus decreases by three orders of magnitude as the cell-matrix contact area, A, increases from 100 to 700 µm(2). In a similar way, the dynamics of formation of the bead-cell contact gets slower as this area increases. This is clear evidence for a strong negative feedback from cell-fibronectin onto cell-cell adhesive contacts, for which we discuss some possible mechanisms.

Spatiotemporal analysis of cell response to a rigidity gradient: a quantitative study using multiple optical tweezers.

We investigate the dynamic response of single cells to weak and local rigidities, applied at controlled adhesion sites. Using multiple latex beads functionalized with fibronectin, and each trapped in its own optical trap, we study the reaction in real time of single 3T3 fibroblast cells to asymmetrical tensions in the tens of pN x microm(-1) range. We show that the cell feels a rigidity gradient even at this low range of tension, and over time develops an adapted change in the force exerted on each adhesion site. The rate at which force increases is proportional to trap stiffness. Actomyosin recruitment is regulated in space and time along the rigidity gradient, resulting in a linear relationship between the amount of recruited actin and the force developed independently in trap stiffness. This time-regulated actomyosin behavior sustains a constant and rigidity-independent velocity of beads inside the traps. Our results show that the strengthening of extracellular matrix-cytoskeleton linkages along a rigidity gradient is regulated by controlling adhesion area and actomyosin recruitment, to maintain a constant deformation of the extracellular matrix.

Power laws in microrheology experiments on living cells: Comparative analysis and modeling.

We compare and synthesize the results of two microrheological experiments on the cytoskeleton of single cells. In the first one, the creep function J(t) of a cell stretched between two glass plates is measured after applying a constant force step. In the second one, a microbead specifically bound to transmembrane receptors is driven by an oscillating optical trap, and the viscoelastic coefficient Ge(omega) is retrieved. Both J(t) and Ge(omega) exhibit power law behaviors: J(t) = A0(t/t0)alpha and absolute value (Ge(omega)) = G0(omega/omega0)alpha, with the same exponent alpha approximately 0.2. This power law behavior is very robust; alpha is distributed over a narrow range, and shows almost no dependence on the cell type, on the nature of the protein complex which transmits the mechanical stress, nor on the typical length scale of the experiment. On the contrary, the prefactors A0 and G0 appear very sensitive to these parameters. Whereas the exponents alpha are normally distributed over the cell population, the prefactors A0 and G0 follow a log-normal repartition. These results are compared with other data published in the literature. We propose a global interpretation, based on a semiphenomenological model, which involves a broad distribution of relaxation times in the system. The model predicts the power law behavior and the statistical repartition of the mechanical parameters, as experimentally observed for the cells. Moreover, it leads to an estimate of the largest response time in the cytoskeletal network: tau(m) approximately 1000 s.

Massive release of extracellular vesicles from cancer cells after photodynamic treatment or chemotherapy.

Photodynamic therapy is an emerging cancer treatment that is particularly adapted for localized malignant tumor. The phototherapeutic agent is generally injected in the bloodstream and circulates in the whole organism as a chemotherapeutic agent, but needs light triggering to induce localized therapeutic effects. We found that one of the responses of in vitro and in vivo cancer cells to photodynamic therapy was a massive production and emission of extracellular vesicles (EVs): only 1 hour after the photo-activation, thousands of vesicles per cell were emitted in the extracellular medium. A similar effect has been found after treatment with Doxorubicin (chemotherapy), but far less EVs were produced, even 24 hours after the treatment. Furthermore, we found that the released EVs could transfer extracellular membrane components, drugs and even large intracellular objects to naive target cells. In vivo, photodynamic treatment and chemotherapy increased the levels of circulating EVs several fold, confirming the vast induction of cancer cell vesiculation triggered by anti-cancer therapies.

Lymphocytes can self-steer passively with wind vane uropods.

A wide variety of cells migrate directionally in response to chemical or mechanical cues, however the mechanisms involved in cue detection and translation into directed movement are debatable. Here we investigate a model of lymphocyte migration on the inner surface of blood vessels. Cells orient their migration against fluid flow, suggesting the existence of an adaptive mechano-tranduction mechanism. We find that flow detection may not require molecular mechano-sensors of shear stress, and detection of flow direction can be achieved by the orientation in the flow of the non-adherent cell rear, the uropod. Uropods act as microscopic wind vanes that can transmit detection of flow direction into cell steering via the on-going machinery of polarity maintenance, without the need for novel internal guidance signalling triggered by flow. Contrary to chemotaxis, which implies active regulation of cue-dependent signalling, upstream flow mechanotaxis of lymphocytes may only rely on a passive self-steering mechanism.

Impact of photosensitizers activation on intracellular trafficking and viscosity.

The intracellular microenvironment is essential for the efficiency of photo-induced therapies, as short-lived reactive oxygen species generated must diffuse through their intracellular surrounding medium to reach their cellular target. Here, by combining measurements of local cytoplasmic dissipation and active trafficking, we found that photosensitizers activation induced small changes in surrounding viscosity but a massive decrease in diffusion. These effects are the signature of a return to thermodynamic equilibrium of the system after photo-activation and correlated with depolymerization of the microtubule network, as shown in a reconstituted system. These mechanical measurements were performed with two intracellular photosensitizing chlorins having similar quantum yield of singlet oxygen production but different intracellular localizations (cytoplasmic for mTHPC, endosomal for TPCS2a). These two agents demonstrated different intracellular impact.

