ABSTRACT To investigate the role of mechanical constraints in morphogenesis and development, we develop 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 (PDMS) 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, and its time evolution.

We study the out-of-equilibrium distribution of stochastic forces generated by molecular motors activity, exerted on a probe attached to the actin cortex of premuscular cells, as a function of ligand density, temperature and biological inhibitors.

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.

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.

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.

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.

We derive a new fluctuation-dissipation relation for non-equilibrium systems with long-term memory. We show how this relation allows one to access new experimental information regarding active forces in living cells that cannot otherwise be accessed. For a silica bead attached to the wall of a living cell, we identify a crossover time between thermally controlled fluctuations and those produced by the active forces. We show that the probe position is eventually slaved to the underlying random drive produced by the so-called active forces.

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.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are neurodegenerative diseases with an inexorably fatal outcome. They affect both human and other mammals, and are especially frequently found in Bovidae and Cervidae families (Aguzzi & Sigurdson, 2004). In the first family, sheep and goats can spontaneously develop the prion disease or scrapie (Grenn et al., 2008), whereas cattle only develop a disease, known as Bovine Spongiform Encephalopathy (BSE), after a contact with the infectious prion protein (Ducrot et al., 2008). In the second family, the chronic wasting disease (CWD) has been described in mule deer and elk. Besides these two Ruminant families, TSE was also retrieved in Carnivora, as Felidae (feline spongiform encephalopathy) and Mustelidae (transmissible mink encephalopathy) (Miller et al., 2008; Sigurdson & Miller, 2003). All these diseases are characterized by the accumulation of PrPSc, the abnormally folded isoform of the cellular prion protein (PrPC) in the brain (Prusiner 1998; Norrby 2011).

We derive a new fluctuation-dissipation relation for non-equilibrium systems with long term memory. We show how this relation allows one to access new experimental information regarding active forces in living cells that cannot otherwise be accessed. For a silica bead attached to the wall of a living cell, we identify a cross-over time between thermally controlled fluctuations and those produced by the active forces. We show that the probe position is eventually slaved to the underlying random drive produced by the so-called active forces.

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.

Using micrometric beads specifically bound to integrins of the cortex and trapped in optical tweezers, we applied a local constant force to myoblasts in culture. The application of a constant force allows to apply a controlled local stress to the cell and to measure its mechanical properties (creep experiment). We followed actin distribution during force application using cells that express GFP-actin. We observed within a few minutes an increase in the viscoelastic modulus of the cell, correlated to both a reinforcement of the cell anchoring to the bead (actin recruitment at the bead-cell contact) and a reinforcement of the entire actin cytoskeleton (actin recruitment up to several micrometers from the force application zone). We show that cell adaptation to mechanical environment can take place at short time scale.

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.