Glycosylation-related gene expression is linked to differentiation status in glioblastomas undifferentiated cells.

Glioblastoma Multiforme (GBM) is the most frequent malignant brain tumor with still poor prognosis. Tumor initiation, growth and recurrences might depend on Brain Tumor Stem Cells (BTSCs) which can promote tumor aggressiveness and potentially affords new therapeutic target. Recent works emphasized aberrant cell-surface glyco-conjugate expression in brain tumors suggesting that altered glycosylation is closely linked to cancer tumor metastasis and invasive process. Post-translational changes might play a key role in determining the fates of most aggressive and undifferentiated cells such as self-renewal, proliferation and differentiation. In order to characterize the glycosylation-related genes involved in differentiation status of the BTSCs, two glioblastoma cell lines, U87-MG and U251 have been cultured according to two conditions leading to undifferentiated floating cells or differentiated adherent cells. The expression level of 559 glycosylation related genes has been analyzed by Taqman Low Density Array (TLDA) analysis and allowed to isolate eight up-regulated genes specific of a subpopulation of undifferentiated cells. Protein expression has been confirmed. Among main selected genes, five are also over-expressed in the undifferentiated condition in primary cultures provided by three GBM freshly isolated from patient. This work suggests that new Glycosylation-related gene signature might improve the characterization of the most aggressive and undifferentiated cells and supports that in future, N-linked glycosylation might provide new target to develop therapeutic strategy for inhibiting tumor growth.

In vivo determination of fluctuating forces during endosome trafficking using a combination of active and passive microrheology.

BACKGROUND: Regulation of intracellular trafficking is a central issue in cell biology. The forces acting on intracellular vesicles (endosomes) can be assessed in living cells by using a combination of active and passive microrheology. METHODOLOGY/PRINCIPAL FINDINGS: This dual approach is based on endosome labeling with magnetic nanoparticles. The resulting magnetic endosomes act both as probes that can be manipulated with external magnetic fields to infer the viscoelastic modulus of their surrounding microenvironment, and as biological vehicles that are trafficked along the microtubule network by means of forces generated by molecular motors. The intracellular viscoelastic modulus exhibits power law dependence with frequency, which is microtubule and actin-dependent. The mean square displacements of endosomes do not follow the predictions of the fluctuation-dissipation theorem, which offers evidence for active force generation. Microtubule disruption brings the intracellular medium closer to thermal equilibrium: active forces acting on the endosomes depend on microtubule-associated motors. The power spectra of these active forces, deduced through the use of a generalized Langevin equation, show a power law decrease with frequency and reveal an actin-dependent persistence of the force with time. Experimental spectra have been reproduced by a simple model consisting in a series of force steps power-law distributed in time. This model enlightens the role of the cytoskeleton dependent force exerted on endosomes to perform intracellular trafficking. CONCLUSIONS/SIGNIFICANCE: In this work, the influence of cytoskeleton components and molecular motors on intracellular viscoelasticity and transport is addressed. The use of an original probe, the magnetic endosome, allows retrieving the power spectrum of active forces on these organelles thanks to interrelated active and passive measures. Finally a computational model gives estimates of the force itself and hence of the number of the motors pulling on endosomes.

The dissipative contribution of myosin II in the cytoskeleton dynamics of myoblasts.

We have determined the microrheological response of the actin meshwork for individual cells. We applied oscillating forces with an optical tweezer to a micrometric bead specifically bound to the actin meshwork of C2 myoblasts, and measured the amplitude and phase shift of the induced cell deformation. For a non-perturbed single cell, we have shown that the elastic and loss moduli G' and G'' behave as power laws f (alpha) and f (beta) of the frequency f (0.01

Probing a nonequilibrium einstein relation in an aging colloidal glass.

We present a direct experimental measurement of an effective temperature in a colloidal glass of laponite, using a micrometric bead as a thermometer. The nonequilibrium fluctuation-dissipation relation, in the particular form of a modified Einstein relation, is investigated with diffusion and mobility measurements of the bead embedded in the glass. We observe an unusual nonmonotonic behavior of the effective temperature: starting from the bath temperature, it is found to increase up to a maximum value, and then decrease back, as the system ages. We show that the observed deviation from the Einstein relation is related to the relaxation times previously measured in dynamic light scattering experiments.

Assessment of mechanical properties of adherent living cells by bead micromanipulation: comparison of magnetic twisting cytometry vs optical tweezers.

We compare the measurements of viscoelastic properties of adherent alveolar epithelial cells by two micromanipulation techniques: (i) magnetic twisting cytometry and (ii) optical tweezers, using microbeads of same size and similarly attached to F-actin. The values of equivalent Young modulus E, derived from linear viscoelasticity theory, become consistent when the degree of bead immersion in the cell is taken into account. E-values are smaller in (i) than in (ii): approximately 34-58 Pa vs approximately 29-258 Pa, probably because higher stress in (i) reinforces nonlinearity and cellular plasticity. Otherwise, similar relaxation time constants, around 2 s, suggest similar dissipative mechanisms.